Development of a targeted HPLC-ESI-QqQ-MS/MS method for the quantification of sulfolipids from a cyanobacterium, selected leafy vegetables, and a microalgae species [original]

RESEARCH PAPER

Development of a targeted HPLC-ESI-QqQ-MS/MS method

for the quantification of sulfolipids from a cyanobacterium, selected

leafy vegetables, and a microalgae species

Judith Fischer

&Mascha Treblin

&Tobias Sitz

&Sascha Rohn

1,2,3

Received: 10 October 2020 /Revised: 27 November 2020 /Accepted: 7 January 2021

#The Author(s) 2021

Abstract

The use of macro- and microalgae, as well as cyanobacteria, becomes increasingly important for human nutrition, even in

Western diets. Health effects, positive as well as negative, are believed to result mainly from minor components in the food.

In macro- and microalgae as well as in certain cyanobacteria, one class of such minor compounds is sulfolipids, more precisely

sulfoquinovosylmonoacylglycerol (SQMG) and sulfoquinovosyldiacylglycerol (SQDG) derivatives. SQMGs and SQDGs con-

sist of a diacylglycerol esterified with varying fatty acid combinations and a sulfoquinovose moiety. Sulfoquinovose is a

sulfonated hexose analogous to D-glucose, but featuring a stable carbon-sulfur bond. With regard to their chemical structure,

SQDGs can be distinguished according to their sn1- and sn2-bound fatty acids. Although there is great interest in SQDGs,

because of their controversially discussed bioactivities, only a negligible number of comprehensive methods for identification

and quantification has been published, so far. Within this work, a sample preparation including a quantitative isolation of SQDGs

from selected raw materials, a clean-up with solid-phase extraction (SPE), and a sensitive liquid chromatography-tandem mass

spectrometry (LC-MS/MS) method for simultaneous identification and quantitation of different, intact SQMGs and SQDGs were

developed and validated. The applicability of the method was further demonstrated by comparing a prominent cyanobacterium

(Arthrospira sp.) with a microalgae preparation (Chlorella vulgaris), and selected leafy vegetables (spinach, basil).

Keywords Cyanobacteria lipids .Sulfoquinovosyldiacylglycerols .HPLC-ESI-QqQ-MS/MS .Method validation

Introduction

As early as in 1974, Arthrospira sp. (filamentous

cyanobacteria) was described by the United Nations World

Food Conference as possibly the best food for the future.

The use of microalgae and cyanobacteria for human nutrition

has a traditional past in several countries and offers many

possibilities and advantages for human nutrition. In Western

diets, they are mostly being consumed for their functional

benefits beyond traditional considerations of nutrition and

health [1]. In Europe, only some algal and cyanobacterial

species are legally on the market as food ingredients.

However, some products have been already legalized accord-

ing to the Regulation (EU) 2015/2283 of the European

Commission as novel food such as oil from the microalgaes

Ulkenia sp. or Odontella aurita [2]. The positive effects on

human health are hypothesized to result from certain macro-

nutrients, but moreover, from their micronutrients. However,

the latter are somehow controversially discussed in the litera-

ture. There is often little known about negative aspects or

minor compounds with adverse properties, although they are

significantly metabolized in the human organism [3].

Sulfoquinovosyldiacylglycerol derivatives (SQDGs) might

represent one of those minor compounds with possible bipolar

properties in algae products. They consist of sulfoquinovose

and a diacylglycerol, whereas sulfoquinovose is a sulfonated

hexose analogous to D-glucose with a stable carbon–sulfur

bond. The meaning of sulfolipids for the human diet is still

*Sascha Rohn

rohn@tu-berlin.de

Hamburg School of Food Science, Institute of Food Chemistry,

University of Hamburg, Grindelallee 117,

20146 Hamburg, Germany

Institute for Food and Environmental Research (ILU) e.V.,

Papendorfer Weg 3, 14806 Bad Belzig, Germany

Department of Food Chemistry and Analysis, Institute of Food

Technology and Food Chemistry, Technische Universität Berlin,

Gustav-Meyer-Allee 25, 13355 Berlin, Germany

https://doi.org/10.1007/s00216-021-03164-3

/ Published online: 22 January 2021

Analytical and Bioanalytical Chemistry (2021) 413:1941–1954

controversially discussed. On the one hand, they are attributed

to some health-promoting effects and are known to have an-

tiviral properties [4]. On the other hand, sulfur compounds can

have a negative impact, because some microorganisms of the

lower gastrointestinal tract can produce hydrogen sulfide as a

(toxic) metabolite during the biotransformation in the gut [5].

