Veterinary Drug Option-MS _review

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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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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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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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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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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


10/19


11/19


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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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.


14/19


15/19


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


16/19


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


17/19


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


18/19


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