Document [original]

Determination of Drugs and Metabolites in Water by use of

Liquid Membrane Systems and HPLC

-Method development and application-

Der Fakultät für Naturwissenschaften

Department Chemie

Der Universität Paderborn

Zur Erlangung des Grades eines

Doktors der Naturwissenschaften

Dr. rer. nat.

Vorgelegte Dissertation

Von

Nabil N. Ahmad AL-Hadithi, M.Sc. in Chemistry

aus AL-Anbar/Irak

Paderborn 2007

The present work has been carried out during April 2004 and April 2007 at the

University of Paderborn, Faculty of Science, Department of Chemistry under

supervision of Prof. Dr. M. Grote.

1. Referent Prof. Dr. M. Grote

2. Referent Prof. Dr. M. Weiß

Eingereicht am 24. 05. 2007

Tag der mündlichen Prüfung: 28. 06. 2007

Acknowledgements

The thesis, the outcome of an intellectual journey over the past three years, would not have

been possible without many people’s support. I wish to convey my most sincere thanks to

those who have helped me in one way or another along the path.

First of all I owe sincere thanks to my doctoral advisor Prof. Dr. Manfred Grote, who

constantly supported this work with both general advice and detailed comments and who

created within his research unit a stimulating and cooperative environment.

I want to express my appreciation and high regard to Dr. Markus Borges for sharing his

knowledge to the synthesis the drug metabolites.

I am also indebted to Dr. Bedia Kutulus, for help with advice in analytical techniques

especially in liquid membrane extraction.

I would like to express my gratitude to Mrs. R. Knaup and the working group: Dipl.-Chem. C.

Schwake-Anduschus, Dipl.-Chem. H. Stevens, M.Sc. I. El Sharaa, Dipl.-Chem.-Ing. M.

Reinhard, Dipl.-Chem.-Ing. D. Hanim Yolcu, Ph.D. G. Abbaszade, Cand.Chem. M. Ewe and

Dipl.-Chem. M. Busse for creating a comfortable working atmosphere and help when need.

Special thanks to Dipl.-Chem.-Ing. H. Korste for his valuable help in LC-MS.

The personal fellowship and financial support of the German Academic Exchange Service

(DAAD) is gratefully acknowledged

Paderborn, June 2007 Nabil AL-Hadithi

Dedicated to:

Mum’s spirit and dad

Mum’s spirit and dadMum’s spirit and dad

Mum’s spirit and dad

Brothers and sisters,

Brothers and sisters,Brothers and sisters,

Brothers and sisters,

My

My My

My wife,

wife, wife,

wife,

My relatives and friends

My relatives and friendsMy relatives and friends

My relatives and friends,

,,

,

Table of Contents

Table of contents

Page

1 Introduction 1

1.1 Preface 1

1.2 Aim of study 2

1.3 Pharmaceuticals in the aquatic environment: Theoretical background 4

1.3.1 Pharmacokinetics 4

1.3.2 Exposure pathways 7

1.3.3 Occurrence and fate 7

1.3.4 Pharmaceuticals under study 8

1.3.4.1 Carbamazepine 8

1.3.4.2 Diclofenac 10

1.3.4.3 Ibuprofen

11

1.3.4.4 Sulfamethoxazole

13

1.4 Analytical extraction techniques

17

1.4.1 Liquid membrane extraction techniques

18

1.4.1.1 Bulk liquid membrane (BLM) 19

1.4.1.2 Supported liquid membrane (SLM) 21

2 Results and discussion

26

2.1 Methodical approach

26

2.2 Three liquid membrane extraction systems

28

2.2.1 BLM system

31

2.2.1.1 Influence of organic solvent

33

2.2.1.2 Influence of pH-gradient between feed and strip phases

34

2.2.1.3 Influence of extraction time 35

2.2.1.4 Influence of carrier in liquid membrane 36

2.2.2 Supported liquid flat membrane (SL-FM) system

40

2.2.2.1 Theoretical approach

41

2.2.2.2 Influence the composition of liquid membrane

44

2.2.2.3 Influence of extraction time

46

2.2.2.4 Influence of TOPO concentration on stripping process 48

Table of Contents

2.2.3 Conclusion

49

2.3 Supported liquid bag membrane (SL-BM) system

51

2.3.1 Extraction efficiency and enrichment factor

52

2.3.2 Extraction of metabolites IBU-2OH and SFM-Ac 53

2.3.2.1 Influence of composition of liquid membrane

54

2.3.2.2 Influence of TOPO concentration

54

2.3.2.3 Influence of extraction time

55

2.3.2.4 Influence of the pH of strip phase

56

2.3.2.5 Influence of strip phase volume 57

2.3.2.6 Influence of metabolite concentration

58

2.3.3

Extraction of active drugs CBZ, DCF, IBU and SFM

58

2.3.4

Comparison between the extractability of active drugs and metabolites by using

SL-BM systems 59

2.3.5

Extraction of IBU-2OH, SFM-Ac, DCF and IBU by SL-4-bag membrane

system (SL-4-BM) system

60

2.3.5.1 Reproducibility of extraction with the SL-4-BM

62

2.3.5.2 Influence of humic acids

63

2.3.6 Solid phase extraction (SPE)

65

2.3.7 Comparative study of SL-4-BM and SPE extraction techniques

67

2.3.8 Conclusion

69

2.4 Application: determination of drug traces in water by means of SL-4-BM

system and HPLC-UV

70

2.4.1 Influence of analyte concentration and matrix 70

2.4.2 Applications of the developed method to a real surface water

71

2.5 Development method based on LC/MS

73

2.5.1 Mass spectrometer parameters 73

2.6 Conclusions and outlook 78

3 Experimental 81

3.1 Synthesis of drug metabolites 81

3.1.1 CBZ-DiOH

81

3.1.2 DCF-4OH 81

Table of Contents

3.1.3 IBU-2OH 83

3.1.4 SFM-Ac

85

3.1.5 SFM-Glu

86

3.1.6

Characteristic data of drug metabolites 89

3.1.6.1 CBZ-DiOH.

89

3.1.6.2 IBU-2OH 90

3.1.6.3 SFM-Ac 91

3.1.6.4 SFM-Ac 92

3.2 Liquid membrane extraction 93

3.2.1 Development of HPLC-UV method 93

3.2.2 Validation of HPLC-UV method 94

3.2.3 BLM

98

3.2.4 SL-FM

98

3.2.5 SL-BM systems

98

3.2.5.1 Preparation of bag-membranes 98

3.2.5.2 SL-BM 99

3.2.5.3 SL-4-BM

100

3.3 SPE

101

3.4 Materials, equipments and chemicals

101

4 References

104

Abbreviations

A

-

Dissociated acidic compound

AH

Undissociated acidic compound

aq.

Aqueous phase

B

Undissociated basic compound

BH

+

Dissociated basic compound

BFR

Biofilm reactors

BLM

Bulk liquid membrane

C Carrier

CE

Capillary electrophoresis

C

F

Initial concentration of analyte in feed phase

C

F,i

Analyte concentration in feed phase at x = 0

C

FM

Analyte concentration in membrane phase at x = 0

C

M

Analyte concentration in membrane phase

C

MS

Analyte concentration in membrane phase at x = h

M

CoA

Acetyl coenzyme A

C

S

Concentration of analyte in strip phase

C

S,i

Initial concentration of analyte in strip phase

D

Diffusion coefficient

DAD

Diode array detector

dC

F

/dt

Mass balance equation in feed phase

dC

S

/dt

Mass balance equation in feed phase

DEHPA Di-(2-ethylhexyl)phosphoric acid

D

F

Diffusion coefficient for active form of analyte in feed phase

DHE Dihexyl ether

D

M

Diffusion coefficient for active form of analyte in membrane phase

D

S

Diffusion coefficient for active form of analyte in strip phase

E Extraction efficiency

ECD Electron capture detector

EC

50

Molar concentration of an agonist, which produces 50 % of the maximum

possible response for that agonist

Abbreviations

ED Electrodialysis

E

e(max)

Maximum enrichment factor

EROD Ethoxyresorufin-O-deethylase

F Feed phase

f

F

Linear flow velocity in feed phase

GC Gas chromatography

HPLC High performance liquid chromatography

h

F

Height (thickness) of feed phase

h

M

Height (thickness) of membrane

h

S

Height (thickness) of strip phase

IC Ion chromatography

J Overall flux

J

F

Flux of active form of the analyte in feed phase

J

F’

Flux of inactive form of the analyte in feed phase

J

M

Flux of analyte in membrane phase

J

S

Flux of active form of the analyte in strip phase

J

S’

Flux of inactive form of the analyte in strip phase

k Overall mass transfer coefficient

K

a

Dissociation constant

k

F

Mass transfer coefficient in feed phase

K

F

Partition coefficient of the analyte between feed and membrane phase

k

S

Mass transfer coefficient in strip phase

K

S

Partition coefficient of the analyte between strip and membrane phase

kV Kilo volt

LC Liquid chromatography

LLE Liquid liquid extraction

Log P

Partition coefficient

MDL Method detection limit

mg Microgram

min Minute

ml Milliliter

mol Mole

n Number of samples

Abbreviations

nm Nanometer

OcSA 1-octanesulfonic acid (sodium salt)

org.

Organic phase

Pc Critical displacement pressure

pH The negative logarithm of the hydrogen ion (H

+

) concentration

PME Polymeric membrane extraction

PP Polypropylene

PTFE Poly(tetra fluoro ethylene)

r Pore radius

S

rel

Relative standard deviation

S Strip phase

SLM Supported liquid membrane

SL-FM Supported liquid flat-membrane

SL-BM Supported liquid bag membrane

SL-4-BM Supported liquid membrane-4-bag membrane

SPE Solid phase extraction

STP Sewage treatment plants

TOF

Time-of-flight

TOPO

Tri-n-octylphosphine oxide

TWA

Time-weighted average

UV

Ultraviolet

V

F

Volume of feed phase

V

S

Volume of strip phase

w/w

Weight/ weight

α

Fraction of analyte in active form

α

F

Fraction of analyte in active form in feed phase

α

S

Fraction of analyte in active form in strip phase

Γ Interfacial tension

Θ Contact angle between the water and the membrane

ξ Membrane tortuosity

Ε Membrane porosity

∆C Concentration difference of neutral extractable analyte between feed and

strip

Abbreviations

Abbreviation of active drugs and metabolites

Active drug Metabolite

CBZ-

DiOH

10,11-dihydroxycarbamazepine

CBZ-

EP

10,11-epoxycarbamazepine

CBZ-

OH

1-hydroxycarbamazepine

CBZ-

2OH

2-hydroxycarbamazepine

CBZ-

3OH

3-hydroxycarbamazepine

CBZ

carbamazepine

CBZ-

10OH

10,11-dihydro-10-hydroxycarbamazepine

DCF-

4OH

4’-hydroxydiclofenac

DCF-

Glu

Diclofenac-N-glucuronid

DCF-

M

3’- methoxydiclofenac

DCF-

MOH

3’-hydroxy-4’-methoxydiclofenac

DCF-3OH

3’-hydroxydiclofenac

DCF-

DiOH

4’, 5-dihydroxydiclofenac

DCF

Diclofenac

DCF-

5OH

5-hydroxydiclofenac

IBU-

2OH

2-hydroxyibuprofen

IBU-

OH

1-hydroxyibuprofen

IBU-

CA

Carboxyibuprofen

IBU Ibuprofen

IBU-3OH 3-hydroxyibuprofen

SFM-

Ac

N4-acetylsulfamethoxazole

SFM-

Glu

Sulfamethoxazole-N1-glucuronide

SFM-

Me

5-methylhydroxysulfamethoxazole

SFM-

MOH

N4-acetyl-5-methylhydroxylsulfamethoxazole

SFM sulfamethoxazole

SFM-

NOH

N-hydroxysulfamethoxazole

*Bold letters: compound was used in this investigation

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1 Introduction

1.1 Preface

The issue of pharmaceuticals in the aquatic environment has raised increasing concern in

recent years. Human and veterinary pharmaceuticals are a group of “emerging” contaminants

[1], some of which are produced and used in increasingly large volume every year. The

amounts produced are reaching quantities similar to those of pesticides and other organic

pollutants. Residues of these biologically active compounds can enter the environment via

transport pathway-emissions during manufacture, disposal of unused or expired medicines,

human and animal excretion in urine and faeces, direct discharge of aquaculture products, and

manure and slurry spreading [2].

The majority of pharmaceutical compounds enter aquatic systems after ingestion and

subsequent excretion in the form of the non-metabolized parent compounds or as metabolites

via the sewage treatment network [3]. Several investigation have shown evidence that sewage

treatment plants (STPs) are not able to remove these drugs and their excretion metabolites

completely and they are discharged to different environmental compartments at

concentrations ranging from ng/L to µg/L [4]. In addition to river and sea water, recent studies

have shown they may even enter drinking water produced from ground water [4, 5]. Many

believe that of all the emerging contaminants, antibiotics are the biggest concern because of

the potential for antibiotics resistance. The increasing use of these drugs in the livestock,

poultry production, and fish farming during the last fives decades has caused a genetic

selection of more harmful bacteria, which is a matter of great concern [6]. However, other

pharmaceuticals compounds, especially polar one, such as anti-epileptics [7], analgesic and

anti-inflammatory drugs [8] also deserve particular attention.

Elimination of these pharmaceuticals in STPs was found to be rather low and consequently

sewage effluents are one of the main sources for these compounds and their recalcitrant

metabolites. Due to their physico-chemicals properties (high water solubility and often poor

degradability) they are able to penetrate through all natural filtration steps and enter

groundwater as well as drinking water [9]. In comparison with conventional pollutants, these

substances are designed to have specific pharmacological and physiological functions and

thus are inherently potent, often with unintended health outcomes in wildlife [10].

Particularly, there is an urgent need for the additional detection of metabolites as has been

demonstrated in the case of clofibrate or erythromycin: in both cases the active compounds

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could not be found in water samples but their main metabolites [10], which may also cause

severe (eco-) toxicological effects. The lack of information on the environmental fate of both

active drugs and their metabolites should be completed for many of the pharmaceuticals

applied. Consequently there is a necessity to monitor the input of pharmaceutical residues in

the different waterways, e.g. surface and groundwater, by means of sensitive chromatographic

methods. Hyphenated techniques such as LC-MS or GC-MS are preferred for the

predominantly polar compounds [11-13]. However, even those sophisticated methods may

need efficient sample pre-treatment to minimise the effects of the sample matrix and to enrich

the analytes.

To achieve the separation of pharmaceuticals from water samples, adsorption techniques such

as solid phase extraction (SPE) are mostly applied, however, the enrichment factors obtained

are not high (<100). Likewise synthetic membranes are often used but not with satisfactory

results particularly to small molecules [14]. In principal the liquid membrane extraction,

provide several advantages compared to other extraction methods. It offers a high selectivity

and, thus, an efficient cleanup from complex matrices by achieving high enrichment factors

and reduced use of organic solvent [15, 16]. Although liquid membrane extraction systems

were found to be effective for the acidic and basic drugs [17, 18], no information is currently

available on liquid membrane extraction of highly polar drugs from water samples. In the

present work attention was focused to develop certain types of liquid membranes: bulk liquid

membranes (BLM) and supported liquid membranes (SLM). Some of these systems have

been already successfully used for the separation of diclofenac and ibuprofen [20].

1.2 Aim of the study

The aim of the present study was to develop analytical methods for the determination of

selected metabolites and the corresponding active drugs of environmental concern in water

samples. The methods will be based on enrichment steps by means of certain liquid

membrane-systems (bulk type and SLM) and liquid chromatography (HPLC-UV or LC-MS).

The target metabolites were: 10,11-dihydroxycarbamazepine (CBZ-DiOH), 4′-

hydroxydiclofenac (DCF-4OH), 4-hydroxyibuprofen (IBU-2OH), N-4-acetylsulfamethoxazol

(SFM-Ac) and sulfamethoxazol-N1-glucuronid (SFM-Glu) and their parent drugs

carbamazepine (CBZ), diclofenac (DCF), Ibuprofen (IBU) and sulfamethoxazole (SFM)

(Table-1.1). The selection of these pharmaceuticals is based on their amounts applied for

medical purposes [21], their relative high concentrations found in the aquatic environment

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[22], their occurrence in the environment [23- 28] and their structural diversity (Table-1.1).

The selected metabolites CBZ-DiOH, DCF-4OH, IBU-2OH, SFM-Ac and SFM-Glu are not

commercially available; however, they are required to perform the membrane tests and to use

them as reference substances for the calibration of the chromatographic systems. Therefore,

the metabolites had to be synthesized. As a consequence the present study is divided into three

scopes:

• Synthesis of the selected metabolites.

• Investigation of the mass transfer of these compounds in liquid membrane systems.

• Development of analytical methods by combining membrane extraction and

determination by HPLC.

Table-1.1: Structure of selected pharmaceuticals

Pharmaceutical class Compound Chemical structure

Carbamazepine (CBZ)

N

H

2

N O

Antiepileptics

Carbamazepine metabolite:

10,11-Dihydroxycarbamazepine

(CBZ-DiOH)

N

HO

O

H

2

N O

Diclofenac (DCF)

Cl

H

N

C

l

O

HO

Diclofenac metabolite:

4′-hydroxydiclofenac (DCF-4OH)

Cl

H

N

C

l

O

HO

O

H

Ibuprofen (IBU)

CH

C

H

3

COOH

H

2

C

CH

H

3

C

CH

3

Analgetics and anti-

inflammatories

Ibuprofen metabolite:

4-Hydroxyibuprofen (IBU-2OH)

CH

3

COOH

H

2

C

H

3

O

H

CH

3

Sulfamethoxazole (SFM)

S

O

N

H

2

NN O

C

H

3

Sulfamethoxazole metabolite:

N-4-acetylsulfamethoxazol

(SFM-Ac)

S

O

ON

H

NN O

C

H

3

H

3

C

O

Antibiotics

Sulfamethoxazole metabolite:

Sulfamethoxazol-N1-glucuronide

(SFM-Glu)

S

O

NH

2

NN O

C

H

3

O

COOH

H

O

OH

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1.3 Pharmaceuticals in the aquatic environment: Theoretical background

1.3.1 Pharmacokinetics and drug metabolism

A major factor determining the occurrence of pharmaceuticals in the aquatic environment is

their pharmacokinetic behavior which describes the times course of a drug and metabolites in

the human body after any kind of administration [33]. Drug metabolism, or biotransformation,

is a major route by which drugs are eliminated from the body [29]. The metabolism of

pharmaceuticals occurs by (phase I) and/ or conjugation (phase II) functionalization reactions,

which usually resulted into polar, water-soluble, and extractable metabolites via urine and

faeces [30].

The clinical response to a therapeutic agent is related to the plasma concentration of the drug

and/ or its metabolism. It has been well established that the metabolism of a drug is often a

major determinant of the duration and intensity of its clinical effects. Metabolic processes are

necessary to convert a lipophilic drug into one or more metabolites, which are more water

soluble than the parent drug, facilitating urinary excretion [30]. Drug metabolism may yield

inactive metabolites, but this is not always true. In some cases, metabolites have

pharmacological activity similar to that of parent drug: well known examples are many

benzodiazepines, whose long-lived active metabolites cause their effects to persist after the

parent drug has been eliminated, or some antidepressants, such as imipramine and

amitriptyline, whose antidepressant action is partially due to their metabolites

desmethylimipramine and nortriptyline [30]. In some cases, a drug referred to as pro-drug,

becomes pharmacologically active only after metabolism: an example is the angiotesin-

converting enzyme inhibitor enalapril that exerts its pharmacological activity through is

metabolite enalaprilat. Metabolism can alter the pharmacological properties of a compound

qualitatively: for example, salicylic acid shares with its parent drug acetylsalicylic acid the

anti-inflammatory, but not the antiplatelet activity. There are also cases in which the

metabolism yields toxic compounds: an example is the hepatotoxicity of paracetamol, caused

by drug metabolising N-acetyl-p-benzoquinone imine [31]. Hence, variability in activity of

drug metabolising enzymes can lead to interindividual differences in drug effects, one of the

major problems in drug therapy [31].

Drug metabolism involves a wide rang of chemical reactions, including oxidation, reduction,

hydrolysis, hydration, conjugation, condensation, and isomerization [30]. The enzymes

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involved are present in many tissues (like brain, lung, intestine and testicle) but generally are

more concentrated in the liver. For many drugs, metabolism occurs in two apparent phases:

Phase I reactions include oxidative, reductive and hydrolytic biotransformation. The purpose

of these reactions is to introduce a polar functional group (e.g. -OH, -COOH, -NH

2

, -SH) into

the drug molecule. This can occur through direct introduction of the functional group (e.g.

aromatic and aliphatic hydroxylation) or by modifying existing functionalities (e.g. reduction

of ketone and aldehydes to alcohols; oxidation of alcohols to acids; hydrolysis of ester and

amides to yield COOH, NH

4

and OH groups; reduction of azo and nitro compounds to give

NH

2

moieties and oxidative -N, -O, -S dealkylation to give -NH

2

, -OH and -SH groups. Phase

I products are often more reactive and sometimes more toxic than the parent drugs [31].

Cytochrome P-450: The most important enzyme system of phase I metabolism is cytochrome

P-450, a microsomal superfamily of isoenzymes that transfer electrons and thereby catalyze

the oxidation of many drugs, which located in the membrane of the smooth endoplasmic

reticulum, mainly in the liver, but also in extrahepatic tissues (e.g. intestinal mucosa, lung,

kidney, brain, lymphocytes, placenta, etc.) [31]. The electrons are supplied by NADPH-

cytochrome P-450 reductase, a flavoprotein that transfers electrons from NADPH (the

reduced form of nicotinamideadenine dinucleotide phosphate) to cytochrome P-450 (see

Figure-1.1) [32]. Cytochrome P-450 enzymes are grouped into 14 mammalian gene families

that share sequence identity and 17 subfamilies. Enzymes in the 1A, 2B, 2C, 2D and 3A are

most important in mammalian metabolism; CYP1A2, CYP2C9, CYP2C19, CYP2D6, and

CYP3A4 are important in human metabolism.

NADPH

NADP

+

O

2

O

2-

H

2

O

Cytochrome P-450

reductase

[oxidized]

Cytochrome P-450

reductase

[reduced]

e

-

Drug-H

P-450[Fe

2+

]

e

-

Drug-H

P-450[Fe

2+

]

Drug-OH

P-450[Fe

2+

]

Drug-H

P-450[Fe

3+

]

Drug-H

2H

+

Figure-1.1: Phase I metabolism pathways (oxidation reactions) [31, 32]

Phase II reactions from water-soluble conjugated products by reaction with polar and

ionisable endogenous compounds such as glucuronic acid, sulphate, glycine and other amino

acids to the functional groups of phase I metabolites. Conjugated metabolites are readily

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excreted in the urine and are generally pharmacologically inactive and non-toxic. Other phase

II pathways, such as methylation and acetylation, serve to terminate biological activity, while

glutathione conjugation serves to protect the body against chemically reactive compounds.

Drugs that already have existing functional groups, such as OH, COOH, and NH

2

, are often

directly conjugated by reactions with phase II enzymes [30].

1- Glucuronidation: Glucuronidation is the most widespread of the conjugation reactions

probably due to the relative abundance of the cofactor for the reaction, UDP-glucuronic acid.