Therefore, knowledge about the impact of SQDGs on human

health might still be a crucial factor. To evaluate potential

physiological effects, it is essential to obtain reference com-

pounds in comparatively high amounts and appropriate purity

and to establish methods enabling a reliable quantification of

SQDGs.

The analytical approaches on SQDGs began in 1959 with

the discovery of novel sulfolipids by Benson et al. They de-

scribed SQDGs for the first time and cultivated photosyn-

thetic microorganisms and higher plants in the presence of

isotopically

S-labeledsulfate.Alipidextractwasseparated

with paper chromatography and they investigated the chem-

ical composition and molecular structure using various

radiochromatographic methods [6]. In the late 1960s, many

chromatographic and crystallographic studies followed to

learn more about the structure and function of the newly

described sulfolipids [7,8]. In 1989, the US National

Cancer Institute in Bethesda, MA, classified sulfolipids as

potential antiviral agents [4]. Many studies on possible phar-

macological relevance were conducted accordingly [9–11].

Generally,anisolationprocedure wasused toobtainSQDGs

as lipid extracts, but more detailed investigation of the indi-

vidual lipid compounds was not carried out [12]. However,

when sulfolipids were quantified, it was only done as sum

parameter with methods that did not differentiate between

the single SQDG. Both thin-layer chromatographic methods

andliquidchromatographywereused[13–17].Inadditionto

the quantification as a sum parameter, some publications

describe identification of selected fatty acids using gas chro-

matography [18,19]. GC analysis of (intact) sulfolipids,

apart from the analysis of their fatty acids only, has not been

done so far, because molecular structure disables evapora-

tion. However, investigations of similar compounds like

monogalactosyldiacylglycerols (MGs) and

digalactosyldiacylglycerols (DGs) using GC-MS have been

performed and published. With the help of GC-MS methods,

itwas also possible todetect andquantifygalactolipids, even

thoughtheyrequireextensivepre-analyticalproceduressuch

as purification and derivatization for yielding volatile

analytes [19,20]. Collision-induced dissociation tandem

mass spectrometry (CID-MS/MS) was used to further inves-

tigate sulfolipids from complex lipid extracts. It provided

structural information about the fatty acids in sn1 and sn2

positions. During fragmentation by CID-MS/MS, similar

fragments(m/z225,165,153,95,and81),beingformedfrom

the sulfolipids, were observed. These fragments result from

the fragmentation of the sulfoquinovose group and are

therefore specific for all sulfolipids. Furthermore, fragments

attributed to the neutral loss of fatty acids were observed, as

well. These allow conclusions to be drawn about the fatty

acid composition of the corresponding sulfolipids [21–23].

It was further determined by CID-MS/MS that the frag-

ment resulting from the fragmentation of the sn1 fatty acid

shows a higher intensity, and thus, the regiochemistry of the

glycerol backbone of the SQDG can be inferred [21]. Recent

studies have focused on the identification of new SQMGs and

SQDGs using lipodomic approaches [24–26]. Nonetheless,

there is actually no method for quantifying multiple intact

SQMGs and SQDGs for evaluating phytochemical or food

chemical questions.

Consequently, the aim of this study was to develop an

accurate and sensitive quantification method for the measure-

ment of different intact SQDGs by liquid chromatography

coupled to mass spectrometry, following the extraction from

cyanobacteria and microalgae. Additionally, selected plant

materials were analyzed. These are believed to synthesize also

sulfolipids to a certain extent, being closely related to chloro-

phyll synthesis. The different matrices are compared for get-

ting an impression about distribution, and also for evaluating,

if formation of SQMGs and SQDGs is differing, depending on

source, specific lipid metabolism, and accompanying sub-

stances/substrates.

