Redefining dilu e and shoo : The e olu ion o he echnique and i s
applica ion in he analysis o oods and biological ma ices by liquid
ch oma og aphy mass spec ome y
B e G ee
a
,
*
, Oli ie Che allie
a
, B ian Quinn
a
, Luis M. Bo ana
b
, Ch is ophe T. Ellio
a
a
Ins i u e o Global Food Secu i y, School o Biological Sciences, Queens Uni e si y Bel as , UK
b
Depa amen o de Fa macología, Facul ad de Ve e ina ia, Uni e sidade de San iago de Compos ela, 27002, Lugo, Spain
a icle in o
A icle his o y:
A ailable online 5 Ap il 2021
Keywo ds:
Dilu e-and-shoo
Liquid ch oma og aphy
Mass spec ome y
Sample p epa a ion
Food analysis
Bioanalysis
Myco oxin
Mul i-class
abs ac
Wi h labo a o ies seeking o expand analy ical capabili ies and c ea e mul i-class, mul i-analy e
me hods, he e has been a shi owa d gene ic sample clean-up echniques such as “dilu e-and-shoo ”.
Ad an ages o his me hodology include i s simplici y, minimal analy e losses, high sample h oughpu
and numbe o analy e classes included. The e olu ion o dilu e-and-shoo has pe mi ed i s use ac oss a
a ie y o ma ices including ood and biological and in a ious scien ificfields such as ood, o ensics
and en i onmen al. The e sa ili y o he echnique pe mi s he expansion o cu en fields o esea ch
wi hou he usual labo ious me hod de elopmen . The e can be issues wi h ma ix e ec s and obus
quan i a ion as analy e numbe inc eases. This e iew p o ides an o e iew o he echnique combined
wi h liquid ch oma og aphy mass spec ome y, highligh ing i s powe in acili a ing mul i-class anal-
ysis. Coupled wi h inc eases in ins umen pe o mance he e is po en ial o employ his me hodology in
expanding analy ical capabili ies in many a eas o li e science esea ch.
©2021 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND
license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
1. In oduc ion
In analy ical chemis y and in pa icula in he field o small
molecule analysis (app ox. <1200 Da) using liquid ch oma og aphy
mass spec ome y (LC-MS), he use o “dilu e-and-shoo ”has
gained much ac ion o e he las en o fi een yea s, wi h i now
employed in many ypes o mul i- esidue LC-MS analysis [1]. The
e m i sel appea s qui e sel -explana o y, e e ing simply o:
“Dilu ion o a sample o sample ex ac be o e ‘shoo ing’in o an
analy ical ins umen o analysis.”
As a s and-alone me hodology, he e m implies ha his
echnique is he only clean-up s ep used be o e sample analysis.
Howe e , dilu e-and-shoo (DnS) is some imes pe o med a e an
ini ial dep o einisa ion s ep dependen on he ma ix being
assayed, wi h i s a ed ha he p o ein p ecipi a ion s ep is
conside ed a clean-up p ocess [2]. I his is he case and dep o ei-
nisa ion is ca ied ou be o e dilu ion o he sample (supe na an ),
i could be a gued ha such me hodology canno be conside ed
DnS. Fu he mo e, ma ices ha a e no liquid mus fi s unde go
an ex ac ion s ep o acili a e mo emen o analy es om he
ma ix o a liquid phase be o e dilu ion and analysis on some o m
o mass analyse . These include ma ices such as animal eed, is-
sues and a ious (solid) oods u s. In hese cases, a sui able
ex ac ion sol en mus be chosen o acili a e mig a ion o analy es
om a solid o a liquid phase which is hen dilu ed p io o analysis.
One such example is in he ecen s udy conduc ed by Sulyok e al.
(2020) measu ing o e 500 seconda y mic obial me aboli es in
a ious ood ma ices using DnS coupled o LC-MS/MS [3]. The ein
lies he issue as o wha ex en a sample clean-up me hod can be
uly classified as DnS. Wi h many publica ions claiming use o he
‘dilu e-and-shoo ’app oach bu which include addi ional s eps
such as solid-liquid ex ac ion o dep o einisa ion, he use o o
defini ion o he echnique appea s o ha e e ol ed. The e o e, due
o he e olu ion o he echnique in he li e a u e o e he las
decade, we ha e expounded on he simple defini ion s a ed abo e
o include solid ma ices.
“The di ec dilu ion o a sample o dilu ion o a sample ex ac
be o e ‘shoo ing’in o an MS pla o m o analysis. This also al-
lows o he addi ion o a sol en o a solid ma ix o ex ac
*Co esponding au ho .
E-mail add ess: b e .g ee @qub.ac.uk (B. G ee ).
Con en s lis s a ailable a ScienceDi ec
T ends in Analy ical Chemis y
jou nal homepage: www.else ie .com/loca e/ ac
h ps://doi.o g/10.1016/j. ac.2021.116284
0165-9936/©2021 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
T ends in Analy ical Chemis y 141 (2021) 116284
un a ge ed o a ge ed compounds in o a liquid phase be o e
dilu ion and analysis. Fil a ion and cen i uga ion a e included
in ou defini ion, as hey a e physical me hods ha emo e solid
ma e ials om liquid samples, he eby making he sample mo e
sui able o LC-MS analyses”.
In using DnS as a sample clean-up echnique, he main pu pose
o he dilu ion s ep is o educe ma ix componen s which can
in e e e wi h he ch oma og aphy and ionisa ion o he a ge
analy es, he eby acili a ing g ea e sensi i i y o pe o mance o
he analy ical me hod. Wi h ha in mind, he dilu ion ac o used is
dic a ed by many ac o s including; concen a ion o he analy es
expec ed, he ma ix analysed, he selec i i y and sensi i i y o he
MS pla o m u ilised and whe he he analysis is quan i a i e o
quali a i e, wi h dilu ion ac o s ypically anging om 1:1 up o
1:100. Some examples include a dilu ion ac o o 1:1 ( : ) in he
analysis o seconda y mic obial me aboli es in ood ma ices; a
dilu ion ac o o 1:10 analysing me aboli es o he myco oxins
deoxyni alenol (DON) and zea alenone (ZEN) in human u ine; a
dilu ion ac o o 1:50 in he analysis o d ugs-o -abuse (DoA) and
pe o mance enhancing d ugs in human u ine; a 1:100 dilu ion o
samples in he analysis o p os hesis- ela ed me als in whole blood
[3e6]. One ca ea o he las wo examples is ha he analysis o
DoA by Alc
an a a-Du
an e al. (2018) employed nanoflow LC
coupled o an O bi ap MS which imp o ed me hod sensi i i y and
pe mi ed a high dilu ion ac o , whe eas he s udy by Bolea-
Fe nandez e al. (2016) was o p os hesis- ela ed me als, i.e. ele-
men s as opposed o in ac molecules and which was ca ied ou on
Induc i ely Coupled Plasma MS (ICP-MS). In he main and o he
pu pose o his e iew, he ocus will be on he analysis o small
molecules by way o LC-MS.
O e ecen yea s he e has been a shi om single class
me hods wi h low analy e numbe s owa d mul i-class, mul i-
analy e me hods. This has been achie ed by mo ing away om
use o adi ional sample clean-up echniques such as solid phase
ex ac ion (SPE) and Immunoa fini y Columns (IAC) o mo e
gene ic echniques such as QuEChERs (Quick, Easy, Cheap, E ec-
i e, Rugged and Sa e), wi h he la e me hodology ou inely
applied in mul i- esidue pes icide analysis in ag icul u al com-
modi ies [7]. Howe e , e en echniques such as QuEChERs s ill
su e om losses o incomple e eco e y o he a ge analy es
when pe o ming mul i-class analysis. These analyses ypically
u ilise ch oma og aphic sepa a ion o aid de ec ion and can be
pe o med on high pe o mance liquid ch oma og aphy (HPLC).