UDP-glucuronic acid (uridine diphosphoglucuronic acid), being part of intermediary

metabolism and closely related to glycogen synthesis, is found in all tissues of the body. The

enzymes involved are located in the cytosol. UDP-glucuronic acid can be considered as an

energy-rich intermediate for the transfer of the glucuronic acid moiety [30].

N-glucuronide conjugation is one of the conjugation reactions which come from the reaction

between amines (mainly aromatic), amides and sulfonamides with UDP-glucuronic acid as

shown in Figure-1.2.

R

Figure-1.2: Phase II metabolism (N-glucuronide conjugation reaction) [30]

R NH

2

O

COOH

OH OH

O-UDP

OH UDP

O

COOH

OH OH

OH

HN

2- Acetylation

: acetylation reactions are typical for aromatic amines and sulfonamides and

require the co-factor, acetyl-CoA (Figure-1.3). Acetylation takes place mainly in the liver and

can be also in the reticuloendothelial cells of the spleen, lung and gut [30].

R

Figure-1.3: Phase II metabolism (acetylation reaction)[30]

SH

N

CH

3

O

R S NH

2

acetyl-CoA CoA-SH

O

1.3.2 Exposure pathway

Production and application of human and veterinary pharmaceuticals lead to a potential

environment exposure and potentially to an accumulation in certain environmental

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compartment. After their use, pharmaceuticals are excreted uncharged and/or as metabolites in

feces and urine and hence are present in wastewater [10].

Similar to other compounds of anthropogenic origin, the fate of the pharmaceuticals residues

during sewage treatment can follow one or a combination of three types of behavior:

a) biodegradation, b) sorption of the residues onto sewage sludge or c) elimination [21]. The

proportion of the pharmaceutical that is retained in sewage treatment either due to

biodegradation or by sorption to sludge strongly depends on its chemical structure and

physico-chemical properties, but also on the specific conditions within the respective plant.

Water temperature, residence times corresponding to flow rates, dilutions with rainwater and

sludge age were found to have influence on elimination efficiencies [10].

Hence, compounds that are not readily degradable enter the environment either with the

digested sludge or as dissolved pollutants in the sewage treatment plant (STP) discharges. The

latter scenario results in the contamination of the receiving waters and finally, the aquatic

environment [21].

1.3.3 Occurrence and fate

Numerous studies have been conducted to investigate various aqueous matrices for the

presence of pharmaceuticals residue, comprising the target compounds and metabolites. In

fact, these residues have been found to be ubiquitous in environmental waters. The main

contributing factor for the occurrence of pharmaceuticals in the aquatic environment is the

elimination efficiency of the STP [34].

As described before, many pharmaceuticals are excreted to a large extent as transformed

phase I metabolites and/or after conjugation to hydrophilic groups as phase II metabolites.

Conjugates are easily cleaved in the STP, causing a re-formation of original pharmaceuticals

[34]. This might lead to higher concentration in the STP effluent than in the raw wastewater.

Residues of various pharmaceuticals are present in the low µg

/

L rang in STP effluents.

Discharge of the STP effluent into the receiving waters leads to a dilution of the

pharmaceuticals residues which occur up to the ng/L range in contaminated surface water.

Once introduced into the surface waters, pharmaceuticals may undergo biodegradation, most

likely due to co-metabolic processes. For some pharmaceuticals, photo induced degradation

may occur from natural solar radiation [35].

The determination of the environmental fate of a compound is a complex issue.

Transformation and distribution processes are strongly dependant on the specific

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environmental conditions, which lead to a sophisticated linkage of individual systems

parameters.

By (continuous) exposure to low concentrations of pharmaceuticals in the theory, the

following negative effects on aquatic organisms are possible:

•

Ecotoxicological effects.

•

Pharmaceuticals effects.

•

Resistance development of micro-organism.

It is clear that during the past few years a wealth of data has become available on the levels of

pharmaceuticals in the environment and their effects on the aquatic and terrestrial organisms.

There are however, still many questions that need to address before we can eventually

determine whether residues in the environment are a threat to human and environment

health.

1.3.4 Pharmaceuticals under study

1.3.4.1 Carbamazepine (CBZ)

Carbamazepine (CBZ) is an established drug for the control of grand and psychomotoric

epilepsy and it is also effective in the treatment of trigeminal neuralgia. Furthermore, it is

presently used in bipolar depression. It is predominantly eliminated in the liver, where it is

metabolised to 10,11-epoxycarbamazepine (CBZ-EP) and other derivatives (Figure-1.4).

CBZ-EP seems to have antiepileptic properties as well as CBZ itself [36, 37].

CBZ undergoes extensive hepatic metabolism by the cytochrome P-450 (CPY) system.

Thirty-three metabolites of CBZ have been identified from human and rate urine. The main

metabolic pathway of CBZ is oxidation to 10,11-epoxycarbamazepine (CBZ-EP), hydration to

10,11-dihydroxycarbamazepine (CBZ-DiOH) and 10-hyroxycarbamazepine (CBZ-10 OH),

then conjugation of these compounds with glucuronide. The second minor distinct pathway

for the biotransformation of CBZ, catalyzed by cytochrome P-450 (CPY), involving oxidation

to 1-hydroxcarbamazepine, 2-hydroxycarbamazepine and 3-hydroxycarbamazepine, and

subsequent conjugation glucuronide see Figure-1.4 [38-41].

In human lymphocytes, CBZ is metabolized by CYP dependant monooxygenase into CBZ-

EP, an active and toxic metabolite which is then transformed into the corresponding CBZ-

DiOH by an epoxide hydrolase (Figure-1.4). A strong and specific CYP1A inhibition was

observed from the CBZ biotransformation into reactive metabolites [42] (Table-1.2).

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Environmental field studies have shown that the CBZ is one of the most frequently detected

pharmaceuticals in sewage treatment plant effluent, in river water, and in seawater [43].

Investigations of influent and effluent samples from different municipal STPs have shown that

CBZ is not significantly removed (less than 10 %) during sewage treatment (Table-1.3). Thus,

CBZ has been detected at concentrations more than 1

µ

g/L in surface water [44- 46]. Also

different studies have shown that CBZ is not biological degradable (Table-1.3), this explains

why CBZ has been detected in a number of groundwater samples [47- 50] (Table-1.4). In

addition all five metabolites of CBZ (Figure-1.4) were detected in the STP influent and

effluent samples. Only CBZ and CBZ-DiOH were detected in the surface water (Table-1.4).

N

CONH

2

N

CONH

2

N

CONH

2

N

CONH

2

N

CONH

2

N

CONH

2

N

CONH

2

OH

CBZ

CBZ-OH

CBZ-2OH

CBZ-EP

CBZ-3OH

OH

O

HO OH

Glucuronid conjugate

CBZ-10OH CBZ-DiOH

Glucuronid conjugate

G

l

u

c

u

r

o

n

i

d

c

o

n

j

u

g

a

t

e

Figure-1.4: Metabolic pathways of carbamazepine

1.3.4.2 Diclofenac (DCF)

Diclofenac is a nonsteroidal anti-inflammatory drug (NSAID). In pharmacologic studies, DCF

has shown anti-inflammatory, analgestic, and antipyretic activity. DCF is indicated for the

acute and chronic treatment of signs and symptoms of osteoarthritis and rheumatoid arthritis.

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In addition, DCF is indicated for the treatment of ankylosing spondylitis and for the

management of pain and primary dysmenorrheal [51].

In human, metabolism of DCF is mediated by both glucuronidation and oxidative

biotransformation [51]. Oxidation of the aromatic rings is mediated by cytochromes P-450

(CYP). Hydroxylation of the dichlorophenyl ring is catalyzed specifically by CYP2C9 to

produce 4

′

-hydroxydiclofenac (DCF-4OH) as the major metabolite [52]. Another five

metabolites of DCF have been identified in human plasma and urine [53]. The metabolites

include 3

′

-hydroxydiclofenac (DCF-3OH), 5-hydroxydiclofenac (DCF-5OH), 4’,5-

dihydroxydiclofenac(DCF-DiOH), 3

′

-hydroxy-4

′

-methoxydiclofenac (DCF-MOH), 3

′

-

methoxydiclofenac (DCF-M), and diclofenac-N-glucuronid (DCF-Glu), [53] (Figure-1.5).

Approximately 65 % of the dose is excreted in the urine, and approximately 35 % in the bile.

Little Conjugates of uncharged DCF account for < 1 % of the dose excreted in the urine and

for less than 5 % excreted in the bile. 15 % of unchanged unconjugated drug is excreted by via

urine see Table-1.3.

The DCF exerted a cytotoxic effect at 500 µM, which is in agreement with previous results on

rat or human hepatocytes [54]. The mechanism of DCF cytotoxicity is not fully understood

but there is some evidence that both uncoupling of mitochondrial oxidative phosphorylation

and CYP-mediated metabolism are involved in human and rat hepatocytes acute toxicity [55].

It was observed a clear inhibition of EROD (Ethoxyresorufin-O-deethylase) at 36 µM,

suggesting a specific interaction between DCF or its metabolites with this enzyme in rainbow

trout [42] (Table-1.2).

Approximately, 86 tons of the prescriptions DCF are annually sold in Germany (Table-1.3). In

long-term monitory investigations of sewage and surface water samples (Table-1.4), DCF

identified as one of the most important pharmaceuticals present in the water-cycle. The

removal rates which have been reported of DCF in STPs were between 9-75 % [56, 57]

(Table-1.3).

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OCOOH

HO OH

OH

Figure-1.5: Metabolic pathway of diclofenac[53]

ClCl NH

COOH

HO

ClCl NH

COOH

OCH

3

OH

ClCl NH

COOH

OH

ClCl NH

COOH

HO

ClCl NH

COOH

HO

OH

ClCl NH

COOH

ClCl N

HOOC

DCF-MOH

DCF-Glu

DCF

DCF-DiOH

DCF-3OH

DCF-4OH

DCF-5OH

1.3.4.3 Ibuprofen (IBU)

Ibuprofen (IBU) is a nonsteroidal anti-inflammatory drug (NSAID). IBU possesses analgetic

and antipyretic activities. Its mode of action, like that of other NSAIDs, is not completely

understood, but may be related to prostaglandin synthetase inhibition. IBU is indicated for

relief of the signs and symptoms of rheumatoid arthritis and osteoarthritis. IBU is also

indicated for relief of mild to moderate pain and for the treatment of primary dysmenorrheal

[51]. It is on the most important pharmaceuticals in term of quantities consumed seeing

Table-1.3.

Oxidative metabolism by using cytochrom P-450 is the major route for biotransformation of

IBU. Four oxidative metabolites have been identified in urine and plasma samples obtained

from human after oral intake of IBU [58] (Figure-1.6). In humans the parent drug, as well as

the metabolites, are found to be conjugated with glucuronic acid [59, 60], and glucuronidation

has in all cases taken place at the carboxyl group in the propionic acid side chain. Studies

have shown that following ingestion of the drug, 45 % to 79 % of the dose was recovered in

urine within 24 hours as metabolites: 2-hydroxyibuprofen (IBU-2OH) 25 %, and 37 % as

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carboxyibuprofen (IBU-CA); the percentages of free and conjugated ibuprofen were 1-8 %

and 14 %, respectively see Table-1.3 [51].

IBU has been shown to significantly affect the growth of several bacterial and fungal [61-63].

Some studies suggests depending with evidences that IBU metabolites are non toxic for

aquatic organisms tested, and may however have growth stimulating properties [64, 65]

(Table-1.2).

Because of large amounts produced and used (Table-1.3), IBU is an environmentally relevant

compound. In sewage water, sewage effluents, and surface water ibuprofen was detected

among other pharmaceuticals residues in the range of ng-

µ

g/L [59] (Table-1.4). IBU is

degradable in the human body to its principal metabolites 1-hydroxyibuprofen (IBU-OH) and

IBU-CA, which are found together with IBU in raw sewage. Some studies observed a

significant removal of IBU and especially of IBU-CA during sewage treatment, whereas the

concentration of IBU-OH in the sewage effluents was almost similar to those in the influents

[44]. Degradation experiment in both biofilm reactors (BFR) and batch experiments with

activated sludge (BAS) reveal IBU-OH as the major metabolite of IBU under oxic conditions,

and IBU-CA under anoxic conditions. Efficient elimination (95-99 %) of all these compounds

(IBU, IBU-CA, and IBU-OH) was found in the municipal STPs [60]. Other studies indicate

that microbial biofilm and other microbial activity play an important role in the degradation of

IBU in the surface water systems and the degradation pathway and resultant metabolism differ

from those observed in human metabolism. Thus, IBU-OH was found in surface water at

much higher concentration than IBU or IBU-CA [66].

OH

O

OH

O

CH

2

OH

O

OH

OOH

O

IBU-3OH

IBU-CA

IBU-2OH

IBU-OH

HO O

HO

HO IBU

Glucuronic acid conjugate Glucuronic acid conjugate

Figure-1.6: Metabolic pathway of ibuprofen [60]

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1.3.4.4. Sulfamethoxazole (SFM)

Sulfamethoxazole (SFM) is a member of the sulfonamide family of antibiotics for human and

animal. It is one of the most widely used anti-bacterial agents [67].

The known metabolism of SFM involves acetylation and oxidation leading to N4-

acetylsulfamethoxazol (SFM-Ac), and N-hydroxysulfamethoxazole (SFM-NOH) (Figure-9).

Hydroxylation take also place to SFM metabolism leading to 5-methylhydroxy-

sulfamethoxazole (SFM-Me), and N4-acetyl-5-methylhydroxylsulfamethoxazole (SFM-

MOH). Moreover SFM is glucuronidated leading to sulfamethoxazole-N1-glucuronide (SFM-

Glu) [68] (Figure-4.4). About 50-60 % of applied dose in human body was excreted as the

inactive metabolite (SFM-Ac), 15 % as the conjugate metabolite (SFM-Glu), and only 15-20

% as the uncharged active compound [6] (Table-1.3).

The SFM was not cytotoxic enough to calculate EC

50

values; it inhibited EROD activity right

from 125 µM (Table-1.2). In human liver microsomes, SFM is described as a selective

inhibitor of CYP2C8 and CYP2C9 that would loose selectivity for the CYP isoforms at

concentrations higher than 500 µM [69]. As a result, SFM must be a selective inhibitor of

CYP1A enzymes in fish hepatocytes [42] (Table-1.2).

Most of antibiotics are metabolized only incompletely by patients after administration and

enter municipal sewage and sewage treatment plants. If they are not eliminated during sewage

treatment plants they are emitted into surface water and may reach drinking water [70].

SFM has been detected in sewage discharge at concentration of 0.62

µ

g/L [71], and detected

SFM in 12.5 % of surface water samples at a median concentration of 0,15

µ

g/L [72, 73].

Also it was reported that SFM could be removed about 67 % during the biological step in

municipal sewage treatment plant [44] as shown in Table-1.3.

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S

O

OH

NNO

H

2

NCH

3

S

O

OH

NNO

HN CH

3

S

O

OH

NNO

H

2

NCH

2

OH

S

O

OH

NNO

HN CH

2

OH

O

CH

3

S

O

OH

NNO

HN CH

3

O

CH

3

S

O

ONNO

H

2

N

C

H

3

SFM-Ac

SFM-Me

SFM-NOH

SFM-Glu

SFM

SFM-MOH

OOH

OH

Figure-1.7: Metabolic pathways of sulfamethoxazole [67]

Table-1.2: Reported effects of active parent drugs on aquatic and terrestrial organisms

Substance Reported effect Reference

Ibuprofen Stimulation of growth of cyanobacteria and inhibition of

growth of aquatic plants [62]

Diclofenac Inhibition of basal EROD activity in cultures of rainbow

trout hepatocytes [42]

Carbamazepine Inhibition of basal EROD activity in cultures trout

hepatocytes.

Inhibition of emergence of

Chironomus riparius

[42, 81]

Sulfamethoxazole

Inhibition of basal EROD activity in cultures of rainbow

trout hepatocytes [42]

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Table-1.3: Active drugs under study: basic properties and ecotoxicological data

a

Germany in 2001

b

UK in 2000

c

Australia in 1998

d

France in 1998

e

Denmark in 1997/1998

Parameter SFM CBZ DCF IBU

Formula [74] C

10

H

11

N

3

O

3

S C

15

H

12

N

2

O C

14

H

11

Cl

2

NO

2

C

13

H

18

O

2

Molecular

weight

(g/mol) [74]

253.28

236.27

296.15

206.28

Solubility in

water (mg/L)

[74]

610

112

242

291

Melting point

[74] 167-169

o

C 190-192

o

C

156-158

o

C 78.87

o

C

Dissociation

constant (p

Ka

)

[74]

5.81±0.5

1.39±0.1

13.94±0.2 4.18±0.2 4.41±0.2

Octanol-water

partition

coefficient

(Log

P

) [74]

0.887±0.419

2.673±0.376 3.284±0.361 3.722±0.227

Estimated

amount used

(tons/year)

[22]

54

a

88

a

, 40

b

, 10

c

, 38

d

86

a

, 26

b

344

a

, 162

b

, 14

c

, 34

e

Activated

sludge Activated

sludge Biologic

filter Activated

sludge Biologic

filter

Total removal

via wastewater

treatment [75]

67 % 7-10 % 69-75 % 9 % 90-99 % 65 %

Predicted

environmental

concentrations

for surface

water (PEC

SW

)

(ng/L) [74]

895

a

1460

a

…. …..

Environmental

risk indicators

[75]

High volumes;

detected in the

environment;

concerns over

toxicity and

antibacterial

resistance

High volumes;

long-term

prescription;

persistent

Very high

prescription and

over-the-counter;

detected in the

environment

Very high

prescription and

over-the-counter;

detected in the

environment

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Table-1.4: Detected concentrations (

µ

g/L) for active drugs and their selected metabolites in

different water sources

Substance Inflow

wastewater Effluent

wastewater Surface

water Ground

water Drinking

water References

3.3- 0.99 0.1, 0.37, 2 [21]

0.37 0.07 [54]

1.642 0.12 n.d. [77]

IBU

1.5, 0.87, 85

2.7 [2]

IBU-2OH 0.92 0.34 [2]

IBU-CA 0.02 [2]

3.55-0.5 3 - 1.18 [77]

3.02 2.51 >1 [2]

0.81 0.15 [54]

DCF

0.359 0.194 [43]

2.410-0.55 1.67-0.73 [77]

2.1 1.075 1.1 0.03 [2]

0.8-0.1 0.25-0.03 [46]

6.3 2.1 [78]

1.78 1.63, 2.1 [21]

CBZ

0.368 0.426 0.0007 [43]

CBZ-EP 0.047 0.0523 n.d. [43]

CBZ-DiOH

1.571 1.325 0.0022 [43]

CBZ-2OH 0.121 0.132 n.d. [43]

CBZ-3OH 0.094 0.101 n.d. [43]

> 1.0 0.1-0.2 0.4 [6]

0.243-0.871 0.008 0.1 [54]

0.1-1.7 0.05-0.09 6 [46]

0.41 [2]

n.d. 0.4, 0.9 [21]

SFM

0.343 0.352 [25]

2.2 [79]

0.316 [80]

2.2-0.690 0.24 [70]

SFM-Ac

0.518 0.082 [25]

n.d.: not detected

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1.4 Analytical extraction techniques

In recent years, several analytical techniques and methods have been developed for analysis of

various pharmaceuticals and corresponding metabolites in environmental and biological

samples. Despite the achievements in analytical science, there are still challenges. One

challenge lies in determining pharmaceuticals in various complex matrices such as

wastewaters, surface waters, sediments, and biological fluids. Most developed analytical

methods require several steps consuming time and solvents. In ecological risk assessment for

chemical pollutants, it is important to quantify the concentrations of freely dissolved in

aqueous samples for approximate characterization of the bioavailable fraction. The challenge

of chemical analysis, especially speciation studies, and determining the freely dissolved

pharmaceuticals in a complex sample is staggering. Moreover, components of interest exist at

trace levels. These challenges have made sample preparation become a key step in modern

chemical analysis. It is essential part of any analytical procedure because of the reasons:

sample preconcentration or enrichment and removal of contaminants [82].

The most widely used sample preparation techniques are liquid-liquid extraction (LLE) [83]

and solid-phase extraction (SPE) [14]. LLE is the traditional technique for extraction of

organic analytes from aqueous solutions. The basis is the partition of the dissolved analytes

between the organic phase (extraction liquid) and the aqueous solution (sample solution)

according to their partition coefficients. Further shifting of the equilibrium toward the organic

phase brings about increased enrichment in the organic phase. The technique is well known

and still widely used, although now it is less attractive and is being replaced by other

techniques. This is because LLE:

•

is tedious and time-consuming especially when extracting aqueous complex samples,

which demands many steps before a clean extraction can be obtained;

•

forms emulsions which at times makes it difficult to separate the two phases;

•

is environmentally unfriendly due to large volumes of organic solvents used.

However, with LLE, large enrichment factors can be obtained despite the cited

drawbacks.

SPE techniques are perhaps the most popular in sample preparation especially for organic

analysis. The principle of SPE is based on sorption of analytes on a sorbent. The aqueous

sample solution passes the SPE column, and the analytes are first trapped on the sorbent and

then eluted with a suitable small volume of organic solvent. Extraction and enrichment of the

analytes is thereby simultaneously achieved. Most sorbents are now available as disks,

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cartridges, or precolumns. Despite its simplicity, it lacks selectivity when extracting analytes

in complex matrices such as plant extracts, foodstuffs, and wastewater [84].

There are a number of different membrane techniques, which have been suggested as

alternative to the SPE and LLE techniques. An area enjoying much attention by various

research groups is developing membrane-based extraction techniques that are simple, cheap,

and miniaturized [85, 86].

1.4.1 Liquid membrane extraction techniques

Generally, a membrane can be classified as a selective barrier between two phases [15, 16].

With a driving force applied across a membrane, analytes can be transferred from one phase

(feed phase) to another (strip phase). The main membrane techniques that have been used for

analytical applications can be classified based on whether membrane is porous or nonporous

during the extraction of the sample solution [84].

In porous membrane systems, the liquids on either side of the membrane are physically

connected through the pores. These membranes are used in dialysis, electrodialysis, and

filtration (Table-1.5) to separate low-molecular-mass analytes from high-molecular-mass

matrix components, leading to an efficient clean-up but no discrimination between different

small molecules. No enrichment of the small molecules is possible; instead the analytes are

diluted since the driving force of the mass transfer process is a simple concentration

difference across the membrane.

Nonporous membrane is utilized in membrane extraction techniques. Such a membrane is

liquid or solid (such as a polymeric) phase that is placed between two other phases-usually

liquid but can also be gaseous [16]. A nonporous membrane may in fact have pores, but these

are usually micropores. The nonporous membrane extraction techniques include bulk liquid

membrane (BLM), supported liquid membrane (SLM), microporous membrane liquid-liquid

extraction, semipermeable membrane devices, polymeric membrane extraction, and

membrane extraction with a sorbent interface [87] see Table-1.5.