Materials and methods

Chemicals

Acetonitrile, chloroform, methanol, acetic acid, phosphoric

acid, and hydrochloric acid were purchased from Carl Roth

GmbH & Co KG (Karlsruhe, Germany). Ammonium acetate,

copper sulfate, and potassium chloride were purchased from

Sigma-Aldrich GmbH (Munich, Germany). The standard sub-

stances SQDG 816 (2-O-hexadecanoyl-1-O-(9Z,12Z,15Z-

octadecatrienoyl)glycerol 3-(6-deoxy-6-sulfo-α-D-

glucopyranoside)) and 3-O-sulfo-D-galactosyl-β1-1′-N-

heptadecanoyl-D-erythro-sphingosine as internal standard

(ISD) were purchased from Avanti Polar Lipids Inc.

(Alabaster, AL, USA). All aqueous solutions were prepared

with deionized water, generated by a Purelab flex water puri-

fication system (Veolia Water Technologies Deutschland

GmbH, Celle, Germany). NH

cartridges (6 mL, 500 mg)

were purchased from Macherey-Nagel GmbH & Co. KG

(Düren, Germany).

Preparation of standard and calibration solutions

Primary standard stock solutions were prepared by dissolving

5 mg of the commercially purchased standard SQDG 816 in

5 mL acetonitrile, resulting in a final concentration of 5 mg/

1942 Fischer J. et al.

mL (5000 ppm). The calibration standard stock solution was

diluted with acetonitrile to achieve equidistant concentration

in the range of 1 to 10 μg/mL. Furthermore, an identical vol-

ume of the internal standard solution was added to each cali-

bration standard. Every calibration point contained ISD at

ultimate concentration levels of 5 μg/mL. Solutions were used

to determine the specific fragmentation pattern for each ana-

lyte. The commercially available SQDG, the internal standard,

and raw extracts of Arthrospira sp. (“Spirulina”)wereused.

The SQDG 816 and ISD stock solution were diluted with

acetonitrile/water (9/1; v/v) with 10 mM ammonium acetate

to achieve a concentration of 100 μM SQDG 816/ISD. All

solutions were kept at −20 °C.

Sample material

Freeze-dried Spirulina powder (Arthrospira sp.) was obtained

from the Institute for Food and Environmental Research

(ILU), Nuthetal, Germany. These cyanobacteria were origi-

nally cultivated photoautotrophic in a 1500-L tubular

photobioreactor PBR 2000 from IGV. Cells were harvested

in a modified Zarrouk’s medium under outdoor conditions,

i.e., under natural sunlight (with day and night changes), with

a pH value of 9.5, and at a temperature over 25 °C. The system

was additionally heated and/or artificially illuminated to bal-

ance the seasonal temperature fluctuations and overnight res-

piration losses. Following lyophilization, material was ground

to a fine powder. Spirulina powder was extracted initially

three times with chloroform/methanol (3/2, v/v). After

vortexing and ultrasonification for 15 min and centrifuging

(5 min, 12,000g), the supernatant was removed and collected.

The resulting eluate was concentrated to dryness and absorbed

into 500 μL methanol. Finally, the mixture was diluted 1:100

(v/v), 1:1000 (v/v), and 1:10,000 (v/v) for further use.

For a screening approach and to compare with contents of

the cyanobacteria, commercially available vegetables and a

prominent microalgae specie were used. Fresh spinach and

basil leaves as well as a Chlorella powder (Chlorella vulgaris)

were purchased froma local organic supermarket in Hamburg,

Germany. Spinach and basil leaves were frozen with liquid

nitrogen, freeze-dried, and ground with mortar and pestle prior

to analysis.