Cu en ly howe e , ch oma og aphy is ou inely pe o med on
ul a-high pe o mance liquid ch oma og aphy (UHPLC o UPLC)
coupled o some o m o mass analyse . The swi ch o UHPLC
acili a ed sho e un imes while allowing he same numbe o
analy es o be esol ed. O la e, me hod un imes ha e become
longe o acili a e he inc ease in he numbe o analy es included
in a single un [3,8]. In ega ds o he mass analyse used, hese
include low esolu ion mass spec ome e s (LRMS), mainly iple
quad upole ins umen s (QqQ o MS/MS), and high- esolu ion
ins umen s such as ion ap o o bi al ion aps (O bi ap),
ime-o -fligh (TOF) and he new gene a ion o hyb id sys ems
such as quad upole-ToF (QToF) and quad upole-O bi ap (Q-
O bi ap), wi h hese HRMS sys ems o e ing di e en esol ing
powe s and he e o e selec i i ies and sensi i i ies. LRMS in-
s umen s a e used mainly in ou ine es ing labo a o ies o high
h oughpu quan i a i e analysis, whe eas HRMS sys ems a e
mo e ypically employed o esea ch pu poses. Thus, he ins u-
men a ion used will be dependen on he ype o analysis
equi ed, whe he i be ully quan i a i e, quali a i e o sc eening
pu poses o simply un a ge ed o “omics”analysis.
The pu pose o his e iew is o highligh he e olu ion in he
use o dilu e-and-shoo in analy ical chemis y which has now
been employed ac oss many di e en ma ices, whils also indi-
ca ing ha he defini ion o he e m should be ex ended o eflec
his. In addi ion, we look a how i s use is expanding pa icula ly in
he a ea o a ge ed analysis, mainly due o he ad ancemen o he
de ec ion pla o ms which has allowed i o anscend many
di e en scien ific disciplines.
2. Dilu e-and-Shoo
2.1. An o e iew o he echnique
DnS e e s o he dilu ion o a sample ma ix wi h a sui able
dilu ion sol en (diluen ) o analysis. The e o e, depending on he
ma ix analysed i can ei he be a s aigh o wa d dilu ion o a
biological fluid such as u ine o o al fluid which con o m o he
‘o iginal’defini ion o he echnique. Howe e , o he mo e complex
fluids such as se um, plasma o milk can equi e an ini ial p o ein
p ecipi a ion s ep, o dilu ion o he ex ac ion sol en used in
ex ac ing analy es o in e es om a solid ma ix such as ood-
s u s, wi h he la e in pa icula included in he ex ended,
upda ed defini ion o he echnique ou lined abo e. Compa ed wi h
o he adi ional sample clean-up echniques which emo e un-
wan ed co-ex ac ed ma ix componen s, DnS simply dec eases
ma ix e ec s while elimina ing ime-consuming and expensi e
ex ac ion p ocedu es and can he e o e be conside ed he mos
s aigh o wa d and as es clean-up me hod a ailable. One ca ea
o his is in he applica ion o solid oods u s whe eby an ex ac ion
s ep is equi ed o acili a e mig a ion o analy es om a solid o a
liquid phase, wi h examples de ailed in Table 1.
Use o DnS in analy ical chemis y has become qui e common-
place due o i s simplici y, limi ed and as sample p epa a ion, low
sol en usage and associa ed low-cos s, as well as educed was e
p oduc ion, wi h he benefi o being mo e en i onmen ally
iendly han mos o he sample clean-up echniques. As well as
being a ela i ely en i onmen ally iendly echnique, DnS lends
i sel well o high h oughpu ou ine analysis which has led o i s
use ac oss a ious disciplines such as in d ugs-o -abuse (DoA) and
an i-doping (pe o mance enhancing d ugs), o ensics and ood
sa e y, and can be used ac oss a ange o “omics”s udies such as
me abolomics. I also lends i sel o mul i-class, mul i-analy e ap-
p oaches as he e a e li le o no analy e losses and i s simplici y
minimises he chance o e o s du ing he ex ac ion s age. How-
e e , he e can be issues wi h he epea abili y, ep oducibili y,
accu acy and p ecision o his echnique, especially in ela ion o
he alida ion o mul i- esidue me hods in oods u s using DnS.
These a o emen ioned issues ha e been add essed somewha in
he s udies ca ied ou by Malacho a e al. (2015) and Sulyok e al.
(2020) [3,9]. Examples o whe e he DnS me hodology has been
employed alongside ei he LC-MS/MS o LC-HRMS a e gi en in
Table 1, wi h examples as o why DnS is such a powe ul analy ical
ool is discussed in mo e de ail la e .
2.2. Issues wi h dilu e-and-shoo : e minology and me hodology
In e ms o he expounded defini ion o DnS, whe eby an
ex ac ion sol en is equi ed o acili a e he mig a ion o analy es
om a solid ma ix in o a liquid phase be o e dilu ion o analysis,
one mus be awa e ha he choice o ex ac ion sol en di ec ly
impac s he ange o analy es ha can be ex ac ed, i.e. he pola i y
o he ex ac ion sol en used will de e mine he pola i y and
he e o e ange o analy es ex ac ed. I is clea ha ce ain
ma ices equi e an ex ac ion s ep be o e dilu ion and an example
o his is add essed in he s udy by Sulyok e al. (2020). The
B. G ee , O. Che allie , B. Quinn e al. T ends in Analy ical Chemis y 141 (2021) 116284
2
Table 1
Applica ion o dilu e-and-shoo alongside LC-MS/MS ac oss a ious scien ific disciplines and sample ma ices.
Compound ype (numbe o
analy es)
Ma ices Me hodology LC-MS Pla o m Analy ical condi ions Re e ence
Myco oxins (39) Whea &maize 0.5 g ma ix ex ac ed wi h 2 mL o ex ac ion
sol en (ACN:H
2
O:CH
3
COOH, 79:20:1 / / ),
350
m
L supe na an dilu ed wi h 350
m
Lo
(ACN:H
2
O:CH
3
COOH, 20:79:1 / / )
Dilu ion ac o 1:2
QTRAP®4000 equipped
wi h a Tu bo Ion Sp ay ESI
sou ce (AB SCIEX) coupled
o a 1100 se ies HPLC
(Agilen )
ESI
þ
&ESI
: MRM
Gemini C
18
column (150 mm 4.6 mm,
5
m
m) þC
18
secu i y gua d ca idge
(4 mm 3mm,5
m
m) @ 25
C, flow a e 1 mL/
min, injec ion olume 5
m
L.
MPA: MeOH:H
2
O:CH
3
COOH (10:89:1 / / ),
MPB: MeOH:H
2
O:CH
3
COOH (97:2:1 / / ), bo h
con aining
5mMNH
4
CH
3
COO
.
[27]
Bac e ial & ungal
me aboli es (295)
Apple pu ee, hazelnu ,
maize &g een peppe
0.5 g ma ix ex ac ed wi h 2 mL o ex ac ion
sol en (ACN:H
2
O:CH
3
COOH, 79:20:1 / / ).
350
m
L supe na an dilu ed wi h 350
m
Lo
(ACN:H
2
O:CH
3
COOH, 20:79:1 / / )
Dilu ion ac o 1:2
QTRAP®5500 equipped
wi h a Tu boV ESI sou ce
(AB SCIEX) coupled o a
1290 se ies UHPLC (Agilen )
ESI
þ
&ESI
: sSRM
Gemini C
18
column (150 mm 4.6 mm,
5
m
m) þGemini C
18
gua d column
(4 mm 3mm,5
m
m) @ 25
C, flow a e 1 mL/
min, injec ion olume 5
m
L.
MPA: MeOH:H
2
O:CH
3
COOH (10:89:1 / / ),
MPB: MeOH:H
2
O:CH
3
COOH (97:2:1 / / ), bo h
con aining
5mMNH
4
CH
3
COO
[28]
Myco oxins &me aboli es
(>500)
Whea , maize, figs, d ied
g apes, walnu s, pis achios
&almonds.
0.25 g ma ix ex ac ed wi h 1 mL o ex ac ion
sol en (ACN:H
2
O:CH
3
COOH, 79:20:1 / / ).
