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B. Le Bizec et al. / J. Chromatogr. A 1216 (2009) 80168034 8017
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8017
2. Mass spectrometric approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8018
2.1. Single MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8018
2.1.1. Basic introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8018
2.1.2. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8018
2.1.3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8023
2.2. Multidimensional MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8023
2.2.1. Basic introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 0232.2.2. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8023
2.2.3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8027
2.3. High-resolution MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8027
2.3.1. Basic introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 027
2.3.2. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8027
2.3.3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8029
2.4. Isotope ratio MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 029
2.4.1. Basic introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 029
2.4.2. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8029
2.4.3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8030
3. Pitfalls and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8030
3.1. Issues related to the application of the 2002/657/EC decision. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8030
3.2. Chromatographic separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8031
3.3. Ionization (matrix effect) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8031
3.4. Ion characterization (resolution, mass accuracy, speed scanning, multiresidue analysis, cross talk). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8032
3.5. Software (acquisition, data analysis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80334. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8033
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8033
1. Introduction
Drug residues regulation in animal-derived food is an inte-
gral component of food safety programmes worldwide. Analytical
methods used to monitor veterinary drugs in feed and food are
essential to help protect human and animal health, monitor con-
sumer exposure to the drugs, reduce the impact of chemicals on
the environment, support the enforcement of laws and regula-
tions and facilitate international trade of animal food products.
Most veterinary drugs are not of acute toxicological concern. Somesubstances, suchas diethylstilbestrol,nitrofuransor chlorampheni-
col, have been banned in most developed countries due to their
demonstrated carcinogenicity. Increased cases of allergic reactions
toantibiotics,as wellas thegrowingcurrent concernforpathogenic
microorganisms becoming antibiotic-resistant, are also important
reasons for setting maximum residue limits in food. Endocrine
disruption properties of residues in food have become another
major issue justifying the regulation of certain veterinary drugs.
The occurrence of unwanted residues in edible productscan be the
result of illegal use, in the cases of prohibited medicines, or of fail-
ureto respect theproper withdrawal timesbefore butchering in the
case of authorized medicines.
The European Union (EU) has strictly regulated controls on the
useof veterinary drugs, including growthpromoters, particularlyinfoodanimalspecies,by publishing differentRegulationsand Direc-
tives. The use of veterinary drugs is regulated through EU Council
Regulation (2377/90/EC) [1], which describes the procedure for
establishing MRLs for veterinary medicinal products in foodstuffs
of animal origin. Four annexes cover substances with MRL values
(I), substances for which it is not considered necessary to estab-
lish MRL values (II), substances with provisional MRL values (III)
and substances for which no MRL values could be established so
that the corresponding compounds are prohibited (IV). The prohi-
bition of the use of growth promoters is laid down in two Council
Directives (96/22/EC, 96/23/EC) [2,3] that contain guidelines for
controlling veterinary drug residues in animals and their products
with all the necessary information to set up national monitoring
plans. For any type of animal or food, there are two main groups
of substances that must be monitored: Group A comprises pro-
hibited substances in conformity with Directive 96/22 and Annex
IV of Regulation 2377/90; Groups B1 and B2 comprise all regis-
tered veterinary drugs in conformity with Annexes I and III of the
2377/90 regulation and Group B3 comprises contaminants of the
environment, such as phytosanitary products, heavy metals, dyes
or mycotoxins.
Incontrast tootherareas of food control or towhat is enforcedin
most non-EUcountries, in the EU thereis no obligation to use stan-
dardizedmethodsin theresiduecontrolof food-producinganimals.Instead, a criteria approach applies, which lays down performance
characteristics, limits and criteria that have to be met by the meth-
odsused.A significant advantage of this approachis the high degree
of flexibility. It allows the ready adaptation of analytical methods
to technical developmentsand offers thepossibility to react rapidly
to newly emerging problems. Recent examples are the presence of
chloramphenicol in shrimps or honey and medroxyprogesterone
acetate in feed. Technical guidelines related to the analytical per-
formance criteria (e.g., detection level, selectivity and specificity)
and validation procedures of methods used for residue control in
the framework of Directive 96/23/EC are described in a dedicated
CommissionDecision(2002/657/EC) [4]. Nextto thegeneralperfor-
mance lines, additional andnew requirementsare described in this
referencedocument, especially forconfirmatory methods,by intro-ducing the concept of identification points (IPs) and unambiguous
identification criteria (maximal variability authorized for ion ratio
intensities). During confirmatory analyses, a specific number of IPs
hastobe collected. For confirmationof the identity of GroupA sub-
stances, a minimum of four IPs is required. For confirmation of the
identity of Group B substances, a minimum of three IPs is required.
Besides the traditional biological measurement approaches
basedon RIA,ELISA or biosensors, which providerapid diagnostic of
compliantversussuspect samples (i.e., screening stage), massspec-
trometry (MS) is today frequently used as confirmatory technique.
Indeed, MS combined either with gas or liquid chromatography
(LC) is a clear and valuable tool of choice to identify and quan-
tify residues in feed and food [58]. Nowadays, analyticalstrategies
based on LCMS supplant those relying upon GCMS, even if they
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B. Le Bizec et al. / J. Chromatogr. A 1216 (2009) 80168034 8019
are sometimes found in the literature for the isolation of this
group of compounds. Stilben conjugated phase II metabolites are
usually hydrolyzed either by enzymatic (glucuronidase) or chem-
ical approaches (solvolysis or methanolysis), especially when the
monitoring is conducted in urine, liver or kidney. This operation
is indeed not required for muscle, hair or faeces. When GCMS
is used, stilbens are quite always derivatized, the most popular
approachbeing silylation, and acylation (acetylation, perfluoroacy-
lation). N-methyl-N-(trimethylsilyl)-trifluoro-acetamide (MSTFA)
is often used for this purpose. When LCMS is exploited, deriva-
tization such as fluoroacylation, even not mandatory, is sometimes
used to enhance the signal and/or improve the specificity. In the
1990s, mass analyzers were mainly single quadrupoles and first
generation ion trap devices with an internal ionization source.
Single MS is nowadays almost systematically replaced by multi-
dimensional approaches, either by triple quadrupole (QqQ) and
more recent generations of ion trap (external sources) either 3D
or 2D (LIT). Few applications on high-resolution mass spectrom-
eters either double sector or time-of-flight instruments are also
reported in the literature, probably because stilbens are not char-
acterized by a remarkable mass defect/excess so that the increase
in resolution does not impactdirectly onto the S/N ratio. One of the
major drawbacks of stilben analysis in GCMS is probably the diffi-
culty to obtain sufficient diagnostic signals especially for hexestrol.Indeed,theTMS-derivativeof thiscompound completelyfragments
when ionized in EI. The major ion is observed at m/z207, which is
a very noisy diagnostic signal as far as the stationary phase is a
methylpolysiloxane derivative. Two strategies are followed in the
confirmatory process, the first being the TBDMSderivatization, the
second one consisting in the modification of the ionization process
or the electron energy. Fig. 1 clearly shows the modification of the
mass spectrum of hexestrol depending on the preferred strategy. A
specificmassaccuracy maybe obtainedfor these target compounds
throughthe introductionof a specific chemical modification so that
the increase of the resolution either on TOF, FTMS or BE instru-
ments hasthe directconsequence of an efficient mass clean-up. For
example, the introduction of several halogen atoms on the target
analytes during the derivatization step was proven to engender asufficientmassdefect, so thatmatrix interferencesare notdetected
at a higher resolution [10,11].
2.1.2.2. Chloramphenicol (GCMS, ITD, NCI, SIM). Chloramphenicol
(CAP) has been widely adopted as an effective broad spectrum
antibiotic to treat many kinds of animal diseases. Because of some
toxicity evidences that have been extensively demonstrated in
humans, chloramphenicol has been prohibited for use in food-
producing animals and the maximum residue limit has been
established at a zero tolerance level in edible tissues in many
countries. For screening purposes, capillary electrophoresis, micel-
lar electrokinetic chromatography and surface plasmon resonance
biosensor assays have been used for the multiresidue analysis of
fenicolsin differentmatrices. Liquidchromatography coupledtoUV
detector as well as GCECD have also been used for the determina-
tion of CAP in bovine, swine, poultry muscle, fish, milk, and shrimp
tissues. However, methods using chromatography coupled to mass
spectrometry remain the current standards to confirm unambigu-
ously the presence of the target analytes in suspect samples. If
GCMS methods based on electron ionization (EI) have historically
been used for this purpose [12], the resulting sensitivity some-
times remains insufficient. Negative chemical ionization (NCI) is
more commonly used because particularly well adapted for these
halogenated substances which exhibit intense electronic capture
properties [1316]. Purified extracts are usually derivatized using
silylating agents prior GC separation. In this case, the trimethylsi-
lyl (TMS) derivative of CAP leads to 4 diagnostic ions (m/z466/468and m/z376/378) which allow complying with the identification
criteria (Council Decision 2002/657/EC) fixed at the European level
(Fig. 2). The detection limits achieved with this approach are typi-
cally in the sub 0.1g kg1 (ppb) range in biological tissues. Eitherquadrupole or ion trap mass analyzers operating in single ion
monitoring (SIM) acquisition mode can be used for this purpose,
usually with methane as reagent gas. Thesame strategycan be suc-
cessfully applied for measuring other related compounds such as
thiamphenicol (TAP) or florfenicol (FF).