High-performance thin-layer chromatography

For the chromatographic separation, silica gel 60 high-

performance thin-layer chromatography (HPTLC) plates were

purchased from Merck KGaA (Darmstadt, Germany). Plates

were pre-washed with methanol and activated at 100 °C for

10 min. The samples were applied as bands (8 mm) using an

HPTLC autosampler (ATS4, CAMAG AG, Muttenz,

Switzerland). Separation was carried out in a twin-trough

chamber with chloroform/methanol/acetic acid/water

(8.5:1.5:1:0.36, v/v/v/v). Chromatography was consistently

performed up to a migration distance of 80 mm. Finally, the

remaining solvents were evaporated. Derivatization was per-

formed by dipping the plate 5% copper sulfate in 8% phos-

phoric acid and afterwards heated for 30 min at 160 °C.

Sample preparation

The freeze-dried Spirulina powder was used for method de-

velopment. 0.5 g of the powder was then ground in a ball mill

for 5 min (frequency 25 Hz, 4 balls ø= 1.5 cm; Retsch MM

400, Retsch GmbH, Haan, Germany). Then, 10 mL

chloroform/methanol (3/2, v/v) was added and the mixture

was ground in a ball mill for 10 min. Ten milliliters

chloroform/methanol (3/2, v/v) was added. After vortexing,

ultrasonification for 15 min, and centrifuging (5 min,

12,000×g), the supernatant was removed and collected in a

10-mL tube. Twenty milliliters chloroform/methanol (3/2,

v/v) was added to the residue. After vortexing,

ultrasonification for 15 min, and centrifuging (5 min,

12,000×g), the supernatant was removed and collected in a

10-mL tube. The last extraction step was repeated three times.

The supernatants were combined and evaporated to dryness

under nitrogen stream. The residue was re-dissolved in 50 mL

chloroform/methanol (3/2, v/v).

Solid-phase extraction

The sample extract was loaded on a NH

-SPE cartridge

(6 mL, 500 mg; Macherey-Nagel GmbH & Co. KG, Düren,

Germany) that was initially conditioned with 5 mL methanol,

5 mL water, 5 mL 0.1 M HCl, 5 mL water, and 5 mL meth-

anol. Cartridges were washed with 10 mL chloroform/

methanol (90/10, v/v) and 10 mL chloroform/methanol (50/

50, v/v). SQDGs were eluted with 10 mL chloroform/

methanol (80/20, v/v) containing 100 mM ammonium acetate

and 2% NH

. For removing salts in the eluent, the solution

was washed prior to the LC-MS/MS measurement. Therefore,

the eluent was mixed with 2 mL 0.9% potassium chloride

solution and mixed for 10 min in the ultrasonic bath, then

centrifuged for 5 min at 12,000g. The organic phase was dried

at 40 °C with a vacuum concentrator (RVC 2-25 CDplus,

Martin Christ GmbH, Osterode, Germany). Then, the residue

was dissolved in 1 mL methanol.

LC-ESI-MS/MS analysis

The development of the new LC-ESI-MS/MS method as well

as the method validation process based on procedures was

already published recently [27]. SQDGs were analyzed on

an Agilent 1260 Infinity Quarternary LC System (Agilent

Technologies Deutschland GmbH, Waldbronn, Germany)

coupled to a triple quadrupole API 4000 QTrap mass

1943Development of a targeted HPLC-ESI-QqQ-MS/MS method for the quantification of sulfolipids from a...

spectrometer (AB Sciex Germany GmbH, Darmstadt,

Germany) equipped with a turbo spray source, operating in

negative ion mode, with the following mass spectrometer set-

tings: ion spray voltage: −4500 V; source gas 1: 16 psi; source

gas 2: 0 psi; curtain gas: 10 psi. Separation of analytes was

achieved using a Kinetex® C18 reversed-phase column

(2.6 μm, 150 mm × 2.1 mm i.d.), equipped with a Kinetex®

C18 security guard column (Phenomenex Inc., Torrance, CA,

USA), using a constant flow rate of 200 μL/min. Eluent A was

water and eluent B acetonitrile/water (9/1; v/v), each with

10 mM ammonium acetate. The elution started with 3% eluent

B for 2 min and linearly increased to 99% eluent B within

4 min, which was kept constant for 34 min. Then, the compo-

sition was readjusted to 3% eluent B within 2 min, followed

by 10 min of re-equilibration.

