LC–MS/MS analysis of organic toxics in food

Trends in Analytical Chemistry, Vol. 24, No. 7, 2005 Trends

LC–MS/MS analysis of organictoxics in foodOscar Nunez, Encarnacion Moyano, Maria Teresa Galceran

A large number of toxic substances, such as pesticides, antibiotics and toxins,

of varying origin and toxicity to human health are found in food. These

compounds are frequently present at low concentration levels in very

complex matrices, so sensitive, selective methodologies are needed for their

analysis. Due to the chemical properties of these compounds, liquid

chromatography (LC) is the separation technique of choice and the coupling

to mass spectrometry (MS) provides the sensitivity and selectivity required.

This article reviews the state of the art of LC–tandem MS (LC–MS/MS) for the

analysis of organic toxics in food products. We also address instrumental

aspects, such as ionization sources and analyzers, as well as confirmation

and quantitation procedures. Moreover, we discuss the application of

LC–MS/MS to compounds, such as pesticides, antibiotics, substances gener-

ated in cooking, and toxins in a range of food products.

ª 2005 Elsevier Ltd. All rights reserved.

Keywords: Antibiotic; Food; LC–MS/MS; Liquid chromatography; Mass spectrometry;

Organic; Pesticide; Tandem mass spectrometry; Toxin

Oscar Nunez,

Encarnacion Moyano,

Maria Teresa Galceran*

Department of Analytical

Chemistry, University of

Barcelona, Diagonal 647, Martı

i Franques, 1-11, E-08028

Barcelona, Spain

*Corresponding author.

Tel.: +34 93 402 12 75;

Fax: +34 93 402 12 33;

E-mail:

[email protected]

0165-9936/$ - see front matter ª 20050165-9936/$ - see front matter ª 2005

1. Introduction

Food products are complex mixturesconsisting of naturally occurringcompounds, such as lipids, carbohydrates,proteins, vitamins, phenolic compounds,organic acids and aromas. In addition,other substances, generally originatingfrom technological processes, agrochemicaltreatments, or packaging materials, arefrequently also present [1]. Althoughtoxic compounds (pesticides, polycyclicaromatic hydrocarbons (PAHs), chlori-nated and brominated compounds,veterinary drugs, toxins, mutagenic com-pounds, migrants from containers, metals,and inorganic compounds of toxicologicalconcern) are present in very smallamounts, they are nonetheless oftendangerous to human health. This fact hasforced legislative bodies to establish strictregulations for maximum residue limits(MRLs). In order to comply with theseregulations, highly selective analyticaltechniques for identifying and character-izing targeted compounds are required.

Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2005.04.012Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2005.04.012

Separation techniques, such as gaschromatography (GC), high-performanceliquid chromatography (HPLC) andcapillary electrophoresis (CE), have largelybeen used for analysis of toxic compoundsin food samples [1]. The complexity of foodmatrices often requires not only extensivesample preparation, but also on-line coup-ling techniques, which are used for theirsuperior automation and high-throughputcapabilities. The detection systems com-monly used in separation techniques canbe more or less selective and sensitive butusually lack information regarding theidentity of compounds. Utilizing sepa-ration techniques in tandem with massspectrometry (MS) or high-resolution MS(time-of-flight, TOF) [2] can overcome thislimitation. Additionally, due to its highsensitivity, sample-preparation procedurescan be simplified, thereby resulting infaster and low-handling methodologies.GC coupled to MS (GC–MS) is now a

routine technique for analysis of non-polar,semi-polar, volatile and semi-volatile foodcompounds, such as PAHs, dioxins andPCBs. In contrast, for polar and non-volatile substances, LC coupled to MS(LC–MS) is the technique of choice, and inrecent years it has experienced anexpanded use in the field of food analysis.Electrospray ionization (ESI) and atmos-pheric pressure chemical ionization (APCI)complement one another in regards topolarity, molecular mass of analytes, andchromatography conditions. However,ESI remains the ionization source mostfrequently used for analysis of toxiccompounds in food products due to thehigh polarity and ionization characteristicsof these substances.While GC–MS provides fingerprint

spectra by electron ionization, atmosphericpressure ionization (API) techniquesusually generate unfragmented spectra.

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Trends Trends in Analytical Chemistry, Vol. 24, No. 7, 2005

Tandem MS (MS/MS), or in-source collision- induceddissociation (CID), is required to obtain structural infor-mation, to improve selectivity and sensitivity, and toconfirm the identity of analytes. Among the analyzerscapable of MS/MS, triple-quadrupole and ion-trapinstruments are the most commonly utilized for theanalysis of toxic compounds. Although MS/MS withhybrid instruments, such as quadrupole-time-of-flight(Q-TOF) can be a good alternative providing good accu-racy inmass determination, no papers have been reportedin the literature applying this technique to food analysis.The aim of this review is to present current state-of-

the-art LC–MS/MS applications in the analysis of toxicsubstances in food samples. It includes a selection of themost relevant papers published from 2000 to the presentregarding instrumental and methodological aspects, aswell as the newest applications. Compounds currentlyanalyzed by LC–MS/MS, such as pesticides, antibiotics,substances generated in cooking processes, and toxins,have been selected for discussion.Although several reviews have been published about

the application of LC–MS in food analysis [3] and aboutdetermination of several specific compounds, such asantibiotics [4] and pesticides [5], the application ofMS/MS is not addressed in depth. In contrast, this publi-cation is specially focused to the use of MS/MS for theanalysis of the above-mentioned type of toxicants in foodproducts. We discuss ionization sources and analyzerscommonly used in this field. Moreover, we also commenton confirmation and quantitation aspects, as well assome selected applications.

2. LC–MS/MS instrumentation

2.1. Ionization sourcesAs toxic substances in food products can originate fromvastly different sources, this family of compoundsembraces a wide variety of chemical structures, fromvery complex molecules, such as mycotoxins [6], tosmall molecules, such as plant-growth regulators [7]. ESIand APCI are the ionization techniques currently usedfor food-sample analysis by LC–MS. Atmosphericpressure photo-ionization (APPI) has recently been pro-posed for complex sample analysis, since it can overcomethe suppression problems encountered in APCI and ESIsources. For example, APPI was recently used in LC–MSanalysis of carbamate pesticides in fruits and vegetables[8], although there are no applications of MS/MScurrently reported in the literature.ESI is the ionization technique recommended for polar,

ionized and high molecular weight compounds, so it isfrequently used for the analysis of toxics in food. Forroutine food analysis, LC-UV methods with columns of3–4 mm i.d. are sometimes adapted to LC–MS using splitdevices to reduce the mobile phase flow-rate required for