2.1.2.3. -agonistic drugs (GCMS, Q, PCI, full scan, reagent gas).-agonist compounds, the so-called -sympathomimetics, arecharacterized by structural and pharmacological properties which
arevery close to those of catecholamines. Thecontrol of-agonistsmisuse received extra attention after several food poisoning cases
in 1990 in Spain caused by consumption of bovine liver, and later
Fig. 1. Different mass spectra obtained for hexestrol depending on the ionization technique used (top left corner EI 70 eV, top right corner PCICH4, bottom left corner
PCINH3, bottom right corner NCINH3).
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Fig. 2. Typical GCNCIMS mass spectrum obtained for chloramphenicol TMS derivative.
on in China (20082009). This was thefirst time that pharmacolog-
ical residues found in slaughtered cattle were found to have caused
acute intoxicationinconsumers. Inmuscle tissue,-agonisticdrugspromote lipolysis. This may resultin a reductionof up to 40% of car-
cass fat (lipolysis) and an increase of up to 40% of carcass protein
(byincreasingprotein synthesisand reducingproteolysisin striated
muscle fibres). Because of their ability to shift nutrients towards
protein instead of lipid anabolism, such molecules were gatheredunder the generic name of repartitioning agents. While the ther-
apeutic treatment of cattle with respiratory diseases is permitted,
the use of-agonists as growth promoters in cattle is forbiddenin the EU. Regarding sample preparation, solid phase extraction
(SPE) is among the first choice for multiresidue -agonist extrac-tion, preferably with mixed phase sorbents such as C8 and cation
exchange stationary phase. Alternatively, immuno-affinity clean-
up (IAC) can be used forefficient purification of theextracts. In this
last case, different antibodies should be coupled to the column so
that the system may capture the widest range of-agonist com-pounds taking into account their difference in terms of chemical
structure, i.e.,from clenbuterolto ractopamine, or fromisoxsuprine
to zilpaterol. Care should be taken for reutilization of IAC one
sample to another to prevent any carry over. Molecular imprintedpolymer (MIP) is a more recent alternative to IAC. MIPSPE is very
selective, butthe production of a constantqualitymaterial is some-
times reported as questionable as is the reproducible extraction of
the analyte from the cartridge, especially when biological matrices
are used.
Nowregarding the measurement techniques, radio and enzyme
immunoassay screening tools were developed in the past and are
still being used in many countries for the control of-agonists.More recent biological tests based on surface plasmon resonance
(SPR), optical biosensor or competition binding assay have been
also proposed for screening -agonists. However, the sensitivityof these tests was sometimes rather limited to comply with the
requirement of low residue levels as found in urine andtissue sam-
ples. Consequently, several methods based on MS instrumentationhave beenset up,amongwhich GCMS andLCMS(often reinforced
by MS/MS or HRMS configurations) have been recognized today
as the most powerful approaches for measuring -agonist com-pounds [1618]. ConcerningGCMS techniques, trimethylsilylation
or tert-butyldimethylsilylation are commonly used derivatization
methods. But extra strategies based on different derivatives and/or
ionization modes have also been set up. For instance, cyclization of
the side chaineither with boroximeor DMCS reagents clearly stabi-
lizesthe compound and provides intense highm/zionseven inEI at
70eV. However, this approachis disappointingly only applicable to
clenbuterol-likecompounds. An alternativeapproach tothederiva-
tizationis theuse of milder ionizationconditions.Positive chemical
ionization (PCI) is indeed an interesting option providing a wide
panel of different mass spectra by varying the natureof thereagent
gas in the ionization source. Fig. 3 illustrates the different mass
spectra for mabuterol when EI, PCI (CH4), PCI (iBu), PCI (NH3) are
operatedrespectively. In the EI mass spectrum, the molecular ionis
missing andthe only valuable ion is the m/z86 characteristic of the
side chain moiety of the compound. According to the same prin-
ciple, detection at sub-ppb levels of most -agonists is achievableon the basis of one single ion, but does not authorize the confident
identificationof theseresidues according to official criteria.Clearly,chemical ionizationconducted in the positive mode provides more
characteristic information especially when the proton affinity in-
between -agonists and reagent gas decreases (from methane toammonia). The energy transfer then drops and the fragmentation
is drastically reduced. The PCIisobutane mass spectrum is proba-
bly the one providing the best combination in-between specificity
(number of high m/zion)andsensitivity (reduced fragmentation so
that the percentage of a given ion is standing for a substantial per-
centage of the total ionic current). The PCINH3 mass spectrum of
mabuterol is almost condensed to the quasimolecular ion (M+H)+,
and hasto be considered as highly attractive on MS/MS instrument
when isolated as precursor ion (all the TIC is concentrated into on
highmass ion)and fragmented inthecollisioncell togenerate prod-
uct ions. If GCMS instruments were historically the more widelyused for various classes of residues, LCMS appears today as the
method of choice andthe majoractual investmentfor many labora-
tories, especially for theanalysisof polarcompounds. Undoubtedly,
reversedphase LC and positive ESI is themethod of choice for most
-agonistic drugs nowadays.
2.1.2.4. Antibiotics (GCMS or LCMS, Q, EICI or ESI, full scan or
SIM). Bioassay techniques which are widely used to screen for
antibiotics in food and tissues do not generally allow a distinc-
tionbetweenmembers of classes of antibiotics, thereforeproviding
a semi-quantitative estimate of total residues level. Suspect sam-
ples consequently need to be analyzed by sufficiently selective and
sensitive confirmatory MS-based methods. Even if the latest MS
instruments present in laboratories allow for MSn
detection, sin-gle MS can still be considered as an efficient acquisition mode
for antibiotics [5]. Few application methods are reported based
on GCMS, mainly intended for chloramphenicol analysis after CI
or EI ionization [19], while in general, the use of RPLCESIMS
is very efficient for several classes of antibiotics. In this con-
text several applications are reported for some antibiotic peptides
(avoparcin, bacitracin, . . .) [20], tetracylines [21], chloramphenicol[22], sulphonamides [2326], -lactams for which sensitive ion-ization is generally achieved by ESI(), however, this ionization
modeis notsuitable fortheamphotericones suchas amoxicillinand
ampicillin forwhich ESI(+) is preferred[27]. Macrolidesarealsoeffi-
ciently analyzed by ESI(+)MS[26,27] on a single quadrupole mass
spectrometer. The mass spectra of some macrolides (spiramycin
(SPI), tilmicosin (TILM), oleandomycin (OLE), erythromycin (ERY),
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B. Le Bizec et al. / J. Chromatogr. A 1216 (2009) 80168034 8021
Fig. 3. Different mass spectra obtained for mabuterol depending on the different ionization modes used (from top to bottom, EI, PCICH 4, PCIiBu, PCINH3). Clearly the
energy involved is decreasing from CH4 to NH3 , whereas the relative abundance of the quasimolecular ion increased.
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8022 B. Le Bizec et al. / J. Chromatogr. A 1216 (2009) 80168034
Fig.4. Typical massspectra obtained formacrolidescompoundsafterESI+ ionization(conevoltage55 V) [28]. SPI:spiramycin,TILM: tilmicosin, OLE:oleandomycinphosphate,
ERY: erythromycin, TYL: tylosin tartrate, KIT: kitasamycin, ROX: roxithromycin, JOS: josamycin.