Method validation

The LC-MS/MS method was validated in absence of matrices

(base calibration). Linearity of the method was determined by

analysis of a 10-point calibration curve (n= 6). Accuracy and

precision of the method were assessed with three different

concentrations of quality control samples (QC 1–4). Five rep-

licates of each QC point were analyzed on 1 day to determine

the intra-day accuracy and precision. This process was repeat-

ed over three different days (n= 3) in order to evaluate the

inter-day accuracy and precision. The recovery rate was cal-

culated as follows: [RE = (c

sample

−c

endogen

) × 100/c

spiked

The limit of detection (LOD) was determined by the cali-

bration method. A calibration curve (n= 4) in the range of the

presumed detection limit was established and measured (n=

3). The parameters of the resulting regression line were then

used to determine LOD and limit of quantitation (LOQ).

Results and discussion

Method development

Previously published methods for determining sulfolipids in-

dicated limitations in their selectivity or sensitivity. Most of

these methods do not differentiate between single SQDGs or

SQMGs, but determine them as sum parameters. In the pres-

ent study, a method development for selective and sensitive

SQDG determination is described. For a sensitive and selec-

tive detection of the SQDGs using mass spectrometry, multi-

ple reaction monitoring (MRM) was applied. Besides sensi-

tive and selective detection of the SQDGs, another advantage

using MRM method was the achievement of higher sensitivity

and better repression of interferences of the complex back-

grounds. MRM transitions of the different SQDGs were ob-

tained by direct flow injection of a crude Arthrospira sp. ex-

tract (acetonitrile/water, 9/1, v/v) with 10 mM ammonium

acetate into the ESI source in positive and negative ionization

modes. The raw extract was used, because there was only one

commercially available SQDG standard and the scope was to

develop a method for several SQDGs. For each precursor ion

being selected based on the full scan (m/z 50–1000), the three

most intense fragment ions (product ions) were determined.

Table 1summarizes the precursor and fragment ions of all

compounds tested in the present study. Mass analyzer settings

were optimized for all analytes to maximize the transmission

and sensitivity of each characteristic mass transition. These

optimizations were acquired automatically by using autotune

mode provided by the Analyst® software 1.6.1 (AB Sciex

Germany GmbH, Darmstadt, Germany). Attention was paid

to the fragmentation pattern and only mass numbers with

fragments typical for SQDGs were included. Such fragments

of SQDGs are m/z 81, m/z 125, m/z 153, m/z 165, m/z 225, and

m/z 255. A comprehensive overview of the fragmentation pat-

tern of selected SQDGs was published by Zhang et al. [23].

Exemplarily, Fig. 1shows the fragmentation of the SQDG

816. The nomenclature of the various SQDGs used in this

work is based on the mass [M-H] . This means, for example,

SQDG 817 has the mass m/z 816 [M-H] as main fragment in

the MRM method. As obvious from Fig. 1, the typical frag-

ments m/z81 and m/z225weredetectedasfullscaninneg-

ative mode. Additionally, the fragments m/z537 and m/z559

represent the masses of the loss of one fatty acid. With the help

of these fragments, a conclusion about the bound fatty acids

can be made. The fragment m/z537 corresponds with the loss

of the fatty acid 18:2 (linoleic acid) and m/z559 with the fatty

acid 16:0 (palmitic acid). A further identification of the fatty

acids was not always possible, because some sulfolipids are

isobaric compounds with combinations of fatty acids equal to

each other, but different in their position. In the negative mode

only, the fragment of the intact fatty acid loss was detected.