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proper LC separation to optimal flow for electrospray[9–15]. As electrospray is concentration-dependent, thesplit of the chromatographic eluent does not greatlyaffect sensitivity. This approach has been used by vanVyncht et al. [12] for the analysis of fluoroquinoloneantibiotics in swine kidney by reversed-phase chromato-graphy using a 4-mm i.d. column and a mobile phaseflow-rate of 1 mL/min. A T-piece splitter (9:1) was usedto reduce the flow-rate down to 100 lL/min beforeintroducing the ESI source.It is generally acknowledged that low-volatility mobile

phases and relatively high-salt-concentration effluentsmust be avoided to prevent instability and poor signals,as well as the possibility of plugging the small orifice ofthe sample cone. Nevertheless, modern ESI sourcespermit the use of such unfavorable conditions by pro-viding different design variables, such as setting the flowof the electrosprayed solution orthogonally to the samplecone, washing the orifice continuously with a small flowof solvent, using curtain gas, or introducing a turbulentgas at high temperature. For example, most of the papersrelated to analysis of toxic compounds and cited in thisreview use the Z-spray configuration source or theTurboIonspray configuration source, with a turbulentgas at high temperature and curtain gas to preventcontamination.As electrospray efficiency depends on eluent com-

position, the optimal mobile-phase composition foradequate chromatographic separation is sometimesunsuitable for obtaining maximal electrospray response.However, the correct choice of a post-column solvent toadd to modify pH, or to increase the percentage oforganic solvent, can enhance ionic evaporation andimprove the signal (e.g., Castro et al. [7] proposed thepost-column addition of methanol to enhance sensitivityin the analysis of the plant-growth regulator chlorm-equat in fruit samples using ion pair reversed-phasechromatography). In another example, Roach et al. [16]proposed the post-column addition of 1% acetic acid in2-propanol to avoid mobile-phase interferent ions duringanalysis of acrylamide in various food products byreversed-phase chromatography.APCI is very sensitive for weakly basic compounds,

and pesticides, such as triazines and phenylureas, can beeasily protonated by gas-phase, mobile-phase ions,according to their proton affinity. APCI is a more ener-getic source than electrospray, since effluent is nebulizedby a coaxial nitrogen flow and rapidly evaporated usinghigh temperatures (350–500�C). These high tempera-tures must be taken into account when working withthermally degradable compounds. For example, oryzalin,the active ingredient in the herbicide Surflan, cannot beanalyzed by LC-APCI-MS because of its instability at hightemperatures; only ESI-MS provides good results withsufficient sensitivity [15]. However, high temperaturescan be an advantage in other applications. For example,


Nakazawa et al. [17] analyzed tetracycline by reversed-phase LC with a mobile phase containing oxalic acid.The authors overcame the presence of non-volatilecomponents in the mobile phase by operating the APCIsource at 475�C, as oxalic acid decomposed to CO2 andwater at temperatures higher than 200�C.In general, ions originating from APCI sources corres-

pond to protonated molecules [M + H]+ in positiveionization mode and deprotonated molecules [M � H]�

in negative ionization mode. However, in some instancesadditional signals for m/z corresponding to Na+, K+ orNHþ

4 adducts appeared in the spectra. Indeed, thesecations are usually present as impurities in organic sol-vents of mobile phases in LC. Sometimes, these adductsappear as the base peak in the spectra. For example, inLC-APCI-MS analysis of carbamates and other polarpesticides in fruits and vegetables [18], ammoniumadduct ions are observed as base peaks. In this particularstudy, some of the pesticides were detected as protonatedmolecules and others as adduct ions.Although APCI techniques represent soft-ionization

modes, ions can sometimes be fragmented in the sourceto provide structural information, although lowersensitivity and more complex spectra are obtained incomparison with MS/MS. Fragmentation strictly dependson potential differences between the sample cone and theskimmer lens. If the target compound does not co-elutewith non-target compounds, the resulting ‘‘in-source’’CID spectra closely resemble that obtained by MS/MS.However, these procedures have not been widely usedfor quantitation purposes in food samples. Nevertheless,this approach is interesting from the perspective ofidentification and characterization. For example, thefragmentation pathways of quaternary ammoniumpesticides [19] and heterocyclic amines (HAs) [20] wererecently studied by in-source CID-MS/MS using a Q-TOFinstrument.

2.2. Mass analyzersAs mentioned in the introduction (Section 1, above),among the mass spectrometers that allow MS/MSexperiments, triple-quadrupole and ion-trap instrumentsremain the most widely coupled to LC for analysis oftoxic compounds in food products. This is principally dueto their easier operating performance, their betterrobustness for routine analysis, and their relatively lowcost when compared to TOF or Fourier Transform-IonCyclotron Resonance (FT-ICR) instruments.Triple-quadrupole analyzers display high sensitivity

when working in multiple reaction monitoring (MRM)mode, and are thus best suited to obtain the strict MRLsregulated for various toxic compounds in different foodmatrices. For example, when analyzing the antibioticchloramphenicol in bovine milk by LC–MS/MS with atriple-quadrupole instrument [21], an LOD of 15 ng/kgwas obtained using the most abundant transition (m/z

321–152). However, for confirmation purposes, at leasttwo transitions must be recorded, and then an increasein LODs occurred because of the second transition wasless abundant. In the case of chloramphenicol in bovinemilk, the LOD increased to 30 ng/kg, although it is not aproblem because this value is 10-fold less than thecorresponding MRL value.When performing MS/MS, ion-trap instruments are

generally less sensitive than triple-quadrupole analyzers,but they have the advantage of working in product-ionscan without losses in sensitivity. Moreover, ion trapsoffer the possibility of performing multiple-stage frag-mentation (MSn). Such advantages are important toolsfor characterizing and correctly identifying toxic com-pounds in complex food matrices [22], and havefrequently been used for screening purposes [23–26]. Forthis application, both product-ion full-scan MSn andselective reaction monitoring (SRM) are used. This latteracquisition mode is especially suited to screeningsamples for a pre-specified set of analytes that are mostlikely to be present. For the detection of contaminantsthat are completely unknown, data-dependent full-scanMS and MSn are proposed by some authors [26]. Iontraps are also useful for differentiating isomer com-pounds, as illustrated by Lehane et al. [6], who proposedan LC–MS3 method for determining azaspiracids inmussels. The structures of azaspiracids, AZA4 andAZA5, are given in Fig. 1. The MS3 spectrum provides acharacteristic fragmentation pattern for each compound,resulting from cleavage of the A-ring at C9–C10. Thisfragmentation permits the distinction between theisomeric hydroxilated azaspiracids, AZA4 and AZA5, ascan be seen in Fig. 1, which shows the LC–MS3 chromato-gram with the separation of both isomers and thecorresponding MS3 spectra.