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B. Le Bizec et al. / J. Chromatogr. A 1216 (2009) 80168034 8023
tylosin (TYL), kitasamycin (KIT) and josamycin (JOS)) obtained in
the full scan mode at the selected conditions (Fig. 4), showed
that the protonated molecular ion [M+H]+ is the predominant ion,
except for SPI and OLE. The base peak for SPI was [M+2H]2+ (m/z
422),whereasthe predominant ionfor OLE was [MC7H13O3+2H]+
(m/z544), which corresponds to the loss of sugar moieties from
[M+H]+ [28]. Sensitive measurements are generally carried out in
SIM acquisition mode focusing on the pseudo-molecular [M+H]+
ion for each macrolide. In this case, the most abundant ion is used
for quantification and the second and third ones are used for con-
firmatory purpose, except in the case of josamycin for which only
two ions are significantly observed in the mass spectra (Fig. 4).
The expected quantification limit of macrolides compounds with
this approach is around 25g kg1 of dry weight of animal muscle(Fig. 5), which is compliant with established MRLs [27].
2.1.3. Conclusion
Single mass spectrometry approaches in the low-resolution
mode are currently almost neglected for trace residue analy-
sis. The release of official identification criteria by means of the
2002/657/EC decision combined with the continuous technology
innovationin thefield ionization interface, iontransmission,mass
analyzer and ion detection gently move forward LRMS. Ion trapdetectors andabove all triple quadrupoles cometo substitute single
quadrupolesin thisexercise,providingincomparable spectrometric
signalswithspecific andsensitive ionchromatogramswith tremen-
dous S/N on at least two diagnostic traces even at the low pg level
loaded onto the system.
2.2. Multidimensional MS
2.2.1. Basic introduction
Tandem (MS2) or multidimensional (MSn) mass spectrometry
techniques today present incomparable advantages in the field
of residue analysis at trace levels in complex biological matrices.
Indeed, the fragmentation of the target compounds for detecting
only specific product ions rather than the entire molecule per-
mits to considerably increase the signal to noise ratio of the target
diagnostic signal by decreasing to a major extent the interfer-
ences due to other compounds present in the final extract with
the same or very close molecular weight as the analyte of inter-
est. Various mass analyzers offering these capabilities are already
used routinely in combination with gas or liquid chromatography,
among whichtriple quadrupoles(QqQs) andion traps(ITDs), are in
common use. More recent technologies are linear ion trap (LITs),
orbital trap (OrbitrapTM) and new-generation of hybrid instru-
ments, e.g., quadrupole time-of-flight (QqTOF), quadrupolelinear
trap (QqLITs) or linearorbital traps (LTQOrbitrapTM), which are
gaining widespread acceptance in several application areas. All
these recent instruments offer advantages such as high scanning
speeds, accurate mass measurement (QqTOF, LTQOrbitrapTM) and
increased sensitivity (LITs and new-generation of QqQs). The appli-
cationrange of multidimensional MS is today extremelywide,both
in terms of target compounds and in terms of possible different
acquisition modes. This last capability authorises not only very
sensitive and specific quantitative target measurements, but also
powerful untargeted fishing approaches based on the detection
of typical product/precursor ions or neutral species belonging to aclass of substances.
2.2.2. Applications
2.2.2.1. Corticosteroids (LCMS/MS, QqQ, ESI, SRM, precursor scan).
Natural corticosteroids (i.e., cortisol, cortisone) are hormones
secreted by the adrenal cortex. Their anti-inflammatory proper-
ties have led to the chemical synthesis of more active artificial
corticosteroids used in many veterinary therapeutic drugs. At the
same time, these compounds increase weight gain (water and fat
retention), reduce feed conversion ratio, and have a synergetic
Fig. 5. Diagnostic SIM chromatograms obtained for different macrolides in a meat sample extract spiked at 100g kg1
[29].
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Fig.6. Specific extractedchromatograms of dexamethasone obtained for a liver sample extract spiked at 0.1 ngg1 acquiredin LC(ESI)MS/MS [MRM](A) or LC(ESI)MS
[neutral loss 90] (B) modes. (Waters UPLC-XEVOTM TQ MS instrument).
effect when combined with other molecules like -agonists oranabolic steroids. Thus, corticosteroids have been illegally used
as growth promoters in cattle, administered through livestockfood or by injection. From a regulatory point of view, some of
themareauthorized for therapeutic treatments (with a withdrawal
period between treatment and slaughtering and established max-
imal residue levels in milk and muscle), but their use as growth
promoters has never been allowed. Many authors have proposed
methods based on gas chromatographymass spectrometry for
the detection of corticosteroids, including chemical oxidation or
derivatization. Although providing good sensitivity, these methods
modify the chemical structure of the molecule, reducing infor-
mation and specificity. Today, liquid chromatography coupled to
multidimensional mass spectrometry (LCMSn) represents a pow-
erful alternative for this class of compounds, combining speed,
specificity and sensitivity, especially when associated to the neg-
ative ionization mode [3037]. Indeed, the main ionic speciesusually observed in negative ESI or APCI is the pseudo-molecular
ion [MH] or a [M+acetate] or [M+formate] adduct if traces of
acetic or formic acid are added in the mobile phase, respectively.
This last ion usually appears very intense and its fragmenta-
tion leads to the [MCH2OH] product ion which corresponds
to a cleavage of the C17 side chain characteristic of this class
of compounds (loss of formaldehyde). This fragmentation path-
way appears extremely efficient for the measurement of a large
number of corticosteroids at trace residue level (i.e., in the sub
0.1g kg1 range) in biological matrices. Triple quadrupole orion trap instruments operating in multiple or selected reaction
monitoring (SRM/MRM) acquisition modes are usually used for
this purpose. But this particular mass spectrometric behaviour of
corticosteroid is also particularly adapted to use alternative acqui-sition modes offered by triple quadrupole devices, such as neutral
loss scan [36,38]. In this case, the loss of 60 or 90 mass units,
which correspond to the fragmentations [M+acetate] > [MH]
or [M+acetate] > [MCH2OH], respectively, represents an effi-
cient strategy to screen potential corticosteroids with unknown
or non-targeted structures. Of course, this strategy appears really
efficient only with the latest generation of instruments reaching
a good sensitivity even in scan mode (Fig. 6). Similarly, precursor
ion scan may also be used fordirect measurement of corticosteroid
phase II metabolites by focusing on fragment ions characteristic of
glucuronides and/or sulphate forms [39].
2.2.2.2. Nitrofurans (LCMS/MS, QqQ, ESI+, SRM). A number of
methods are currently available for the analysis of nitrofurans
in a variety of matrices. Nitrofurans are characterized by their
rapid metabolism within a few hours after administration, leading
to protein-bound metabolites which may persist for a consider-able period of time in edible animal tissues. Therefore, detection
methods for nitrofurans should focus on metabolites of the
parent drugs such as 3-amino-2-oxazolidinone (AOZ), 3-amino-
morpholinomethyl-2-axozolidinone (AMOZ),semicarbazide(SEM)
and 1-aminohydantoin (AHD), corresponding respectively to
the metabolites of furazolidone, furaltadone, nitrofurazone and
nitrofurantoin. Most methods described in the literature are
based on the release of protein-bound nitrofuran metabo-
lites under acidic conditions followed by derivatization with
2-nitrobenzylaldehyde (2-NBA) and determination by liquid
chromatography coupled to mass or tandemmass spectrome-
try [4042]. RPLCESI(+)QqQ is in this context the preferred
technique to confirm the identity of nitrofuran metabolites
which are monitored using the SRM transitions correspond-ing to the fragmentations of the [M+H]+ ion of the derivatized
metabolite (Fig. 7): NPSEM: 209.1 > 165.9, 209.1 > 191.9; NPAOZ:
236.0> 133.6, 236.0> 130.6; NPAMOZ: 335.1> 291.1, 335.1 > 262.0;
NPAHD: 249.0> 133.6, 249.0 > 130.6 [5,4245]. Labelled internal
standards such as d4AOZ, d5AMOZ,13C3AHD or
13C, 15N2SEM,
are available and have been recently introduced in the methods to
overcomeproblemssuch asmatrixsuppressionduringelectrospray
ionization [43,45].