300
m
L supe na an dilu ed wi h 300
m
Lo
(ACN:H
2
O:CH
3
COOH, 20:79:1 / / )
Dilu ion ac o 1:2
QTRAP®5500 equipped
wi h a Tu boV ESI sou ce
(AB SCIEX) coupled o a
1290 se ies UHPLC (Agilen )
ESI
þ
&ESI
: sMRM
NB: Two sepa a e uns o
ESI
þ
&ESI
Gemini C
18
column (150 mm 4.6 mm,
5
m
m) þC
18
secu i y gua d ca idge (4 3mm
i.d.) @ 25
C, flow a e 1 mL/min, injec ion
olume 5
m
L.
MPA: MeOH:H
2
O:CH
3
COOH (10:89:1 / / )
MPB: MeOH:H
2
O:CH
3
COOH (97:2:1 / / ), bo h
con aining 5 mM
NH
4
CH
3
COO
.
[3]
Myco oxins (10) and
pes icides (6)
Rice 5 g o ice was ex ac ed and dilu ed wi h 10 mL
ACN:H
2
O (8:2 / ) con aining 2% CH
3
COOH ( /
) hen fil e ed.
Dilu ion ac o 1:3
QTRAP®6500 (AB SCIEX)
coupled o a 1290 se ies
UHPLC (Agilen )
ESI
þ
&ESI
: MRM
Zo bax RRHD C
18
column (50 mm 2.1 mm,
3
m
m) wi h p e-column @ 30
C, flow a e
350
m
L/min, injec ion olume 5
m
L.
MPA:H
2
O, MPB: MeOH bo h con aining 0.5%
CH
3
COOH ( / )
[29]
Pes icides (185) and
myco oxins (12)
Wine ( ed) A 500
m
L aliquo o wine was dilu ed wi h
500
m
L o MeOH:ACN (1:1, / ) and fil e ed.
Dilu ion ac o 1:2
Xe o®TQ-S (Wa e s)
coupled o an ACQUITY H-
Class UPLC®wi h
qua e na y sol en
manage (Wa e s)
ESI
þ
: MRM
ACQUITY UPLC®BEH C
18
column
(100 mm 2.1 mm, 1.7
m
m) @ 60
C, flow a e
450
m
L/min.
Pes icide analysis:
MPA: 5 mM NH
4
HCOO
(aq)
,MPB: MeOH
Myco oxin analysis:
MPA:H
2
O and MPB: ACN, bo h con aining 0.1%
HCOOH ( / )
[17]
P esc ip ion d ugs, DoA and
me aboli es (37)
U ine (human) 100
m
L o cen i uged u ine dilu ed wi h
700
m
LH
2
O and 200
m
LIS
mix
.
Dilu ion ac o 1:10
TQ-S Mic o (Wa e s)
coupled o an ACQUITY
UPLC (Wa e s)
ESI
þ
: MRM
CORTECS C
18
column (2.1 mm 50 mm, 1.6
m
m)
@30
C.
NB: Injec ion olume no s a ed.
MPA: Wa e , MPB: MeOH (bo h con aining 0.1%
HCOOH / ).
[16]
Myco oxins and
me aboli es (37)
U ine (human) 100
m
L o cen i uged u ine dilu ed wi h 900
m
L
o diluen (H
2
O:ACN:HCOOH, 94:5.:1 / / )
Dilu ion ac o 1:10
QTRAP®6500 (AB SCIEX)
coupled o a 1260 Infini y
se ies HPLC (Agilen )
ESI
þ
&ESI
: sMRM
NUCLEODUR®C
18
Py amid column
(150 mm 2 mm, 3
m
m) wi h a C
18
gua d
column (2 mm 4mm),flow a e 600
m
L/min,
injec ion olume 10
m
L.
MPA: ACN, MPB: Wa e (bo h con aining 0.1%
HCOOH / ).
[70]
S a ins (8) U ine (human) 500
m
L o u ine spiked wi h 60
m
LIS
mix
and
made up o 1.5 mL o al olume wi h H
2
O.
Dilu ion ac o 1:3
API 3200 Q-T ap equipped
wi h a Tu bo V ESI sou ce
(AB SCIEX) coupled o an
Agilen 1200 se ies.
ESI
þ
: MRM
Kine ex C
18
column (50 mm 3.0 mm, 2.6
m
m)
wi h a C
18
gua d column (4 mm 3 mm) @
20
C, flow a e 350
m
L/min, injec ion olume
10
m
L.
MPA: Wa e (0.1% CH3COOH), MPB: ACN.
[92]
(con inued on nex page)
B. G ee , O. Che allie , B. Quinn e al. T ends in Analy ical Chemis y 141 (2021) 116284
3
Table 1 (con inued)
Compound ype (numbe o
analy es)
Ma ices Me hodology LC-MS Pla o m Analy ical condi ions Re e ence
Neu o ansmi e s and
me aboli es (10)
U ine (human) Dilu e 1 mL o u ine wi h 1 mL o ACN. Mix,
cen i uge hen dilu e 1 mL supe na an wi h
0.25 mL IS
mix
and 1.25 mL 0.2 M ace ic acid.
Dilu ion ac o 1:5
6490 TQ MS (Agilen )
coupled o a 1290 infini y
se ies LC sys em (Agilen )
ESI
þ
: MRM
Agilen Po oshell 120 Bonus-RP LC column
(2.1 mm 100 mm, 2.7
m
m), flow a e 180
m
L/
min.
NB: Injec ion olume no s a ed.
MPA: Wa e (0.2% HCOOH, / ). MPB: ACN (0.1%
HCOOH, / ).
[31]
(i) Myco oxin DON and i s
glucu onide me aboli es
(ii) Myco oxins and
me aboli es (15)
(iii) Myco oxins DON, ZEN
and me aboli es
NB: All o he abo e s udies
use he same ex ac ion
and analysis.
U ine (human) Dilu e 100
m
L o cen i uged u ine wi h 900
m
L
dilu ion sol en (H
2
O:ACN, 90:10 / ).
Dilu ion ac o 1:10
QTRAP®5500 equipped
wi h a Tu bo V ESI sou ce
(AB SCIEX) coupled o a
1290 se ies UHPLC (Agilen )
ESI
þ
&ESI
: SRM
A lan is®T3 column (150 mm 3mm,3
m
m)
wi h a C
18
secu i y gua d ca idge @ 35
C, flow
a e 600
m
L/min, injec ion olume 5
m
L.
MPA: Wa e , MPB: ACN (bo h con aining 0.1%
CH
3
COOH)
[4,10,11]
ZEN and me aboli es (9*)
*known me aboli es
U ine (po cine) U ine samples dilu ed o 0.2 mM c ea inine
wi h H
2
O hen cen i uged.
Dilu ion ac o unknown
QTRAP®6500 equipped
wi h an Ion D i e Tu bo V
ESI sou ce (AB SCIEX)
coupled o a 1290 se ies
UHPLC (Agilen )
ESI
: SRM
Kine ex Biphenyl column (150 mm 3 mm,
2.6
m
m) @ 30
C, flow a e 400
m
L/min, injec ion
olume 5
m
L.
MPA: Wa e , MPB: ACN (bo h con aining 0.1%
CH
3
COOH)
[72]
No el Psychoac i e
Subs ances (826)
U ine (human) 100
m
L o u ine spiked wi h IS
mix
and dilu ed
wi h 400
m
LH
2
O.
Dilu ion ac o 1:5
Enzyma ic hyd olysis:
100
m
L o u ine mixed wi h 100
m
L
glucu onidase solu ion.
Dilu ion ac o 1:2
Agilen 6460 QqQ MS wi h
Je
S eaming Technology
coupled o a 1290 Infini y
se ies LC (Agilen )
ESI
þ
: dMRM
Zo bax RR HD Eclipse Plus C
18
column
(100 mm 3 mm, 1.8
m
m) wi h p e-column @
40
C, flow a e 300
m
L/min, injec ion olume
5
m
L.
MPA:5mMNH
4
HCOO
(aq)
(0.1% HCOOH, / ),
MPB: MeOH (0.1% HCOOH, / ).
[30]
Chemical wa a e agen
me aboli es (6)
U ine (human) 500
m
L o u ine mixed wi h 500
m
Lo H
2
O.