3. Aspects of identification and quantitation

To comply with established regulations, both accurateidentification and quantitation of target analytes at tracelevel are mandatory. While MS/MS exhibits a highersensitivity and selectivity, confirmation of analytes is notalways completely achieved. Most applications ofLC–MS/MS found in the literature for food-sampleanalysis monitor only one transition for each targetcompound, although current legislation requires morethan one to confirm the presence of the analyte. Indeed,the European Union (EU) Council Directive regardinganalytical methods and interpretation of results [27]requires a minimum of three identification points (IPs)for correct LC–MS/MS confirmation, although, for sub-stances having anabolic effect and unauthorized sub-stances, at least four IPs are needed. The four IPs can beobtained in low resolution by monitoring two transitionsfrom the same precursor ion for each compound

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Figure 1. (a) Structure of the isomers AZA4 (R1 = OH, R2 = H, R3 = H, R4 = H) and AZA5 (R1 = H, R2 = H, R3 = H, R4 = OH). The hashed lineindicates a characteristic MS fragmentation pathway. (b) (A) Chromatogram showing the separation of isomers obtained using LC–MS3; AZA4(3.73 min) and AZA5 (4.63 min). (B) and (C) are the mass spectra corresponding to AZA4 and AZA5, respectively. Chromatographic conditions:Luna-2-C18 column (5 lm, 150 · 2.0 mm) at 40�C, acetonitrile–water (46:54) with 0.5 mM ammonium acetate, and 0.05% TFA as mobile phase.ESI in positive ionization mode (reprinted from [6] with permission).


[10,28–30]. The relative abundance of product ions usedfor confirmation has to match with those of a standardand be higher than 2%. Moreover, the retention timemust be within 2% of standards and the chromato-


graphic peak must have a signal-to-noise ratio (S/N)higher than 3. Ions present in isotopic clusters can alsobe used for confirmation, serving as additional IPs. Forexample, Sancho et al. [31] analyzed plant-growth


regulator paclobutrazol in pears by exploiting thepresence of chlorine to monitor both the transitioncorresponding to the 37Cl-paclobutrazol as well as thequantitative transition corresponding to the 35Cl isotope.Since two precursor ions were used for confirmationpurposes, five IPs were achieved, thereby fulfilling therequirements of the EU Directive.Even when using LC–MS/MS, matrix effects must be

taken into account, particularly when studying complexsamples, such as food. In general, multi-residue deter-mination methods with simple sample treatment areroutinely used in laboratories to reduce analysis time.However, simplification of clean-up procedures canresult in dirty extracts with high co-extractive substancecontent. The main problem of analyzing these samplesby LC–MS is the occurrence of signal suppression,especially with electrospray sources. For example, a10–50% decrease in signal for two pesticides, carben-dazim and thiabendazole, was observed by Jansson et al.[32] using a multi-residue method for pesticide analysisof fruits and vegetables. These authors analyzed straw-berries spiked with a mixture of five pesticides usingsimple extraction with ethyl acetate, and then injectingthe extract directly into the LC–MS/MS system. Fig. 2compares the chromatogram obtained after extractionwith that of a standard mixture prepared in methanol atthe same concentration level, showing the decrease inresponse for some of the compounds.To overcome matrix effects when quantifying, several

approaches are available. Isotopic dilution is the bestoption if labeled target compounds are available. Thisapproach allows signal suppression to be corrected, asboth labeled and native compounds will suffer the samesuppression effect. This method is frequently used whenonly one analyte must be determined, as happens, for

Figure 2. Example of pesticide suppression. Injection of pesticideat 0.05 mg/kg in strawberry, overlaid with the same pesticide inmethanol and a blank chromatogram of strawberry. Retention timeof pesticides in strawberry (- - -) was manually moved by 0.3 min;true retention time was identical. Only peaks no. 2 and no. 3 aresuppressed. LC-ESI-MS/MS in positive ionization mode. Peak 1:ethiofencarb-sulphoxide; 2: carbendazim; 3: thiabendazole; 4:propoxur; 5: carbaryl (reprinted from [32] with permission).

example, in the analysis of acrylamide [16], chlormequat[7] or chloramphenicol [33]. As an example, Ortelli et al.[33] reported significant signal suppression (63–82%,depending on sample) during analysis of chloramphen-icol in honey, but, by using a deuterated internal stan-dard (IS), this effect was efficiently corrected andaccurate results were obtained. The availability of iso-topically labeled analogues as ISs for a relatively highnumber of target analytes is frequently difficult. In thesecases, alternative calibration methods are used. Quanti-tation by standard addition is one possibility to correctmatrix suppression and has been used, for example, forthe determination of HAs in meats [34]. Nevertheless,this method is not very convenient when a high numberof samples must be analyzed, and is therefore not usedfrequently for the analysis of toxic compounds in food.Matrix-matched calibration is another way to overcomematrix effects and is the method used in most of thepapers included in this review. However, for its appli-cation, blank matrices must be available. For example, ithas been used for the analysis of some penicilliummycotoxins in food products, such as fruits, vegetables,cheese and grain [35]. Nevertheless, this method isinconvenient when a large number of samples withdifferent matrices have to be analyzed, since a blank ofeach kind of sample is needed. External calibration seemsto be the less suitable method but it is currently used forthe analysis of matrices, such as fruits and vegetables,although extensive clean-up procedures must be appliedto obtain sufficiently clean extracts. If analyte responsesin standard solution and extracts are in agreement, thismethod can be used; this is the case with most of thepapers on pesticide analysis cited in Table 1. An alter-native calibration technique is that proposed by Hajslovaet al. [36,37]; that simulates the use of an IS. With thistechnique, the unknown sample and a standard solutionof the target analyte are injected within a short time.Nevertheless, this procedure seems to be difficult toimplement for samples with a high number of analytes,since sufficient separation is needed to avoid coelutionbetween standards and analytes. Moreover, differentmatrix effects on analyte and standards would occur.