A wide variety of membrane materials are available. Porous synthetic membranes, such as

polypropylene, polysulphone and cellulose derivative, are most widely employed. Ion-

exchange membranes and nonporous membrane are commonly used as well. The pore sizes of

the membranes vary with the technique applied. In dialysis, for example, the pore sizes range

from a few nanometers to 100 nm, while in SLM from 0.1-10

µ

m. Most of the applications

involve the use of a planar flat-sheet membrane, but hollow fibre membranes are also

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available. The liquid membrane techniques have been coupled with liquid chromatography

and gas chromatography and they have been applied to gaseous and liquid samples [84].

Table-1.5: Different major membrane techniques used in analytical applications [85]

Abbr.*: Abbreviation

1.4.1.1 Bulk liquid membrane (BLM)

It is a liquid phase, usually organic, interposed between two miscible aqueous solutions. At

one side of the membrane (feed phase) the material to be transported is extracted, while at the

other side (strip phase) re-extraction occurs. Since in each of the aqueous phase some specific,

and different for each of them, thermodynamic conditions exist, the extraction and re-

extraction occur simultaneously.

The BLM technique requires a simple design of the cell for the transport process to be

realized (Figure-1.8). The membrane liquid is placed above the feed and strip solutions,

separated with a solid barrier, thus being in contact with both of these solutions [88-91].

Name Abbr.* Type Membrane

contents

Phase combinations

used

feed/membrane/strip

Dialysis ….. porous

membrane porous

membrane aq/membrane/aq

Electrodialysis ED porous

membrane porous

membrane aq/membrane/aq

Filtration …. porous

membrane porous

membrane aq/membrane

Bulk liquid

membrane BLM nonporous

membrane organic liquid aq/org/aq

Supported liquid

membrane SLM nonporous

membrane porous polymer

soaked with

organic liquid

aq/membrane/aq

Microporous

membrane

liquid-liquid

extraction

MMLLE nonporous

membrane porous polymer

soaked with

organic solvent

aq/membrane/org

Semipermeable

membrane

devices

SPMDs nonporous

membrane polymer aq/polymer/org

Polymeric

membrane

extraction

PME nonporous

membrane polymer aq/polymer/aq,

org/polymer/aq,

aq/polymer/org

Membrane

extraction with

sorbent interface

MESI nonporous

membrane polymer gas/polymer/gas,

liquid/polymer/gas

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There are two basic principle how the BLM operate (Figure-1.9). In the first, simple case, the

analyte, A, is transferred from the feed phase into the membrane, because of its larger

solubility in the organic phase, and then from the membrane into the strip phase, where

conditions prevent back-extraction of the analyte (Figure-1.9a), this is the so-called

“unfacilitated transport”. For the second case, “facilitated transport” (Figure-1.9b), the

appropriate solubility of the analyte A in the membrane phase is not required since the analyte

interacts with the carrier molecule, C, dissolved in the membrane phase. This carrier should be

totally insoluble in both feed and the strip phases and should react and reversibly with the

analyte [88, 92, 93].

A

D

A

D

AAA

Figure-1.9: Schematic representation of transport mechanisms of analyte through bulk

liquid membrane (a) unfacilitated transport, (b) facilitated transport [88]

Membrane

Feed phase Strip phase

C A

C D

MembraneFeed phase Strip phase

-

a

-

b

-

1.4.1.2 Supported liquid membrane (SLM)

In an SLM extraction, an organic solvent is immobilized in the pores of an inert support

material, separating the aqueous feed and the strip phases (Figure-1.10). The analytes are

partitioned from the aqueous sample stream into the organic membrane and are then re-

extracted into the aqueous strip phase. The driving force is the difference of the analyte

concentration between the feed and strip phases. In order to maintain the concentration

gradient across the two phases, the solutes must be able to exist in two forms: in a nonionic

form on the feed side to extract into the membrane and in an ionic form on the strip side in

Figure

-

1

.

8

:

Bulk liquid membrane, (1) feed phase (2) membrane

(organic solvent) and (3) strip phase

3

1

2 2

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order to irreversibly trapped [15, 16, 82, 84, 85]. This is most simply achieved by pH

adjustments in the two phases, and the method is, therefore, particularly well suited for

ionizable compounds such as medium-to-weak acids and bases [94- 97]. SLM extraction can

provide very selective enrichment. Selectivity can be fine-tuned by proper choice of the

conditions in the three phases as seen in Figure-1.10 [98]. This creates a selectivity window

such that by the time the analytes are enriched in the strip phase, an indirect structural

recognition is achieved and only analytes belonging to the same family are generally trapped

at a time. Macromolecules are discriminated on the basis of their size while charged

compounds are too polar dissolve into the organic liquid. Natural molecules merely distribute

between the three phases without any enrichment.

A

AHAH

AH OH A

B

BH

OH B

BH

Figure-1.10: Principle of SLM extraction of an acidic analyte A, a basic analyte B [98]

Feed Low pH

Strip High pH

_

__

Feed High pH

Strip Low pH

+

Liquid

Membrane

_

Liquid

Membrane

Often, selective transport based on relative differences in solubility in the membrane and

trapping in the strip phase may be difficult to achieve. In another case, the solubility of the

analyte may be too low to give efficient extraction even when the trapping in the strip can

easily be realized. A good approach in such a case is to incorporate a mobile carrier into the

membrane that selectively binds the analytes. The idea of incorporating a carrier also allows

SLM extraction to be a variety of compounds such as permanently charged chemical species

like metal ions. It also gives different versions of carrier-mediated transport mechanism such

as simple carrier transport (with chemical reaction in the strip), coupled co-transport [15, 99,

100], and coupled counter-transport [83]. In simple carrier transport, the carrier in the

membrane forms a complex with the analyte in the feed that diffuses to the strip, where the

analyte is converted to a non-extractable form. This type of transport was used in the

extraction of short chain aliphatic carboxylic acidic feed solution to an alkaline strip solution

with liquid membrane containing tri-

n

-octylphosphine oxide (TOPO) as a neutral carrier

[100]. Changed carriers can be used, such as the anionic di-(2-ethylhexyl)phosphoric acid

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(D2EHPA) and octansulfonic acid (OcSA) [19, 101]. In such case, dissolution of the analyte

into the membrane occurs through ionic interactions with the charge carrier. Once the analyte

reaches the strip phase, it is exchange for a proton and converted to a non-extractable form.

The proton gradient across the membrane in this case is the driving force [102].

Stability of the membrane

The reason for SLM to become instable is the loss of liquid phase (solvent and/or carrier) out

of the pores of the membrane. This loss can be due to several parameters, such as a pressure

difference over the membrane, solubility of membrane solvent and carrier in adjacent feed and

strip solutions, wetting of membrane pores by the aqueous phases, blockage of membrane

pores by precipitation of the carrier or by water, the presence of an osmotic pressure gradient

over the membrane or the emulsion formation of the liquid membrane phase in water induced

by lateral shear forces [103].

An organic solvent filling the pores of a hydrophobic membrane cannot be displaced by water

due to the surface tension of water against the membrane material and the organic solvent. It

prevents the surface deformation of the water caused by penetration into the pores. The

critical displacement pressure

P

c

for an SLM can be defined as the minimum transmembrane

pressure required to displace the impregnating out of the largest pore.

For a cylindrical capillary, this can be quantified by the equation of Young and Laplace:

r

P

c

θ

γ

cos.2

= (1)

with P

c

the critical displacement pressure (lowest pressure needed to force water through the

pores) (Pa),

γ

the interfacial tension (Nm

-1

),

θ

the contact angle between water and the

membrane (dimensionless) and r the pore radius (m) [104].

Recent directions in SLM

Recently, there has been a shift towards designing simple and easily miniaturized SLM

extraction modules. For the most part the miniaturized SLM extraction designs have been a

strip-phase volume of less than (1 mL) to a few microliters. These new designs are very much

suited for speciation studies. Many of the reported miniaturized SLM extractions to this point

have been used to extract the bioavailable fraction of organic compounds in water or

biological fluids [105]. Most of miniaturized SLM have been based on polypropylene fibers,

but some efforts with porous polyvinyldene difluoride tubing have also been reported.

I

ntroduction

23

Polypropylene was selected because it is highly compatible with a broad range of organic

solvents. In addition, with a pore size of approximately 0.2

µ

m, polypropylene strongly

immobilizes the organic solvents used in SLM. This strong immobilization is important for

ensuring that the organic phase does not leak during extraction, as that may alter extraction

performance and the characteristics of the system. Jönsson and Mathiasson introduce SLM

flat membrane extraction module as an enrichment technique for polar and ionisable analytes.

SLM flat membrane is based on a three phase system, with the organic phase being

immobilized in the pores of polypropylene membrane sandwiched between two aqueous

phases with 10

µ

L volume of strip channel (Figure-1.11). This module of SLM extraction is

usually constructed as flow system (on-line), where the strip flow is directly interfaced to

HPLC [15, 106, 107].

The on-line coupling of SLM extraction units to analytical instruments poses the problem of

controlling memory effects. In order to avoid this problem, Pedersen-Bjergaard and

Rasmussen established a liquid-phase microextraction technique (LPME) based on a

disposable porous polypropylene hollow fibre [15, 108]. The fibre is impregnated with an

organic phase, filled with a (1- 25

µ

L) volume of strip phase solution and place in a small

volume of biological or environmental samples within a (4 mL) vial (Figure-1.12). The device

is agitated and the extract is finally transferred to an autosmapler vial. No memory effects

occur, as each membrane is used only for once. Extraction is carried out off-line, but, because

of the simplicity of the extraction devices, high sample throughput can be achieved by

performing many extractions in parallel. Similar device have been described by Lee et al.

[109], Müller et al. [110], and Hauser et al [111]. This method has been used successfully to

trap and enrich of various types of organic pollutant such as acidic drugs like DCF and IBU,

basic drugs and metabolites from water samples as listed in Table-1.6.

The technique of SL-bag membrane (SL-BM) was introduced by Kurtulus et al. [112]. It is

base of a three phase liquid membrane with a bag-type membrane geometry separating the

aqueous sample (feed phase) with a volume 500 mL (or higher) from the strip phase with a

volume of 300- 400

µ

L. This technique was successfully applied to extract and enrich DCF

and IBU from real water samples [112]. Efficient preconcentration was achieved by

immobilizing dihexyl ether (DHE) loaded with octansulfonic acid (OcSA) inside the pores of

polypropylene membrane, resulting in enrichment factors of up to 1100 [112]. Like LPME,

the SL-BM technique is carried out within a vial and off-line system. Despit the many

advantages of SL-BM, the technique failed to extract CBZ and SFM from real water samples.

I

ntroduction

24

Different ways can be used to overcome this problem: In case of acidic and basic compounds

the enrichment can be achieved by adjusting the pH of the feed and strip phase to appropriate

values [94, 95]. In order to extract highly polar compounds (like target metabolites), it is

necessary to use a carrier incorporated into the membrane organic phase. Of great importance

is the specific of carrier-analyte interactions allowing highly selective separations. The choice

of the carrier will be made, therefore, considering the analyte to be transported, as well as the

experimental conditions [19, 100]. This technique may therefore be of vital important in the

current situation where much attention has been focused on the presence of selected

pharmaceuticals that also tend to contaminate the aquatic environment.

Figure-1.11: On-line SLM flat membrane module with 10

µ

L volume of strip

channel [106]

Figure-1.12: Off-line liquid-phase microextraction (LPME) with 1- 100

µ

L of

strip phase[108]

Syringe needles for handling

of strip phase

Hollow fibre containing

strip phase

Sample solution

(Feed phase)

Magnetic

stirrer

I

ntroduction

25

Table-1.6: Applications of SLM to the determination of trace pollutants in natural water

Liquid membrane Type of analyte Enrichment

factor Organic solvent Carrier Detection

method Ref.

Acidic Pharmaceuticals:

Piroxicam

Ketorolac

Clofibric acid

Naproxen

Bezafibrate

Fenoprofen

Ibuprofen

Diclofenac

Indomethacin

38- 234

1-octanol

…..

LC-MS-

MS

113

Ibuprofen

2-(4-chlorophenoxy)-2-

methylpropionic acid

15000

1-octanol

…..

LC-UV

109

Ibuprofen

Naproxen

Ketoprofen

100

DHE

…..

CE

115

34-79

1-octanol

CE

Basic pharmaceuticals:

2-amino-1-phenylethanol

Norephedrine

Pindolol

Atenolol 19- 55 DHE CE

115

60 DHE 5 %

(w/w)

TOPO

LC-UV Haloacetic acids

3000 DHE DEHP LC-UV

113

Fungicides 67 DHE 15 %

(w/w)

TOPO

LC-UV 113

400 1-octanol …... LC-UV

100 hexane ….. LC-UV

Phenols

300 heptane-toluene

(1:1) ….. LC-UV

113

Phenoxy herbicides 490 1-octanol ….. LC-UV 113

6000 benzyl alchohol-

ethyl acetate (8:2)

….. LC-UV

500 DHE ….. LC-UV

Aromatic amines

250 DHE ….. LC-UV

113

Triazine herbicides 390 DHE ….. LC-UV 113

Trihalomethanes 62 1-octanol ….. GC-ECD 116

Dinitrophenols 300 undecane-

toluene(1:1) ….. HPLC-

DAD 113

Ref.: References

Results and discussion

26

2. Results and discussion

2.1. Methodical approach

This study is divided into three steps:

At first metabolites were synthesized and characterized according to the methods described in

literature. In the second part the mass transfer of metabolites and active drugs in the liquid

membrane systems was investigated. In the third part analytical methods were developed by

combining membrane extraction and HPLC-UV to determine metabolites and active drugs in

surface water.

Synthesis of metabolites

The metabolites 10,11-dihydroxycarbamazepine (CBZ-DiOH), 4`-hydroxydiclofenac (DCF-

4OH), 4-hydroxyibuprofen (IBU-2OH), N-4-acetylsulfamethoxazole (SFM-Ac), and

Sulfamethoxazol-N1-glucuronide (SFM-Glu) are not commercially available. They were

synthesized and identified according to common spectroscopic methods (see section 3.1).

Mass transfer of metabolites and active dugs in liquid membrane systems

The mass transfer of metabolites through the liquid membrane systems was carried out in a

three-compartment transport cell and supported liquid membrane-chambers which have been

developed at the University of Paderborn [17-21].

The different liquid membrane systems consist of an aqueous (acidified or alkaline) feed

solution containing the analytes, a bulk (organic solvent with and/or without a dissolved

mobile carrier) and an aqueous stripping solution, e.g. mineral acids or alkalis. For the

preliminary experiment further solvent systems were tested as organic bulk phase. Organic

solvent and mixtures were varied systematically to increase transport efficiency. Acidic or

neutral carriers (such as OcSA and TOPO) were admixed to the organic phase to support the

forward and back-extraction. pH-gradients between the aqueous feed and strip phase were

optimized to increase the effectiveness of transport processes.

Optimal combinations of liquid phases were transferred to the SLM technique. The supported

flat-membrane impregnated with certain selected water-immiscible organic phase is mounted

between two PTFE-chambers of equal size. Moreover, direct influences on the extraction

efficiencies of the metabolites like concentration of selected analytes, extraction time and

concentration of carrier in liquid membrane are very important to investigate.

Results and discussion

27

Finally miniaturized SL-BMs were prepared and employed as enrichment devices for

metabolites and their parent drugs. The following steps were carried out:

•

Optimization of SL-BM extraction conditions

•

Development of enrichment procedures for metabolites and active drugs

•

Comparison of enrichment and clean up properties: SL-BM and SPE (Solid Phase

Extraction)

Development of analytical methods by combining membrane extraction and determination

by HPLC-UV

The final stage of this study the enrichment SLM-devices was tested for the analysis of real

water samples: tap water and surface water. For this purpose the extraction procedure adjusted

and modified to chromatographic conditions of the HPLC-UV or LC-MS system.

The surface water was sampled from the river Ruhr by the Institut für Wasserforschung (IFW)

Dortmund. The water quality is affected by effluents of a sewage treatment plant (STP).

Results and discussion

28

2.2 Three liquid membrane extraction systems

a) Extraction principles

The enrichment principle of three phase liquid membrane systems is depending on the liquid

extraction and back extraction of the analytes from agitating aqueous feed phase, through a

hydrophilic or hydrophobic liquid membrane, into a stagnant aqueous phase. By careful

choice of solvent for the liquid membrane, combined with a proper composition of feed and

strip phases, a selective preconcentration and efficient clean-up of the sample can be achieved

simultaneously. The organic solvent has to satisfy the following variety of requirements:

1)

its water solubility and also volatility should be low in order to separate it from the

aqueous phases (feed and strip) and also to prevent solvent loss during extraction;

2)

it should have a low tendency to dissolve or take up interferences existing in the feed

phase;

3)

it should low viscosity because this promotes high analyte fluxes through the

membrane;

4)

and finally it is better that the solvent have low toxicity due to occupational health and

safety[130].

Considering the extraction of acidic and basic compounds, the selectivity in the process is

achieved by adjusting the pH of the two phases so that the uncharged species pass the liquid

membrane by diffusion and are trapped by ionization in a stagnant acceptor phase [131]. In

case of highly polar compounds like selected metabolites and some of active drugs, molecules

are charged over the pH range and thus, are not directly extractable. As a consequence, it is

necessary to use carrier dissolved in the membrane phase to facilitate the analyte transport.

Typically, for polar analytes, diffusion through the organic membrane can be enhanced by

doping the organic solvent with several carriers. Thus, tri-n-octylphosphine oxide (TOPO), a

strong H-bonding carrier [130, 131], has been used to promote the extractability of polar acids

[131], bases [94] and some of fungicide metabolites (see Table-1.6). 1-octanesulfonic acid

sodium salt monohydrate (OcSA) as an ion-pair carrier can also be employed for extraction of

polar compounds [19, 132, 133]. The ion pairs formed are extracted into the organic interface

and broken by selecting the appropriate pH in the strip solution, which releases the free

analytes. In Table-2.1, some physical and toxicological data of TOPO and OcSA are listed.

Results and discussion

29

Table-2.1: Physical properties and toxicological data of OcSA and TOPO [74]

Carrier

Molecular

weight Melting

point (

o

C)

p

K

a Log

P

(Octanol/water) Toxicological data

OcSA 194.29 …….. 1.86 1.829±0.394 Not available

TOPO 386.63 51.0- 51.5 ……

9.398±0.552 ORAL (LD50): >10000

mg/kg [Rat]

DERMAL (LD50): 2830

mg/kg [Rabbit]

Carrier transport model:

The transport model of carrier in three liquid membranes is based

on five different steps which can be identified during the extraction of analyte [134]:

1)

Diffusion of analyte through the feed side boundary layer to reach the feed- liquid

membrane interface;

2)

complexation reaction between the analyte and the carrier as a carrier present in the

liquid membrane at feed-membrane interface to form a carrier-analyte complex;

3)

diffusion of the carrier-analyte complex through liquid membrane;

4)

de-complexation reaction between the carrier-analyte complex and the strip agent at

the strip-membrane interface and release of analyte;

5)

diffusion of analyte through the strip side boundary layer to reach the bulk of the strip

phase.

Anionic carrier OcSA

With OcSA as an anion carrier solved in liquid membrane a proton gradient between the strip

and feed phase is the driving force for the mass transfer of polar analytes in three liquid

membranes as shown in Figure-2.1. This type of carrier is associated with counter-ions to

maintain electroneutrality in the polar membrane phase [19, 132, 133].

H

M

S OH

O

S O M

O

H

M

Figure-2.1: Mechanisum transport of polar compounds (M) across a liquid membrane

containing OcSA as carrier

F

e

d

p

h

a

s

e

M

e

m

b

r

a

n

e

p

h

a

s

e

S

t

r

i

p

h

a

s

e

M + OcSA OcSA M

OcSA M + H OcSA + M

OcSA

Complex

Results and discussion

30

Neutral carrier TOPO

TOPO is known as an efficient carrier for polar compounds [16], owing to the two lone

electron pairs on the oxygen atom. TOPO has the ability to form hydrogen-bond complexes of

various compositions [131, 136]. Therefore, it is particularly useful in the extraction of acidic

and basics, especially highly polar. Moreover, TOPO is very stable when used in a liquid

membrane, as it is soluble in organic solvents but insoluble in water (see Table-2.1). The

whole reaction scheme for polar acidic and basic drugs by TOPO can be presented as follows:

Extraction of acidic drug

[130]:

A

-

+ H

+

(aq)

HA

(aq)

(2)

HA

(aq)

+ TOPO

(org)

[TOPO..HA]

(org)

(3)

[TOPO..HA]

(org)

+ OH

-(aq)

A

-

(aq)

+ TOPO

(org)

+ H

2

O (4)

O H

O

O H

O

P O

O

TOPO

Figure-2.2: Mechanisum transport of polar acidic drug across liquid membrane

containing TOPO as carrier

Feed phase

(aqueous)

M

e

m

b

r

a

n

e

p

h

a

s

e

(organic) Strip phase

(aqueous)

Acid Base

HO

Drug

Complex

Drug

Extraction of basic drug

[131]:

BH

+

+ OH

-

(aq)

B

(aq)

(5)

B

(aq)

+ TOPO

(org)

[TOPO..B]

(org)

(6)

[TOPO..B]

(org)

+ H

+(aq)

BH

+ (aq)

+ TOPO

(org)

+ H

2

O (7)

Results and discussion

31

P O

NH

3

NH

2

N

HH

NH

3

Acid

P O

TOPO

Feed phase

(aqueous)

M

e

m

b

r

a

n

e

p

h

a

s

e

(organic) Strip phase

(aqueous)

Base

HO

OH

Figure-2.3: Mechamisum transport of polar basic drug across liquid membrane

containing TOPO as carrier

Drug

Complex

Drug

b) Development of HPLC-UV methods

In order to monitor the transport of the metabolites and active drugs through the membrane

systems, aliquots were taken from the liquid phases (feed and strip) in intervals by means of a

micro-liter syringe which is introduced through self-closing seals (bulk liquid membrane) or

directly from the open SLM-chambers and then analyzed by HPLC.

The samples are introduced into the chromatographic systems by an autosampler and an

isocratic or gradient pump. UV-Vis-PAD-Detector was connected with a chromato-integrator.

RP 18 analytical column was used (details see section 3.2.1).

2.2.1 Bulk liquid membrane (BLM)

The equipments used are based on earlier developments [17-20]. As shown schematically in

Figure-2.4, the three-phase system was established in a home-made glass cell equipped with

an agitator (PTFE) which allows extraction and back-extraction in one unit. The cell consists

of two concentric chambers dividing it into separate compartments. Thus, the feed is allowed

to contact the bulk membrane and the strip solution to contact the membrane. The whole cell

covered by a fitting glass lid in order to minimize loss of solvent by evaporation.

Results and discussion

32

Figure-2.4: Three-phase liquid bulk membrane system (1: strip phase, total volume:25 mL,

2: feed phase 32 mL, 3: membrane phase 20 mL) [19]

The efficiency and selectivity of membranes will be influenced by the composition of organic

phase. Therefore, we used common water-immiscible organic solvents of different polarities

as shown in Table-2.2. The different combinations of feed and strip phases, which were used

in the BLM systems, are summarized in Table-2.3.