2.2.2.3. Malachite green (LCMS/MS, QqQ, ESI+, SRM). Confirmatory
methodsdevelopedfor malachite green (MG)and its mainmetabo-
lite leuco-malachite green (LMG) in fish tissues originally involved
in the mid 1990s GCMS detection methods. Selected ion monitor-
ing was then performed based on 5 diagnostic ions (m/z330, 329,253, 210 and 165 in the case of LMG) [46]. Morerecently, the devel-
opment of LCMS coupling combined with instruments allowing
specific acquisition through tandem mass spectrometry has led
to efficient detection methods which are now widely reported in
the field. Chromatographic separation of MG and LMG is gener-
ally performed on phenyl phases using either a gradient of acidic
acetonitrile (0.1% FA)/water or an isocratic mixture of ACN/acetate
buffer(70/30,v/v) as mobilephases [47,48]. C18 phases with 50mM
ammonium acetate/ACN or acidic water/ACN as eluents have also
been reported [4951]. Atmospheric pressure chemical ionization
has shown tobe very efficientfor MG ionization which is recovered
as [M]+ with a molecular ion at m/z329 [52]. Electrospray ioniza-
tion also allows monitoring MG as [M]+ while LMG is recovered
as [M+H]+
. The use of ion trap (3D or linear) as mass analyzer is
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Fig.8. Typical SRMdiagnostic ionchromatogramsobtainedfor malchitegreen(MG),
d5MG, leuco-malachite green (LMG) and13C6-LMG in a fish sample extract (cor-
responding to 2g kg1 MG and 7g kg1 LMG) monitored on a LCESI+QTrapinstrument. [55].
including-zearalanol (-ZAL),-zearalenol (-ZEL),-zearalenol(-ZEL), and zearalanone (ZAN), are referred to as resorcylic acidlactones (RALs). As a consequence of their occurrence in the food
chain, considerable attention has been paid to their potential risk
for human health. Zeranol has been widely used as a growth pro-
moter in the United States since 1969 to improve fattening rates
of cattle. Its application has been banned in the European Union
(EU) since 1985 (Group A of Council Directive 96/23/EC). Methods
for urine and tissue have been published [5459]. Several meth-
odsusinggas chromatographywith massspectrometry(GCMS) or
tandem mass spectrometry (GCMS/MS) for the analysis of resor-
cylic acid lactones in biological samples have been reported but
requiredchemicalderivatization[60]. Liquid chromatography com-
bined with tandem mass spectrometry (LCMS/MS) has proven
to be the ad hoc technique for the determination of the whole
range of resorcylic acid lactones. Triple quadrupole instruments
with electrospray (ESI) or atmospheric pressure chemical ioniza-
tion (APCI) interfaces either in the negative or the positive mode
are applied. Zeranol and zearalenone are known to give identical
metabolites which explains why these metabolites, including zer-
anol itself, can also occur naturally in ovine urine and bovine bile
after metabolism of zearalenone. To differentiate an illegal use of
zeranolfrom consumptionof foodcontaminated withFusariumspp.
Toxin, an exhaustive monitoring of main RALs is necessary. The in
vivo metabolism of zearalenone and zeranol has been investigated
in several animal species and in humans. It has been shown that
the anabolic agent zeranol is predominantly metabolized into its
diastereoisomer-zearalanol (taleranol) and to a minor extent into
zearalanone. The mycotoxin zearalenone is preferentially trans-formed into - and -zearalenol [61]. The objective of the NaturalZeranolproject FAIR5-CT-1997-3443 wasto study and to establish
criteria for discriminating between illegal treatment with zeranol
containing preparations and the presence ofFusarium spp. toxins.
A statistical model wasdevelopedafter screening and confirmation
Fig. 9. Compared HPLC separation of zeranol metabolites and precursors (mycotoxin origin). On the left, a C18 (X) column 50 mm2 mm, 3m has been used; on the right,a Hypersil Gold C18 column 100 mm2.1mm, 1.9m shows its ability to separate the complex mixture of resorcylic acid lactones. Positive ESI has been used to ionize theanalytes, andSRM acquisitionon a QqQwas found themost adapted toreachthesub-ng mL1 in urine, and to fulfilidentificationcriteriaas fixedby the 2002/657/EC decision.
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B. Le Bizec et al. / J. Chromatogr. A 1216 (2009) 80168034 8027
of 8008samplescollectedfrom differentpartsof Europe. Themodel
developed is a tool based on comparing the sum of the zeranol and
taleranol concentrations with the sum of zearalenone and its two
major metabolites, namely - and -zearalenol. Validated quanti-tativemethodsarenecessaryfor practical applications.An example
of a LCMS/MS characterization is proposed in Fig. 9, pointing out
that crucial importance has to be paid to the stationary phase for
efficientseparation betweenmetabolites(close chemical structure)
aswellas withothersecondary metabolites andinterferences. Elec-
trospray ionization can be successfully applied for this class of
compounds both in the positive or negative mode. SRM acquisi-
tionmode providesthe adhocspecificity (at least fouridentification
points) and guarantees the necessarysensitivity (CC150,000) and excellent mass accuracy (specified as 25 ppm, but
demonstrated to be as low as 0.2ppm under favourableconditions)
significantly reduces false positive identification.
2.3.2. Applications
2.3.2.1. Boldenone (LCHRMS, Orbitrap, ESI). The applicability of
the OrbitrapTM technology to the measurement of steroid-related
compounds in general, and conjugated phase II metabolites of
boldenone in particular, in complex biological matrices has already
been demonstrated [62,63]. Boldenone (androsta-1,4-dien-17-ol-3-one) is an anabolic steroid synthesized by dehydrogenation
of testosterone. Its use as growth promoter for cattle fattening
is banned within the EU. Until 1996, the identification of either
17- or 17-boldenone in urine was considered exclusively as theresult of an exogenous administration of boldenone or analogues.
Nevertheless, some observations reported by official laboratories
underlying the almost systematic presence of 17-boldenone inurine with values in the low g L1 range [64], have led to con-
sider a possible endogenous production/excretion of boldenone inbovine/ovine. Then a non-unambiguous piece of evidence became
necessary to allow the competent authorities to take appropri-
ate action. A screening criterion based on concentration level of
17-boldenone levels (>2g L1) in urine has been set up. Forconfirmatory needs, the presence of 17-boldenone conjugates(glucuronide or sulphate metabolite) in urine has been recognized
as a definite indicator to differentiate treated from untreated ani-
mals. LCMS approaches became necessary to point out these
hydrophilic residues, based eitheron MS/MS or HRMS signal acqui-
sition after electrospray ionization operated in the negative mode.
For high-resolution acquisition on the OrbitrapTM system (Fig. 10),
FTMS resolution was setat 30,000(FWHMat 400m/z). Mass spec-
tra were recorded from m/z 100 to 400. The method validation
focused on blankcalf urinesamples was runto assessthe specificity
Fig. 10. Ion chromatograms (ESI, LCHRMS, R 30,000 FWHM) for 17-boldenone sulphate in (a) a blank urine sample, (b) a spike urine at 1g L1 and (c) a urine sample
collected 4 h after administration of boldenone to a calf. [62].
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of the method, which was concluded satisfactory since no inter-
ferences were eluted at the retention time (extended both sides
to 5 times the peak width at half height of 17-boldenone sul-phate, 11.60.5min). A linear regression-based approachprovided
estimated values of decision limit (CC) and detection capability(CC) equal to 0.2 and 0.4g L1, respectively. Response linearitywas found adapted to the needs (R2 = 0.9925). Identification relies
uponthe monitoringof twoions(precursorsand products) acquired
in high-resolution mode (R = 30,000 FWHM), which provided four
identification points according to EU Decision 2002/657/EC. Com-
parable performances have been observed on a last generation but
moreconventional triple quadrupole instrument (QqQ, SRM acqui-
sition) in terms of sensitivity, specificity andpotential identification
points. The main advantage of QqQ was its ability to provide fast
scanninginformation. Whenthe OrbitrapTM is limited to78 acqui-
sition points (30,000 resolution), the triple quadrupole was able to
generate 15 to 20 points without any significant loss in sensitivity,
making fast LC approach achievable.