4. Applications

4.1. PesticidesAgrochemicals such as insecticides, fungicides andherbicides, which are known generically as pesticides,consist of a multitude of substances with myriad chemi-cal structures. The spectrum of pesticide toxicity rangesover:

� the acutely toxic, such as organochlorine,organophosphate and carbamate insecticides;

� those posing a significant risk for chronictoxicity, such as fungicides;


Table 1. Analysis of pesticides in food samples by LC–MS/MS

Compound Foodproduct

LC conditions Extraction Clean-up Recoveries Ionizationsource

Analyzer Acquisitionmode

LODs References

Atraton, atrazin, diuron,linuron, metalachlor,diazinon, DNOC,dichlorprop

Carrots,potatoes

Zorbax SB-C18

methanol/10 mMammoniumformate solution

ACN – – ESI Triplequadrupole

MRM 0.5–2 lg/kga [10]

Chlormequat Pears Kromasil C8

ACN/ammoniumformate buffer/15 mM HFBA

Methanolicammoniumformate-formicacid buffer

SPE: C18 86–92% ESI Ion trap Product-ionscan

30 lg/kg [7]

38 Pesticide residues(carbofurans, etc.)

Fruits,vegetables

Hypersil C18

BDS methanol/10 mMammoniumacetate solution

Ethylacetate – 63–96% ESI Triplequadrupole

MRM 5 lg/kgb [11]

Paclobutrazol Pears Discovery C18

ACN/formicacid solution

Methanol – 82–102% ESI Triplequadrupole

MRM 0.7 lg/kg [31]

Benzimidazoles, azoles,carbamates,phenylureas,benzoylphenyl-ureas

Apples,apricots

Discovery C18

methanol/waterACN SPE: ion

exchange/polymeric

75–122% ESI Ion trap Product-ionscan

0.1–10 lg/kg [40]

Dichloran, flutriafol,prochloraz, tolclofosmethyl, o-phenylphenol

Fruits Luna C18

methanol/waterAcetone/water(5:1) SPME:fused silica fibercoated withCarbowax/template resin

– 10–60%c APCI Ion trap MRM 0.5–10 lg/kgb [43]

24 Pesticide residues(azoles, etc.)

Processedfruits andvegetables

Synergy Polar-RP ACN/formicacid solution

Acetone LLE:ethylacetate-cyclohexane

76–106% ESI Triplequadrupole

MRM 1–10 lg/kg [30]

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Oryzalin Citrus fruits,stone fruits

Zorbax SB-C8

methanol/0.1%acetic acidsolution

Methanol SPE: Strata-Xpolymeric


MRM 0.3–10 lg/kg [15]

Carbamates Fruits,vegetables,cereals

Genesis C18

methanol/ammoniumacetate buffer

Methanolicammoniumacetate-aceticacid buffer

– 70–125% ESI Triplequadrupole

RM 10–20 lg/kg [18]

Benzimidazoles,carbamates,N-methylcarbamates,organophosphoruscompounds

Fruits,vegetables

Genesis C18

methanol/ammoniumformate solution

Ethylacetate – 70–100% ESI Triplequadrupole

RM 10 lg/kg [32]

Methoxyfenozide Fruits,vegetables,mint

Restek AllureC18 methanol/ammoniumacetate buffer

Methanol-aqueous 0.1 NHCl (9:1, v/v)

LLE: hexaneand dichloro-methane


RM 5–25 lg/kg [42]

74 Pesticide residues(carbamates, conazole,pyrimidine fungicides,insecticides)

Fruit andvegetables

Nucleosil C18

formic acid inwater/methanolsolution

Ethylacetate – – ESI Triplequadrupole

RM 10 lg/kgb [41]

Imidacloprid,carbendazim,thiabendazole,methiocarb, imazalil,hexythiazox

Oranges Luna C18

methanol/waterEthylacetate – 72–94% APCI Ion trap RM 1–300 lg/kgb [22]

aLarge volume injection-LC–MS/MS.bLOQ.cAt LOQ levels.

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� those virtually non-toxic to mammals, such asplant growth regulators.

Residues from pesticides can sometimes find their wayto human consumption, and, to safeguard consumers,MRLs for some pesticides found in foodstuffs have beenset at levels of 10–100 lg/kg [38,39].Although GC has traditionally been applied for pesti-

cide-residue analysis, the use of LC has rapidly grown inthe last years. Agrochemicals of carbaxamide, pyrimi-dine, triazol, and carbamate classes, among others, arerepresentative of such molecules [30]. Recent applica-tions of LC–MS/MS for pesticide analysis in fruits, vege-tables, and processed products are included in Table 1,where separation conditions, sample treatment, MS/MSworking conditions and limits of detection (LODs) areindicated. In general, reversed-phase (C18, C8) andmobile phases with methanol or ACN as organic modi-fiers, and volatile buffers, such as acetic acid/ammoniumacetate or formic acid/ammonium formate, are used.Ion pair LC has also been coupled to MS for the

analysis of ionic compounds. In one study, this tech-nique was applied to the analysis of cationic herbicides,such as chlormequat, using a relatively volatile ion-pairreagent (heptafluorobutiric acid) [7]. Although areduction in signal occurred, resulting from the presenceof the reagent, an acceptable LOD of 30 lg/kg wasobtained.For monitoring pesticide residues in fruits and vege-

tables, multi-residue methods are preferred, as severalanalytes can be determined in a single extraction. Mostof the applications involving pesticide-multi-residuemethods [10,11,18,30,32,40,41] place emphasis onsimplifying the sample treatment for direct injection ofthe extract into the LC–MS/MS system [10,11,18,32],taking advantage of the superior selectivity of thistechnique. In general, these multi-residue methods areperformed using triple-quadrupole instruments thathave the capacity to determine simultaneously a largenumber of pesticides in front of ion trap that requires theuse of several retention windows. Several extraction orclean-up procedures, such as liquid–liquid extraction(LLE) [30,42], solid-phase extraction (SPE) usingreversed-phase [7], polymeric sorbents [15,40], or ionexchange [40], and recently solid-phase microextraction(SPME) [43], have been used.An interesting example of a fast and simple clean-up

method has been set forth by Sannino et al. [30],whereby 24 new pesticides in processed fruit and vege-tables were analyzed using a triple-quadrupole instru-ment. The authors proposed an acetone-basedextraction, partitioning with ethylacetate–cyclohexanein a single step. To comply with EU pesticide regulations,they monitored 55 simultaneous MS/MS transitions(two or three for each pesticide) in MRM mode usingonly one retention time window and 25 ms dwell time.


Under these conditions, LODs of 1–10 lg/kg were ob-tained. Fig. 3 shows the typical MRM profile of an applepuree fortified with those 24 pesticides.By contrast, in a study of 17 polar pesticides,

Zrostlikova et al. [40] proposed grouping the compoundsinto two families for independent analysis using ion trapand product-ion-scan acquisition mode. To obtain suffi-cient data points per chromatographic peak, no morethan three analytes in a time windowwere acquired sinceion trap requires longer cycle-times than triple-quadru-pole instruments for simultaneous acquisition of MS/MStransitions. For this reason, two clean-up procedureswere proposed, one using an Oasis HLB cartridge for non-ionic and less polar-like compounds, and a second, usingan anion-exchange cartridge for polar compounds, basicbenzimidazoles and azoles. Nevertheless, it remainsinteresting to report that lower LODs (0.1–0.3 lg/kg forbenzimidazole and azole fungicides and carbamateinsecticides, and 3–10 lg/kg for urea insecticides) wereobtained with the method proposed by Zrostlikova et al.,although it is known that product-ion scan in ion-trapinstruments provides higher LODs than MRM in triple-quadrupole instruments, as can be seen for example inTable 3 for HAs [44]. This may result from the extremeacquisition conditions used in the triple-quadrupolemethod, which allows detection of 24 compounds [30]but with significant reduction in sensitivity.