Table-2.2: Physical properties of organic solvents used as liquid membrane [74]

Solvent Log

P

(octanol/water) Density

(g/mL)

1-pentanol 1.407±0.176 0.811±0.06

DHE 5.232±0.206 0.799±0.06

Undecane 6.600±0.166 0.743±0.06

Decane 6.069±0.166 0.734±0.06

Results and discussion

33

Table-2.3: Composition of three-phase membrane system used

Feed phase

Volume: 32 mL Membrane phase

volume: 20 mL Carrier Strip phase

volume: 25 mL

0.1 mol/L NaOH 1-pentanol - 0.1 mol/L HCl

0.1 mol/L HCl 1-pentanol - 0.1 mol/L NaOH

0.1 mol/L HCl DHE - 0.1 mol/L NaOH

0.1 mol/L HCl undecane - 0.1 mol/L NaOH

0.1 mol/L HCl decane - 0.1 mol/L NaOH

0.1 mol/L HCl DHE 0.025 g/L OcSA 0.1 mol/L NaOH

0.1 mol/L HCl DHE 1 % (w/w) TOPO 0.1 mol/L NaOH

0.1 mol/L HCl undecane 1 % (w/w) TOPO 0.1 mol/L NaOH

0.1 mol/L HCl decane 1 % (w/w) TOPO 0.1 mol/L NaOH

2.2.1.1 Influence of organic solvent

By using organic solvents (Table-2.2), the distribution of the metabolites governs the extent

of extraction from the aqueous feed matrix into the organic bulk phase. As can be expected

from other classes of compounds, the magnitude of the calculated partition coefficients in the

octanol/water systems (Log P) of the individual drugs corresponds to the extraction yields

determined (Table-2.4) [19]. Obviously the metabolites with high Log P such as IBU-2OH

and SFM-Ac lead to different extractabilities in polar and nonpolar liquid phases. These

compounds were highly dissolved in 1-pentanol and too much lower yields in DHE and

undecane were obtained (Table-2.4).

By contrast, SFM-Glu and CBZ-DiOH with very low partition coefficient gave drastically

much lower extraction efficiencies in polar and nonpolar membrane as seen in Table-2.4.

Table-2.4: Extraction efficiency E (%) of metabolites in liquid membrane phase after 4 hours

of extraction by using BLM (feed: 0.1 mol/L HCl, strip: 0.1 mol/L NaOH)

E

(%) after 4 hours Metabolite p

Ka*

Log

P*

1-pentanol DHE Undecane

IBU-2OH 4.44 1.690±0.242 ~100 19.7 19.3

SFM-Ac 5.60 1.478±0.436 ~100 40.7 45.2

SFM-Glu 2.70 (pK

a1

)

0.36 (pK

a2

) 0.561±0.624 22.6 9.9 11.7

CBZ-DiOH 12.62 0.132±0.405 54.8 11.8 17.6

*Dissociation constants (pK

a

) and octanol-water partition coefficients (Log P) are taken from

reference [74]

Results and discussion

34

2.2.1.2 Influence of pH-gradient between feed and strip phase

Besides the polarity of the organic bulk phase the acid-base properties of the metabolites were

utilized to facilitate their transfer from the feed into the strip phase. pK

a

values of the

metabolites correspond from acidic (like: SFM-Glu, IBU-2OH, and SFM-Ac) to basic

compounds (like; CBZ-DiOH) as shown in Table-2.4. Since the analytes should be in their

neutral state in order to be extracted into the organic phase, and to trap charged analytes into

the strip phase. Different pH values of feed and strip were used to adjust the optimum

extraction efficiency (Table-2.3) of metabolites in strip phase. The best results were found

when the pH of the feed phase was kept at pH ~1 by using 0.1 mol/L HCl and the strip at pH

~13 by using 0.1 mol/L NaOH. About 50 % of IBU-2OH and ~35 % of SFM-Ac are released

into the strip solution after 4 hours of extraction (Figure-2.6).

Feed phase

0

10

20

30

40

50

60

70

1 2 3 4

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

Strip phase

0

10

20

30

40

50

60

70

80

90

100

1234

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

a) b)

Figure-2.5: Transport of metabolite mixtures in BLM, by using basic feed phase and acidic

strip phase, a) Forward-extraction, b) back-extraction (Extraction conditions: feed 1 mg/L of

metabolites in 32 mL of 0.1 mol/L NaOH, strip: 25 mL of 0.1 mol/L HCl, and liquid

membrane: 1-pentanol)

Results and discussion

35

Feed phase

0

20

40

60

80

100

120

1234

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

Strip phase

0

10

20

30

40

50

60

1 2 3 4

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

a) b)

Figure-2.6: Transport of metabolite mixtures in BLM by using acidic feed phase and basic

strip phase, a) Forward-extraction, b) back-extraction (Extraction conditions: feed 1 mg/L of

metabolites in 32 mL of 0.1 mol/L HCl, strip: 25 mL of 0.1 mol/L NaOH and liquid

membrane: 1-pentanol)

2.2.1.3 Influence of extraction time

The influence of extraction time on the transport of metabolites was investigated, 2, 4, and 6

hours were applied. Complete extraction of IBU-2OH and SFM-Ac from feed solution

achieved within 2 hours. The extracted amount of IBU-2OH and SFM-Ac in the strip phase

increased with increasing extraction time and maximum extraction was found after 4 hours

(Figure 2.7). After then the extraction efficiencies were slightly decreased as some of the

analytes re-entered to the membrane phase, therefore a time interval of 4 h was chosen for

further experiments.

Results and discussion

36

Feed phase

0

20

40

60

80

100

120

0 2 4 6

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

Strip phase

0

10

20

30

40

50

60

0 2 4 6

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

a) b)

Figure-2.7: Transport of metabolite mixtures in BLM with different extraction times, a)

Forward-extraction, b) back-extraction (Extraction conditions: feed 1 mg/L of metabolites in

32 mL of 0.1 mol/L HCl, strip: 25 mL of 0.1 mol/L NaOH, and liquid membrane: 1-pentanol)

2.2.1.4 Influence of carrier in liquid membrane

Since all the metabolites are polar (Table-2.4) and are less extractable by DHE, undecane and

decane as expected [130]. OcSA dissolved in DHE was already successfully used as liquid

membrane to extract active drugs (IBU and DCF) from water samples [19]. In another

application, TOPO was used as a carrier to increase the dissolution into the liquid membrane

of DHE and undecane in the extraction of polar acidic and basic compounds [94, 131]. In

order to enhance the dissolution into the liquid membrane and thus the extraction efficiency,

the added amounts of 0.025 g/L of OcSA or 1 % (w/w) of TOPO as carrier were applied

(Table-2.3). Generally, the results indicate a significant increase of extraction efficiency for

IBU-2OH and SFM-Ac. OcSA with DHE as a liquid membrane was not able to extract the

metabolites into the strip phase (see Figure-2.9).

TOPO has a good but not unlimited solubility in organic solvents as in some cases

precipitations were observed in liquid membranes at a concentration higher than 1 % (w/w)

after 2 h of extraction.

Results and discussion

37

Feed phase

0

5

10

15

20

25

30

35

40

45

1 2 3 4

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

Strip phase

0

10

20

30

40

50

60

70

80

90

100

1234

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

a) b)

Figure-2.8: Transport of metabolite mixtures in BLM using DHE as liquid membrane, a)

Forward-extraction, b) back-extraction (Extraction conditions: feed 1 mg/L of metabolites in

32 mL of 0.1 mol/L HCl, strip: 25 mL of 0.1 mol/L NaOH)

Feed phase

0

5

10

15

20

25

30

35

40

45

50

1234

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

Strip phase

0

10

20

30

40

50

60

70

80

90

100

1234

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

a) b)

Figure-2.9: Transport of metabolite mixtures in BLM using DHE with 0.025 g/L OcSA as

liquid membrane, a) Forward-extraction, b) back-extraction (Extraction conditions: see

Figure-2.8)

Results and discussion

38

Feed phase

0

20

40

60

80

100

120

1234

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

Strip phase

0

5

10

15

20

25

30

35

40

1 2 3 4

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

a) b)

Figure-2.10: Transport of metabolite mixtures in BLM using DHE with 1 % (w/w) TOPO as

Liquid membrane, a) Forward-extraction, b) back-extraction (Extraction conditions: see

Figure-2.8)

Feed phase

0

5

10

15

20

25

30

35

40

45

50

1 2 3 4

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

Strip phase

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

a) b)

Figure-2.11: Transport of metabolite mixtures in BLM using undecane as liquid membrane,

a) Forward-extraction, b) back-extraction (Extraction conditions: see Figure-2.8)

Results and discussion

39

Feed phase

0

20

40

60

80

100

120

1234

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

Strip phase

0

5

10

15

20

25

30

1 2 3 4

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

a) b)

Figure-2.12: Transport of metabolite mixtures in BLM using undecane with 1 % (w/w)

TOPO as liquid membrane, a) Forward-extraction, b) back-extraction (Extraction conditions:

see Figure-2.8)

Feed phase

0

5

10

15

20

25

30

35

40

45

50

1 2 3 4

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

Strip phase

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

a) b)

Figure-2.13: Transport of metabolite mixtures in BLM using decane as liquid membrane, a)

Forward-extraction, b) back-extraction (Extraction conditions: see Figure-2.8)

Results and discussion

40

Feed phase

0

20

40

60

80

100

120

1 2 3 4

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

Strip phase

0

5

10

15

20

25

30

35

1 2 3 4

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

a) b)

Figure-2.14: Transport of metabolite mixtures in BLM using decane with 1 % (w/w) of

TOPO as liquid membrane, a) Forward-extraction, b) back-extraction (Extraction conditions:

see Figure-2.8)

2.2.2 Supported liquid flat-membrane (SL-FM) system

This SLM technique is base on a polypropylene (PP) membrane (pore size 0.1 µm, total

thickness 90 µm). It is impregnated with a water-immiscible organic solvent (e.g. DHE,

undecane or decane) containing the carrier (e.g. TOPO) which is held by capillary forces

placed between two aqueous phases, feed and strip.

The supported liquid flat-membrane (SL-FM) system 150:150 (v/v) (Figure-2.15) has been

used to extract the metabolites. Certain membranes used for the bulk liquid membrane were

selected to test their applicability in the SL-FM system, applying different concentrations of

TOPO as a carrier.

Figure-2.15: SL-FM device, 1 strip-chamber, 5 feed-chamber (volume of both

chambers 150 mL), 2 contact window, 3 fixing plants, 4 membrane [19]

Results and discussion

41

2.2.2.1 Theoretical approach

SLM-transport of compounds is based on two processes: extraction of an analyte from the

feed phase into an organic solvent situated in membrane pores, and a simultaneous back-

extraction from the organic phase into an aqueous stripping phase, where the analyte is

converted to a non-extractable form and thus trapped.

The schematic transport mechanism of an analyte from an bulk agitating aqueous feed phase

to a bulk stagnant strip phase (Figure-2.16) (where the volume of both phases is equal)

through an organic phase in the microporous polymeric membrane is shown in Figure-2.17

[137].

Figure-2.16: SLM device , (1) feed phase, (2) strip phase, (3) membrane, (4)

agitator

h

F

h

S

h

M

Feed Strip

12

3

4

C

F

C

FM

C

MS

C

S

Figure-2.17: Schem atic transport of analyte inSLM [137]

h

F

h

M

C

F,i

C

S,i

M em brane

Concentraction

Distance

x = 0 x = h

M

Feed Strip

h

S

Interface boundary layers

Results and discussion

42

Overall mass transfer

The overall mass transfer under steady-state condition, consist of three mass transfer

processes: the mass transfer in feed phase, in the membrane phase and in the strip phase.

To obtain an expression for mass transfer coefficient, k, Fick’s first law and definition of mass

transfer coefficients are applied [138]:

[ ]

CtCk

dx

dC

DJ −== =)(0h x

(8)

Where J = mass flux of analyte per unit area per unit time

D = diffusion coefficient

C

0

(t) = analyte concentration at t = 0

∫

=

h

Cdx

h

C

0

1

dC/dx = spatial concentration gradient along the axis of flow path

Assuming equilibrium at feed-membrane interface, and between the active and inactive forms

of analytes, the mass transfer in feed phase is described as following equation (Eq.) [139]:

)(

,iFFFFF

CCkJ

−

=

α

(9)

))(1(

,iFFFFF

CCkJ

−

′

=

′

α

(10)

Where, J

F

and J’

F

are the mass transfer of the analyte in active and inactive form in feed

phase respectively.

F

α

is the fraction of total analyte that is in active form in the feed phase,

i.e., in such a form that it may transverse the membrane

)10(

≤

F

α

. It is assumed that the

conditions are such that

F

α

is constant throughout the feed phase, also in the vicinity of

membrane surface.

The mass transfer coefficient for active form in the feed phase (k

F

) is derived from the

penetration theory as [138]:

x2

fD3

k

FF

F

π

=

(11)

Where, D

F

is the diffusion coefficient of analyte in active form in feed phase, and f

F

, is the

linear flow velocity in feed phase.

The fluxes of membrane (J

M

) and strip phase (J

S

) can be written analogously to Eq. (9), noting

that no inactive form of the analyte exists in the membrane phase:

)(

MSSSFMFFMM

CKCKkJ

α

−

=

(12)

)(

,SiSSSS

CCkJ

−

=

α

(13)

Results and discussion

43

))(1(

,SiSSSS

CCkJ

−

′

=

′

α

(14)

The distributions constant, K

F

and K

S

are the partition coefficient of feed and strip phases

respectively.

α

S

is assumed to be constant throughout the strip phase, even at high degree of

enrichment.

For the mass transfer in the membrane phase (k

M

), the film theory for mass transfer gives

[139]:

M

h

D

k

ξ

ε

= (15)

Where, D

M

is the diffusion coefficient for active form of analyte in membrane phase,

ε

, the

membrane porosity and

ξ

is the membrane tortuosity.

An expression of the mass transfer coefficient in the stagnant strip phase (ks) can be derived

from the film theory as [139]:

S

h

D

k= (16)

Where, Ds is the diffusion coefficient for active form of analyte in strip phase, and hs is the

thickness of strip phase.

The total flux of analyte from bulk of feed phase to bulk of the stripping phase (J) is described

by [139]:

)( S

F

S

SFF C

K

CkJ

αα

−= (17)

Where, k is the overall mass transfer coefficient.

Implicit in this equation is zero flux

)0(

=

J between all bulks at equilibrium. Thus, at steady

state transfer it holed that:

SSMFF

JJJJJJ

′

+

=

′

+

=

(18)

From Eqs. (8), (9), (12), (13), (17), and (18) an expression for the overall mass transfer

coefficient can be derived [140]:

FS

SS

FMF

F

K

k

K

k

α

++= 11 (19)

, where K

F

is the partition coefficient between the membrane and the feed, and K

S

is the

partition coefficient between the membrane and the strip [140].

Results and discussion

44

Mass balance equations

With the agitating feed phase, the mass balance equation in the feed phase is:

)(

SSFF

F

CC

V

k

dx

dC

f

dt

dC

αα

−−−= (20)

And for the stagnant stripping phase, the mass balance equation in the strip phase as following

[139]:

)(

SSFF

S

CC

V

k

dt

dC

αα

−= (21)

The rate of mass transfer

The rate of mass transfer across the liquid membrane is proportional to the concentration

difference ∆C of neutral extractable analyte between feed and strip phase, which can be

written as [141]:

SSSFFF

KCKCC

α

−

=

∆

(22)

In most applications of supported liquid membrane, the conditions as shown in Figure-2.18

are set so that the second term in Eq. (22) is negligible ( S

α

~ 0) and F

α

is closed to 1.

RCOOH

RCOO RCOO

RNH3RNH2RNH3

C

(A)

(B)

Figure-2.18: The optimum extraction conditions of an acidic analyte (A),

and basic analyte (B) by SLM [141]

acid base

base acid

F

e

d

(

a

q

)

M

e

m

b

r

a

n

e

(

o

r

g

)

S

t

r

i

p

(

a

q

)

pH = pKa - 2 pH = pKa + 3.3

pH = pKa + 2 pH = pKa - 3.3

~ 2 log P ~ 4

α

αα

αF

=

==

=~ 1 α

αα

αS

=

==

=~ 0

2.2.2.2 Influence of the composition of liquid membrane

The certain liquid membranes which have been used in BLM systems were also tested in an

SL-FM system. As shown in Table-2.5, the extraction efficiencies of metabolites in SL-FM

membrane phase were approximately the same as in BLM systems. By increasing the

Results and discussion

45

concentration of TOPO from 1 to 10 % (w/w) as carrier in DHE, undecane and decane

(Table-2.6) gave a quantitative separation of IBU-2OH and SFM-Ac as can see in Figures-

2.19, 2.20, and 2.21.

Table-2.5:

Extraction efficiency, E, (%) of the metabolites after 8 hours by using SL-FM

system (with feed: 0.1 mol/L HCl, and strip: 0.1 mol/L NaOH)

E

(%) after 8 hours

Metabolite

1-pentanol DHE with

1 % (w/w) of

TOPO

Undecane with

1 % (w/w) of

TOPO

Decane with

1 % (w/w) of

TOPO

IBU-2OH ~100 ~100 ~100 ~100

SFM-Ac ~100 ~100 ~100 ~100

SFM-Glu ~0 ~0 ~0 ~0

CBZ-DiOH 33.4 ~0 ~0 ~0

Table-2.6:

Compositions of liquid membranes in SL-FM systems

Membrane Carrier ( w/w)

1-pentanol …..

DHE 1 , 3 , 5 , and 10 % of TOPO

Undecane 1 , 3 , 5 , and 10 % of TOPO

Decane 1 , 3 , 5 , and 10 % of TOPO

Feed phase

0

20

40

60

80

100

120

2468

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH

IBU-2OH SFM-Ac

Strip phase

0

20

40

60

80

100

2 4 6 8

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

a) b)

Figure-2.19:

Transport of metabolite mixtures in SL-FM, DHE with 10 % (w/w) of TOPO as

liquid membrane, a) Forward-extraction, b) back-extraction (Extraction conditions, feed: 1

mg/L of metabolites in 150 mL of 0.1 mol/L HCl, and strip: 150 mL of 0.1 mol/L NaOH)

Results and discussion

46

Feed phase

0

20

40

60

80

100

120

2 4 6 8

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

Strip phase

0

20

40

60

80

100

2 4 6 8

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

a) b)

Figure-2.20:

Transport of metabolite mixtures in SL-FM, undecane with 10 % (w/w) of

TOPO as liquid membrane, a) Forward-extraction b) back-extraction (Extraction conditions:

see Figure-2.19)

Feed phase

0

20

40

60

80

100

120

2 4 6 8

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

Strip phase

0

20

40

60

80

100

2 4 6 8

Time (hour)

Extraction (%)

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

a) b)

Figure-2.21:

Transport of metabolite mixtures in SL-FM, decane with 10 % (w/w) of TOPO

as liquid membrane, a) Forward-extraction b) back-extraction (Extraction conditions: see

Figure-2.19)

2.2.2.3 Influence of extraction time

SL-FM is a three-phase extraction system with two liquid-membrane interfaces; as a result,

the metabolite molecules need time to diffuse through each phase and cross all interfaces to

get into strip phase. Hence, the influence of extraction time on the extraction efficiency of the

Results and discussion

47

metabolites in SL-FM was studied. SFM-Glu and CBZ-DiOH remain in the membrane phase

as shown in (Figure-2.22

a

and

b

), while IBU-2OH and SFM-Ac were simultaneously

extracted from the feed and back-extracted into the strip phase see (Figure-2-22

c

and

d

) and

the extracted amounts of both compounds increase with extended extraction time. The

maximum extraction efficiencies of IBU-2OH and SFM-Ac were obtained 8 hours and was

chosen for further experiments. The extraction time in this regard is relatively long, but it is

possible (as the membrane is more stable) to achieve the best extraction efficiency.

2468

Feed phase

Strip phase

0

0,2

0,4

0,6

0,8

1

1,2

1,4

Extraction (%)

Time (hour)

SFM-Glu

2468

Feed phase

Strip phase

0

0,5

1

1,5

2

2,5

3

Extraction (%)

Time (hour)

CBZ-DiOH

a b

2468

Feed phase

Strip phase

0

10

20

30

40

50

60

70

80

90

100

Extraction (%)

Time (hour)

IBU-2OH

2468

Feed phase

Strip phase

0

10

20

30

40

50

60

70

80

90

100

Extraction (%)

Time (hour)

SFM-Ac

c d

Figure-2.22:

Time effect on the extraction efficiencies of metabolites by using SL-FM

system and DHE with 5 % (w/w) of TOPO as liquid membrane, a) SFM-Glu, b) CBZ-DiOH

c) IBU-2OH, d) SFM-Ac (Extraction conditions: see Figure-2.19)

Results and discussion

48

2.2.2.4 Influence of TOPO concentration on the stripping process

The effect of the TOPO concentration in the membrane phase (DHE, undecane and decane)

on the re/ back-extraction/ stripping of IBU-2OH and SFM-Ac can be seen in Figures-2.23

and 2.24. For a low carrier concentration (≤10 % w/w) the extraction efficiency of IBU-2OH

and SFM-Ac increase with increase of TOPO concentration in the liquid membrane. These

observations are the result of the influence of two factors on the mass transfer of analyte

through the liquid membrane, namely the concentration gradient of the analyte-carrier

complex and the viscosity of the organic liquid membrane phase. If it is assumed, that the flux

of the compound through the membrane is (J

M

) related to the concentration gradient ( C

∆

)

and the membrane thickness (h

M

) through Fick’s first law, where D

M

is the diffusion

coefficient

C

h

D

J

M

M∆×=

(23)

In this case, high fluxes can be achieved when the high diffusion coefficient is maintained.

From the other side, the diffusion is dependent on the viscosity of the organic phase (

η

) and

the radius (r) of the species according to the Stokes-Einstein relationship [142]:

r

kT

D

M

πη

6

=

(24)

Therefore, as an increase of the carrier concentration generally increases the driving force as

well as the viscosity of the liquid membrane (see Tables-2.1 and 2.2).

13510

DHE

Undecane

Decane

0

10

20

30

40

50

60

70

80

90

Extraction (%)

TOPO % (w/w)

Figure-2.23:

Stripping of IBU-OH as a function of TOPO concentration in DHE, undecane

and decane (Extraction conditions: see Figure-2.19)

Results and discussion

49

13510

DHE

Undecane

Decane

0

10

20

30

40

50

60

70

80

90

Extraction (%)

TOPO % (w/w)

Figure-2.24:

Stripping of SFM-Ac as a function of TOPO concentration in DHE, undecane

and decane (Extraction conditions: see Figure-2.19)

2.2.3 Conclusion

The metabolites 10,11-dihydroxycarbamazepine (CBZ-DiOH), 4-hydroxyibuprofen (IBU-

2OH), N-4-acetylsulfamethoxazole (SFM-Ac) and sulfamethoxazol-N1-glucuronide (SFM-

Glu) were synthesized and characterized by IR,

1

H-,

13

C-NMR and mass spectroscopy see

section 3.1. The synthesis steps of 4`-hydroxydiclofenac (DCF-4OH) have faced problems,

leading at the end to incapability to synthesize this compound (section 3.1.2).

In order to carry out experiments with BLM and SL-FM systems a HPLC-UV method was

developed and validated as shown in section 3.2.2.