2.3.2.2. Thyrostats. Specific detection procedures for the analysis
of this group of drugs have been described in the literature involv-
ing a longtimeago TLC protocols [6567]. Then GCMS and LCMS
approaches appeared[6870], andmorerecentlyLCMS/MS[71,72]and GCMS/MS methods which allowed an increase in the per-
formance of thyrostat detection in various biological matrices and
extending the monitoring to a wide range of compounds. The char-
acterization of thyrostats in biological matrices remains a current
difficult challengebecause of theirlow molecularweight,their high
polarity and the existence of several tautomeric forms. The deriva-
tization step prior to any MS analysis was considered until today
as the most efficient way to extract and analyze the compounds;
derivatization agents like benzylchloride [73]. Pentafluorobenzyl-
bromide [67,7275], NBDCl [77] or 3-BrBBr or 3-IBBr [78] have
been described for this purpose. Such derivatization induces a
stabilization of the thyrostats chemical structure, a reduction of
their polarity improving their separation, and an increase of their
molecular weight, all these elements allowing lower decision limitand detection capabilities [72]. The newly developed protocols
are efficient for the detection and identification of thyrostat com-
pounds in biological fluids and edible tissues in the g kg1 org L1 range which is in accordance with the requirements of
the European Union provisional minimum required performance
limit (MRPL) suggested at 10g L1 (CRL guidance paper, 2007).Since high urine concentrations of residues (100g L1) wouldbe generated upon drug administration aimed at increasing ani-
mal weight, the occasional occurrence in the range 110 g L1
of thiouracil in urines collected on food-producing animals raised
the question of the origin of the molecule and/or the origin
of the associated signals [76,78]. Therefore resorting to high-
resolution mass spectrometry enabled deep investigation of the
target compound proving beyond doubt its identity in the urine
sample. Confirmatory analysis were performed by gas chromatog-
raphy coupled to either negative chemical ionization or electron
ionization high-resolution mass spectrometry (GC(NCI)HRMS)
or (GC(EI+)HRMS) following derivatization with pentafluo-
robenzyl bromide (PFB). The first approach (GCNCIHRMS) led
to an intense fragment ion at m/z 199.0361 corresponding to
the elemental composition C7H11ON2SSi (Fig. 11). The second
approach (GCEIHRMS, PFB/TMS derivative) led to a molecular
ion M+ appearing at m/z380.0438 corresponding to the elemen-
tal composition C14H13OF5N2BrSSi. Both ions led to unambiguous
identification of thiouracil and can therefore be considered as fur-
ther diagnostic ions for the identification of thiouracil in urine
samples (Fig. 11).
2.3.2.3. Antibiotics. HRMS is becoming more popular in laborato-
ries, particularly in the form of time-of-flightmass spectrometry
(TOFMS) technology which can be implemented as an alternative
to MS2 instruments providing high specificity due to both mass
accuracy and high mass resolution. HRMS can also be found in
magnetic sector, Fourier transform(FT)MS and OrbitrapTM exhibit-
ing similar mass accuracy compared to TOF but a higher resolving
power. In the field of antibiotic residues analysis, this particu-
lar property might for example be of interest to distinguish two
anthelminthic drugs with different regulatory LODs and exhibit-
ing the same nominal molecular weight [i.e., albendazole sulphone
(MW= 297.328) and hydroxyl mebendazole (MW= 297.313)] which
are co-eluted and therefore undistinguishable using the clas-sical RPLCMS2 methods implemented in control laboratories
[79]. More generally, and as an emerging trend in veterinary
drug residue analysis, several applications based on HRMS mea-
surements have recently been reported for the development of
Fig. 11. GCNCIMS full scan massspectrumof the PFB/TMS thiouracilderivative (left) and GCNCIHRSIM (R = 10,000) diagnostic ion chromatogramobtained for a standard
solution (TU 10g L1
) and a urine sample.
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Fig. 12. Isotopic deviation measurement (expressed in 13CVPDB in ) of ERC (DHEA and 5-androstene-3,17-diol) and metabolites of testosteroneand 4-androstenedione (etiocholanolone, 5-androstane-3,17-diol and 17-testosterone) during 10 days after testosterone enanthate administration (250 mg,
IM injection) and 12 days after 4-androstenedione injection (100 mg).
the robustness of the analytical methodology and the good homo-
geneity of the endogenous steroid isotopic deviation (when the
diet, i.e., hay in this case, is kept constant). Fig. 12 illustrates thedepletion of the metabolite isotopic deviation following injection
of 17-testosterone and shows a difference in-between ERC andmetabolites(etiocholanoneor 5-androstane-3,17-diol) foroveroneweek after injection. Buisson et al.[83] studied theefficiency of
such an approach to demonstrate oestradiol administration to cat-
tleafteroestradiolvalerateinjection[83]. The13CVPDB valuesof theboth ERC (i.e., DHEA and 5-androstene-3,17-diol) and the mainoestradiol metabolite (17-oestradiol) were measured in urinesamples collected in different animals, treated versus non-treated,
gender (male, female versus castrated), age (sexually mature and
immature)and feedings (grassor maize). TheERC 13C/12C ratio was
not affected by the oestradiol treatment and found very repeatable
one animal to another when feed was remained constant (maize
17.8). The () was found equal to 14 after treatment; asthe relative concentration of endogenous 17-oestradiol wasweakcompared to exogenous residues, the() remained almost con-stant over the period of time. This difference is substantial and
far above the 3 threshold (uncertainty of measurement + inter-
animal variability) and unambiguously allowed the differentiation
in-between treated and non-treated animals.
2.4.2.2. GH. Somatotropin, also known as Growth Hormone, is a
protein hormone exhibiting a molecular mass around 22 kDa and
containing 191 amino acids in the bovine specie. Several phar-
maceutical companies have developed large scale production of
this protein using recombinant techniques. In several countries
somatotropine is thus available on the drug market and used in
food-producinganimalsasa generalgrowth promoter ortoincreasemilkproductionin dairycattle.The useof somatotropin in theEuro-
pean Union is forbidden and therefore efficient control methods
have to be set up in order to differentiate between endogenous and
recombinantforms of thehormone.One way foranalysisthese sub-
stances is based on the mass difference between endogenous and
recombinantsomatotropine andsuccessful strategies have recently
been developed to solve this issue [8487]. However and since one
of the available recombinant growth hormones is the same as the
natural product, only isotope ratio analysis can be used for the
determination of the origin of the growth hormones. Stable iso-
tope ratio analysis can provide distinctive fingerprints in order to
determine the originof natural materials and to authenticate phar-
maceuticals. If this strategy is now very efficient for natural steroid
hormones identification through GCCIRMS, the implementation
of such a strategy for growth hormones characterization come up
against several critical points such as the thermolability of thecon-
sidered macromolecule and the trace level at which it occurs in
biological matrices. The only attempt in this way was reported
by Karlsen et al. [88] where the 13C/12C isotope ratio of recombi-
nant bovine somatotropine (rbST) and of the endogenous growth
hormone have been analyzed via on-line determination of13C/12C
isotope ratios after HPLC separation and isotope ratio MS measure-
ment via the LC IsoLink interface [88]. Basically, the samples are
oxidized withinthe aqueous solvent eluting from the HPLC and the
generated CO2 is separated from the liquid phase and fed into the
isotope ratio MS. The measured delta 13C/12C values of endogenous
and recombinant bovinesomatotropine analyzed by LCIRMS were
shown significantly different on standards, 13C/12C ()=20.94and 16.69, respectively. The analysis of high level spiked plasma
samples (10g100L1) showed lower difference in themeasure-ments (13C/12C () =24.66 and 22.94 for bST and rbST,respectively) andthereforethe influenceof thematrix andtheneed
for improved purification before this strategy can be considered as
successful.
2.4.3. Conclusion
The IRMS methodology is nowadays becoming more and morepopular for confirmation purpose regarding the determination
of natural occurring growth promoters in cattle. The method is
officially applied in antidoping and food safety to control testos-
terone, oestradiol, and research studies are currently running for
nandrolone, boldenone and cortisol. An official threshold is now
proposed for testosterone and oestradiol in cattle, but it has not
beenaccepted officially bytheEuropeanauthorities.Improvements
are expected in the sensitivity of the instrument to facilitate the13C/12C measurement of other natural steroids and to allow the
technique to be applied to other biological matrices such as tissue
samples. Extension to other isotopes would be beneficial to ensure
the unambiguous character of the conclusion; 2H/1H is probably
thenext item. A possiblefurtherdevelopmentwouldbetheGCGC
approachto improvethe chromatographicseparation, as it remains
a critical limitation in therobust determinationof isotopecomposi-
tion. Finally, for otherclass of compounds such as growth hormone
(somatotropine)new couplingsuch asLCIRMS would be beneficial
to the domain.
3. Pitfalls and future trends
3.1. Issues related to the application of the 2002/657/EC decision
The validation of analytical methods has been subjected to var-
ious debates for decades among analytical chemists, but not only.