4.2. Veterinary drugsDirect treatment with antibiotics or use of contaminatedfeed may introduce drug residues into animal-foodproducts. Moreover, the intensive use of antibiotics inveterinary medicines, as well as in industrial forming(food additives), has led to significant increases in anti-microbial resistance, with important consequences forpublic health. Safe MRLs have therefore been establishedfor antibiotics used in animal tissues and derived food-stuffs entering the human food chain [45,46].Of chromatographic separation techniques, and in

contrast to pesticide analysis, GC and GC–MS are notsuitable for direct analysis of antibiotics. Consequently,LC–MS techniques have been selected for many classes ofantibiotics used in veterinary medicine. Table 2 sum-marizes recent LC–MS/MS applications for antibioticanalysis of food products of animal origin. Generally, ESIremains the preferred API ionization source, although,in some instances, APCI is utilized. Nakazawa et al. [17],for example, used an APCI interface to analyze tetra-cyclines in several fortified (50–300 lg/kg) foodproducts, while Sorensen et al. [21], determined chlor-amphenicol at 0.2–1 lg/kg in bovine milk.Sample treatments for antibiotic-residue analysis

include extraction procedures involving different sol-vents, such as acetonitrile [9,12,24,28,29,47], ethyl-acetate [33,48,49] and water [50]. In some cases, a

Figure 3. Typical MRM profiles of an apple puree fortified at 2–10 lg/kg. Chromatographic conditions: Synergy Polar-RP column (150 · 2 mm i.d.); gradient elution with 0.1% aqueous formicacid solution and acetonitrile (from 40% to 96%) as mobile phase. ESI source in positive ionization mode (reprinted from [30] with permission).

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Table 2. Analysis of antibiotics in food samples by LC–MS/MS

Compound Foodproduct

LC conditions Extraction Clean-up Recoveries Ionizationsource


LODs References

Tetracyclines Eggs, milk,honey, fish,animal tissues

Bakerbond C8

methanol/ACN/5 mMoxalic acid

0.1 M Na2 EDTA-McIlvaine buffer(pH 4.0)

SPE: C18 60.1–88.9% APCI Triplequadrupole

MRM 1–4 lg/kg [17]

Gentamicin,neomycin

Milk Zorbax C18 methanol/water with 20 mMHFBA

SPE: cation-exchange

– 44–70% ESI Ion trap Product-ionscan

30 lg/kgd [23]

Ionophoreantibiotics

Eggs, animaltissues

Luna C18

ACN:MeOH:THF:water:TFA(67:10:10:13:0.1 v/v)

ACN SPE: silica 93–106% ESI Triplequadrupole

MRM 0.4 lg/kg [29]

Sulfonamides,tetracyclines,flumequine

Honey Nucleosil 100-5 C18

ACN/formic acidsolution

Hydrolysis (HCl) SPE: polymeric 65–125% ESI Triplequadrupole

MRM 0.4–11 lg/kg [51]

b-Lactam antibiotics Milk Luna C18 methanol/water with 1% aceticacid

ACN SPE: polymeric 85–115% ESI Ion trap Product-ionscan

0.2–2 lg/Lc [47]

Sulfonamide residues Eggs ODA-AQHydrophilic modifiedC18 methanol/formicacid solution

ACN SPE: C18 60% ESI Ion trap Product-ionscan

5–10 lg/kgd [24]

Fluoroquinolones Swine kidney Nucleosil 100-5 C18

ACN/formic acidsolution

ACN SPE: mixed mode(C18-cationexchange)


MRM 10–20 lg/kg [9,12]

Coccidiostats Eggs Symmetry C18 ACN/0.1% formic acidsolution

ACN – 42–113% ESI Triplequadrupole

MRM 0.9–8.3 lg/kg [28]

Streptomycin Honey Zorbax C18 20 mMHFBA with 30% CAN

SPE: cationexchange


MRM 2 lg/kg [52]

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FluoroquinolonesPhenicols

Aquaculturedproducts

Inertsil phenyl ACN/2% formic acidsolution

Fluoroquinolones:SPE: cationexchangePhenicols: ACN/ethylacetate

– 70% ESI Ion trap MRM 10–40 lg/kgd [26]

b-Lactam antibiotics Kidney tissue Waters C18 MeOH/water 0.1% formicacid

ACN/water SPE: C18 – ESI Ion trap MRM 10–100 lg/kgd [25]

Chloramphenicol Shrimp, crabmeat Luna C18 ACN/ammonium acetatebuffer

Ethylacetate LLE: hexane 83–103%a ESI Triplequadrupole

MRM 0.03 lg/kg [48]

Chloramphenicol Bovine milk NovaPak C18 ACN/ammonium acetatebuffer

SPE: Bond-ElutC18

SPE: neutralalumina

90% APCI Triplequadrupole

MRM 0.03 lg/kg [21]

Chloramphenicol Honey Nucleosil 100-5 C18

ACN/waterEthylacetate SPE: C18 58–74%b ESI Triple

quadrupoleMRM 0.2 lg/kg [33]

Nitrofurans Pork Symmetry C18 ACN/0.1% acetic acidsolution

Ethylacetate – 11.5–48.6% ESI Triplequadrupole

MRM 0.02–0.09 lg/kg [49]

Quinolones Bovine muscle,milk,aquaculturedproducts

Symmetry C18

methanol/water with0.1% TFA

Water SPE: C18 86–110%b ESI Ion trap Product-ionscan

36–110 lg/kg [50]

aInterlaboratory study. Recovery depends on laboratory.bDepending on the sample.c10:1 Signal-to-noise ratio.dLimit of confirmation.

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preliminary hydrolysis step is required as, for example,occurs in the analysis of sulfonamides in honey [51] toliberate those compounds bound to glucose.SPE is also frequently used for both extraction of liquid

samples, such as milk [21], and clean-up procedures.Several sorbents have been utilized, depending on thecharacteristics of the antibiotic in question. Silica hasbeen proposed for ionic antibiotics [29], while C18 orpolymeric sorbents are used for neutral or ionizablecompounds working at a pH lower than the pKa of theanalytes [17,33,47,51]. For compounds with variedchemical properties, mixed-mode sorbents are recom-mended.van Vyncht et al. [12] proposed a sorbent C18-cation