Dilu ion ac o 1:2
QTRAP®4000 (AB SCIEX)
coupled o an Ul ima e
3000 LC (Dionex)
ESI
þ
&ESI
: MRM
Acclaim 120C
18
column (150 mm 2.1 mm,
2.2
m
m) @ 30
C, flow a e 450
m
L/min, injec ion
olume 20
m
L.
MPA: Wa e , MPB: ACN (bo h con aining 0.5%
HCOOH)
[83]
Cannabinoids and acid
p ecu so s (8)
O al fluid, se um and u ine
(human)
100
m
L o sample mixed wi h 100
m
LM3®
eagen , 200
m
LIS
mix
and 200
m
L ace one:ACN
(8:2 / )
Dilu ion ac o 1:6
Xe o®TQ-S (Wa e s)
coupled o an ACQUITY
UPLC®I-class (Wa e s)
ESI
þ
: MRM
ACQUITY UPLC®BEH C
18
column
(50 mm 2.1 mm, 1.7
m
m) @ 50
C, flow a e
400
m
L/min, injec ion olume 10
m
L.
MPA: Wa e con aining 50 mM NH
4
HCOO
pH3,
MPB: MeOH
[73]
Psychoac i e d ugs (13) O al fluid (human) 500
m
L o o al fluid was mixed wi h 10
m
LIS
mix
and dilu ed u he wi h 1 mL H
2
O.
Dilu ion ac o 1:3
Xe o®TQ-S (Wa e s)
coupled o an ACQUITY
UPLC®I-class (Wa e s)
ESI
þ
: MRM
ACQUITY UPLC®BEH C
18
column
(75 mm 2.1 mm, 1.7
m
m) @ 50
C, flow a e o
350
m
L/min, injec ion olume 2
m
L.
MPA: Wa e , MPB: ACN (bo h con aining 0.1%
HCOOH)
[15]
B oad spec um d ugs o
abuse (61)
U ine (human) 200
m
L aliquo o u ine dilu ed wi h 400
m
LH
2
O,
300
m
L
b
-Glucu onidase solu ion and 100
m
L
IS
mix
.
Dilu ion ac o 1:2
Xe o®G2 ToF (Wa e s)
coupled o an ACQUITY
UPLC (Wa e s)
ESI
þ
Resolu ion: 20 000 FWHM @
m/z 400
Mass ole ance: ±5 and
20 ppm
ACQUITY UPLC®BEH C
18
column
(150 mm 2.1 mm, 1.7
m
m) @ 50
C, flow a e
400
m
L/min, injec ion olume 20
m
L.
MPA:5mMNH
4
HCOO
(aq)
(pH 3), MPB: ACN
(0.1% HCOOH, / )
[93]
B. G ee , O. Che allie , B. Quinn e al. T ends in Analy ical Chemis y 141 (2021) 116284
4
Anabolic s e oids and
me aboli es (32)
U ine (human) 200
m
L aliquo o u ine dilu ed wi h 200
m
LIS
mix
in MeOH.
Dilu ion ac o 1:2
Exac i e O bi ap®
(The mo Scien ific) coupled
o an Accela 1250 LC
(The mo Scien ific)
ESI
þ
&ESI
Resolu ion: 50 000 FWHM @
m/z 100 o 2000
Mass ole ance: ±5 ppm
Va ian Omnisphe ™C
18
column
(100 mm 2 mm, 3
m
m) þCh omSep gua d
column (10 mm 2 mm, 5
m
m) @ 35
C, flow
a e 250
m
L/min, injec ion olume 25
m
L.
MPA:H
2
O and MPB: MeOH, bo h con aining
1mMNH
4
CH
3
COO
and 0.1% CH
3
COOH / .
[94]
Spo s d ugs and
me aboli es (27)
U ine (human) 270
m
L aliquo o u ine dilu ed wi h 100
m
Lo
100 mM NH
4
HCOO
, 700
m
L ACN and 30
m
L
IS
mix
.
Dilu ion ac o 1:11
Q Exac i e Hyb id
O bi ap®coupled o an
Accela LC (The mo
Scien ific) and a 1100 se ies
LC (Agilen )
ESI
þ
&ESI
Resolu ion: 17 500 FWHM @
m/z 100e500 Da
Mass ole ance: ±5 ppm
Nucleodu HILIC column (100 mm 2mm,
1.8
m
m) þNucleodu HILIC apping column
(20 mm 2 mm, 3
m
m), flow a e 250
m
L/min,
injec ion olume 20
m
L.
MPA:H
2
O, MPB: ACN and MPC: 200 mM
NH
4
CH
3
COO
(aq)
(0.15% glacial CH
3
COOH, / )
[95]
Spo d ugs (46) U ine and plasma (equine) Plasma: 75
m
L aliquo o dep o eina ed plasma
dilu ed wi h 75
m
L MPA.
Dilu ion ac o 1:3
U ine: 20
m
L aliquo spiked wi h 20
m
LIS
mix
and
dilu ed wi h 960
m
L MPA.
Dilu ion ac o 1:50
Q-Exac i e™O bi ap
(The mo Scien ific) coupled
o an ACQUITY UPLC®
(Wa e s)
ESI
þ
Resolu ion: 140 00 FWHM @
m/z 200
Mass ole ance: ±5 ppm
ACQUITY UPLC®BEH C
18
column
(100 mm 2.1 mm, 1.7
m
m) @ 55
C, flow a e
350
m
L/min, injec ion olume 5
m
L.
MPA: 5 mM NH
4
HCOO
(aq)
(pH 3), MPB: ACN
(0.1% HCOOH, / ).
[96]
Pes icide and e e ina y
d ugs (>350)
Mea (bee , po k &chicken) 2.5 mL o H
2
O added o 2.5 g o homogenised
mea hen dilu ed wi h 7.5 mL ACN (1% HCOOH,
/ ).
Dilu ion ac o 1:4
Exac i e™O bi ap
(The mo Scien ific) coupled
o a T anscend 600 UHPLC
(The mo Scien ific)
ESI
þ
&ESI
Resolu ion: 25 000 FWHM @
m/z 70 o 1000
Mass ole ance: ±5 ppm
Hype sil GOLD aQ C
18
column
(100 mm 2.1 mm, 1.7
m
m) @ 30
C, flow a e
300
m
L/min, injec ion olume 10
m
L.
MPA: 4 mM NH
4
HCOO
(aq)
(0.1% HCOOH, / ),
MPB: MeOH (0.1% HCOOH, / ).
[80]
Pes icides, myco oxins,
p ocess-induced
oxican s &packaging
Con aminan s (32)
Tea (b ew and lea es) Tea b ew: 2 mL dilu ed wi h 8 mL H
2
O:ACN
(3:1, / ) acidified wi h 0.1% HCOOH ( / ) hen
0.5 mL o he mix u e fil e ed (0.2
m
m).
Dilu ion ac o 1:5
Tea lea es unde wen a “dilu e, shoo &e apo a e
app oach”.
Xe o®G2-S ToF (Wa e s)
coupled o an ACQUITY H-
Class UPLC®wi h
qua e na y sol en
manage (Wa e s)
ESI
þ
&ESI
Resolu ion: 30 000 FWHM @
m/z 200
Mass ole ance: no gi en
C
18
PFP column (150 mm 2.1 mm, 2
m
m) @
30
C, flow a e 400
m
L/min, injec ion olume
10
m
L.
MPA:H
2
O, MPB: ACN bo h con aining 0.1%
HCOOH ( / ) and MPC: MeOH
[97]
DoA and spo d ugs (81) U ine (human) Aliquo o u ine dilu ed wi h H
2
O:ACN (95:5 /
) and fil e ed.
Dilu ion ac o 1:50
Q-Exac i e™O bi ap
(The mo Scien ific) coupled
o an EASY-nLC 1000 nano-
LC sys em (The mo
Scien ific)
ESI
þ
&ESI
Resolu ion: 70 000 FWHM @
m/z 195
EASY-Sp ay PepMap®C
18
column
(150 mm 75
m
m, 3
m
m) @ 25
C, flow a e
200 nL/min, injec ion olume 1
m
L.