The mass transfer of the drug metabolites through the liquid membranes was carried out in a

three-compartment transport cell and supported liquid membrane-chamber. The three-phase

bulk liquid membrane system (BLM) consisted of an aqueous feed solution, an organic

solvent (1-pentanol, DHE, undecane, and decane) with and without a dissolved tri-n-

octylphosphine oxide as a liquid bulk membrane and an aqueous stripping solution. The BLM

employed offers a tool to separate selectively and efficiently various metabolites from

aqueous solution. It extracts most efficiently IBU-2OH and SFM-Ac by combining TOPO as

carrier, the appropriate solvent and a pH-gradient between feed and strip. In general the

extractability of SFM-Glu and CBZ-DiOH by the liquid membrane is lower. Maximum

extraction yield were obtained with 1-pentanol as a polar membrane.

Results and discussion

50

The supported liquid flat-membrane (SL-FM) system 150:150 (v/v) based on a porous

polypropylene (PP) membrane (pore size 0.1

µ

m, total thickness 90

µ

m) was impregnated

with an appropriate water-immiscible organic membrane phase. It contained the solvent with

or without a dissolved carrier held by capillary forces placed between two aqueous phases,

feed and strip.

Certain three-phase compositions were tested in SL-FM. Different factors have been studied

to find the best extraction conditions for these metabolites:

•

strip phase: adjusted to pH >13 by using 0.1 mol/L NaOH;

•

liquid membrane: DHE with 10 % (w/w) TOPO or undecane with 10% (w/w) TOPO,

and decane with 10 % (w/w) TOPO;

•

Time of extraction: 8 hours.

This condition gave ~ 90 % of IBU-2OH and ~ 85 % of SFM-Ac transported from the feed

into the strip solution at a concentration of 1 mg/L.

By contrast, SFM-Glu and CBZ-DiOH which have a very lower partition coefficient (octanol/

water) value can not be transported to the strip phase by using both BLM and SL-FM

systems.

High enrichment factors are possible by employing certain SLM-cells, so that even traces of

metabolites residues or drugs can be separated and enriched. Therefore, the investigations are

continued to develop particular SLM devices which should be suitable for trace analysis.

Results and discussion

51

2.3 Supported liquid bag membrane (SL-BM) systems

In SLM extractions, a high enrichment is a result of high solubility of the analytes in the

membrane and complete trapping into the stripping phase. Theoretically infinitely high

enrichment factors can be obtained. In practice, however, enrichment factors are limited by

several factors such as the processing time, the adjustable volume ratio of sample and strip

phase, and affectivity of extraction and stripping.

The objectives of the present experiments are two-fold. The first one is to develop a method

to extract drug traces from water samples. The second one is to compare the efficiency and

applicability of this method with common solid-phase extraction.

The original version of SL-bag membrane was developed at the University of Paderborn

[112]. Special polypropylene-bags (PP-bags) were produced from PP-sheets by a special

technique used as a support for the liquid membrane. The method provides high enrichment

factors (up to 1100) for a limited number of analytes employing a DHE/ OcSA-based SLM-

system [112].

The SL-BM device is shown in Figure-2.25. The PP-bag soaked with the liquid membrane

and filled with 0.3 - 0.6 mL of 0.1 mol/L NaOH as the strip phase was placed in 500 mL of

the aqueous, acidified (pH 1) sample, containing the analytes of drug traces dissolved. A

magnetic stir bar was used to agitate the sample solution during the extraction. At the end of

the extraction process (4 h), the strip solution was drawn into a syringe and transferred into a

vial insert for HPLC-UV or LC-MS analysis.

Figure-2.25:

Schematic diagram of SL-BM; A represents the analyte

=

SL-BM loaded

with strip phase

Stir bar

A

Feed phase

Results and discussion

52

2.3.1 Extraction efficiency and enrichment factor: Theoretical approach

As shown in Figure-2.26, the analytes (IBU-2OH, SFM-Ac, IBU and DCF) in their

undissociated form first diffuse from the bulk feed solution to the surface of the membrane

and then partitioned into the membrane liquid. After migration across the membrane, they

were extracted into the strip via deprotonation. In the ionized form they can not re-enter the

membrane. The two processes occurred simultaneously, and the overall extraction can be

highly efficient [143].

Figure-2.26:

SL-BM device; (1) bag membrane filled with strip phase 0.3 - 0.6 mL; (2)

sample solution with 500 mL of feed phase; (3) stir bar [112]

At equilibrium, the mass balance in the SL-BM system can be written as [144]:

SSMMFiFFF

VCVCVCVC

+

=

,

(25)

C

F

is the initial analyte concentration in the feed phase, C

F,i

, C

M

, and C

S

are the equilibrium

analyte concentrations in the feed, membrane, and strip respectively. V

F

, V

M

, and V

S

are the

volumes of the feed, membrane, and strip, respectively.

K

F

is the partition coefficient between the membrane and the feed, and K

S

is the partition

coefficient between the membrane and the strip [143]:

FF

M

F

C

K

α

=

(26)

SS

M

S

C

K

α

=

(27)

The extraction efficiency, E, is defined as the fraction of the analytes extracted, and is given

as:

Results and discussion

53

FF

SS

V

C

VC

E

=

(28)

The enrichment factor, E

e

, is defined as the ratio of the analyte concentration in the strip and

to that in feed phase:

)(

S

F

S

e

V

E

C

E

==

(29)

When (V

F

/V

S

) is fixed, according to Eq. (29), E

e

is proportional to E. Combining Eqs. (25) -

(29), the E at equilibrium can be written as:

1V)VK()KV()KV(

1

E

SMSSFSFSFS

+α+αα

= (30)

Combining Eqs. (25)- (27) and (29), the E

e

at equilibrium can be written as:

FSFFFSS

e

VVVKK

E++

=)VK()((

1

MSS

ααα

(31)

Because

F

α

is approximately 1, and if K

S

and K

F

are assumed to be similar, Eqs. (30) and (31)

can be simplified to calculate maximum enrichment E

(max)

and extraction efficiency E

e(max)

as:

1)()(

1

(max)

++

=

SMSSSFS

VVKVV

E

αα

(32)

FSFMSSS

e

VVVVK

E++

=)(

1

(max)

αα

(33)

Eq. (32) indicates that in order to achieve a high E,

S

α

should be low. Theoretically the

maximum possible E is 1 corresponding to an extraction yield of 100 %. According to Eq.

(33), a small

S

α

is also necessary for a high E

e

-value. E

e

decreases with increase of V

S

, and

increase with the increase in V

F

. The maximum possible E

e

can be calculated by:

S

F

e

V

E=

(max)

(34)

A high E

e

-value

is desired to obtain lower detection limits. In case of the SL-BM used the

theoretically highest E

e

-value is 1250, in case of quantitative extraction (E

(max)

= 1)

2.3.2 Extraction of the metabolites IBU-2OH and SFM-Ac

For the efficient enrichment of metabolites, factors that control the transfer of the analytes

from the feed phase to the strip phase across the bag membrane and the entrapment of the

analytes to the strip phase were optimized. For an efficient enrichment analytes in the feed

phase need to be non-ionic or in an uncharged form before they diffuse across the membrane.

The partition coefficient K

F

(see Eq.26) of the analyte molecules between the organic solvent

Results and discussion

54

and the aqueous feed phase has to be as high as possible for the target molecules IBU-2OH

and SFM-Ac. For the interfering compounds it has to be low. Also an efficient trapping or

conversion of analytes into the inactive form which in turn prevents back-diffusion into the

feed phase, should take place from the stripping phase. Therefore, a number of parameters

were optimized in order to achieve the objective of efficient trapping of the analyte.

2.3.2.1 Influence of composition of the liquid membrane

The compositions (solvent/carrier) used in the SL-FM (section 2.2.2) which provided best

extraction results (see Table-2.7) were selected to test their applicability in SL-BM systems.

Table-2.7:

Composition of liquid membranes used for SL-BM systems

Solvent Carrier

DHE 1, 3, 5 and 10 % (w/w) of TOPO

Undecane 1, 3, 5 and 10 % (w/w) of TOPO

Decane 1, 3, 5 and 10 % (w/w) of TOPO

Liquid membranes consisting of undecane or decane and higher concentrations of TOPO were

not stable; the bag membrane leaked the strip solution into the feed phase after 1 hour of

extraction. By contrast, DHE with concentration in the range of 1- 10 % (w/w) of TOPO was

more stable. Consequently, DHE with dissolved TOPO as carrier was qualified for further

experiments with the SL-BM system.

The low drug concentrations (

≤

1 mg/L) chosen, do not allow the determination of the

extraction yield from the feed into the SL-BM. Therefore, the judgment of the membrane

performance is exclusively based on the determination of the trapped pharmaceuticals in the

strip.

2.3.2.2 Influence of TOPO concentration

The influence of the carrier on the extraction efficiency for metabolites was studied by

varying the TOPO concentration in DHE from 1 to 15 % (w/w). As demonstrated in Figure-

2.27, the re-extraction for both compounds increased with increasing TOPO-concentration

and reaches a maximum at 10 % (w/w). At a higher carrier concentration, the higher stripping

yield determined could be attributed to an increased formation of aggregate complexes with

low diffusion constants [145]. As a consequence, for further experiment, the TOPO

concentration in the membrane was adjusted to 10 % (w/w).

Results and discussion

55

0

2

4

6

8

10

12

14

16

18

20

Extraction (%)

1 3 5 10 15

TOPO (w/w) concentration

IBU-2OH SFM-Ac

Figure-2.27:

Stripping of IBU-2OH and SFM-Ac as a function of TOPO concentration in

DHE (Extraction conditions: feed 0.01 mg/L of metabolites in 500 mL of 0.1 mol/L HCl,

strip: 0.4 mL of 0.1 mol/L NaOH, 4 h of extraction)

2.3.2.3 Influence of extraction time

Figure-2.28 shows effect of time on the stripping process. Stripped amounts increased over a

period of 4 h and then they declined. This effect may be attributed to the influence of the pH

on the stripping reaction. As IBU-2OH and SFM-Ac continue to be trapped, the pH value

adjusted decreased slightly from pH 13 to pH 11 during the process and this may cause the

analyte to back extract. In comparison to SL-FM systems, the pH of the strip phase is more

stable (as the volume is comparatively bigger) thus, the analyte is continuously trapped into

the strip phase and the extraction efficiency increased with increasing time.

Results and discussion

56

0

3

6

9

12

15

18

21

24

Extraction (%)

1 2 3 4 5

Time (hour)

IBU-2OH SFM-Ac

Figure-2.28:

Stripping of IBU-2OH and SFM-Ac as a function of extraction time

(Extraction conditions: see Figure-2.27)

2.3.2.4 Influence of the pH of the strip phase

The pH of the strip phase also plays an important role on the extraction efficiency.

Theoretically, for a nearly complete trapping of IBU-2OH and SFM-Ac, the pH on the strip

side should be at least 3.3 pH units higher than the pK

a

of these compounds (Figure-2.18).

Different buffer solutions (Figure-2.29) and different concentrations of NaOH in the strip

phase (Figure-2.30) were used. Figure-2.30 shows that optimal stripping of IBU-2OH and

SFM-Ac occurred at pH ~ 13 by using 0.1 mol/L NaOH.

Results and discussion

57

0

2

4

6

8

10

12

14

16

Extraction (%)

7 8 9 10

pH in strip phase (buffer)

IBU-2OH SFM-Ac

Figure-2.29:

Stripping of IBU-2OH and SFM-Ac as a function of pH of buffer in strip

phase, (Extraction conditions: see Figure-2.27)

0

5

10

15

20

25

Extraction (%)

0,01 0,1 1

Concentration of NaOH in

strip phase (mol/L)

IBU-2OH

SFM-Ac

Figure-2.30:

Stripping of IBU-2OH and SFM-Ac as a function of the concentration of

NaOH in strip phase, (Extraction conditions: see Figure-2.27)

2.3.2.5 Influence of strip phase volume

In the present work, the volume of the strip phase was varied in the range of 0.3- 0.6 mL,

while the concentration of metabolites in the feed phase was kept at 0.01 mg/L. The volume

of the feed phase was 500 mL. The results show that the maximum extraction efficiencies for

IBU-2OH and SFM-Ac were obtained at a strip phase volume of 0.4 mL (Figure-2.31).

Results and discussion

58

0

5

10

15

20

25

Extraction (%)

0,3 0,4 0,5 0,6

Strip phase volume (mL)

IBU-2OH SFM-Ac

Figure-2.31:

Stripping of IBU-2OH and SFM-Ac as a function of the volume of strip phase

(0.1 mol/L NaOH), (Extraction conditions: see Figure-2.27)

2.3.2.6 Influence of metabolites concentration

Three concentrations 1, 10, and 100

µ

g/L of metabolites were investigated. In this range of

concentration the effect on the extraction efficiency is negligible for IBU-2OH and SFM-Ac

(Table-2.8).

Table-2.8:

Extraction efficiency of SL-BM for IBU-2OH and SFM-Ac as function of

concentration (Extraction conditions: feed phase 500 mL of 0.1 mol/L HCl; membrane: DHE

with 10 % (w/w) TOPO; strip phase 0.4 mL 0.1 mol/L NaOH; n = 3)

Metabolite Concentration of feed

(

µ

g/L)

Cs

(mg/L)

E

(%)

E

e

(

E

e(max)

=1250)

1 0.228 18.3 228.7

10 2.325 18.6 232.5

IBU-2OH

100 23.875 19.1 238.8

1 0.212 16.96 212.5

10 2.105 16.84 210.0

SFM-Ac

100 22.050 17.64 220.5

2.3.3 Extraction ability of the active drugs CBZ, DCF, IBU and SFM

The SL-BM system was also employed to extract the active drugs SFM, CBZ, DCF, and IBU

from spiked water samples. Different liquid membranes with and without carrier, e.g. DHE,

DHE with 0.025 g/L OcSA, and DHE with 10 % (w/w) TOPO were tested. As shown in

Table-2.9, the maximum extraction yield for DCF (~ 64.3 %) and for IBU (~ 70.1 %) were

Results and discussion

59

obtained by using DHE with OcSA as liquid membrane, whereas SFM and CBZ remain not

extractable.

Table-2.9:

Extraction efficiency of SL-BM for DCF and IBU as function of liquid membrane

composition, (Extraction conditions: feed phase 0.01 mg/L of active drugs in 500 mL of 0.1

mol/L HCl; strip phase 0.4 mL 0.1 mol/L NaOH; n = 3)

Active drug Membrane

Cs

(mg/L)

E

(%)

E

e

(

E

e(max)

=1250)

DHE 5.10 40.8 510

DHE with 0.025 g/L OcSA 8.03 64.3 803

DCF

DHE with 10 % (w/w) of TOPO 3.425 27.4 342

DHE 5.71 45.7 571

DHE with 0.025 g/L OcSA 8.76 70.1 876

IBU

DHE with 10 % (w/w) of TOPO 5.812 46.5 581

2.3.4 Comparison between the extractability of active drugs and metabolites by using

SL-BM systems

The extraction of active drugs and metabolites were compared to find out the different

behaviour of these compounds in SL-BM extraction systems. For this purpose the same

extraction conditions with three different liquid membrane systems (DHE, DHE with 0.025

g/L OcSA and DHE with 10 % (w/w) TOPO) were applied in a SL-BM cell.

As shown in Figure-2.32, a co-relation between the extraction efficiency and partition

coefficient of the acidic compounds is obvious. DHE as a liquid membrane gave a satisfactory

extraction efficiency only for the compounds with a high partition coefficient like DCF and

IBU. By using DHE admixed with OcSA, the extraction efficiency for DCF and IBU

increased, however, the membrane is not able to extract the compounds with lower partition

coefficient. DHE with TOPO as a carrier extracts DCF, IBU and to some extend, also the

compounds having lower partition coefficients such as IBU-2OH and SFM-Ac. It is to assume

that TOPO forms hydrogen–bonded associates with the carboxyl and/or hydroxyl substituents

of DCF, IBU, IBU-2OH and SFM-Ac, thus enhancing the analyte-DHE interactions so that

transport of these compounds across the liquid membrane occurred. On the other hand, adding

of TOPO to DHE facilitates the transport into the membrane (see Table-2.1). However, the

release of these compounds (DCF, IBU, IBU-2OH, and SFM-Ac) into the strip phase is

negligible. Therefore, the overall extraction efficiency is low. A similar behavior was found

for SFM-Glu and SFM which can not be extracted at all.

Results and discussion

60

Also the basic compounds CBZ and CBZ-DiOH were not transported into the strip phase of

the different liquid membranes employed.

Furthermore, the extraction of active drugs and metabolites depend in a different way on the

pH of the feed phase. For the metabolites, the extraction efficiency of IBU-2OH and SFM-Ac

decrease with a decreasing acidity of the feed phase in the range from acidic (pH ~1, by 0.1

mol/L HCl) to neutral water sample. The pH value of the feed phase has no effect on the

extraction efficiency of the active drugs DCF and IBU.

SFM-Glu 0,56

SFM 0,88

SFM-Ac 1,47

IBU-2OH 1,69

DCF 3,28

IBU 3,72

DHE

DHE + OcSA

DHE + TOPO

0

10

20

30

40

50

60

70

80

Partition coefficient (Log P)

Extraction (%)

Figure-2.32:

Stripping of SFM-Glu, SFM, SFM-Ac, IBU-2OH, DCF, and IBU as a

function of the extraction efficiency with Log P of these compounds, membrane: DHE,

DHE with 0.025 g/L OcSA, and DHE with 10 % (w/w) TOPO, (Extraction conditions: feed

0.01 mg/L of selected pharmaceuticals in 500 mL of 0.1 mol/L HCl, strip: 0.4 mL of 0.1

mol/L NaOH, and 4 hours of extraction)

Results and discussion

61

2.3.5 Extraction of IBU-2OH, SFM-Ac, DCF and IBU by using a SL-4-bag membrane

(SL-4-BM) system

In order to improve the extraction yields and the extraction efficiencies, the number of bag

membranes in the same extraction device was raised. It was found that a higher number of

bags in the same extraction device increased the extraction efficiencies markedly. As

presented by the data in Table-2.10: ~ 44 % of IBU-2OH, ~ 58 % of SFM-Ac, ~ 30 % of DCF

and ~ 52 % of IBU were trapped in to the strip phase by using four bags as SLM-support

(Figure-2.33).

Figure-2.33:

SL-4-BM device; (1) bag membrane filled with strip phase with volume 0.3 -

0.6 mL for every bag; (2) sample solution with volume 500 mL of feed phase; (3) stir bar

Table-2.10:

Extraction efficiency of SL-BM systems as a function of number of bag

membranes (Feed phase: 0.01 mg/L of IBU-2OH, SFM-Ac, DCF, and IBU in 500 mL of 0.1

mol/L HCl; strip phase: 0.4 mL of 0.1 mol/L NaOH for every bag; liquid membrane: DHE

with 10 % TOPO (w/w); n = 3)

IBU-2OH SFM-Ac DCF IBU Number of

bags

Cs

(mg/L)

E

(%)

Cs

(mg/L)

E

(%)

Cs

(mg/L)

E

(%)

Cs

(mg/L)

E

(%)

1 2.28 18.3 2.1 16.8 0.71 5.7 1.83 14.7

2 3.46 27.7 2.9 23.2 1.08 8.7 2.81 22.5

3 4.98 39.9 4.82 38.6 2.38 19.1 4.56 36.5

4 5.58 44.7 7.27 58.2 3.8 30.4 6.52 52.2

The data of table Table-2.10 clearly reveal that the increased number of bags increases the

overall extraction yield drastically.

Results and discussion

62

2.3.5.1 Reproducibility of extraction with the SL-4-BM system

The reproducibility of the SL-4-BM system was tested by a series of tests under the same

conditions. The total relative standard deviations (S

rel

) were found to range from 0.3 to 1.2 for

IBU-2OH, SFM-Ac, DCF, and IBU as listed in Tables-2.11 -2.14. The methods show

reasonable reproducibility of the extraction of low concentration of analytes (0.01 mg/L).

Table-2.11:

Reproducibility for SL-4-BM system for extraction of IBU-2OH

4 Bags

number Bag I Bag II Bag III Bag IV Total

Cs

(mg/L)

1.17 1.65 1.13 1.72

5.67

E

(%)

9.4 13.2 9.1 13.8

45.5

Cs

(mg/L)

1.0 1.5 1.47 1.61

5.58

E

(%)

8.0 12.0 11.8 12.9

44.7

Cs

(mg/L)

1.08 1.56 1.43 1.53

5.6

E

(%)

8.7 12.5 11.5 12.3

45

Cs

(mg/L)

0.88 1.58 1.4 1.86

5.72

E

(%)

7.1 12.7 11.2 14.9

45.9

Cs

(mg/L)

0.9 1.61 1.3 1.8

5.61

E

(%)

7.2 12.9 10.4 14.4

44.9

S

rel

0.9 0.4 1.0 1.0 0.5

Feed phase 0.01 mg/L of IBU-2OH in 500 mL of 0.1 mol/L HCl, strip phase: (4 x 0.4 mL) of

0.1 mol/L NaOH, liquid membrane: DHE with 10 % (w/w) TOPO, extraction time: 4 hours, n

= 5,S

rel

: standard deviation.

Table-2.12:

Reproducibility for SL-4-BM system for extraction of SFM-Ac (extraction

conditions see Table-2.11)

4 Bags

number Bag I Bag II Bag III Bag IV Total

Cs

(mg/L)

1.7 2.02 1.53 2.15

7.41

E

(%)

13.6 16.2 12.3 17.2

59.3

Cs

(mg/L)

1.71 2.11 1.3 2.15

7.27

E

(%)

13.7 16.9 10.4 17.2

58.2

Cs

(mg/L)

1.73 2.11 1.46 2.15

7.45

E

(%)

13.9 16.9 11.7 15.8

58.3

Cs

(mg/L)

1.78 2.03 1.48 1.97

7.26

E

(%)

14.3 16.3 11.9 16.5

59

Cs

(mg/L)

1.68 2.01 1.45 2.13

7.27

E

(%)

13.5 16.1 11.6 17.1

58.3

S

rel

0.3 0.3 0.7 0.6 0.5

Results and discussion

63

Table-2.13:

Reproducibility for SL-4-BM system for extraction of DCF (extraction

conditions see Table-2.11)

4 Bags

number Bag I Bag II Bag III Bag IV Total

Cs

(mg/L)

1.0 1.08 0.8 0.71

3.59

E

(%)

8.0 8.7 6.4 5.7

28.8

Cs

(mg/L)

0.93 1.07 0.82 0.73

3.55

E

(%)

7.5 8.6 6.6 5.9

28.9

Cs

(mg/L)

1.08 1.02 0.87 0.72

3.69

E

(%)

8.7 8.2 7.0 5.8

29.7

Cs

(mg/L)

1.03 1.02 0.85 0.72

3.62

E

(%)

8.3 8.2 6.8 5.8

29.1

Cs

(mg/L)

0.97 1.11 0.76 0.77

3.61

E

(%)

7.8 8.9 6.1 6.2

29

S

rel

0.4 0.3 0.3 0.1 0.3

Table-2.14:

Reproducibility for SL-4-BM system for extraction of IBU (extraction conditions

see Table-2.11)

4 Bags

number Bag I Bag II Bag III Bag IV Total

Cs

(mg/L)

1.61 2.81 0.47 1.83

6.72

E

(%)

12.9 22.5 3.8 14.7

53.9

Cs

(mg/L)

1.36 2.72 0.55 1.81

6.44

E

(%)

10.9 21.8 4.4 14.5

51.6

Cs

(mg/L)

1.46 2.56 0.77 1.72

6.51

E

(%)

11.7 20.5 6.2 13.8

52.2

Cs

(mg/L)

1.26 2.82 0.52 1.83

6.43

E

(%)

10.1 22.6 4.2 14.7

51.6

Cs

(mg/L)

1.56 2.65 0.7 1.83

6.74

E

(%)

12.5 21.2 5.6 14.7

54

S

rel

1.1 0.8 1.0 0.3 1.2

It is remarkable, that the overall standard deviation (S

rel

) of the extraction data of the SL-4-

BM system were found to be relatively lower (IBU-2OH) or at a similar level compared to the

individual bags (DCF, IBU and SFM-Ac).