exchange for clean-up of fluoroquinolones extractedfrom swine kidney with good recoveries (�100%). Thesecompounds possess a very wide pKa range, making itdifficult to pre-concentrate on C18 in a single step. Ionexchange permits high recovery but a mixed-mode sor-bent must be used to obtain cleaner extracts. Fig. 4shows the reconstituted ion chromatogram for a swinesample spiked at the MRL/4 level with 11 fluoroquino-lones, using a Nucleosil C18 column and acetonitrile anddiluted formic acid (pH 2.5) as the mobile phase. Only asingle transition for each compound was monitored andlimits of quantification (LOQs) below 50 lg/kg wereobtained, considerably lower than the EU imposed MRLsof 150–300 lg/L. In a posterior study, this sameresearch group [9] validated the proposed method,obtaining excellent accuracy and precision for ampho-teric quinolones, whereas acidic quinolones showedrelatively poor reproducibility (20–40%). Such problemsare explained by the weak protonation of these acidiccompounds in the ESI(+) mode, so, while the authorsrecommended the proposed method for simultaneousquantitation of amphoteric species, only estimatedvalues could be obtained for acidic species.While most of the publications listed in Table 2 are

focused on determining a high number of antibiotics in asingle run, some are devoted to the analysis of one spe-cific antibiotic, such as chloramphenicol, in differentmatrices (honey [33]; bovine milk [21]; shrimp andcrabmeat [48]), or streptomycin in honey [52].The maximum allowable level of chloramphenicol in

food has recently been reduced because of its un-predictable effects on the human population, so methodswith LODs less than 0.3 lg/kg must be implemented. Aninteresting contribution to the capability of LC–MS/MSto achieve this low LOD is the paper published byHammack et al. [48]. Their results validate a methodusing LLE, LC on a Luna C18 column, ESI in positiveionization mode, and MRM for the determination ofchloramphenicol in shrimp and crabmeat. In this study,three laboratories participated using different triple-quadrupole instruments. The method meets both theconfirmation criteria recommended by the US Food and


Drug Administration [53] as well as the four-IP criterionestablished by the EU [27]. Results from two of theparticipating laboratories showed a variability between2% and 9% depending on the concentration level.Although variability in the third laboratory was higher(7–35%), the authors concluded that the method issufficiently precise for analysis of chloramphenicol inseafood matrices.While most of applications related to analysis of anti-

biotics in food utilize triple-quadrupole instruments inMRM acquisition mode, ion-trap instruments are alsoused in some instances [47,50]. For example, van Hoofet al. [50] analyzed eight quinolones in bovine muscle,milk, and aqua-cultured products using ESI in positiveionization mode with an ion-trap analyzer. In this study,detection was performed using three segments in whichonly 2–4 analytes were acquired, with LODs rangingfrom 36 to 110 lg/kg using product-ion scan. Foraccurate confirmation of two quinolones, oxolinic acidand flumequine, which exhibit only the product ion m/z244 in the corresponding MS/MS spectrum, MS3 spectrawere obtained in an additional run.Ion trap has also been used for screening and confir-

mation of drug residues in different foods [23–26], andin some cases following the guidelines used in the USAfor positive confirmation of drug residues [54]. Thepresence of antibiotics in eggs and milk has been identi-fied using product-ion scan MS/MS with limits ofconfirmation at low ppb level [23,24].A comparison of product-ion scan MSn and selected

reaction monitoring (SRM) MSn was performed byFagerquist and Lightfield [25]. They found that the latteracquisition mode (SRM MSn) allows a rapid, un-ambiguous identification of b-lactam antibiotics inkidney more easily than using full-scan MSn mode thatrequires manual mass filtering.Another interesting approach is that proposed by

Turnipseed et al. [26], who screened the presence ofquinolones in aquacultured products using data-dependent scan. The advantage of this acquisition modeis that eliminates the needs to set static time segments orto scan for each compound continuously, allowing theconfirmation at low levels.

4.3. Substances generated in cookingIt is well known today that not all the toxic substances infood products have an anthropogenic origin. Compoundsthat have some toxicity, such as acrylamide and HAs,can originate during thermal treatment or cooking ofsome food products.Acrylamide (2-propenamide) is a potent cumulative

neurotoxin in both animals and man [55]. Recentstudies in Sweden reported the presence of acrylamide ina wide range of fried and oven-cooked foods [56]. Thesefindings have provoked considerable interest worldwide,since acrylamide has been classified as ‘‘probably

Figure 4. Reconstituted ion chromatogram obtained by LC–MS/MS analysis of a swine-kidney sample spiked with 11 (fluoro)quinolones atMRL/4. Deuterated norfloxacine was used as an internal standard. MRL values ranged between 150 and 300 lg/kg when defined (1500 lg/kgfor flumequine antibiotic). Chromatographic conditions: Nucleosil 100-5 C18 column (70 · 4 mm i.d.); gradient elution with formic acid(pH 2.5) and acetonitrile (from 2% to 70%) as mobile phase (reprinted from [9] with permission).


carcinogenic to humans’’ by the International Agencyfor Research on Cancer (IARC) [57]. The general findingis that acrylamide is likely to be found in foods that havebeen heated by means other than boiling.LC–MS/MS has been used for analysis of polar com-

pounds in different food samples to include bread,cereals, coffee, chocolates and fried foods, such as pota-toes (Table 3). The broad variety of food products whereacrylamide is present complicates development of asingle method suitable for all purposes and matrices. Formost samples, water extraction remains the most reliabletreatment due to the high solubility of acrylamide.

Although Soxhlet extraction with methanol has alsobeen used, it is not recommended, since a study byTanaka et al. [58] detected an enormous amount(6887 lg/kg) of acrylamide in methanol extracts,following Soxhlet extraction of freeze-dried and powderedraw potatoes. Nevertheless, the addition of smallamounts of methanol to an extractant does not seem tocause acrylamide formation [59]. For fat matrices, ade-fatting step with iso-hexane prior to water extractionhas been suggested by some authors to improve swellingproperties [60]. A clean-up protocol using LLE [60,61] orSPE (Table 3) is currently used. Nevertheless, these


Table 3. Analysis by LC–MS/MS of toxic substances generated in cooking

Compound Food product LC conditions Extraction Clean-up Recoveries Ionizationsource


LODs Reference

Acrylamide Potatoes, bread,oat, rye, friedfoods

Hypercarb water Water SPE: mixed mode(C18 and ionexchange)


MRM 5 lg/kg [63]

Acrylamide Potatoes, cereals Hypercarb water Water SPE: mixed mode(C18 and ionexchange)

– ESI Triplequadrupole

MRM 10–30 lg/kg [64]

Acrylamide Potatoes, bread Primesphere 5 C18

ACN/0.1% aceticacid solution(7:93 V/v)

Water SPE: mixed mode(C18 and ionexchange)


MRM 10 lg/kg [65]

Acrylamide Potatoes, bread,biscuits, cereals,etc.