MPA:H
2
O, MPB: ACN bo h con aining 0.1%
HCOOH ( / )
NB: Nano-flow used as opposed o LC
[5]
ACN: Ace oni ile,CH
3
COOH: Ace ic acid, DoA: D ugs o Abuse, DON: Deoxyni alenol, dMRM: Dynamic Mul iple Reac ion Moni o ing, ESI: Elec osp ay Ionisa ion, ESI
-
: Elec osp ay Ionisa ion (nega i e mode), ESI
þ
: Elec osp ay
Ionisa ion (posi i e mode), FWHM: Full Wid h a Hal Maximum ( esolu ion), HCOOH: Fo mic acid, HILIC: Hyd ophilic In e ac ion Liquid Ch oma og aphy, HPLC: High Pe o mance Liquid Ch oma og aphy, IS
mix
:In e nal
S anda d mix u e, LC: Liquid Ch oma og aphy, MeOH: Me hanol, MPA: Mobile Phase A, MPB: Mobile Phase B, MPC: Mobile Phase C, MRM: Mul iple Reac ion Moni o ing, MS: Mass Spec ome y, MS/MS: T iple Quad upole Mass
Spec ome y (QqQ), m/z: Mass o Cha ge Ra io, NH
4
CH
3
COO
: Ammonium ace a e, NH
4
HCOO
: Ammonium o ma e, ppm: Pa s Pe Million, QTRAP®: T iple Quad upole Linea Ion T ap, QuEChERS: Quick, Easy, Cheap, E ec i e,
Rugged, and Sa e, sMRM: Scheduled Mul iple Reac ion Moni o ing, SPE: Solid Phase Ex ac ion, SRM: Selec ed Reac ion Moni o ing, UHPLC: Ul a-High Pe o mance Liquid Ch oma og aphy, UPLC®: Ul a-Pe o mance Liquid
Ch oma og aphy, ZEN: Zea alenone.
Dilu ion ac o exp essed as sample: o al ( olumes), i.e. 500
m
L sample mixed wi h 500
m
L diluen equa es o a dilu ion ac o o 1:2.
B. G ee , O. Che allie , B. Quinn e al. T ends in Analy ical Chemis y 141 (2021) 116284
5
wo kflow employed was highly fi o pu pose and being classed as
DnS is a good example o he ambigui y o he classic DnS defini-
ion. In his s udy, he au ho s looked a se e al ood ma ices wi h
a ying deg ees o complexi y, om whea and maize o se e al
classes o nu s. A 250 mg aliquo was ex ac ed wi h 1 mL o
ex ac ion sol en and a e mixing and cen i uga ion, a 300
m
L
aliquo o supe na an was dilu ed 1:1 ( / ) wi h dilu ion sol en
gi ing a dilu ion ac o o 1:2, a e which a po ion was injec ed o
analysis. Compa e his o he DnS p o ocol employed by Wa h e al.
(2012) whe eby a 100
m
L aliquo o sample ma ix, in his case
human u ine, was simply dilu ed wi h 900
m
L o dilu ion sol en
a e cen i uga ion, gi ing a dilu ion ac o o 1:10 be o e analysis
[10,11]. Al hough bo h me hods employ sample dilu ion, he
simplici y o he la e leans mo e owa d he ype o applica ion
ha fi s wi h he DnS concep , i.e. no o he ex ac ion o sample
clean-up echnique was used, wi h he key di e ences ou lined in
he wo kflows shown in Fig. 1. The e a e also publica ions ha
men ion use o DnS as hei clean-up app oach, howe e on ex-
amina ion o he me hodology employed, some ha e used an
addi ional clean-up s ep o educe ma ix in e e ences u he
including QuEChERS o SPE and a e he e o e no DnS [12e14].
A po en ially mo e igo ous defini ion o DnS is ha i is he only
echnique used p io o analysis by LC-MS, hus he echnique only
eally applies o liquid ma ices such as u ine, o al fluids and liquid
oods u s such as wine which can simply be dilu ed be o e analysis
[4,5,11,15e17]. Howe e , as ou lined abo e, he defini ion mus be
ex ended o include dilu ion o a sample ex ac such as ha pe -
o med in he s udy by Sulyok e al. (2020). Fu he mo e, o he
biological ma ices such as blood (whole blood, se um o plasma)
mus fi s unde go a s ep o acili a e emo al o p o eins due o he
ac hey can block he column, injec o and/o componen s o he
LC sys em. This p o ein p ecipi a ion s ep is ypically pe o med by
addi ion o an o ganic sol en such as ace oni ile, me hanol,
e hanol o ace one which p ecipi a es he p o ein con en as well as
pa i ioning he analy es o in e es om he ma ix in o he
o ganic sol en laye . Howe e , his p o ein p ecipi a ion s ep can
be conside ed an addi ional clean-up p ocess. A e dep o einisa-
ion and cen i uga ion, an aliquo o supe na an is dilu ed o
analysis, o no u he dilu ion is pe o med due o he sol en
olume added a he p o ein p ecipi a ion s ep being su ficien .
The e o e, in his case we ha e o ex end he defini ion o DnS o
include his addi ional s ep. Examples o his me hodology include
he analysis o pes icides and ca o enoids in human se um using
HPLC-DAD and HPLC-MS/MS espec i ely [18,19].
Howe e , use o DnS can be p oblema ic o de ec ion o blood
me aboli es ound in low concen a ions, due o he dilu ion caused
by addi ion o sol en equi ed o dep o einisa ion. This can be
emedied by d ying he supe na an unde a gen le s eam o ni-
ogen be o e econs i u ion in an app op ia e olume o acili a e
de ec ion o lowe concen a ion me aboli es. This echnique has
been e e ed o as a ‘dilu e, e apo a e and shoo ’app oach [20].
One such example o his me hodology was employed in he s udy
by De eese e al. (2012) in he analysis o pig plasma o myco-
oxins and me aboli es and which is ou inely employed in his
ype o analysis [20e23]. One no e o cau ion in his me hodology is
ha he d ying s ep can lead o some loss o analy es, pa icula ly in
complex ma ices such as blood. The e o e, his me hodology does
no all unde he expounded DnS defini ion, and as such should be
e med ‘dilu e, e apo a e and shoo ’.
2.3. Ins umen a ion: low esolu ion (LRMS/QqQ) e sus high
esolu ion mass spec ome y (HRMS)
The lack o selec i i y associa ed wi h DnS is due o i s gene ic
sample clean-up and his can be o se by he selec i i y a o ded by
iple quad upole (QqQ) mass spec ome e s ha gene ally a o d
Fig. 1. Example o he bes p ac ice wo kflows ac oss di e en ma ices such as oods u s (solid and liquid) and biological o bo h quan i a i e and quali a i e (sc eening) analysis
using LC-MS (MS/MS o HRMS).
1
Dilu e and shoo app oach;
2
Dilu e, e apo a e and shoo app oach; *S o e in da kness o e nigh o allow sol en e apo a ion and analy e
in e ac ion wi h ma ix; *Rep esen a i e sol en ; IS
mix
: In e nal s anda d mix.
B. G ee , O. Che allie , B. Quinn e al. T ends in Analy ical Chemis y 141 (2021) 116284
6
highe sensi i i y han HRMS sys ems. The e a e many MS in-
s umen s on he ma ke compa ible wi h HPLC/UHPLC and which
ha e been comp ehensi ely e iewed in ela ion o hei sui abili y
o he in ended analy ical me hod [24e26]. As shown in Table 1
whe eby DnS was employed alongside LC-MS/MS, he majo i y o
analys s used an AB SCIEX QTRAP [27e29]. Wi h DnS dilu ing
a he han emo ing ma ix, as well as dilu ing he analy es o
in e es he ein, hese sys ems appea o educe he influence o co-
elu ing ma ix compounds and o e be e sensi i i y due o he
enhanced selec i i y a o ded by he QTRAP echnology and sou ce
design. This is no o s a e ha hese a e he only QqQ ins umen s
capable o being used alongside DnS, wi h o he QqQ ins umen s
also used success ully alongside DnS such as he Wa e s Xe o TQ-
XS and Agilen 6460 and 6490 T iple Quad upole LC-MS sys ems
[15,17,30,31].