2.3.5.2 Influence of humic acids

Humic substances are widespread in the aquatic environment, i. e., natural waters, lakes and

sediments, in both soluble and insoluble forms. These macromolecular substances are formed

as a product of chemical and biological transformations of animal and plant residues. The

Results and discussion

64

concentration of humic acids in groundwater is in the range of 10 ng/L -100

µ

g/L. The

principal properties of humic acids and their subsequent potential application depend strongly

on their origin and the isolation procedure [146].

Humic acids are considered natural polyelectrolytic organic compounds of complex structures

including condensed aromatic rings with a large number of attached carboxylic and hydroxyl

groups. The structure of these biopolymers is still not clearly defined. Humic acids are

organic ligands and play a crucial role in speciation, transport and deposition of a variety of

compounds ranging from metal ions to lipophilic compounds [147].

It is very common that surface waters contain humic acids. Therefore, the effect of the

presence of humic acids on the extraction efficiency of IBU-2OH, SFM-Ac, DCF, and IBU

were studied by adding 18 mg/L of humic acids (HUS-Standard Hohlohsee 13) [112] to the

sample solution (feed phase). Figure-2.34 shows, that the extraction efficiency of IBU-2OH

and SFM-Ac were slightly increased in present of humic acid in the feed phase. By contrast,

the extraction efficiency of DCF and IBU decreased after adding 18 mg/L humic acids in feed

phase as shown in Figure-2.34.

IBU-2OH

SFM-Ac

DCF

IBU

without humic acids

with humic acids

0

10

20

30

40

50

60

70

Extraction(%)

Figure-2.34:

Stripping of IBU-OH , SFM-Ac, DCF, and IBU in SL-4-BM as a function

with humic acids adding in feed phase, [Extraction conditions: feed: 0.01 mg/L of drugs

with or without 18 mg/L humic acids (HUS-Standard Hohlohsee 13) in 500 mL of 0.1

mol/L HCl, liquid membrane: DHE with 10 % (w/w) TOPO, 4 bag membranes filled with

(4 x 0.4 mL) 0.1 mol/L NaOH as strip phase, after 4 hours of extraction]

As pointed out in section 2.4, the second object is to compare the analytical applicability of

SL-4-BM with another standard method such as SPE that proved to be useful to extract the

selected analytes from water samples.

Results and discussion

65

2.3.6 Solid phase extraction (SPE)

Solid-phase extraction (SPE) is widely used in the determination of pharmaceuticals from

environmental water samples [148, 149]. SPE is essentially a three-step process. A sample is

initially passed through the sorbent bed, and analytes present in the sample are exhaustively

extracted from the sample matrix to the solid sorbent. In the second step, unwanted analytes

or matrix components are selectively desorbed from the solid sorbent by washing with a

solution or appropriate solvent. In the final step, an eluting solvent is able to desorb analytes

of interest. The resulting eluent may then be concentrated by evaporation to the desired

volume [150, 151]. Depending on the choice of sorbent, a wide range in polarity and chemical

class may be covered. For the extraction of high polar analytes SPE with polymeric sorbents

often proved to be superior to alkyl-bonded silica (e.g., octadecasilane). A variety of hyper-

crosslinked sorbents are commercially available, differing in the degree of linkage, porosity

and surface area. They are either co-polymerisates of styrene or a polar component (e.g.,

methacrylate or N-vinylpyrrolidone) or the function groups are introduced after

polymerization (e.g., by sulfonation). This functionalisation results in mainly two effects:

improved wetting characteristics for better mass transfer and additional possibilities for

interaction with functional groups of the analytes and thus a higher degree of retention.

At the first stage of this study, a variety of cartridges were investigated such as polystyrol-

DVE-copolymer (Isolute ENV) [152, 153], octadecasilane (Bakerbond C-18) [152, 153],

[poly(divinylbenzene-co-N-vinylpyrrolidone)] (Oasis HLB) [152, 153] and C18-material

endcapped (Strata C18-E) [154] with the extraction procedure presented schematically in

Figure-2.35.

Table-2.15 shows the results of a comparative study on the recovery efficiencies of the

analytes in question obtained by means of the four cartridges applied to prepared water

samples. At sample volume 500 mL with 0.01 mg/L the concentration of analytes, the higher

recoveries were obtained with Oasis-HLB cartridge.

Results and discussion

66

Table-2.15:

Analyte recoveries obtained with various SPE cartridges (sample volume: 500

mL, concentration of analytes: 10

µ

g/L, n = 3)

Recovery (%) SPE-Material IBU-2OH SFM-Ac DCF IBU

Bakerbond 56.7 % 60.7 % 92.8 % 80.3 %

Isolute ~ 0 % ~ 0 % 5.2 % 23.7 %

Strata 73.5 % 71.4 % 76.3 % 80.0 %

Oasis 74.6 % 77.4 % 76.6 % 82.4 %

The Oasis-HLB cartridge is classified as a mixed mode; it features two retention mechanism

mainly strong cation exchange and reversed-phase. The most attractive features of Oasis-HLB

cartridge are its hydrophilic-lipophilic-balanced composition that is responsible for both

strong reversed-phase retention and water-wetability. In addition, these sorbent are stable

from pH 1 to 14 due to the use of pH wash systems [155]. Therefore Oasis-HLB cartridge was

finally chosen as the best cartridge for extraction of selected analytes.

Figure-2.35:

SPE extraction procedure

500 mL sample volume (10

µ

g/L analytes in

distilled water)

Filtration: 0.45

µ

m Nylon-Filter

Solid phase Extraction:

Conditioning:

5 mL MeOH, 5 mL distilled water

Loading:

at 5 mL/min

Washing:

10 mL 5% (v/v) MeOH

Drying:

for 10 min under water vacuum

Elution:

0.8 mL MeOH

Addition of MeOH to final volume (1 mL)

HPLC analysis

Results and discussion

67

2.3.7 Comparative study of SL-4-BM and SPE extraction techniques

The comparison between Oasis-HLB of SPE cartridge (Figure-2.35) and SL-4-BM system

(Figure-2.36) in extraction of IBU-2OH, SFM-Ac, DCF and IBU compounds from prepared

water samples has the following results:

•

Recovery:

the recovery obtained with SPE (70% for IBU-2OH, 71.7% for SFM-Ac,

75.1% for DCF and 81.5% for IBU) were higher than with SL-4-BM (44.7 for IBU-

2OH, 58.2 % for SFM-Ac, 30.6 % for DCF and 52.7 % for IBU) as listed in Table-

2.16.

•

Enrichment factor:

the enrichment factor obtained with SL-4-BM (558 for IBU-

2OH, 727 for SFM-Ac, 383 for DCF and 658 for IBU) were higher than with SPE

(350 for IBU-2OH, 355 for SFM-Ac, 375 for DCF and 407 for IBU) (see Table-2.16).

•

Solvent consumption:

SL-4-BM has a very low consumption of organic solvent (only

organic solvent needed is used to fill the pores of porous support) comparing to SPE

(more than 15 mL); as a result the former is more environmentally friendly.

•

Extraction time:

the extraction time for SL-4-BM is 4 hours, while SPE need more

than 6 hours.

•

Clean up:

SL-BM gave a considerably more efficient clean-up than SPE [112].

•

Selectivity:

there are many

examples of analytical applications to aquatic environment

system which show nearly complete selectivity of SLM systems. For example, triazine

herbicides were extracted from spiked river water both with SLM and with SPE [14].

The results show that in SLM extract virtually only the expected compounds are

found, while a typical “humic hump” from humic acids together with disturbing from

unknown compounds is seen in the SPE extract.

Table-2.16:

Comparison of performance between SL-4-BM and SPE, (concentration of

metabolites: 1

µ

g/L, and n = 3)

IBU-2OH SFM-Ac DCF IBU

Extraction

technique

R

(%)

C

S

µ

g/L

E

e

R

(%)

C

S

µ

g/L

E

e

R

(%)

C

S

µ

g/L

E

e

R

(%)

C

S

µ

g/L

E

e

E

e(max)

SPE 70 350 350

71.7

355 355

75.1

375 375

81.5

407 407

500

SL-4-BM 44.7 558.7

558

58.2

727.5

727

30.6

383.7

383

52.7

658.7

658

1250

Results and discussion

68

Step-1

Step-2

Step-3

Figure-2.36:

Extraction procedure with the SL-4-BM system

4 bag membranes (PP)

Washing: 3 times with

water

Drying: for 30 min at

room temperature

Soaking: 2 h in liquid

membrane (dihexyl ether

with 10 % (w/w) TOPO)

Filling: 0.4 mL (0.1

mol/L NaOH) every bag

500 mL water acidified

with 6.5 mL of

25% HCl

Filtration: 0.45

µ

m

Nylon-Filter

Extraction

.

Dipping of SL-4-BM device

in feed at room temperature

.

Stirrer setting:

at 360 rpm

.

Extraction time:

4 h

Neutralizing the strip phase of every

bag with 0.4 mL of 0.1 mol/L HCl

HPLC analysis

of each strip

Mounting of 4 bag

membranes to holding

device

Preparation of SLM-system

Results and discussion

69

2.3.8 Conclusion

SLM bag membrane systems were developed as a sample enrichment device to extract and

enrich metabolites and drugs from spiked water samples. The liquid membrane used to trap

these compounds consisted of DHE with TOPO and OcSA as carrier.

Several factors affecting the extraction efficiency during SL-BM enrichment were studied.

These factors can be structure or operator dependent.

In the first case, it can be assumed that the transport of all investigated compounds is

influenced by its acid-base (pK

a

, pK

b

) properties and partition coefficient (octanol/water)

Log P.

In the second case, it can be concluded that from the obtained results, the most effective

conditions for membrane transport are: 10 % (w/w) TOPO as carrier dissolved in DHE, low

pH of the feed phase (0.1 mol/L HCl), high pH of the strip phase (0.1 mol/L NaOH), a 4-bag

membrane system, and 4 hours of extraction to create a driving force of the process which

sufficient by produces a high mass transfer of IBU-2OH and SFM-Ac.

SL-4-BM system was used to enrich and determine the target metabolites from prepared

water sample by means of HPLC-UV. High enrichment factors were achieved for both acidic

metabolites IBU-2OH and SFM-Ac (Table-2.16). Extraction efficiency was 44 % for IBU-

2OH and 58 % for SFM-Ac.

DCF and IBU were also separated and enriched under the same conditions. The extraction

efficiency of these compounds was 27 % for DCF and 50 % for IBU.

Solid phase extraction (SPE) was used to extract the target drug metabolites from prepared

water samples. The achieved recoveries with SPE (80 % IBU-2OH, 71 % SFM-Ac, 96.8 %

DCF, and 91.5 % IBU) were higher than SL-4-BM (44 % IBU-2OH, 58 % SFM-Ac, 27 %

DCF, and 50 % IBU), but the novel SL-4-BM offer more advantages due to higher

enrichment factors, low consume of organic solvents and time.

Results and discussion

70

2.4 Application: determination of drug traces in water by means of SL-4-BM system

and HPLC-UV

2.4.1 Influence of analyte concentration and matrix

To judge to what extent the developed method can be affected by parameters having direct

influence on the extraction recoveries and signal response, e.g. sample concentration and

matrix effects were studied.

Three sample concentrations (300, 500, and 1000 ng/L) were chosen to study the

concentration effects at a constant sample volume of 500 mL. To insure the robustness of the

developed method, spiked tap water of Paderborn (see Table-3.14) was analyzed.

The percentage recovery of the analytes spiked into each type of tested sample was calculated

as follows:

100(%) ×

−

=

S

OCAS

R (35)

Where AS is the peak area of spiked sample, OC is the peak area of unspiked sample and S is

the peak area of non enriched standard [160, 161].

Recoveries obtained by varying the sample concentrations in spiked distilled water and tap

water showed no significant differences (Table-2.17 and 2.18).

Table-2.17:

Extraction recoveries (R) of selected analytes from

distilled water

Compound Concentration

Spiked

C

S

µ

g/L

C

F

µ

g/L

R

(%) S

rel

0.3

µ

g/L 155.2 0.124 40.6 8.1

0.5

µ

g/L 269.3 0.215 43.1 6.1

IBU-2OH

1

µ

g/L 528.7 0.422 42.3 6.5

0.3

µ

g/L 199.5 0.159 53.2 7.7

0.5

µ

g/L 343.7 0.274 55.0 6.5

SFM-Ac

1

µ

g/L 718.7 0.574 57.5 6.8

0.3

µ

g/L 96 0.076 25.6 4.5

0.5

µ

g/L 185 0.148 29.6 2.5

DCF

1

µ

g/L 392 0.313 31.4 4.5

0.3

µ

g/L 177.3 0.141 47.3 7.1

0.5

µ

g/L 323.1 0.258 51.7 8.7

IBU

1

µ

g/L 685 0.548 54.8 8.0

Results and discussion

71

Table-2.18:

Extraction recoveries (R) of selected analytes from

tap water

(city of Paderborn)

Compound Concentration

spiked

C

S

µ

g/L

C

F

µ

g/L

R

(%) S

rel

0.5

µ

g/L 266.8 0.213 42.7 4.2

1

µ

g/L 543.7 0.434 43.5 6.2

IBU-2OH

1.5

µ

g/L 860.6 0.688 45.9 5.2

0.5

µ

g/L 341.8 0.272 54.7 7.2

1

µ

g/L 692.5 0.554 55.4 6.9

SFM-Ac

1.5

µ

g/L 1038.7 0.830 55.4 6.6

0.5

µ

g/L 183.1 0.146 29.3 2.5

1

µ

g/L 380 0.304 30.4 4.1

DCF

1.5

µ

g/L 613.1 0.490 32.7 2.9

0.5

µ

g/L 309.3 0.247 49.5 8.9

1

µ

g/L 632.5 0.506 50.6 6.2

IBU

1.5

µ

g/L 997.5 0.798 53.2 5.8

2.4.2 Application of the developed method for real surface water samples

The developed methods were tested for applicability to real water samples by analyzing the

surface water from river Ruhr affected by effluent STP. The water samples were spiked and

non-spiked with the metabolites synthesized and the parent drugs to determine recoveries.

The samples were treated by the developed separation procedure. The strip phase contained

the enriched analytes separated from the surface water matrix.

For trace analysis of real samples the extraction procedure was modified and the

chromatographic conditions were adjusted to HPLC-UV analysis. The method detection

limits (MDLs) were determined by spiking the analytes into 500 mL of surface water in the

concentration range 0.3-1

µ

g/L. MDLs are distributed between 0.071- 0.185

µ

g/L.

Recoveries obtained by varying the sample concentrations in surface water and distilled water

showed no significant differences (Table-2.17 and 2.19). As a result, no target analytes were

found in the surface water sample and the water was evidently not polluted.

Results and discussion

72

Table-2.19:

Extraction recoveries (R) of selected analytes from

surface water

(river Ruhr)

Compound

OC*

Concentration

spiked

C

S

µ

g/L

C

F

µ

g/L

R

(%)

MDL

µ

g/L

0.3

µ

g/L 171.6 0.137 44.8

0.5

µ

g/L 261.1 0.208 41.7

IBU-2OH

< 0.001

1

µ

g/L 484.1 0.387 38.7

0.185

0.3

µ

g/L 202 0.161 53.8

0.5

µ

g/L 355.4 0.284 56.8

SFM-Ac

< 0.001

1

µ

g/L 591.4 0.473 47.3

0.094

0.3

µ

g/L 125 0.100 33.3

0.5

µ

g/L 190 0.152 30.4

DCF

< 0.001

1

µ

g/L 335 0.268 26.8

0.071

0.3

µ

g/L 197.4 0.157 52.6

0.5

µ

g/L 304.2 0.243 48.6

IBU

< 0.001

1

µ

g/L 640.2 0.512 51.2

0.122

OC*

:

the peak area of unspiked surface water sample

Results and discussion

73

2.5 Development method based on LC/MS

Mass spectrometry (MS) is widely used detection technique that provides quantitative and

qualitative information about the components in mixture. In qualitative analysis it is very

important to determine the molecular weight of an unknown compound and MS is a technique

capable for that. MS is also generally more sensitive than an UV-detector for quantification.

An MS detector consist of three main parts: the ionization source (interface) where the ions

are generated, the mass analyzer (separation), which separates the ions according to their

mass-to-charge ration (m/z), and the electron multiplier (detector). There are several types of

ion sources, which utilize different ionization techniques for creating species.

Three popular ionization techniques are: electrospray ionization (ESI), atmospheric pressure

chemical ionization (APCI) and matrix-assisted laser desorption (MALDI). Electrospray is

the most widely used ionization technique when performing LC-MS, and has proved to be a

most versatile tool for soft ionization of a large variety of analytes [156, 157].

Common mass analyzers that are commercially available are: quadrupole, ion-trap, time-of-

flight (TOF) and magnetic sector analyzers. Ion trap MS is especially useful for advanced

qualitative analysis due to the ability of technique to provide scans up to the MS

5

range. The

power of ion trap MS stems from the fact, that several generations of daughter ions can be

generated providing MS

n

spectra. This capability provides a rich source of structural

information [157].

Previously published methods for analysis of pharmaceuticals in natural water samples

commonly used gas chromatography coupled to mass spectrometry (GC-MS). However,

recently, the application of liquid chromatography/ mass spectrometry (LC-MS) rapid growth,

in part due to the instrumental developments afforded in this field. With the development of

atmospheric-pressure ionization techniques, the combination of liquid chromatography and

tandem mass spectrometry (LC-MS/MS) has become the method of choice for high-

sensitivity quantitative analysis of drugs and metabolites in water samples. This because LC-

MS/MS with ESI and APCI offers [158]:

•

versatility: most drugs and metabolites can be effectively analyzed by LC-MS/MS;

•

specificity: the combination of liquid chromatography and tandem mass spectrometric

detection can provide unparalleled specificity;

•

sensitivity: ESI and APCI often permit accurate measurement of analyte at sub-ng/L;

Results and discussion

74

•

rapid analysis: no derivatisation of analyte is required, and run times are often a few

minutes long;

•

quantitative accuracy and precision: by the use of stable isotope-labeled analogs as

internal standards one can avoid problems resulting from differences in extraction and

chromatographic behavior between the analyte and the internal standard, and can

correct for analyte losses due to chemical instability;

•

Ease of operation: LC-MS/MS analyses are often easier to set up and perform than

GC-MS analysis.

Therefore, LC-MS was chosen for this study. The LC-MS/MS system used was a LCQ

Advantage in ESI-mode (Thermo Finnigan, electron, Egelsbach, Germany), connected with a

gradient pump (Spectra SYSTEM P4000) was employed.

2.5.1 Mass spectrometer parameters

Several mass spectrometric parameters must be optimized in order to obtain the highest

possible abundance of the analytes in MS.

Electrospray operation

:

electrospray operation parameters were optimized by direct infusion

of the analytes by means of a syringe pump at flow rate of 10

µ

g/min into the ESI source.

Nitrogen was used as sheath gas at a flow rate of approximately 47.0 L/min, the spray voltage

was set to 5 kV in the positive ionization mode and to 4.5 kV in the negative ionization mode,

and the transfer capillary was set to 250

o

C.

Capillary voltage:

the properties of the functional groups define the ionization mode; IBU-

2OH, DCF and IBU contain carboxylic acid groups, so they will favor the negative mode

while, SFM-Ac prefer the positive mode because of its amine functionality. The capillary

voltage, ion optics parameters were optimized for all compounds based on mass peak

intensity. The optimal collision energy was between 5 and 10 V in negative ion mode.

Analyte fragmentation pattern:

all analytes were measured in full scan mode under MS/MS

conditions. The chemical structure and typical mass spectra of the analytes are shown in

(Figures-2.37- 2.40) and the selected ions are listed in Table-2.20.

Results and discussion

75

Table-2.20:

LC-ESI-MS/MS parameters for analysis of the selected drugs

Analyte Mode Collision

energy

[V]

Normalized collision

energy

[%]

Fragment ion

(m/z)

IBU-2OH -ve 5 30 221/177

SFM-Ac +ve 10 35 296/197.9

DCF -ve 5 30 293.8/250

IBU -ve 5 28 204.9/159

m/z = 159

[M-H]

-

m

/

z

=

2

0

5

COO

CH

3

H

3

C

CH

3

CH

3

H

3

C

CH

3

- CO

2

a)

b)

Figure-2.37:

(a) LC-ESI/MS spectrum of IBU and (b) the MS/MS product ion spectrum of

the [M-H]

-

at m/z 205 (Concentration of analyte: 1 mg/L in methanol)

Results and discussion

76

H

N

O-

O Cl

Cl

H

N

O-

O Cl

Cl

-CO

2

m

/

z

=

2

9

4

m/z = 250

[M-H]

-

a)

b)

Figure-2.38:

(a) LC-ESI/MS spectrum of DCF and (b) the MS/MS product ion spectrum of

the [M-H]

-

at m/z 294 (Concentration of analyte: 1 mg/L in methanol)

Results and discussion

77

S N

O

N

O

CH

3

HN

CH

3

O

S

O

N

CH

3

O

H

N

H

5

O

OH

2

N

CH

[M+H]

+

m/z = 296

m/z= 65

m

/

z

=

1

0

8

m

/

z

=

1

3

4

m/z= 198

H

a)

b)

Figure-2.39:

(a) LC-ESI/MS spectrum of SFM-Ac and (b) the MS/MS product ion spectrum

of the [M+H]

+

at m/z 296 (Concentration of analyte: 1 mg/L in methanol)

Results and discussion

78

m/z = 177

[M-H]

-

m

/

z

=

2

1

COO

CH

3

HO

H

3

C

CH

3

CH

3

HO

H

3

C

CH

3

- CO

2

Figure-2.40:

(a) LC-ESI/MS spectrum of IBU and the MS/MS product ion spectrum of the

[M-H]

-

at m/z 221 (Concentration of analyte: 1 mg/L in methanol)

The extraction steps by SL-4-BM end up in a solution of 0.1 mol/L NaCl. NaCl is reported

potentially to cause signal suppression during the ESI process [159]. It was experienced that a

salt film built up quickly on the spray needle and curtain plate, resulting in a decrease in

signal intensity and stability of the mass spectra, when the analyte sample contained NaCl.

Therefore, it was decided to avoid NaCl (see section 3.2.5.3).