In each application field, specific discussions arise. As far as food
safety is concerned and chemical hazard involved, the application
of a specific decision is mandatory in Europe (2002/657/EC deci-sion), except when a more specific regulation hasbeen publishedas
it is forotherresidues such as PCDD/F, PCB-dl(see 1883/2006 regu-
lation). The Decision 2002/657/EC defines the performance criteria
for analytical residue methods both for validation of the method
and identification of the target analytes. Other specific expecta-
tions in term of performances have been published by CODEX,
IUPAC, ISO, FDA, AORC, or WADA. Often met in these institutions,
the limit of detection, limit of identification and limit of quantifi-
cation, have been replaced by the concept of decision limit (CC)anddetection capability (CC). For GroupA compounds, i.e., forbid-den substances, theconcept of theminimum requiredperformance
limit (MRPL) has been introduced; it corresponds to the minimum
content of an analyte in a sample that has to be detected and con-
firmed. The Decision introduces as well the concept of IPs, which
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corresponds to the minimal number of credits to be reached before
any conclusion compliant/non-compliant for a sample. When 4 IPs
are necessary to prove the identity of forbidden compounds, 3 IPs
only are expected for Group B substances. These numbers of cred-
its depends mainly on the specificity of the signals generated by
the analytical method used; for all these reasons, chromatography
coupled to mass spectrometry is unavoidable. The attribution of
IP relies upon tolerable variability in the relative ion-abundance
ratios; the tolerance is wider in positive chemical ionization mode
and atmospheric pressure ionization thanin electron ionization for
instance.
Necessary discussion and possible improvements may concern:
- Mass accuracy. The recent introduction of TOF and FTMS in the
field makes urgent a discussionregarding the minimal exigencies
linked to the definition of mass accuracy. A strong link with MS
resolution hastobekeptin mind,as itis knownthatan insufficient
resolution leads to inappropriate mass accuracy andso directand
clear consequence onto false compliant results.
- High-resolution. The HR criteria for mass spectrometer is appli-
cable when resolution is greater than 10,000 for the entire mass
range at 10% valley definition (approximately 20,000 FWHM). It
hasbeendetermined at theorigin formagneticsectorswherelock
mass were used; in the 2002/657/EC decision, nothing refers toTOF, or FTMS instruments.
- Very highresolution, i.e.,R above equal or above 80,000100,000.
It concerns mainly FTMS instruments.
- Fingerprinting approaches. Severalresearchprojects arecurrently
on-going, and finalresults are expectedsoon. Thesemetabolomic
approachesaremainly conducted to fight againsttheillegal use of
forbiddengrowth promoters. Topreparetheintroductionof these
new screening strategies in theofficial control,newspecific crite-
ria should be discussed. Special attention should be paid at least
to the approved definition given to a biomarker, the number of
target molecules to build a robust metabolomic model, etc. Some
new validation criteria for this multiparametric approaches will
be also tobe invented andimplemented,as well as some methods
of estimation for false positive and negative rate.- Isotoperatio mass spectrometry. Theneeds aregrowingup in the
field of natural hormones control; the approach is now officially
used in at least one National Reference Laboratories in Europe.
Some guidelines have to established regarding the 13C/12C deter-
mination; it may concern at least the apparatus calibration, the
signal integration, the linearity of the response, thecalculation of
the isotopic deviation difference between endogenous reference
compounds and target metabolites, etc.
- Minor additional points should be discussed, and they may con-
cern numerous items, e.g., the consequence of the introduction
of last generation of MS instruments for which the noise is
not always measurable (zero amplitude), or the definition of a
reference sample (spiked, standard . . .) to be used for final iden-
tification of an analyte.
Anotherimportant issuenotcoveredin thisreference document
is the quantitative aspect associated by definition to the concept
of CC and CC. Indeed, even with qualitative methods, the com-parison of a measurement result with the decision limit value
determined during thevalidation process imply a quantitative esti-
mation of the concentration present in theconsidered sample. And
noofficialindicationis givenregardingthisquantitativeestimation:
howmany andwhatconcentration levels for thecalibration curve?,
what fortified sample (representative sample, mixture of different
sample)?, whatquality controland procedures (preparationof stan-
dard solution, metrology)? . . . The quantitative verification of ionratio is also in some extend incompletely discussed, considering
that no precise indication is given regarding thesample natureand
concentrationlevelthathaveto be usedas reference value (ionratio
are largely dependent of either matrix effects and analyte concen-
tration so that thereference samplehave tobe representativeof the
analyzed sample with a similar concentration).
3.2. Chromatographic separation
Chromatographictechniques play a significant role in the deter-
mination of an analyte in a complex biological matrix. For residue
control in food, gas chromatography and liquid chromatography
are the two main chromatographic techniques in use for routine
analysis. A growing interest in fast GC and LC separations is cur-
rently observed. As the run time is continuously decreasing, the
detector ability to scan faster and faster is expected. GC and LC
methods can be speeded up by employing higher mobile phase
flows, shorter or dedicated columns such as shorter megabore or
microbore forGC purposes or smaller particle size (1.7 and 1.8m)or monolithic columns for LC purposes. Fast separation plays an
important role in two-dimensional (2D) separations, particularly
in comprehensive 2D chromatography which represents a major
improvementin comparison to GCMS techniques [89,90]. GCGC
present the advantages to increase peak capacity, increased sen-
sitivity and selectivity, independently from retention processes,
and finally provide two independent retention times for each ana-lyte. Basically, theseparation of many unresolved components from
the first dimension column is achieved in the second dimension.
Primary columns typically used in these systems are generally
1530m0.25mm0.251.0m film thickness. The first columnis often a non-polar stationary phase but not always. The second
dimension separation must be very fast and performed with a
stationary phase that is different from the one used in the first
dimension. Typical dimension characteristics for the secondary
column is 0.51.5 m0.1 mm0.10.25m. To be able to char-acterise, the multitude of narrow peaks generated by the system,
the MS detector must be characterized by a high scanning rate;
TOF instruments are appropriate to achieve this exercise [91,92].
The GCGCTOFMS system presents not only a superb sepa-
ration power, but also reliable data for identification, which isobtained from continuous acquisition of full mass spectra. Very
recently, Silva et al. [91] demonstrated theidentification of anabolic
agents (clenbuterol, norandrosterone, epimetendiol, two methyl-
testosterone metabolites and 3-hydroxystanozolol) contained in
a spiked urine sample at 2 ngmL1. Special emphasis was given to
3-hydroxystanozolol, mainly considering thedifficultyin its detec-
tion. In contrast to conventional GCMS approaches that must use
single ion monitoring, the GCGCTOFMS method enabled the
identification of that metabolite through the deconvolution of the
full mass spectrum and also resolved the co-eluted peaks of 3-
hydroxystanozolol and an endogenous component (Fig. 13).
3.3. Ionization (matrix effect)
During a first development period (1980searly 1990s), a com-
mon enthusiasm was shared by the new users of LCMS-related
techniques. Limited or no sample preparation was often presented
as a possibility even for complex matrices with a guarantee of
repeatable results even with high-throughput ambitions. At the
early stage of LCAPIMS, the chromatographic system was often
considered as a loading system only. However, during a sec-
ond period (1990stoday), various studies started to report some
troubleshooting associated to these techniques. Overall, the main
source of analytical problems encountered by LCMS users was
related to matrix effects problems. For many years, the composi-
tion of a sample extractand thepresence of interfering compounds
havebeen recognized tohavemajorinfluence on the analyte signal,
whatever the detection technique used. But in the specific case of
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Fig. 13. GCGCTOFMS TIC chromatogram of a human urine sample spiked
with anabolic agents and with three-dimensional (3D) expanded regions of the
anabolic agents in the spiked and in the blank urine for the key compounds
(clenbuterol, stanozolol, 17-methyl-5-androst-1-en-3-17-diol, epimetendiol
(EMD), 17-methyl 5-androstane-3,17-diol (methyltestosterone M1), 17-methyl 5-androstane-3,17-diol(methyltestosteroneM2)) [from Silva etal. [91]].
mass spectrometry, the so-called ion suppression phenomenon
appears as one particular tricking manifestation of matrix effect. It
represents certainly one of the main source of pitfall for the ana-
lyst, affecting many aspects of the method performances such as
detection capability, repeatability, or accuracy. The possible origins
of ion suppression are multiple [93,94]. The main one universally
reported is the presence of endogenous substances, i.e., organic or
inorganic molecules present in a sample and still present in the
final extract. Among this first group of ion suppressor agents,
ionic species can be included (inorganic electrolytes, salts), but
highly polar compounds (phenols, pigments), and various organic
molecules including carbohydrates, amines, urea, lipids, peptides,analogous compoundsormetabolites witha similarchemical struc-
ture shouldbe considered aswell.Finally,a wide range ofmolecules
can lead to ion suppression, especially when they are present in
high concentration in the extract and eluted in the same elution
range than the analyte of interest. A second source of difficulty is
the presence of exogenous substances, i.e., molecules not present
in the sample but coming from various external sources during
the sample preparation. Among this second group, plastic and
polymer residues [95] phthalates, detergent degradation products
(alkylphenols), ion pairingreagents[9698], protonexchangespro-
moting agents such as organic acids [98100], calibrationproducts,
buffers, or material released by the solid phase extraction (SPE),
LC or GC stationary phases can be listed. Different mechanisms
have been proposed to explain ion suppression [101,102]. In thecase of LCMS, the main phenomenon corresponds to the decrease
of the evaporation efficiency due to the presence of matrix com-
ponents. Indeed, the presence of interfering compounds in high
concentration can increase the viscosity and the surface tension
of the droplets produced in the ESI or atmospheric pressure chem-
ical ionization (APCI) interfaces, and may reduce the capability of
the analytesto reachthe gas phase. Theco-precipitation of theana-
lytes with non-volatile material such as macromolecules can also
limit their transfer in the gas phase. Another proposed mechanism
is the competition between analytes and interfering compounds
regarding the maximal ionization efficiency of the technique [103].