The measurement was carried out in negative mode, be-

cause sulfolipids are easily deprotonated to form the quasi-

molecular ion [M-H] , due to their strongly acidic sulfate

group. The negative and the positive ion modes were used

during this study, but the results of the ionization in the neg-

ative mode were much more meaningful. The negative ion

mode is particularly suitable for electrophilic substituents such

as the sulfonic acid group, which was detected a hundred

times more sensitive compared to the positive ion mode. The

intensity in the positive mode was so low that the software was

not able to record the data as an MRM approach. It is possible

that the formation of the positive adducts was not stable

enough so that no suitable fragments could be recorded, as

the formation of the sulfonic acid ion with the cleavage of a

proton yielded more stable fragments. This was already ob-

served by Keusgen et al. [28]. Xu et al. [29] observed the same

phenomenon for SQDGs when using positive ionization

mode. Although they tried to use different modifiers for sta-

bilizing the SQDG fragments in positive mode, they did not

1944 Fischer J. et al.

Table 1 MRM transitions and MS parameters in negative ion mode for all SQDGs and the internal standard (ISD)

Analyte Rt (min) M (g/mol) MRM transition (m/z) DT (ms) DP (V) EP (V) CE (P) CXP (V)

792 (ISD) 20.5 794 792.0→96.0 150 −155 −10 −128 −15

792.0→58.0 150 −155 −10 −130 −1

792.0→80.0 150 −155 −10 −128 −3

555 9.23 555 555.0→80.0 150 −130 −10 −100 −3

555.0→94.0 150 −130 −10 −80 −5

555.0→224.0 150 −130 −10 −64 −15

765 19.7 765 765.0→80.0 150 −110 −10 −120 −11

765.0→224.0 150 −110 −10 −64 −15

765.0→94.0 150 −110 −10 −112 −5

787 15.4 787 787.3→80.0 150 −155 −10 −112 −11

787.3→224.0 150 −155 −10 −64 −1

787.3→164.0 150 −155 −10 −11 −11

789 17.4 789 789.2→80.8 150 −165 −10 −130 −1

789.2→224.7 150 −165 −10 −64 −17

789.2→164.7 150 −165 −10 −14 −11

791 11.7 791 791.0→81.0 150 −160 −10 −104 −3

791.0→225.0 150 −160 −10 −64 −5

791.0→165.0 150 −160 −10 −80 −1

793 25.7 793 793.0→80.8 150 −135 −10 −130 −9

793.0→224.8 150 −135 −10 −17 −17

793.0→79.9 150 −135 −10 −1−1

801 16.6 801 801.3→81 150 −150 −10 −130 −11

801.3→152.9 150 −150 −10 −70 −9

801.3→224.9 150 −150 −10 −62 −15

803 18.9 803 803.4→80.6 150 −185 −10 −128 −3

803.4→224.8 150 −185 −10 −64 −17

803.4→164.5 150 −185 −10 −80 −11

805 23.1 805 805.0→81.0 150 −65 −10 −114 −3

805.0→80.0 150 −65 −10 −126 −1

805.0→224.0 150 −65 −10 −68 −17

807 29.7 807 807.2→80.6 150 −130 −10 −126 −1

807.2→224.9 150 −130 −10 −66 −11

807.2→94.8 150 −130 −10 −108 −3

813 15.7 813 813.3→80.9 150 −160 −10 −130 −1

813.3→152.7 150 −160 −10 −72 −13

813.3→94.7 150 −160 −10 −126 −1

815 18.4 815 815.4→80.9 150 −205 −10 −118 −3

815.4→152.7 150 −205 −10 −64 −15

815.4→94.7 150 −205 −10 −106 −5

817 21.3 817 817.0→81.0 150 −230 −10 −120 −11

817.0→225.0 150 −230 −10 −66 −17

817.0→165.0 150 −230 −10 −72 −9

819 26.1 819 819.3→80.8 150 −150 −10 −116 −1

819.3→225.1 150 −150 −10 −68 −3

819.3→164.7 150 −150 −10 −72 −13

821 36.0 821 821.5→80.8 150 −140 −10 −130 −1

821.5→225.1 150 −140 −10 −72 −1

821.5→95.0 150 −140 −10 −120 −5

837 14.6 837 837.2→80.9 150 −80 −10 −124 −3

1945Development of a targeted HPLC-ESI-QqQ-MS/MS method for the quantification of sulfolipids from a...

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