Wi h imp o emen s in QqQ ins umen a ion elec onics acili-
a ing as e cycle and dwell imes, he numbe o analy es ha can
be included in a single un can be inc eased. One example o his is
in he analysis conduc ed by Sulyok e al. (2020) in which mo e
han 500 seconda y mic obial me aboli es we e analysed in a single
un using a QTRAP 5500 (AB SCIEX), wi h ch oma og aphy pe -
o med on HPLC as opposed o UHPLC esul ing in a un ime o
app oxima ely 20 min. One ca ea o his me hod and o he s wi h
mul iple analy es such as ha o S eine e al. (2020), is ha wi h
inc easing analy e numbe and subsequen MRM ansi ions, cycle
and dwell imes ha e o be educed in o de o acqui e enough da a
poin s pe analy e o obus quan i a ion. Fu he o his, al hough
pola i y swi ching can be pe o med as apidly as 5 msec acco ding
o some endo s, his along wi h he se ling ime would be
de imen al o he numbe o da a poin s acqui ed a any gi en
ime, and he e o e, wo injec ions may be equi ed. Ano he mul i-
analy e me hod was ha conduc ed by Kimble and DeCap io
(2019), analysing mo e han 800 psychoac i e subs ances using an
Agilen 6460 MS/MS wi h Je S eaming Technology coupled o
UHPLC. As wi h he a o emen ioned me hods, due o he dwell and
cycle imes a o ded by he ins umen , he ac ual numbe o ana-
ly es in a single un was app oxima ely 400, wi h samples analysed
wice o co e all 800-plus compounds. The analysis ime o each
un was 20 min and he e o e 40 min o e all [30]. Thus, due o he
enhanced selec i i y and s abili y a o ded by QqQ pla o ms, hese
ins umen s a e gene ally used o quan i a i e analysis.
A new gene a ion o hyb id HRMS sys ems has imp o ed hei
capabili ies, such as he quad upole-ToF (QToF) and quad upole-
O bi al ion ap and which can now compe e wi h QqQ in-
s umen s. These la es HRMS ins umen s ha e significan ly be e
sensi i i y specifica ions han p e ious HRMS ins umen s, ach-
ie ed by ad ances in de ec ion echnology and inc eased esol ing
powe , ul ima ely leading o a educ ion in in e e ences wi h
ma ix compounds. The addi ion o he quad upole (Q) o hese
ins umen s allows hem o pe o m bo h quali a i ely and quan-
i a i ely, wi h one such s udy using one o he new gene a ion
hyb id HRMS sys ems ca ied ou by Ma inez-Dominguez e al.
(2016). In his mul i-class, mul i-analy e me hod, hey analysed 257
pes icides and myco oxins using a Q-Exac i e O bi ap bo h quali-
a i ely and quan i a i ely. Al hough hei me hodology included
an ex a clean-up s ep a e pe o ming DnS, by way o flo isil SPE
ca idges, hey epo ed simila esul s o DnS alone and only
added SPE o main ain ins umen pe o mance [13].
One d awback in he use o QToF analyse s is ha pola i y
swi ching is no possible o p ac ical and al hough his can be
achie ed wi h an O bi ap, i is o se by a educ ion in he in-
s umen s esol ing powe . Howe e , wi h use o UHPLC as
opposed o HPLC p o iding as e un imes, his allows injec ion o
each sample in posi i e and nega i e ion modes wi h he benefi
ha ionisa ion can be op imised o bo h, he e o e inc easing he
numbe o de ec ed compounds [32]. O e all, he numbe o
me hods employing DnS alongside LC-MS/MS compa ed o LC-
HRMS o e he las 10 yea s is highe as indica ed in he numbe
o examples shown in Table 1. One hing o no e is ha no all a -
icles using he DnS app oach men ion he me hodology in he i le,
abs ac o keywo ds. The e o e, he numbe o publica ions
employing he echnique a e highe han indica ed when a li e a-
u e sea ch is pe o med wi h he keywo ds ‘DnS’combined wi h
ei he ‘ andem MS’,‘high esolu ion MS’o ‘O bi ap’. Use o QqQ
o e HRMS is in pa due o he enhanced selec i i y a o ded by
QqQ ins umen s as well as hei sensi i i y which compensa e o
he gene ic sample clean-up echnique. Wi h he ma ix being
educed a he han emo ed, he selec i i y o QqQ ins umen s
allows analy es o be iden ified based on he di e en ia ion be-
ween analy e and unwan ed ma ix, al hough his is also due o
he sensi i i y as selec i i y alone does no a o d adequa e de ec-
ion limi s. Howe e , wi h many o he echnical limi a ions a ound
HRMS ins umen s ha ing been esol ed wi h ad ancemen s in
echnology, as well as he in oduc ion o he hyb id quad upole
HRMS sys ems, he e may well be a ise in he numbe o analys s
using HRMS o ou ine labo a o y es s.
3. Ma ix e ec s
In o de o ex ac analy es om a liquid ood ma ix o bio-
logical ma ices such as u ine o blood, liquid-liquid ex ac ion
(LLE) can be used. Al e na i ely, solid-liquid ex ac ion is used on
solid ma ices such as eed and ood. In ei he case, he p ocess
using a single o mul iple componen liquid ex ac ion mix u e
(o ganic sol en s, wa e , acids, bases, e c.) se es o mig a e he
analy es o in e es om he ma ix in o he added liquid phase.
The addi ion o wa e o he o ganic (ex ac ion) sol en can help
he o ganic sol en pene a e he ood ma ix in he case o solid
oods u s, while also acili a ing ex ac ion o some o he mo e
pola analy es. The addi ion o acid se es o help b eak bonds
be ween he analy es and some o he ma ix componen s such as
p o eins and suga s, aiding hei ex ac ion [33]. When an ex ac-
ion echnique is chosen, he e will always be co-ex ac ion o
ma ix componen s gi ing ise o ma ix e ec s, wi h he amoun
dependen on he ex ac ion sol en and complexi y o he sample.
Some o he mos challenging issues in analy ical chemis y a e
ma ix e ec s caused by co-ex ac ed ma ix compounds. These a e
unp edic able and can cause a ious issues such as: ion supp ession
(o in some cases enhancemen ), poo ch oma og aphy, alse pos-
i i es due o he p esence o isoba ic compounds, poo analy e
eco e y due o chemiso p ion and in e e ences wi h he ion a io
o he p oduc ions in a ge ed analysis [34,35]. Use o in e nal
s anda ds is one o he mos commonly used ways o compensa e
o ma ix e ec s, howe e , as a ious analy es can exhibi di e en
beha iou s in he same ma ix, ideally an in e nal s anda d is
equi ed o each analy e. This is no easible due o he ac hey a e
expensi e and also ha comme cial a ailabili y is limi ed. S anda d
addi ion is ano he way o compensa e o ma ix e ec s and is
o en used in ou ine analysis. Howe e , i is mo e sui ed o single-
analy e me hods a he han mul iple-analy e de e mina ion due
o i being labo ious and cos ly because o i s high consump ion o
s anda ds [1]. Some o hese issues can be o e come by imp o ed
ch oma og aphy, howe e , his has limi a ions as he e has o be a
benefi o imp o ed ch oma og aphy agains he need o as un
imes, especially in high- h oughpu labo a o ies.
E en wi h he ad ancemen in de ec ion capabili ies a o ded by
LC-MS, he e may s ill be a need o sample clean-up o emo e
unwan ed ma ix compounds using mo e adi ional echniques
such as SPE, IACs, LLE o QuEChERS when me hod sensi i i y is
pa amoun [26]. Use o hese echniques is eally only applicable o
B. G ee , O. Che allie , B. Quinn e al. T ends in Analy ical Chemis y 141 (2021) 116284
7
a ge ed me hods whe e he e ficacy o he clean-up echnique can
be assessed. This has limi a ions in ega ds o he numbe o ana-
ly es ha can be inco po a ed and can ge di ficul as he numbe o
analy es inc ease due o se e al ac o s, which include bu a e no
limi ed o: Di e ing physiochemical p ope ies, selec i i y/speci-
fici y o he clean-up echnique, ins abili y o some analy es in he
wo king s anda d solu ion, ionisa ion e ficiency, a ailabili y o
e e ence ma e ial and sui abili y o he clean-up pla o m chosen.