Alltima C18 5% ACNin 5 mM formic acidsolution

5% Methanol inwater

– – ESI Triplequadrupole

MRM 30–60 lg/kg [59]

Acrylamide Breakfast cereals,crackers

Shodex Rspack DE-613 polymethacrylategel column methanol/formic acid solution

Water SPE: mixed mode(ion exchangeand C18) and ionexchange


MRM 15 lg/kg [13]

Acrylamide Potatoes, cereals,bread

Hypercarb 15%methanol inammonium formatesolution

Water LLE:dichloromethane


MRM 0.6 lg/kg [61]

SPE: Ionexchange andcarbon-based

Acrylamide Potatoes, cereals,coffee, bread,chocolate, etc.

Synergi Hydro-RP0.5% methanol, 0.1%acetic acid in water

Water SPE: polymericand ion exchange

95% ESI Triplequadrupole

MRM 10 lg/kga [16]

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Acrylamide Coffees Synergi Hydro-RP0.5% methanol inwater

Water SPE: polymericand ion exchange


MRM 10 lg/kg [62]

Acrylamide Potatoes, cereals,bread, coffee,cacao, malt, etc.

Merck LiChrospher100 CN ACN/1%acetic acid solution

Water LLE: iso-hexaneEthyl acetateb


MRM 10 lg/kg [60]

HAs Meat extracts,bacon

Vydac C18 ACN/ammonium acetatesolution

Dichloromethane(diatomaceousearth)

SPE: ionexchange andC18

80% APCI Triplequadrupole

MRM 0.02–0.05 lg/kg [70]

HAs Lyophilized meatextract

TSK-gel ODS ACN/ammonium formatebuffer



75–98% APCI Ion trap Productionscan

0.8–10 lg/kg [71]

HAs Hamburgers,chicken

TosoHaas C18 ACN/formic acid solution

Dichloromethane SPE: ionexchange andC18


MRM 0.1 lg/kgc [80]

HAs Grilled meat Aquasil C18 ACN/ammonium formatebuffer


SPE: ionexchange


MRM – [72]

HAs Lyophilized meatextract

Symmetry C18 ACN/ammonium acetatebuffer

Ethyl acetate(diatomaceousearth)


– ESI Ion trap Product-ionscan

0.1–3.6 lg/kg [44]

Triplequadrupole

MRM 0.02–0.1 lg/kg

HAs Hamburgers,chicken, porkloin, lamb,sausages, etc.

Symmetry C8 ACN/ammonium acetatebuffer




MRM 0.02 lg/kgc [34]

aLOQ.bAfter extraction with water. Only on difficult matrices, such as cacao, soluble coffee, molasses, or malt.cThe lowest amount detected in food.

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extraction procedures often prove insufficient for deter-mination of acrylamide at low levels in very complexmatrices. Indeed, even acquiring in MRM mode requiresan SPE clean-up step with ion exchange, C18 sorbents ora combination of both (Table 3). For example, coffeeextracts that are among the most challenging samplesrequire two clean-up steps due to co-extractives in thefinal extract [16,62].LC–MS/MS analysis of acrylamide in food samples is

generally performed using carbon-based [61,63,64] andC18 [16,59,62,65] columns, although CN [60] andpolymethacrylate [13] columns are also used. An ESIsource in positive mode, in combination with triple-quadrupole analyzers, is the instrument most frequentlyutilized to obtain LODs in the range 5–60 lg/kg,depending of the food sample. To achieve lower LODs(0.6 lg/kg), Becalski et al. [61] used extensive clean-upprocedures, wherein anion-exchange, cation-exchange,and carbon-based cartridges were connected in tandem.Acrylamide protonated ion is obtained in the API

source, and fragmentation of this precursor ion in acollision cell reveals a prominent product ion of m/z 55due to loss of NH3, while the other transitions exhibitvery low intensities (0.2–2%). In complex food samples,such as soluble coffee or cocoa powder, high backgroundlevels of interfering co-extractives undergoing the sametransition as acrylamide (m/z 72 fi 55) are observed. Inthese settings, the transition m/z 72 fi 54 proved to bean alternative due to its higher specificity. However, itshould be noted that higher injection volumes or analyteenrichment are necessary to achieve adequate sensitivity[13].To establish acrylamide exposure in humans, analysis

of this compound in several foods has been performed.Konings et al. [59] analyzed 344 food products andfound acrylamide concentration levels of <30–3100lg/kg. These results were then used to estimate acryl-amide exposure in consumers who had participated in a1998 National Food Consumption Survey (NFCS) in TheNetherlands (n = 6250). Mean acrylamide exposure ofNFCS participants was 0.48 lg/kg bw/day, from whichthe authors concluded the risk of neurotoxicity to benegligible, although the risk of cancer was not neces-sarily negligible.Another family of compounds generated during food

processing are HAs, the major mutagens found incooked foods [66]. HAs are formed during high-temperature cooking (125–250�C) of protein-rich foods,such as meat and fish. Constant exposure to thesechemicals over many years may pose a health hazard tohumans. In fact, several epidemiological studies indicatea link between consumption of cooked foods containingHAs and incidence of colorectal [67], breast [68], andprostate [69] cancer.LC–MS/MS remains the optimum choice for low-level

detection of these polar and ionizable compounds in


complex matrices. The parameters used in these variousmethods for theanalysis ofHAsare also included inTable3.Most studies utilize reversed-phase chromato-graphywith C18 [44,70–72] and C8 [34] columns. Generally, acoupled LLE and SPE are used. In this procedure, anextraction is performed with diatomaceous earth permit-ting direct coupling to SPE clean-up. Dichloromethane isfrequently used as an extractant, although ethyl acetate[44] or dichloromethane/toluene [72] has also beenproposed. The extract eluted is then directly transferredto cartridges used in tandem for clean-up (ion exchangeand C18) that allows the cleaning of the extract and thepre-concentration of the amines.Both ESI and APCI ionization techniques have been

used for analysis of HAs in food, with ESI generallyproviding better LODs. Barcelo-Barrachina et al. [44]reported LODs of 0.1–3.6 lg/kg for the analysis of HAsin a lyophilized meat extract using ESI. However, thesame sample under APCI analysis yielded LODs in therange 0.8–10 lg/kg [71]. In both cases, an ion-trapinstrument in product ion scan was used to conductanalysis. Triple-quadrupole instruments are typicallypreferred for analysis of HAs, since low LODs can beattained in MRM mode. As an illustration, Fig. 5 displaysthe LC–MS/MS chromatogram obtained for a sample ofgriddled chicken, where a significant number of HAswere identified. A comparison of both triple-quadrupoleand ion-trap instruments in the analysis of HAs usinglyophilized meat extract as a sample matrix showed thatLODs (0.1–0.02 lg/kg) in triple-quadrupole instrumentswere 19–360 times lower (depending on the amine)than those with the ion-trap instrument [44]. None-theless, the product-ion scan mode with an ion trap canbe used to prevent false peak identification.Recently, a feasibility study and two interlaboratory

exercises on the determination of selected HAs in beefextract, under the aegis of a European working group,were completed [73]. Some LC–MS/MS methods wereevaluated during this exercise using both APCI and ESIionization sources, with triple-quadrupole and ion-trapinstruments. The results of this study showed thatLC–MS/MS remains the optimum technique, at least forsamples with very low amounts of amines.