A last possible mechanism involves neutralization processes linked
to the relative basicity in the gas phase of the analytes and interfer-
ingsubstances, as well as to the stabilityof the produced ions in the
gas phase. The consequences of ion suppression are numerous, all
affectingthedifferentaspects of theanalytical result. The detection
capability is clearlyreduceddue tothe decreaseof theanalyte signal
intensity. The repeatability is also affected, because the degree of
suppression may vary in a large extentfrom one sampleto another.
Ion ratio, linearity, and quantification, are also affected due to the
variability of this unpredictable and not always repeatable phe-
nomenon. Another side-effect of ion suppression is the difficulty
to perform database searching, because of the modification of the
typical mass spectra patterns. Finally, ion suppression may lead to
the non-detection of a given analyte, to the weak estimation of
its real concentration, or to the non-fulfilment of the identifica-
tion criteria, with immediate consequences on the false compliant
score. If affecting the internal standard rather than the analyte,
ion suppression may also lead sometimes to an overestimation of
the analyte concentration with a clear risk on false non-compliant
results; it concerns mainly maximum residue limit compounds. A
first possible action to overcome ion suppression troubles would
consist into the modification of the mass spectrometric conditions,
when possible. Indeed, the occurrence of ion suppression may
differs between different ionization techniques (ESI, APCI, APPI),
ionization modes (positive or negative), or between equipments
with different source design [95,97,104106]. A second possibility
is to improve thechromatographic separation efficiencyin order toshift the retention time of the analytes of interest far away from
the area affected by ion suppression [105,107,108]. A third level
to overcome this problem is to use adequate internal standard
[109111]. 2H- or better 13C-labelled corresponding standards per-
mitto reduceto a greatextent the signalvariability observed forthe
analyte and consequently to improve the repeatability of the mea-
surement. The previously described action levels should permit to
limit the consequences of ion suppression, but not to eliminate the
risk as the cause is not deeply treated. Obviously, the best strat-
egy is to take care of the sample preparation and purification to
limit the presence of interfering compounds in the final extract.
Numerous authors demonstrated the evidence of such approach
[108,112116]. Therefore, it should be strongly suggested to check
thematrix effects resultingfrom differentsample treatment proce-dures systematically during method development. In other word,
the usual tendency to consider the recovery of thetarget analyte as
a main performance indicator should be moderated by the neces-
sity to evaluate also the method efficiency in terms of removing
interfering compounds.
3.4. Ion characterization (resolution, mass accuracy, speed
scanning, multiresidue analysis, cross talk)
Many significant improvements have appeared since the last
couple of years in the field of mass spectrometric data acquisition,
withdirect positiveimpact on the instrumentation capabilities and
performances. Some of these latest improvements may have some
clear advantages in the specific field of residue and contaminantanalysis. Besides the introduction of new types of mass analyz-
ers such as linear ion trap or orbital trap [117], a first noticeable
tendency is the increase of both resolution and mass accuracy.
Current resolution better than to 30,000 FWHM may be today
achieved either on the very last generation of time-of-flight or
orbital trap instruments, which offers new capabilities for sep-
arating very complex mixtures containing isobaric compounds.
Continuous progresses on electronic and computing devices also
permits to increase the scanning speed of the corresponding MS
instruments, which direct consequence on multi residue monitor-
ing, and coupling with fast, high-resolution or two-dimensional
chromatography.These improvements arebeneficialboth fortarget
classical approaches (focused approaches on already known target
compounds), but to more global mass spectrometric approaches
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[51] L.A. Perez-Estrada, A. Aguera, M.D. Hernando, S. Malato, A.R. Fernandez-Alba,Chemosphere 70 (2008) 2068.
[52] L. Valle, C. Diaz, A.L. Zanocco, P. Richter, J. Chromatogr. 1067 (2005) 101.[53] D.R. Doerge, M.I. Churchwell, T.A. Gehring, P. Yu Ming, S.M. Plakas, Rapid
Commun. Mass Spectrom. 12 (1998) 1625.[54] K.C. Lee, J.L. Wu, C. Zongwei, J. Chomatogr. B 843 (2006) 247.[55] Z. Hall, C. Hopley, G. OConnor, J. Chromatogr. B 874 (2008) 95.[56] E.O. van Bennekom, L. Brouwer, E.H.M. Laurant, H. Hooijering, M.W.F. Nielen,
Anal. Chim. Acta 473 (2002) 151.[57] F.M. Launay, P.B. Young, S.S. Sterk, M.H. Blokland, D.G. Kennedy, Food Addit.
Contam. 21 (2004) 52.
[58] F.M. Launay,L. Ribeiro, P. Alves, V. Vozikis,S. Tsitsamis, G.Alfredsson, S.S.Sterk,M. Blokland, A. Iitia, T. Lovgren, M. Tuomola, A. Gordon, D.G. Kennedy, FoodAddit. Contam. 21 (2004) 833.
[59] J. Jodlbauer, J. Zllner, W. Lindner, Chromatographia 51 (2000) 681.[60] M.H. Blokland, S.S. Sterk, R.W. Stephany, F.M. Launay, D.G. Kennedy, L.A. Van
Ginkel, Anal. Bianal. Chem. 384 (2006) 1221.[61] M. Kleinova, P. Zllner, H. Kahlbacher, W. Hochsteiner, W. Lindner, J. Agric.
Food Chem. 50 (2002) 4769.[62] B. Destrez,E. Bichon, L. Rambaud, F.Courant, F.Monteau, G. Pinel, J.P. Antignac,
B. Le Bizec, Steroids 74 (2008) 803.[63] B.Destrez, G.Pinel,E. Bichon,F. Monteau,R. Lafont, B.Le Bizec, RapidCommun.
Mass Spectrom. 22 (2008) 4073.[64] H.F. De Brabander, S.S.Poelmans,R. Schilt,R.W. Stephany,B. Le Bizec, R. Draisci,
S.S. Sterk, L.A. Van Ginkel, D. Courtheyn, N. Van Hoof, A. Macri, K. De Wash,Food Addit. Contam. 21 (2004) 515.
[65] H.F. De Brabander, R. Verbeke, J. Chromatogr. 108 (1975) 141.[66] H.F.De Brabander, The determinationof thyreostaticdrugs in biological mate-
rial, PhD Thesis, University of Ghent, Ghent, Belgium, 1986.[67] H.F. De Brabander,P. Batjoens,J. VanHoof, J. PlanarChromatogr. 5 (1992) 124.
[68] W.J. Blanchflower, P.J. Hughes, A. Cannavan,M.A. McCoy, D.J.Kennedy, Analyst122 (1997) 967.
[69] B. Le Bizec, F. Monteau, D. Maume, M.-P. Montrade, C. Gade, F. Andr, Anal.Chim. Acta (1997) 340.
[70] J.W. Pensabene, S.J. Lehotay, W. Fiddler, J. Chromatogr. Sci. 39 (2001) 195.[71] K. De Wasch, H.F. De Brabander, S. Impens, M. Vandewiele, D. Courtheyn, J.