An example o some o he issues encoun e ed when employing
DnS o a mul i-class, mul i-analy e me hod was highligh ed in he
s udy conduc ed by Malacho a e al. (2014), wi h alida ion da a
acqui ed o 295 o he 331 a ge analy es due o some o he
a o emen ioned issues. One o he main issues was ha o signal
supp ession/enhancemen (SSE) due o ma ix e ec s and which
was obse ed o all ou ma ices s udied. G een peppe was
conside ed he mos complex ma ix o he ou , wi h only 10% o
analy es no su e ing om SSE [28]. In his example, samples we e
un in bo h posi i e (ESI
þ
) and nega i e ionisa ion modes (ESI
). A
ecen s udy by S eine e al. (2020) in es iga ed ma ix e ec s o
80 ungal me aboli es, 11 pes icides, and 9 pha maceu ical ac i e
agen s in complex eed using DnS and LC-MS/MS. Al hough he
majo i y su e ed signal supp ession, wi h pola analy es in ESI
þ
pa icula ly suscep ible o his, apola analy es in ESI
we e p one
o signal enhancemen , bo h caused by co-ex ac ed ma ix com-
pounds [8].
O he ma ices such as black ea (highly complex and d y) and
ocke (high wa e and chlo ophyll con en ) ha e shown o be
somewha p oblema ic in pes icide analysis (39) using dilu ion o a
sample ex ac [36]. Black ea and ocke indica ed ha only 4% and
7% o he analy es espec i ely we e ee o ma ix e ec s a a 10-
old dilu ion, whe eas he majo i y we e comple ely ee o ma-
ix e ec s a a 100- old dilu ion, indica ing ha a “dilu e-and-
shoo ”app oach may be success ul. Howe e , depending on he
concen a ion o analy e(s) p esen , his dilu ion ac o may p o e
oo much o analy e de ec ion. The e o e, a comp omise could be
sough , wi h a 25- old dilu ion p o ing su fice i ma ix e ec s we e
no g ea e han 20%. Ano he pes icide s udy in ui and ege ables
indica ed ha wi h no dilu ion, he ma ices a ied in he pe cen
o analy es showing no supp ession, defined as being <20%. Leek
p o ed he mos p oblema ic, wi h only 9% o pes icides no
sup essed and 37% o analy es highly sup essed (>50%). O e all, a
dilu ion ac o o 15- old was enough o elimina e ma ix e ec s,
pe mi ing quan ifica ion wi h sol en s anda ds o he majo i y
[37]. The e o e, ma ix e ec s and analy ical sensi i i y will be a
majo ac o in whe he DnS can be employed, as well as he
numbe and classes o analy es o be inco po a ed in he me hod. In
some ins ances, he complexi y o he sample ma ix can lead o
se e e ma ix e ec s and depending on he sensi i i y equi ed,
DnS may no be sui able and o he sample clean-up s a egies may
need o be employed, such as hose lis ed in sec ion 4.
Taking in o accoun complex ma ices such as black ea, ocke ,
leek and g een peppe , as well as o he ma ices s udied such as
u ine and oods u s, all o he a o emen ioned can lead o signal
supp ession o he analy es he ein, caused by co-elu ion o un-
wan ed ma ix componen s and he esul ing con amina ion o he
MS sou ce and ion op ics. Thus, al hough use o DnS can gi e
excellen esul s wi h li le o no sample p epa a ion, MS de ec o s
may well equi e egula en ing and cleaning o he sou ce com-
ponen s a e mul iple injec ions o main ain ins umen sensi i i y
leading o unwan ed ins umen down ime. The e o e, when using
DnS, he down ime is dependen on he MS sys em used and sou ce
design, wi h ABSCIEX QqQ ins umen s appea ing o be he in-
s umen o choice o a ge ed analysis using DnS as indica ed in
Table 1. O la e, he e has been imp o emen s in sou ce design wi h
a ious endo s implemen ing di e en designs o echnologies o
imp o e sensi i i y while also educing he necessi y o egula
main enance. Examples o his include di e en ial mobili y spec-
ome y (DMS) wi h SCIEX in oducing he SelexION DMS ech-
nology and he Agilen Je S eam Ion Sou ce wi h he addi ion o
VacShield. These echnologies se e o enhance he sys ems
obus ness wi h he bonus o spending less ime on main enance
and inc easing sample h oughpu .
4. Sample clean-up echniques o educe ma ix e ec s
4.1. Solid phase ex ac ion (SPE)
SPE is a use ul ool o educing ma ix e ec s ac oss a ious
complex ma ices such as oods u s, animal eed, aqueous samples
and o he biological ma ices, and usually a o ds low LOQs. How-
e e , a d awback in i s sui abili y o sample clean-up is he numbe
o analy es inco po a ed is usually limi ed due o he selec i i y o
he s a iona y phase, wi h se e al ac o s o conside when
choosing his such as: a ying physiochemical p ope ies o he
analy es and he loading, washing and elu ion s eps which can lead
o losses o incomple e eco e y o analy es. All o he a o emen-
ioned can lead o ime consuming and labo ious me hod de el-
opmen . Some examples in he use o SPE include he analysis o
myco oxins in pig se um, plasma and u ine and in he analysis o
eshwa e oxins in aquacul u e ponds and fish issues [38e41],
he analysis o myco oxins in baby ood, analysis o ne e agen s in
human u ine and he analysis o e e ina y d ug and pes icide
esidues in animal eed [12,42,43]. Fu he mo e, wo o mo e SPE
columns can be used in andem o analyse se e al compound
classes, o an analy e (ZEN) and i s me aboli es ac oss a ious
ma ices [44].
4.2. Mic o-ex ac ion echniques: so ben -based
Simila o SPE, mic o-ex ac ion echniques based on solid
phases o so ben s a e now commonplace o use in sample p ep-
a a ion and clean-up. These echniques include solid-phase
mic oex ac ion (SPME), s i -ba so p i e ex ac ion (SBSE) and
mic o-ex ac ion by packed so ben (MEPS) [45].
SPME is based on pa i ioning o analy es be ween a coa ed fib e
and sample, achie ed by placing he SPME fib e abo e a solu ion
(ma ix) o cap u e he ola ile apou . Howe e , SPME is mo e
ou inely employed alongside GC-MS analysing ola ile o ganic
compounds (VOCs) o ola ile me aboli es (VOMs). Use o di ec
imme sion SPME (DI-SPME) has acili a ed i s compa ibili y wi h
LC-MS in he analysis o non- ola iles [46]. One main d awback is
i s use o s a iona y phases coa ing he fib e [47], and along wi h
DI-SPME showing poo eco e ies in he analysis o pola mole-
cules, he capaci y o his echnique in he c ea ion o mul i-class,
mul i-analy e me hods o analysis by LC-MS is limi ed. Simila o
SPME, SBSE is sui ed o analysis o medium o apola molecules
ac oss a ious ma ices using GC-MS. When combined wi h a
so p i e phase, mul i- esidue me hods ha e been de eloped o he
analysis o endoc ine dis up o s (77) and pes icides (15) in wa e
samples using GC-MS and LC-MS espec i ely [48]. Analysis o pola
molecules by SBSE equi es de i i isa ion which is labo ious,
al hough new ma e ials could be de eloped in o de o di ec ly
analyse pola molecules by LC-MS, making hem compa able o SPE
ca idges [48]. Once again howe e , he limi a ions o SBSE a e
simila as hose men ioned o bo h SPME and SPE.
MEPS has been de eloped as a minia u ised e sion o SPE and is
ully compa ible wi h LC-MS, wi h he added benefi s o being ully
au oma ed, apid and educed sol en usage in compa ison o SPE.