Mobile phase

: in HPLC, the pH-value of the samples or the mobile phases can have a great

influence in the retention time for the analytes having an acidic group (IBU-OH, SFM-Ac,

DCF and IBU). In all cases, a comparison has to be made between the improvement of

separation and deterioration of the ionization process. Therefore, many mobile phase

compositions with different pH values were studies to achieve suitable chromatographic

separation and a stable ionization spray. Methanol is a better protic solvent and generally

produces more ions than acetonitrile. Whereas, acetonitril lead to better HPLC separation and

a lower column back pressure. Initially, both acetonitrile and methanol were tested as organic

mobile phase for LC separation. Different methods have been tested to find out the better

separation and resolution of IBU-2OH, SFM-Ac, DCF and IBU. Due to the widely varying

properties of these pharmaceuticals, no single method was capable of measuring all of these

Results and discussion

79

compounds. Hence, further work is needed to optimize the mass spectrometer parameters and

to acquire best separation and resolution of the selected analytes by LC-ESI-MS.

2.6 Conclusions and outlook

Residues of pharmaceutical products reaches wastewater treatment plants via human and

veterinary urinary or fecal excretion and from pharmaceutical manufacturing discharges.

Wastewater treatment plants influent constituents have to face a complex mixture of various

organic and inorganic substances and detailed information on potential wastewater

composition are often scarce. Pharmaceutical compounds are not totally eliminated in the

wastewater treatment plants and due to this fact, variable concentrations of pharmaceutical

drugs and their metabolites can reach surface, ground water and exceptionally drinking water.

The concentrations of drugs and metabolites, which have been detected in aquatic

environment, are in the range from ng to µg/L and this level would be sufficient to induce

estrogenic response and cause reproductive and development effects in wildlife.

Consequently, there is an urgent need to improve the techniques for water and wastewater and

to develop sensitive analytical methods for monitoring the drugs and metabolites input into

the aquatic environment. The analytical techniques usually used such as HPLC-UV-MS or

GC-MS still afford an efficient sample pretreatment to enrich and separate the analytes from

complex matrix. The aim of this study was to investigate the applicability of certain types of

liquid membranes to extract efficiently selected drugs of environmental concern and some of

their metabolites from water samples.

For this purpose certain types of liquid membranes were used such as: bulk liquid membranes

(BLM) and supported liquid membranes (SLM).

The metabolites 10,11-dihydroxycarbamazepine (CBZ-DiOH), 4

′

-hydroxydiclofenac (DCF-

4OH), 4-hydroxyibuprofen, (IBU-2OH), N-4-acetylsulfamethoxazole, (SFM-Ac),

sulfamethoxazol-N1-glucuronide (SFM-Glu) and their parent drugs carbamazepine (CBZ),

diclofenac (DCF), ibuprofen, (IBU) and sulfamethoxazole (SFM) were selected due to their

high quantity applied in medicine and their relative high concentrations found in the aquatic

environment in previous studies. Except 4

′

-hydroxydiclofenac (DCF-4OH), the metabolites

could be synthesized and characterized by IR,

1

H-,

13

C-NMR and mass spectroscopy as they

are not commercially available.

The transport of the analytes in membrane chamber was monitored by HPLC-UV detection.

Aliquots were taken from the liquid phases (feed or strip) at intervals by means of a micro-

Results and discussion

80

liter syringe. The validation was achieved by analyzing the detection system parameters:

linearity, sensitivity, limit of detection, and limit of quantitation

The mass transfer of metabolites through the liquid membranes was carried out in a three-

compartment transport cell and various supported liquid membrane-chambers. The three-

phase liquid bulk membrane system (BLM) consisted of an aqueous feed solution, an organic

solvent like: 1-pentanol, dihexyl ether (DHE), undecane and decane with and without

dissolved tri-n-octylphosphine oxide (TOPO) as a liquid bulk membrane and an aqueous

stripping solution. The transport of metabolites shows some differences, which can be

attributed to their acid/basic-behavior and their partition coefficients Log P (octanol/ water).

The supported liquid flat-membrane system (SL-FM) has been also used to extract the

metabolites. SL-FM based on a porous polypropylene membrane (PP) flat sheet impregnated

with a water-immiscible organic membrane phase placed between two PTFE-chambers of

equal size, one filled with feed sample solution and another filled with the strip phase. The

feed solution was stirred by a magnetic stirrer.

Certain membrane conditions used for bulk

liquid membrane were selected to test their applicability in SL-FM system, with different

concentrations of TOPO as a carrier.

By means of the SL-FM-chamber 4-hydroxyibuprofen, (IBU-2OH), and N-4-

acetylsulfamethoxazole, (SFM-Ac) were efficiently extracted by combining an organic

solvent (DHE, decane, undecane with TOPO as a carrier), and a pH-gradient between feed

and strip phases. Maximum extraction yields (~ 90 %) of IBU-2OH and (~ 85 %) of SFM-Ac

were obtained by using DHE with 10 % (w/w) of TOPO.

A SL-bag membrane (SL-BM) has been developed as a miniaturized sample enrichment tool

for analytical purposes. The SL-BM device consists of PP-bag soaked with the liquid

membrane and filled with 0.3 - 0.6 mL of 0.1 mol/L NaOH, as the strip phase placed in 500

mL of the aqueous, acidified sample, containing the analytes of drug traces dissolved. A

magnetic stir bar used to agitate the sample solution during the extraction. Several factors

affecting the extraction efficiency during SL-BM enrichment were studied. These factors can

be structure or operator dependent. It can be concluded that from the obtained results, the

most effective conditions for membrane transport are: 10 % (w/w) TOPO as carrier dissolved

in DHE, low pH of the feed phase (0.1 mol/L HCl), high pH of the strip phase (0.1 mol/L

NaOH), a 4-bag membrane system, and 4 hours of extraction to create a driving force of the

process which sufficient by produces a high mass transfer of IBU-2OH and SFM-Ac.

Results and discussion

81

SL-4-BM system was used to enrich and determine the target metabolites from water samples

by means of HPLC-UV. High enrichment factors (558 for IBU-2OH and 727 for SFM-Ac)

with an extraction efficiency of 45 % for IBU-2OH and 58 % for SFM-Ac were achieved in a

concentration range from 1-100

µ

g/L.

DCF and IBU were also separated and enriched by using the SL-4-BM device with DHE and

10 % (w/w) of TOPO as a liquid membrane. The extraction efficiency of these compounds

was 27 % for DCF and 46 % for IBU with an enrichment factor of 383 for DCF and 658 for

IBU at concentration range from 1-100

µ

g/L in 500 mL sample volume.

Solid phase extraction (SPE) was applied to extract IBU-2OH, SFM-Ac, DCF and IBU from

water samples. The achieved recoveries with SPE (70 % IBU-2OH, 71 % SFM-Ac, 75 %

DCF and 81 % IBU) were higher than SL-4-BM (45 % IBU-2OH, 58 % SFM-Ac, 30 % DCF

and 52 % IBU), but the novel SL-4-BM offer more advantages due to higher enrichment

factors, efficient clean-up-effects, low consume of organic solvents and time.

To prove if the SL-4-BM is robust against interferences, parameters

,

e.g. sample

concentration and matrix effect were studied. It can be recognized that neither an increasing

sample concentration nor the matrixes loaded (distilled or tap water) had a noticeable

influence in the extraction recoveries of selected analytes.

Finally the SL-4-BM method was applied to real aqueous samples from the river Ruhr. No

target analytes have been detected and the surface water sample was evidently not polluted.

These results are encouraging in terms of improving the capability of the SL-4-BM system to

extract polar pharmaceuticals from water samples, by choosing adequate liquid membrane

and operating parameters. More work is required to fully understanding the extraction process

of the analytes cross the membrane, and with this, the process may be important in future

analytical chemistry for isolation or pre-concentration. Especially in miniaturized analytical

systems, this new concept may have future.

Experimental

82

3 Experimental

3.1 Synthesis of drug metabolites

3.1.1 CBZ-DiOH

CBZ-DiOH was successfully prepared by using the oxidation

procedure of carbamazepine with

potassium permanganate as shown in Scheme-3.1. The product of CBZ-DiOH purified by

column chromatography gave white crystals with yield 20 % and m.p. 244-246

o

C [117, 118].

N

H

2

N O

N

H

2

N O

KMnO

4

, MgSO

4

MeOH

HO OH

Scheme-3.1: Synthesis of CBZ-DiOH

(0.004 mole) of carbamazepine was dissolved in dry methanol (15.2 mL). The mixture was

stirred and cooled at (-2 to 0

o

C). A solution of KMnO

4

(0.003 mole), MgSO

4

(0.5 g) and (15

mL) of water was added drop wise to the main solution of carbamazepine with methanol. The

resulting mixture was stirred at (5

o

C) for (3 hours), then leaved to be at room temperature and

filtered under vacuo. The product was collected on a filter and washed with MeOH, acetone,

and CH

2

Cl

2

, then dried under vacuo at (40

o

C) to constant weight. Finally the product was

purified by chromatography over silica gel eluted with CH

2

Cl

2

MeOH (9:1). The 10,11-

dihydroxy-carbamazepine was obtained as a white solid with yield (20 %) and m.p. (244- 246

o

C) (literature data [134]: m.p. 247

o

C)

3.1.2 DCF-4OH

The procedure was contained a multistep as shown in Scheme-3.2 [119]. Starting with 3,5-

dichlorophenol which treated with sodium nitrite in sulphuric acid gave the required nitro

compound (I) together with the o-nitro product. The isomeric nitro compounds were not

easily separate hence; the mixture was methylated with metallic tin and then acetylated. The

regioisomeric acetanilides were readily separated chromatographically to give the desired

isomer of acetamide compound (IV) as a mauve solid, m.p. 179-182

o

C, in 12% overall yield

from first compound (3,5-dichlorophenol). Copper-mediated N-arylation of acetamide with

bromobenzene proceeded smoothly to give, after basic hydrolysis, an excellent yield of

Experimental

83

diphenylamine (VI). Acylation of amine with chloroacetyl chloride yielded crude chloroamide

(VII) as a purple solid, which was then cyclised with alumimium chloride. The reaction

conditions for this cyclisation were so critical; and gave a mixture of indolone (VIII) and ether

form (IX) together with a more polar impurity. The mixture was not separable. Repeating of

these steps gave the same problem; therefore we decided to stop the synthesis steps for this

compound.

OH

ClCl

NaNO

2

, H

2

SO

4

OH

ClCl

NO

2

DMF

OCH

3

ClCl

NO

2

Sn

HCl

OCH

3

ClCl

NHCOCH

3

OCH

3

ClCl

+

NHCOCH

3

OCH

3

ClCl

NH

2

Ac

2

O, AcOH,H

2

SO

4

OCH

3

ClCl

NHCOCH

3

OCH

3

Cl Cl

NH

Bromobenzene

Cu, K

2

CO

3

OCH

3

Cl Cl

NO

Cl

ClCH

2

COCl

AlCl

3

OH

Cl Cl

N

COOH

NaOH aq

EtOH

OCH

3

Cl Cl

N

O

OH

Cl Cl

N

O

+

III

IIIIV

V

IV VI VII

VIIIIX

X

Scheme-3.2: Synthesis of DCF-4OH [119]

Me

2

SO

4

, K

2

CO

3

Experimental

84

3.1.3 IBU-2OH

The compound was prepared in a multistep procedure starting with IBU, which was converted

to 2-(p-(1-bromo-2-methylpropyl)-phenyl)propionic acid (I) with N-bromosuccinimide. Then

lithium bromide dissolved in dimethylformamide was reacted with (I) (see Scheme-3.3) to

achieve 2-(p-(2-methylprop-l-ene)phenyl)propionic acid (II). Compound (II) was converted

with m-chloroperbenzoic acid to 2-(p-(2-methyl-1,2-epoxypropyl)phenyl)propionic acid (III).

In the next step, product (III) gave 2-(p-(2-methyl-2-hydroxypropyl)phenyl)propenoic acid

(IV) by the action of potassium tert-butoxide in tetrahydrofuran under nitrogen and final

quenching by dilute HCl. Finally, compound (IV) was hydrogenated in tetrahydrofuran under

50 psi of H

2

at room temperature in the presence of 10 % (w/w) Pd/C, yielding the final 2-(p-

(2-methyl-2-hydroxypropyl)phenyl)propionic acid (IBU-2OH) as shown in scheme-6.3. The

solid product of IBU-2OH was filtered with fresh cyclohexane, and dried to constant weight

at 50

o

C in vacuo which gave white crystal with yield 45 % and m.p. 122-124

o

C [120-122].

COOH COOH COOH

NBS LiBr

III III

Br

COOH COOH COOH

OOH

OH

KO-t-Bu H

2

IV VVI

Scheme-3.3: The synthesis steps of IBU-2OH [120]

m-CPBA

Pd/C

2-(

p

-(1-Bromo-2-methylpropyl)-phenyl)propionic acid (II)

A solution of (1.16 mole) of ibuprofen (I), and (2.5 L) of CCl

4

was refluxed for (10 min)

under nitrogen, cooled to room temperature, and the treated with (1.06 mole) of N-

bromosuccinimide and 300 mg of benzoyl peroxide. The mixture was refluxed for (6 hours),

stirred overnight at room temperature, and filtered. The filtrate was concentrated to a reddish-

brown oil which was diluted with (1.5 L) of hexane to give crystals. The product was

collected on a filter and washed four times with (200 mL) portions of hexane and then dried to

constant weight to afford 185 g of (II), m.p. 112-117

0

C.

Experimental

85

2-(

p

-

(

2-Methylprop-l-ene

)

phenyl)propionic acid (III)

A solution of (0.35 mole) of 2-(p-(1-Bromo-2-methylpropyl)-phenyl)propionic Acid (II),

(0.83 mole) of lithium bromide, and (1.5 L) of DMF was heated under nitrogen between (80-

100

0

C) for (5 hours). The solution was cooled to room temperature, diluted with (5 L) of

water and extracted with three (1 L) portions of ether. The ether extracts were combined and

washed with two (1 L) portions of water and (500 mL) of brine. After the solution was dried

over MgSO

4

, the ether was removed in vacuo to give 73 g (100 %) as yellow oil.

2-(

p

-

(

2-Methyl-1,2-epoxypropyl

)

phenyl)propionic acid (IV)

To a well-stirred solution of (0.35 mole) of 2-(p-(2-Methylprop-l-ene)phenyl)propionic Acid

(III), in (750 mL) of CH

2

Cl

2

at room temperature under nitrogen was added a slurry of (0.37

mole, 85 % quality) of m-chloroperbenzoic acid in (700 mL) of CH

2

Cl

2.

The reaction was

slightly exothermic and was maintained below (35

o

C) with a water bath. The mixture was

reduced to (500 mL) in vacuo. Upon dilution with an equal volume of hexane, m-

chlorobenzoic acid (m-CBA) precipitated. The solids were filtered and washed with hexane to

dissolve any 2-(p-(2-Methyl-1,2-epoxypropyl)phenyl)propionic Acid (IV). The filtrate was

concentrated to an oil and again diluted with (500 mL) of hexane. More m-chlorobenzoic acid

was removed by filtration. The filtrate was concentrated to oil which was used in the next step

without further purification.

2-(

p

-

(

2-Methyl-2-hydroxypropyl

)

phenyl)propenoic acid (V)

To a solution of 2-(p-(2-Methyl-1,2-epoxypropyl)phenyl)propionic Acid (IV), obtained in the

previous steo(0,3 mole) in (1 L) THF at (15

0

C) under nitrogen was added dropwise over (2

hours) (550 mL) of (20 %) potassium tert-butoxid in THF. As the pH became neutral, a milky

slurry resulted, and as the pH became basic an orange mixture was observed. After

disappearance of starting material the reaction was quenched by the dropwise addition of (1

N) HCl over a (20 min) period at (15

o

C). The two-phase, acidic system was diluted with (700

ml) of the aqueous phase was removed. The organic phase was washed twice with (1 L) of

water and once with (500 mL) of saturated sodium chloride and dried over MgSO

4

.The

mixture was filtered, and the filtrate concentrated to a yellow oil. The oil was azeotroped with

(500 mL) of cyclohexane and then slurried with another (500 mL) of cyclohexane until

crystallization occurred. The solids were filtered, washed twice with fresh cyclohexane, and

dried to constant weight to give (52.4 g) as a first crop and (7.3 g) as a second crop. The yield

Experimental

86

from (III) to (V) was (78 %). Recrystallization of (50 g) of (V) from acetone/cyclohexane

afforded (30.9 g) of material, m.p. (110-114

o

C).

2-(

p

-

(

2-Methyl-2-hydroxypropyl

)

phenyl)propionic acid (VI)

A mixture of (0.12 mole) of 2-(p-(2-Methyl-2-hydroxypropyl)phenyl)propenoic Acid (V),

(250 mL) of THF, and (1 g) of 10 % Pd/C was placed in a Parr hydrogenation apparatus and

reduced under 50 psi of H

2

at room temperature for (1.5 hour). The resulting mixture was

filtered through a Celite pad, and the filtrate concentrated to yellow oil. The oil was slurried in

(400 mL) of cyclohexane for a few minutes and crystallization of a white solid was observed.

The solids were filtered, washed with fresh cyclohexane, and dried to constant weight at

(50

o

C) in vacuo to give (27.2 g) with yield was (80 %) of 2-(p-(2-Methyl-2-

hydroxypropyl)phenyl)propionic acid (VI), and m.p. (122-124

o

C) (literature data [128]: m.p.

122

o

C).

3.1.4 SFM-Ac

SFM was reacted with acetylchloride in pyridine as described in Scheme-3.4.

Recrystallisation from acetonitrile yield the title compound (65 %) as a yellow amorphous

solid m.p. (205-210

o

C) [123-125].

H

2

N S NH

O

ONO

CH

3

H

N S NH

O

ONO

CH

3

H

3

C

O

Acetylchlorid

Pyridin

Scheme-3.4: Synthesis of SFM-Ac

To a stirred solution of sulfamethoxazole (0.1 mole) in pyridine (300 mL) was added

dropwise acetylchloride (0.1 mole) at (0-5

o

C). The reaction mixture was stirred for 8 hours at

room temperature; the solution was concentrated to about (40 mL) and poured into excessive

water. The precipitate was washed with (1 M) hydrochloric acid and water successively, and

then dried to constant weight. Recrystallisation from acetonitrile yield the title compound (65

%) as a yellow amorphous solid m.p. (205-210

o

C) (literature data [133]: m.p. 207

o

C).

3.1.5 SFM-Glu

Initially, SFM was coupled in a 2-phase system (aqueous dilute NaOH, chloroform) with

methyl-2,3,4-tri-O-acetyl-

α

-bromoglucuronate in presence of tetrabutylammonium

Experimental

87

hydrogensulfate. As shown in Scheme-3.5, the resulting sulfamethoxazole-N-(methyl-tri-O-

acetyl-

β

-D-glucuronide) was converted to the final SFM-Glu by the action of sodium

methoxide and careful neutralization by means of carbon dioxide [125, 126-129].

HO O

OH OH

OH

H

3

COOC

AcO O

AcO OAc

OAc

H

3

COOC

AcO O

AcO Br

OAc

H

3

COOC

III

H

2

N S NH

O

ONO

CH

3

III

H

2

N S N

O

ONO

CH

3

OCOOCH

3

OAc

AcO

H

2

N S N

O

ONO

CH

3

OCOOH

OH

HO NaOMe IVV

Scheme-3.5: Synthesis of SFM-Glu

Acetyl anhydrid

Pyridine

HBr/HAC

CHCl

3

(abs.)

+

CHCl

3

tetrabutylammoniumsulfate

NaOH

MeOH (abs.)

β

-Tetraacetylglucuronic acid methyl ester (II)

(3.2 g) of the methyl ester glucuronic acid (I) were dissolved in (12 mL) of pyridine and (8

mL) of acetic anhydride at (0

o

C). The mixture was allowed to stand for (3 hours) in ice bath.

The solvent was evaporated by distillation in vacuo until crystals of

β

-tetraacetylglucuronic

acid methyl ester separated. The mixture was cooled to (0

o

C) and filtered. The crystals were

washed with cold absolute ethanol and ether [136].

Methyl 2,3,4-tri-O-acetyl-

α

-bromoglucuronate (III)

(0.133 mole) of methyl

β

-tetraacetylglucuronic acid methyl ester (II) was dissolved in (200

mL) of (30 %) hydrobronic acid in acetic acid and the mixture, after solution, allowed to stand

in the refrigerator overnight. Solvent was removed under reduced pressure and the residue

dissolved in (100 mL) of chloroform The chloroform layer was separated and washed twice

with aqueous sodium hydroxide (1.25 M, 3 mL) and dried (with sodium sulphate). The

Experimental

88

solvent was removed under reduced pressure. Recrystallization from ethanol yielded the

product (85 %) [137, 138].

Sulfamethoxazol-N-(methyl-tri-O-acetyl-

β

-D-glucuronide) (IV)

By dissolving sulphamethoxazole (0.002 mole) and benzyltriethylammonium bromide (1

mole) in aqueous sodium hydroxide (1.25 M, 2 mL). The resulting solution was added to a

solution of methyl 2,3,4-tri-O-acetyl-

α

-bromoglucuronate(0.001 mole) in chloroform (5 mL).

The resulting mixture was stirred vigorously and heated under reflux (3 hours). After cooling

water (5 mL) was added. The chloroform layer was separated and washed twice with aqueous

sodium hydroxide (1.25 M, 3 mL) and dried (with sodium sulphate). The solvent was

removed yielding a yellow amorphous solid. Recrystallization from ethanol yielded the

product (64 %) [139].

Sulfamethoxazol-N1-glucuronide (V)

(0.001 mole) of sulphamethoxazol-N-(methyl-tri-O-acetyl-

β

-D-glucuronide) (IV) was

dissolved in dry methanol (5 mL) and sodium methoxide. The mixture stirred at room

temperature for (2 hours) and then condensed to (1 mL). To the solution was added aqueous

sodium hydroxide (1 M, 2 mL) and the resulting mixture was stirred at room temperature for

(2 hours). The solution was adjusted to (pH 3). After filtration, the solution was removed

yielding a pale yellow solid. Recrystallization from ethanol yielded the product (15 %) [139]

and m.p. (112-114

o

C) (literature data [140]: m.p. 109

o

C).