Chromatogr. A 912 (2001) 311.[72] G. Pinel,E. Bichon, K. Pouponneau, D. Maume,F. Andr,B. Le Bizec,J. Chroma-
trogr. A 1085 (2005) 247.[73] S. Floberg, K. Lanbeck, B. Lindstrm, J. Chromatogr. 489 (1980) 63.[74] Q.-H. Zou, Y. Liu, M.-X. Xie, J. Han, L. Zhang, Anal. Chim. Acta 551 (2005) 184.[75] L. Zhang, Y. Liu, M.-X. Xie, Y.-M. Qiu, J. Chromatogr. A 1074 (2005) 1.[76] G. Pinel, S. Mathieu, N. Cesbron, D. Maume, H. De Brabander, F. Andr, B. Le
Bizec, Food Addit. Contam. 23 (2006) 974.[77] P. Batjoens, H.F. De Brabander, K. De Wasch, J. Chromatogr. A 750 (1996) 127.[78] G. Pinel,D. Maume,Y. Deceuninck, F.Andr, B. Le Bizec,Rapid Commun. Mass
Spectrom. 20 (2006) 3183.[79] S.J. Lehotay, K.Mastovska,A. Amirav,A.B.Fialkov, T.Alon,P.A.Martos,A. DeKok,
A.R. Fernandez-Alba, TrAC Trends Anal. Chem. 27 (2008) 1070.[80] A. Kaufmann, P. Butcher, K. Maden, M. Widmer, J. Chromatogr. A 1194 (2008)66.
[81] M. Becchi,R. Aguilera,Y. Farizon,M.-M.Flament,H. Casabianca,P.James,RapidCommun. Mass Spectrom. 8 (1994) 304.
[82] E. Bichon, F. Kieken, N. Cesbron, F. Monteau, S. Prvost, F. Andr, B. Le Bizec,Rapid Commun. Mass Spectrom. 21 (2007) 2613.
[83] C. Buisson, M. Heberstreit, A. Preiss-Weigert, K. Heinrich, H. Fry, U. Flenker, S.Banneke, S. Prevost, F. Andr, W. Schanzer, E. Houghton, B. Le Bizec, J. Chro-matogr. A 1093 (2005) 69.
[84] G. Pinel, F. Andr, B. Le Bizec, J. Agric. Food Chem. 52 (2004) 407.[85] L. Bailly-Chouriberry, G. Pinel, P. Garcia, M.-A. Popot, Y. Bonnaire, B. Le Bizec,
Anal. Chem. 80 (2008) 8340.[86] M.-H. Le Breton, S. Rochereau-Roulet, G. Pinel, G. Rychen, T. Goldmann, B. Le
Bizec, Rapid Commun. Mass Spectrom. 22 (2008) 3130.[87] M.-H. Le Breton, S. Rochereau-Roulet, G. Pinel, N. Cesbron, T. Goldmann, B. Le
Bizec, Anal. Chim. Acta 637 (2009) 121.
[88] C. Karlsen, G. Balizs, D. Juchelka, A. Lampen, Proceeding of the 17th Interna-tional Mass Spectrometry Conference, Praha, 2006.
[89] P. Mariott, R. Shellie, TrAC Trends Anal. Chem. 21 (2002) 573.[90] R. Ong, P.J. Marriott, J. Chromatogr. Sci. 40 (2002) 276.[91] A.I. Silva, H.M.G. Pereira, A. Casilli, F.C. Conceicado, F. Aquino Neto, J Chro-
matogr. A 1216 (2009) 2913.[92] J. Dallge, R. Vreuls, J. Beens, U.A.Th. Brinkman, J. Sep. Sci. 25 (2002) 201.[93] J.-P. Antignac, K. de Wasch, F. Monteau, H. De Brabander, F. Andr, B. Le Bizec,
Anal. Chim. Acta 529 (2005) 129.[94] T.M. Annesley, Clin. Chem. 49 (20 03) 1041.[95] H. Mei, Y. Hsieh, C. Nardo, X. Xu, S. Wang, K. Ng, W.A. Korfmacher, Rapid
Commun. Mass Spectrom. 17 (2003) 97.[96] S.A. Gustavsson, J. Samskog, K.E.Markides,B. Langstrm, J. Chromatogr.A 937
(2001) 41.[97] M. Holcapek, K. Volna, P. Jandera, L. Kolarova, K. Lemr, M. Exner, A. Cirkva, J.
Mass Spectrom 39 (2004) 43.[98] C.R. Mallet, Z. Lu, J.R. Mazzeo, Rapid Commun. Mass Spectrom 18 (2004) 49.[99] J. Eshraghi, S.K. Chowdhury, Anal. Chem. 65 (1993) 3528.
[100] A. Apffel, S. Fischer, G. Goldberg, P.C. Gooley, F.E. Kuhlmann, J. Chromatogr. A712 (1995) 177.
[101] R. King, R. Bonfiglio, C. Fernandez-Metzler, C. Miller-Stein, T. Olah, J. Am. Soc.Mass Spectrom. 11 (2000) 942.
[102] P.T.A. Reilly, A.C. Lazar, R.A. Gieray, W.B. Whitten, J.M. Ramsey, Aerosol Sci.Technol. 33 (2000) 135.
[103] P. Kebarle, L. Tang, Anal. Chem. 65 (1993) 972A.[104] E.T. Gangl, M. Annan, N. Spooner, P. Vouros, Anal. Chem. 73 (2001) 5635.[105] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Anal. Chem. 70 (1998)
882.[106] Y. Hsieh, M. Chintala, H. Mei, J. Agans, J.M. Brisson, K. Ng, W.A. Korfmacher,
Rapid Commun. Mass Spectrom. 15 (2001) 2481.
[107] P.R. Tiller, L.A. Romanyshyn, Rapid Commun. Mass Spectrom. 16 (2002)92.
[108] C. Mller, P. Schfer, M. Strtzel, S. Vogt, W. Weinmann, J. Chromatogr. B 773(2002) 47.
[109] R. Kitamura, K. Matsuoka, E. Matsushima, Y. Kawaguchi, J. Chromatogr. B 754(2001) 113.
[110] M.J. Avery, Rapid Commun. Mass Spectrom. 17 (2003) 197.[111] L. Gomidez Freitas, C.W. Gtz, M. Ruff, H.P. Singer, S.R. Mller, J. Chromatogr.
A 1028 (2004) 277.[112] M.W.J. van Hout, H.A.G. Niederlnder, R.A. de Zeeuw, G.J. de Jong, Rapid Com-
mun. Mass Spectrom. 17 (2003) 245.[113] R. Bonfiglio, R.C. King, T.V. Olah, K. Merkle, Rapid Commun. Mass Spectrom.
13 (1999) 1175.[114] D.L. Buhrman, P.I. Price, P.J. Rudewicz, J. Am. Soc. Mass Spectrom. 7 (1996)
1099.[115] G. Shi, T.L. Lloyd,S.K.B. Sy, Q. Jiao, A. Wernicki, A. Mutlib,T.A.Emm,S.E. Unger,
H.J. Pieniaszek Jr., J. Pharmaceut. Biomed. 31 (2003) 937.[116] C.H.P. Bruins, C.M. Jeronimus-Stratingh, K. Ensing, W.D. van Dongen, G.J. de
Jong, J. Chromatogr. A 863 (1999) 115.[117] R.J. Qizhi Hu, R.J. Noll, A. Hongyan Li, A. Makarov, M. Hardman, R.G. Cooks, J.Mass Spectrom. 40 (2005) 430.
[118] J. Nicholson, J. Lindon, E. Holmes, Xenobiotica 29 (1999) 1181.[119] D. Wishart, Trends Food Sci. Technol. 19 (2008) 482.[120] G. Theodoridis, H. Gika, I. Wilson, Trends Anal. Chem. 27 (2008) 251.[121] W. Lu, B. Bennett, J. Rabinowitz, J. Chromatogr. B 871 (2008) 236.[122] B. Warrack, S. Hnatyshyn, K.-H. Ott, M. Reily, M. Sanders, H. Zhang, D. Drexler,
J. Chromatogr. B 877 (2009) 547.[123] D. Yuan,Y. Liang, L. Yi,X. Qingsong,O. Kvalheim,Chemometr. Int. Lab.Syst. 93
(2008) 70.[124] S. Mahadevan, S. Shah, T. Marrie, C. Slupsky, Anal. Chem. 80 (2008) 7562.[125] J. Trygg, E. Holmes, T. Lundstedt, J. Proteome Res. 6 (2007) 469.[126] H. Lu, Y. Liang, W. Dunn, H. Shen, D. Kell, Trends Anal. Chem. 79 (2007)
2745.[127] C. Smith, E. Want, G. OMaille, R. Abagyan, G. Siuzdak, Anal. Chem. 78 (2006)
779.