Due o i s size, MEPS a e pe ec ly sui ed o small olumes o bio-
logical fluids such as plasma/se um, o al fluids and u ine [45]. Wi h
B. G ee , O. Che allie , B. Quinn e al. T ends in Analy ical Chemis y 141 (2021) 116284
8
hei abili y o be u ilised o small sample olumes and low con-
cen a ion o analy es he ein, hey concen a e he analy es o
in e es whils simul aneously emo ing unwan ed ma ix com-
pounds, gi ing ise o mo e sensi i e analy ical me hods. Howe e ,
as wi h he o he a o emen ioned echniques, he selec i i y o he
so ben limi s he numbe o analy es inco po a ed.
4.3. Immunoa fini y Columns (IACs)
IACs wo k on he basis ha he s a iona y phase con ains an
an ibody o cap u e he analy e(s) o in e es and can acili a e
analysis a low le els, gene ally a o ding low LOQs. Al hough IACs
a e e y selec i e, his has d awbacks such as lack o specifici y o
analy es o di e en classes o analogues, and hey can exhibi
some non-specific binding wi h o he p o eins. IACs a e mainly
used in he analysis o one o wo compounds due o he c oss
eac i i y p ofile o he an ibody, such as in he analysis o chlo -
amphenicol in ood o i amin B12 in g een algae [49,50]. IACs can
be u ilised in he analysis o se e al compounds such as in he s udy
o mic ocys ins (15) and he s uc u ally ela ed oxin nodula in,
wi h a compa ison be ween IAC and SPE indica ing a lowe me hod
de ec ion limi using IAC [51]. They ha e also been de eloped wi h
he capaci y o bind se e al myco oxins wi h a ying physico-
chemical p ope ies ac oss a ange o biological ma ices, such as
he 11þMyco MS-PREP IAC (R-Biopha m: h ps:// ood. -biopha m.
com/p oduc s/11myco-ms-p ep/). IACs can acili a e quan ifica ion
wi h sol en calib a ion cu es, al hough his is no always he case
as some eed ma ices ha e been shown o cause SSE e en wi h use
o IACs [52]. In o de o analyse a wide ange o analy es, IACs wi h
di e en specifici ies can be used in andem [53], and hey can also
be used in conjunc ion wi h SPE, wi h he main d i e in doing so o
inc ease analy e numbe [54,55]. Howe e , he selec i i y o hese
echniques do no lend hemsel es o c ea ion o mul i-analy e
me hods.
4.4. QuEChERs
QuEChERs was in oduced in 2003 and has apidly expanded
om use in pes icide analysis o applica ions in ood, pha maceu-
ical, en i onmen al and biological analysis. Due o i s success, wo
in e na ional s anda d o ganisa ions adop ed e sions o he o ig-
inal echnique o quan i a ion o pes icides in bo h ui and eg-
e ables; he Eu opean Commi ee o S anda disa ion (CEN) and he
Associa ion o O ficial Analy ical Chemis s (AOAC) In e na ional
[56]. The me hod combines liquid ex ac ion and pa i ioning using
sal s, ollowed by a clean-up s ep using dispe si e SPE (dSPE). As
well i s simplici y, i is ela i ely cheap in compa ison o o he
echniques as well as being apid.
QuEChERs has been used ex ensi ely in pes icide esidue anal-
ysis in ui and ege ables, wi h 451 pes icide esidues analysed
along wi h a Q-Exac i e O bi ap, pe mi ing bo h iden ifica ion
and quan i a ion [7]. Wo k has been conduc ed o e ol e he
me hod o a eas o he han pes icide analysis in ag icul u al com-
modi ies, wi h hese discussed in wo comp ehensi e e iews
looking a i s e olu ion, applica ions and ends [56,57]. Su fice o
say, i is now used in he analysis o myco oxins, an ibio ics, e -
e ina y d ugs and he bicides ac oss a a ie y o ma ices. One such
example is he analysis o 630 mul i-class ood con aminan s using
UHPLC-QToF including, e e ina y d ugs, ood-packaging con am-
inan s and myco oxins, al hough he majo i y we e pes icides (426)
[58]. Al hough a e y e ec i e and e ol ing echnique, i like o he
clean-up echniques has disad an ages due o incomple e o low
eco e ies o he sui e o analy es due o he choice o dSPE chosen,
as well as being mainly pes icide-cen ic.
4.5. Liquid-liquid ex ac ion (LLE)
Used as an ex ac ion echnique o some liquid ma ices, LLE is
conside ed o be a sample clean-up s ep. Apa om acili a ing
p o ein p ecipi a ion in some ma ices, he pa i ioning sepa a es
compounds based on hei solubili y in wo di e en immiscible
liquids, such as wa e and an o ganic sol en , wi h analy es ypi-
cally ex ac ed in he o ganic laye lea ing ma ix componen s in
he o he , wi h some ma ix co-ex ac ed in he o ganic laye and
ice e sa. As men ioned p e iously, he choice o ex ac ion sol en
dic a es he pola i y o he analy es ex ac ed and he e o e p e-
sen ed o analysis. This is pa icula ly impo an o un a ge ed
quali a i e analysis o me abolomic s udies using LC-HRMS. In
gene al, mos o hese ex ac ions a e conduc ed on biological
ma ices wi h examples gi en below.
4.5.1. LLE applied o blood samples (plasma o se um)
This in ol es addi ion o an o ganic sol en o bo h dep o ei-
nisa ion and mig a ion o analy es om ma ix o he sol en ,
which may be acidified. This echnique has limi ed ad an ages due
o only pa ial emo al o ma ix componen s which a e espon-
sible o signal supp ession [21]. Fu he mo e, in mos ins ances
ins ead o dilu ion, he sample is d ied and econs i u ed o
concen a e he analy es o in e es , wi h he ca ea ha i also
concen a es any ma ix p esen oo. This echnique, e e ed o as
‘dilu e, e apo a e and shoo ’has benefi s simila o DnS whe e he
same ex ac ion p ocedu e can be employed o a ge ed and
un a ge ed analysis due o minimal o no analy e losses. Examples
include he s udies ca ied ou by Ca euw e al. (2019), De eese
e al. (2012) and Lauwe s e al. (2019) on pig plasma [21,22,59] and
o he s conduc ed in human plasma [60,61].
4.5.2. LLE applied o u ine samples
Unlike blood, u ine can simply be dilu ed o analysis as i does
no usually equi e dep o einisa ion. In some ins ances, he addi-
ion o an o ganic sol en is needed o acili a e mig a ion o he
analy es om he pola , aqueous u ine phase in o he o ganic
phase, whils emo ing some ma ix, be o e d ying and econ-
s i u ing he o ganic laye . Examples o his include he analysis o
myco oxins whe e he u ine was ex ac ed wi h ace oni ile/wa e /
o mic acid (52/45/3, / / ) o e hyl ace a e in human and pig u ine,
espec i ely [23,62]. In bo h cases, use o bo h a ge ed and
un a ge ed me hods can be employed, howe e , some cau ion has
o be obse ed o he la e ex ac ion i analysing by HRMS o
me aboli es o po en ial bioma ke s as some analy es had low e-
co e ies in e hyl ace a e a a ying pH le els.
4.6. Dilu e-and-shoo
In compa ison o he a o emen ioned echniques ba LLE, DnS is
a gene ic analy ical me hod wi h i s ad an ages and disad an ages
ou lined in Table 2. The e olu ion o DnS has acili a ed he c ea ion
o mul i-analy e me hods which we e p e iously limi ed due o he
di e ing physiochemical p ope ies o he analy es in ques ion and
he sample clean-up echnique chosen. One can no e ha wi h he
e olu ion o DnS o include oods u s, he ini ial solid-liquid
ex ac ion can lead o SSE due o co-ex ac ion o ma ix, as well
as low eco e ies o some o he a ge analy es based on he sol-
en chosen and possible chemiso p ion. Howe e , due o he
absence o s a iona y phase and chemis y he ein, he echnique is
conside ed no selec i e and pe mi s he c ea ion o mul i-class,
mul i-analy e me hods. Al hough in compa ison echniques such
as SPE, mic oex ac ion (SPME, SBSE and MEPS) and IAC can
gene ally achie e lowe de ec ion limi s, hey a e hampe ed by
hei espec i e selec i i ies and specifici ies, limi ing he numbe
B. G ee , O. Che allie , B. Quinn e al. T ends in Analy ical Chemis y 141 (2021) 116284
9