Experimental

89

3.1.6 Characteristic data of drug metabolites

3.1.6.1 CBZ-DiOH

1- Molecular Formula

: C

15

H

14

N

2

O

3

1

2

3

4

56

7

8

1

23

4

5

67

N

O NH

2

OHHO

g

N

O NH

2

OHHO

aa

bb

c c

dd

ee ff

2-

1

H-NMR, and

13

C-NMR data:

Table-3.1

:

1

H-NMR (D

6

-DMSO)

H-Atom cis-Diol (Lit. [01])

δ

[ppm] cis-Diol (Synthese)

δ

[ppm] trans-Diol (Lit. [01])

δ

[ppm]

B 5.01 (d, J= 4.3Hz, 2H)

A

5.01 (d, J= 4.8, 2H) 4.02 – 5.02 (very br., 2H)

A 5.37 (d, J= 4.3Hz, 2H)

B

5.43 (d, J= 5.3

C

, 2H) 5.74 (d, J= 4.2, 2H)

B

G 5.73 (br, s, 2H)

B

5.76 (br, s, 2H) 5.81 (br, s,

2H)

B

C – f 7.20 – 7.60 (m, 8H) 7.32 – 7.23 (m, 6H

c-e

) 7.15 – 7.60 (m, 8H)

7.49 (dd, 2H

f

)

A

~Becomes s after exchange with D

2

O

B

~Exchangeable with D

2

O

C

~Resolution of the NMR-Device 0.5 Hz

Table-3.2

:

13

C-NMR (D

6

-DMSO)

C-Atom cis-Diol

δ

[ppm]

1 71.06

2 – 7 127.15 – 130.3

8 So difficult to detected

3- IR-data:

3550 – 3200 cm

-1

(s, OH, NH

2

), 1690cm

-1

(s, C=O)

N

O

N

H

2

H

O

OH

130.3 142.0

142.0

130.3

73.0

128.2

118.2

128.3

118.1

118.1 128.3

118.2

128.2

(152)

Experimental

90

3.1.6.2 IBU-2OH

1- Molecular Formula

: C

13

H

18

O

3

15

C

H

2

CH

3

CH

3

OH

CH

3

HOOC

2

34

6 7

8 9

2

6 7

C

H

2

CH

3

CH

3

OH

CH

3

HOOC a

e

a

b

c

d

e

f

f10

2-

1

H-NMR, and

13

C-NMR data:

Table- 3.3:

1

H-NMR and

13

C-NMR data:

H-Atom

∆

[ppm]

δ

* [ppm]

Multiplicity

number

H C-

Atom

δ

[ppm]

Number

C

A 1.25 1.2 S 6 1 18.13 1

B 1.53 1.5 D 3 2 29.13 2

C 2.77 2.7 S 2 3 44.95 1

D 3.74 3.7 Q 1 4 49.26 1

E 7.19 7.1-7.3 d/m (4 H)* 2 5 70.99 1

F 7.28 D 2 6 127.43 2

7 130.78 2

8 136.82 1

9 138.04 1

10 180.10 1

3- IR-data:

3402 cm

-1

s, (OH) 2978 cm

-1

, 2923 cm

-1

s, (C-H) 1694 cm

-1

s, (C=O)

Experimental

91

3.1.6.3 SFM-Ac

1- Molecular Formula:

C

12

H

13

N

3

O

4

S

O

N

NO

CH

3

a

b

c

d

H

N

H

C

O

CH

3

e

2-

1

H-NMR, and

13

C-NMR data

:

Table-3.4:

1

H-NMR data

1

H-Atome

δ

[ppm] J [Hz]

J [Hz]*

∆

[ppm]*

2H, d, H

a

-H

a’

7.53

J

ab

= 8.9

7.52

J

ab

= 8.8

2H, d, H

b

-H

b’

6.76

J

ab

= 8.9

6.61

J

ab

= 8.8

1H, s, H

d

6.19

6.08

3H, s, H

c

2.40

2.39

3H, s, H

e

2.1

2.15

•

Literature data

H

N

H

C

O

CH

3

S

O

N

NO

CH

3

87

32

56

41

10 12

911

Table-3.5:

13

C-NMR data

13

C-Atome

∆

[ppm]

δ

[ppm]*

Aromat

C

1

156.7 156.8

C

4

171.0 170.3

C

2

+C

6

130.3 129.9

C

3

+C

5

114.5 112.7

Isoxazol

C

9

123.7 123.7

C

10

86.6 86.9

C

11

153.2 152.6

C

12

11.9 11.8

Acetyl

C

7

158 160

C

8

24.5 27.9

* Literature data

Experimental

92

3.1.6.4 SFM-Glu

1- Molecular Formula:

C

16

H

19

N

3

O

9

S

O

S

H

2

N

O

N

NO

CH

3

COOH OH

OH

a

b

c

d

efg

h

i

O

SH

2

N

O

N

NO

CH

3

COOH OH

OH

1

2

3

4

56

2

'

3

'

4

'

5

'

1

''

2

''

3

''

4

''

5

''

6

''

2-

1

H-NMR, and

13

C-NMR data:

Table-3.6:

1

H-NMR data

1

H-Atome

δ

[ppm] J [Hz]

J [Hz]*

δ

[ppm]*

2H, d, H

a

-H

a’

7.53

J

ab

= 8.9

7.52

J

ab

= 8.8

2H, d, H

b

-H

b’

6.76

J

ab

= 8.9

6.61

J

ab

= 8.8

1H, s, H

d

6.19

6.08

1H, d, H

i

5.25

J

hi

= 9.3

5.25

J

hi

= 9.2

1H, d, H

e

3.76

J

ef

= 9.9

3.91

J

ef

= 9.4

1H, t*, H

g

3.55

J

fg

= 9.1, J

gh

=9.1

3.54

J

fg

= 9.0, J

gh

=9.0

1H, t*, H

f

3.36

J

fg

= 9.4, J

ef

= 9.6

3.47

J

fg

= 9.0, J

ef

= 9.4

1H, t*, H

h

3.21

J

gh

= 9.1, J

hi

= 9.1

3.21

J

gh

= 9.0, J

hi

= 9.2

3H, s, H

c

2.37

2.39

Table-3.7:

13

H-NMR data

13

C-Atome

∆

[ppm]

δ

[ppm]*

Aromat

C

1

156.7 156.8

C

4

171.0 170.3

C

2

+C

6

130.3 129.9

C

3

+C

5

114.5 112.7

Isoxazol

C

2’

123.7 123.7

C

3’

86.6 86.9

C

4’

153.2 152.6

C

5’

11.9 11.8

Glucuronid

C

1’’

102.5 102.3

C

2’’

76.7 76.4

C

3’’

76.1 76.3

C

4’’

71.4 70.6

C

5’’

69.6 69.4

C

6’’

173.2 170.9

Further information by GC/MS and LC/MS data for all these metabolites see [80].

Experimental

93

3.2 Liquid membrane Extraction

3.2.1 Development of HPLC-UV method

The transport of analytes was monitored by HPLC and UV-detection. Aliquots were taken

from the liquid phase (feed or strip) at intervals by means of a micro-liter syringe. The HPLC-

UV development methods for metabolites and active drugs are described in chromatogram

(Figure-7.1). The stock solutions of metabolites and active drugs were prepared by dissolving

an appropriate amount of the drugs in methanol. The external calibration curve were built

from 8 concentrations (n = 3) in a concentration range 0.5- 10 mg/L obtained by diluting of

aliquot of appropriate stock solution with 0.1 mol/L NaCl.

The analytes were introduced into the chromatographic system by an autosampler connected

with UV-Vis Detector. Detection was performed at 225 nm. The analytical column used was

LichroCART RP 18. Deferent mobile phases were tested to determine the calibration curve of

metabolites. The best mobile phase was consisted a mixture of 8.295 mmol/L KH

2

PO

4

:

acetonitrile 80:20 (v/v). The flow rate of the mobile phase was 1.0 mL/min. Retention data

(R

t

) of analytes: SFM-Glu.:3.57 min, CBZ-DiOH: 5.33 min, IBU-2OH: 17.88 min, SFM-Ac:

20.43 min.

For the active drug, the best mobile phase was consisted a mixture of 0.6 mmol/L NaH

2

PO

4

:

acetonitrile 50:50 (v/v). The flow rate of the mobile phase was 1.0 mL/min. Retention data

(R

t

) of analytes: SFM: 3.55 min, CBZ: 4.60 min, DCF: 6.34 min, IBU: 7.73 min.

In addition the LC-MS/MS system LCQ Advantage in ESI-mode (Thermo Finnigan,

Egelsbach, Germany), connected with a gradient pump (SpectraSYSTEM P4000) was

employed for metabolite trace analysis. The separation was performed on a Nucleosil 100 C

18

ec-column (5

µ

m, 250

x

5 mm i.d., Macherey-Nagel, Düren, Germany).

Experimental

94

a)

b)

Figure-3.1:

HPLC-UV chromatogram for the drug metabolites and active drugs

a) 1. SFM-Glu, 2. CBZ-DiOH, 3. IBU-2OH and 4. SFM-Ac,

b) 1. SFM, 2. CBZ, 3. DCF and 4. IBU

The stock solutions of active and metabolite drugs were prepared by dissolving an

appropriate amount of the drugs in methanol. HPLC separation was achieved with

LichroCART RP 18 (5

µ

m, 250 x 4 mm; Merk) column at wavelength of 225 nm

3.2.2 Validation of HPLC-UV method

Method validation has received considerable attention in literature, from industrial

committees and regulatory agencies. In recent years an increasing number of laboratories have

applied for accreditation according to EN 45000 series. Quality management and quality

assurance systems according to ISO 9000ff are now certified in analytical laboratories;

therefore, the validation of HPLC-UV was based on the methods of analytical quality

assurance (AQA), which have many possible solutions for routine practice analysis by

providing quality data. The validation characteristics of analytical quality assurance methods

are:

•

The range tested

•

The coefficients of the calibration function:

•

in the case of a first order calibration function (y = a + bx): axis intercept a and slop b

(characteristic of sensitivity of the analytical procedure);

1

2

3

4

1

2

3

4

Experimental

95

•

in the case of a second order calibration function(y = a + bx + cx

2

): axis intercept a,

coefficient b of the linear term, as the coefficient c of the quadratic term, the

sensitivity E of the analytical process determined from the function;

•

the standard deviation of the procedure S

xo

as an absolute measure of precision for the

calibration and;

•

the process variation coefficient V

xo

as a relative measure of precision.

•

In addition, the general evaluation of the analytical process also documents the:

•

Limit of detection LOD

•

Limit of quantitation LOQ

1. Sample preparation

During the preparation of the standard samples we tried to concern that:

- The precision of the balance and the volumetric equipment have been taken into account.

- No successive dilution since during the preparation of standard samples

The stock solutions of drug metabolites (SFM-Glu, CBZ-2OH, IBU-OH, and SFM-Ac) were

prepared at 1 mg/mL in methanol. Appropriate dilutions of stock were made with 0.1 mol/L

NaCl to prepare of standard solutions for calibration at concentrations of 0.025, 0.05, 0.1, 0.5,

1, 2, 3, 4 and 5 mg/L.

2. Fundamental calibration

The preliminary first and second-degree calibration functions are calculated from the

measured of these standard samples and listed in Tables-3.9 and 3.9.

Table-3.8:

First order calibration function

Compound

Calibration function

first order A B

R

2

S

y

SFM-Glu 0.4446x + 0.026 0.026 0.4446 0.9986 0.0341

CBZ-DiOH

1.1363x – 0.0275 - 0.0275 1.1363 0.9993 0.0645

IBU-2OH 1.0307x - 0,0358 - 0.0358 1.0307 0.9995 0.0490

SFM-Ac -0.0105 + 0.6806x - 0.0038 0.6806 0.9997 0.0310

Experimental

96

Table-3.9:

Second order calibration function

Compound

Calibration function

second order A b C

R

2

S

y

SFM-Glu 0.0069x

2

+ 0.4148x +

0.0341 0.0341 0.4148 0.0069 0.999 0.0361

CBZ-DiOH

0.0066x

2

+ 1.1054x +

0.0179 - 0.0179 1.1054 0.0066 0.999 0.0680

IBU-2OH 0.0025x

2

+ 1.0264x +

0.0309 - 0.0309 1.0264 0.0025 0.999 0.0532

SFM-Ac 0.0044x

2

+ 0.6598x +

0.0065 0.0065 0.6598 0.0044 0.999 0.0316

3. Sensitivity

The measure of sensitivity results from the change in the measured value caused by a change

in the concentration values. If the calibration function for an analytical procedure is linear,

then the sensitivity is constant over the entire range and is equivalent to the regression

coefficient b. From the sensitivity the process of standard deviation S

xo

, and relative process

of standard deviation V

xo

can be calculate, and the results for the drug metabolites are listed in

Table-3.10.

Table-3.10:

The standard deviation and the coefficient of variation for HPLC-UV method

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

S

xo

0.0715 0.0601 0.0523 0.0456

V

xo

[%] 3.650 3.07 2.67 2.33

4. Linearity

a) Mandel’s fitting test: The Mandel’s fitting test is recommend for the mathematical

verification of linearity. For this, the first order and second- order calibration function

including their respective residual standards deviation S

y

are used. The testing value TV is

compared with the value obtained from the table F(f

1

=1, f

2

= N-3, P = 99%) as shown in

Table-3.11.

Table-3.11

: Mandel’s fitting test for HPLC-UV method

SFM-Glu

(N = 9) CBZ-DiOH

(N= 9) IBU-2OH

(N=9) SFM-Ac

(N=9)

DS

2

1.8 x 10

-4

9.14 x 10

-4

- 6.036 x10

-4

6.981 x10

-4

TV 0.1381215 0.1976643 - 0.2132711 0.6990485

F(f

1

=1, f

2

= N-3,

P = 99%) 3.7047 3.7047 3.7047 3.7047

Experimental

97

b) Variance homogeneity test

The linear regression calculations described assume a constant (homogeneous) imprecision

(variance of measured values) over the range. Inhomogeneity leads not only to a higher

imprecision, but also to a higher inaccuracy through possible change in the linear slope. In

order to verify the homogeneity of variances, n = 10 standard samples of each of the lowest

(S

21

) and the highest (S

2N

) concentrations of the preliminary range are analyzed separately.

The variances of both of measurement are checked for homogeneity using an F-test. As listed

in Table-3.12, the TV for the metabolites are so bigger than F , and to solve this problem we

choose narrow range of concentrations for all drug metabolites from 0.5 to 5 mg/L .

Table-3.12:

The homogeneity test for HPLC-UV method

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

S

21

3.131789 x 10

-5

3.222964 x 10

-5

4.070281746 x 10

-6

5.725999 x 10

-5

S

2N

0.01010725 0.00625666 0.0014724 0.006018844

TV 322.7315 194.1275 361.7440 105.1143

F(f

1

=1, f

2

=

N-3, P =

99%)

5.35 5.35 5.35 5.35

5. The limit of detection and limit of quantitation

The limit of detection LOD is the lowest of substance concentration that produces a response

detectable above the noise level of the system. The limit of quantitation LOQ for a basic

analytical process is defined as the smaller concentration of substance that can be determined

using a given analytical precision. The values of LOD and LOQ for metabolites are listed in

Table-3.13.

Table-3.13

: The capability of detection and limit of quantitation for HPLC-UV method

SFM-Glu CBZ-DiOH IBU-2OH SFM-Ac

LOD [mg/L] 0.13 0.11 0.09 0.08

LOQ [mg/L] 0.35 0.33 0.27 0.24

Experimental

98

3.2.3 BLM

Extraction procedure

As shown Figure-2.4, the three-phase system was established in a home-made glass cell

equipped with an agitator (PTFE) which allows extraction and back-extraction in one unit.

The cell consists of two concentric chambers dividing it into separate compartments. Thus,

the feed is allowed to contact the bulk-membrane while the strip solution contacts the

membrane. The three liquid phases stirred at a frequency of 60 rpm. The whole cell is covered

by a fitting glass lid in order to minimize the loss of solvent by evaporation.

3.2.4 SL-FM

Extraction procedure

The SL-FM is based on a porous polypropylene membrane (pore size 0.1 µm, total thickness

90 µm, Membrana GmbH, Wuppertal, Germany) impregnated with a water-immiscible

organic membrane phase containing neutral carrier (tri-n-octylphosphinoxid) or acidic carrier

(octane sulfonic acid) dissolved in an appropriate solvent (1-pentanol, DHE, undecane and

decane), which is held by capillary forces placed between two aqueous phases, the feed and

the strip. The SL-FM as shown in Figure-2.15 was mounted in a window between two PTFE-

chambers, one for the sample input (feed phase), one for the permeate uptake (strip phase).

Both chambers were filled up to 150 ml. The size of membrane which used in every batch of

extraction was (5 x 5 cm), and the time of soaking was (2 hours) in liquid membrane. The

feed solution was stirred by magnetic stirrer at 360 rpm.

3.2.5 SL-BM systems

3.2.5.1 Preparation of bag membrane

Bag membranes were prepared by using the procedure developed in Paderborn University

[112]. The procedure steps are:

1-

Two layers (10 x 17 cm) of polypropylene (PP) membrane (pore size 0.1 µm, total

thickness 90 µm) were held between two aluminum layers which designed to be

identical to each other and to prepare simultaneously several as shown in Figure-3.2.

Experimental

99

Figure-3.2:

Two aluminum plates which used to prepare the bag membrane [112]

2-

The aluminum plates with PP-membrane were heated and pressed by using iron plate

sees Figure-3.3.

Figure-3.3:

Heating and pressing with iron plate to prepare the bag membrane

3-

Heating and pressing with iron plate for at least 2 minutes, the two PP-membrane

layers will be melted, bended and take the shape of aluminum plate then the bag

membrane can be removed from the aluminum plates.

4-

After checking and washing the bag membranes were soaked in the liquid membrane

for 2 hours then used for SL-BM extraction

3.2.5.2 SL-BM

Extraction procedure

Figure-2.26 illustrates the

SL-BM system. The bag membrane (1) was filled with volume

between 0.3- 0.6 mL of strip phase, and the beaker down (2) was filled with volume 500 ml of

feed phase. The feed solution was stirred by magnetic stirrer at 360 rpm.

Experimental

100

3.2.5.3 SL-4-BM

Extraction procedure

After preparation of bag membranes (see section 3.2.5.1), the bags were washed 3 times by

distilled water and methanol and dried for 30 min at room temperature. 4 bags were soaked in

liquid membrane for 2 hours. After soaking, 0.4 mL of strip solution 0.1 mol/L NaOH was

injected into every bag with a microlitre syringe and the bags were subsequently placed in the

sample solution (feed phase) as shown in Figure-2.36. Sample solution contained the analytes

with 500 mL of 0.1 mol/L HCl or 6.5 mL of 25 % (w/w) HCl in 500 mL of real water

samples: tap water from Paderborn city (see Table-3.14) and surface water from river Ruhr. A

magnetic stir plate was used to agitate the sample solution during the extraction. After 4 hours

of extraction, the strip solution from every bag was drawn separately and neutralized with the

same volume of 0.1 mol/L HCl, and then the neutralized samples were combined in one test

tub. To remove the NaCl, the sample was dried under reducing pressure and the solid residue

was washed with 3 mL of absolute diethyl ether. After decantation the sample solution was

transported to another test tub and dried under reducing pressure. The residue was dissolved

in 0.4 mL of methanol. After filtration, the filtrate was transferred into a vial then injected to

HPLC-UV analysis.

Table-3.14:

Tap water quality of Paderborn city

(Wasserwerk Aabach) [162]

Parameter Dimension Limit value Analysis value

pH-value …. 6.50 – 9.50 7.72

Electrical conductivity (20°C) µS/cm 2.500 315

Calcium mg/L … 60.0

Magnesium mg/L … 5.70

Sodium mg/L 200 5.90

Potassium mg/L … 1.15

Iron mg/L 0.20 < 0.01

Manganese mg/L 0.05 < 0.005

Ammonium mg/L 0.50 < 0.05

Nitrite mg/L 0.50 < 0.005

Nitrate mg/L 50.00 7.00

Chloride mg/L 250.00 10.00

Sulfate mg/L 240.00 31.00

Fluoride mg/L 1.50 < 0.1

Hydrogencarbonate mg/L … 160

Acid capacity up to pH 4,3 mmol/L … 2.68

Carbonate hardness °dH … 7.50

Experimental

101

3.3 SPE

Extraction procedure

As shown in Figure-2.35, the SPE cartridges were conditioned with methanol (5 mL) and

distilled water (5 mL), at a flow rate of 5 mL/min. 500 mL of prepare water sample was

filtrated by 0.45

µ

m (Nylon-Filter). Sample loading was also performed at 5 mL/min.

Afterwards the cartridge was washed with 10 mL/min of water containing 5 % methanol, at 5

mL/min, and dried by pumping air through the cartridge for 15 min. Elution was performed

with 0.8 mL of methanol. After one fold dilution with methanol to complete the total volume

to 1 mL, the extract was injected to HPLC-UV system.

3.4 Materials, equipments and chemicals

The materials, equipments and chemicals which have been used in the present work are listed

in Tables-3.15- 3.17.

Table-3.15:

Materials used in this work

Material Supplier

Polypropylene membrane (pore size 0.1

µ

m,

total thickness 90

µ

m) Membrana GmbH Wuppertal Germany

Bakerbond SPE cartridge Merck

Isolute ENV SPE cartridge Merck

Strata C18-E SPE cartridge Merck

Oasis-HLB SPE cartridge Merck

Cellulose membrane filter (0.45

µ

m) Merck

Parameter Dimension Limit value Analysis value

Basic capacity up to pH 8,2 mmol/L … 0.10

Free carbonic acid mg/L … 4.40

Water temperature °C … 13.7

Color (SAK 436) 1/m 0.50 0.07

Turbidity FTU 1.0 0.05

Oxygen mg/L … 10

Oxidizability mg/L 5.00 0.90

Phosphate mg/L 6.70 < 0.01

Aluminum mg/L 0.20 < 0.01

Lead mg/L 0.025 < 0.001

Copper mg/L 2.00 0.01

Total organic carbon (TOC) … … 1.48

Experimental

102

Table-3.16:

Equipments used in this work

Equipment Supplier

Autosampler GINA 50 Gynkotek/ Dionex Idsten

Isocratic pump P580 Gynkotek/ Dionex Idsten

UV-Vis Detector 655 A Merk-Hitachi

Analytical column LichroCART RP 18 (5

µm, 250 x 4 mm) Merk

Digital-pH-Meter 766 Calimatic Knick/Berlin

Vortex-Mixer VXR IKA VIBRAX

Magnetic stirrers H+P Labortechnik AG

Table-3.17:

Chemicals used in the present work

Chemical Supplier

Pyridine Aldrich

Acetic anhydride Fluka

Methyl ester glucuronic acid Fluka

Hydrochloric acide Fluka

Sodium hydroxide Fluka

Sodium sulphate Merck

Benzyltriethylammonium bromide Aldrich

Sodium methoxide Merck

Acetyl chloride Fluka

N-bromosuccinimide Fluka

Benzyl peroxide Fluka

Lithium bromide Fluka

Magnesium sulphate Fluka

m-chloroperbenzoic acid Merck

Potassium tetr-butoxid Merck

Sodium chloride Fluka

Potassium permanganate Merck

Silica gel Aldrich

Methanol Aldrich

Chloroform Merck

Ethanol Merck

Acetone Aldrich

Carbon tetrachloride Fluka

Hexane Fluka

Dimethylformamide Fluka

Diethylether Merck

Dichloromethane Merck

Tetrahydrofuran Fluka

Cyclohexane Merck

Acetone Fluka

Heptane Fluka

Experimental

103

Chemical Supplier

1-pentanol Fluka

Decane Fluka

Undecane Fluka

DHE Fluka

Potassium dihydrogen phosphate Fluka

Diclofenac sodium salte Fluka

Sulfamethoxazole Fluka

Carbamazepine Fluka

Ibuprofen Fluka

Tri-n- octylphosine oxide Merck

1-octanesulfonic acid Sodium salt monohydrate Merck

Buffer solution Titrisol pH 7 Merck

Buffer solution Titrisol pH 8 Merck

Buffer solution Titrisol pH 9 Merck

Buffer solution Titrisol pH 10 Merck

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