4.4. ToxinsThe study and characterization of toxins produced byliving organisms posing a significant threat to humanhealth is a problem of tremendous importance, and bothLC–MS and LC–MS/MS have been used to identify anddetermine these compounds in food. There are manytypes of toxins produced by living organisms. For exam-ple, microbial toxins may result from microbial growth infoods or, as in the case of fungal toxins, also known asmycotoxins, from mold growth during the harvesting ofagricultural crops. Other toxins from different origins,such as phycotoxins or algal toxins, such as azaspiracids,

Figure 5. Chromatogram of HAs from a sample of griddled chicken breast obtained by LC-ESI-MS/MS. Chromatographic conditions: Symmetry C8 column (150 · 2.1 mm i.d.); gradient elutionwith 30 mM acetic acid–ammonium acetate (pH 4.5) buffer and acetonitrile (from 5% to 60%) as mobile phase (reprinted from [34] with permission).

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Table 4. Analysis of mycotoxins in food samples by LC–MS/MS

Compound Food product LC conditions Extraction Clean-up Recoveries Ionizationsource


LODs Reference

Azaspiracid Mussels C18 l-HPLCcolumn ACN/water (0.03%TFA)

Acetone – – ESI Triplequadrupole

MRM 20 lg/kga [75]

Azaspiracid Mussels Luna-2 C18

ACN/ammoniumacetate solution(0.05% TFA)

Acetone–ethylacetate

– – ESI Ion-trap Product ionscan (MS3 )

0.4–3.2 lg/kg [6]

Penicilliummycotoxins

Food products Symmetry C18

methanol/ammoniumacetate solution

ACN–waterwith 0.1%formic acid (9:1v/v)

– 75–116% APCI Ion-trap MRM 5–20 lg/kg [35]

Trichothecenemycotoxins

Maize Alltima LC-18 ACN:water(75:25 v/v)

SPE: carbon-based sorbent


MRM 1.5–10 lg/kg [14]

Alternariamycotoxins

Fruit juices andbeverages

Inertsil-ODS2water/methanol/ACN

SPE: Bond-ElutC18 andaminopropyl


MRM 0.5 lg/L [79]

Polyethermarine toxins

Mussels, marinephytoplankton

Luna C18 ACN/water withammoniumacetate

Methanol–waterchloroform

– – ESI Triplequadrupole

MRM 0.48 lg/kg [78]

Trichothecenemycotoxins,zearalenone

Maize Aquasil RP-18methanol/ammoniumacetate solution

ACN–water(84:16 v/v)

SPE: MycoSep226 column

73–89% APCI Triplequadrupole

MRM 0.3–3.8 lg/kg [77]

aEstimated from instrumental LOD.

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which are produced by marine dinoflagellates [6,74,75],are also important. For some of them, such as severalfungi mycotoxins (Penicillium and Aspergillus species), arestrictive MRL value (0.1 lg/kg) has been established bythe EU for baby foods and processed cereal-based foods forinfants and young children [76].Table 4 summarizes applications of LC–MS/MS in the

determination of some toxins in food products. As withmost of the compounds previously discussed, separationof toxins is basically conducted by reversed-phasechromatography using C18 columns. Extraction of toxinsis currently performed using organic solvents, such asacetone [6,75], ethylacetate [6], different amounts ofACN:water [14,35,77] or methanol:water and chloro-form [78], although SPE procedures have been directlyused in the case of liquid samples. For example, for fruitjuices and other beverages, the use of two sorbents,reversed-phase and aminoprolyl, is recommended [79].In addition to an extraction step, in some cases, clean-upprocedures based on SPE are suggested in order to obtainextracts without interference [14,77], as happens in thepaper published by Lagana et al. [14], who used carbon-based SPE cartridges for clean-up during the analysis oftrichothecene mycotoxins in maize to eliminate lipidsand pigments. This system permitted the pre-concen-tration of slightly polar trichothecenes as well as thosewith higher polarities, such as nivalenol, which containsfour hydroxyl groups. Good recoveries for each of thecompounds were obtained (81–98%), which are similarto those of Berthiller et al. (78–89%) [77] using a multi-functional sorbent especially designed for mycotoxinanalysis.Both ESI and APCI ionization sources have been used

for coupling to MS. ESI is recommended for analysis ofazaspiracids [6,74,75], compounds easily ionized at lowpH in positive mode, as well as for alternaria mycotoxinsin beverages [79]. In the latter, comparison of APCI andESI in both positive and negative modes was performed,and ESI was recommended, as it offers greater sensitivity.Moreover, negative mode is preferable, since a significantnumber of adducts are formed in positive mode. Forother compounds, APCI provides greater sensitivity thanESI, as exemplified in the aforementioned study byBerthiller et al. [77], who analyzed trichothecenemycotoxins in maize using APCI and a triple-quadrupoleinstrument and obtained a better LOD for deoxynivalenol(0.8 lg/kg) than that reported by Lagana et al. [14](1.5 lg/kg) using ESI in negative ionization.As for the other toxic compounds commented on

before, triple-quadrupole instruments seem best suitedfor mycotoxin quantitation in food samples. Neverthe-less, ion-trap analyzers have also been used for toxindetermination in food [6,35] and, in some cases, resultssimilar to those achieved by triple-quadrupole instru-ments are obtained. As an example, Lehane et al. [6],using a selective LC–MS3 method with an ion trap for

analysis of azaspiracids, obtained LODs of 0.4–3.2 lg/kgin mussels. The same authors have recently identifiedfive new hydroxyl analogues of azaspiracids in shellfishusing the previously proposed method [74].

5. Conclusions

The relatively high number of publications on theanalysis of toxic substances in food samples by LCcoupled to MS/MS shows that this technique has becomea powerful tool in the quality control of food productsand the safeguarding of human health. However, anal-ysis of these compounds is not a simple task due to thecomplexity of the matrices, which require exhaustiveclean-up procedures if accurate sample quantitations areto be obtained. Generally, an extraction step followed byclean-up using SPE is performed, and only in rareinstances is direct injection of the initial extract possible.ESI is the most frequently utilized ionization source

due to its higher sensitivity for toxic compounds, most ofwhich are readily ionizable in liquid phase. However, forsome compounds, better results are obtained using APCI,suggesting that both API sources should be consideredwhen establishing new methodologies of food analysis.Although both ion-trap and triple-quadrupole instru-ments are frequently used in this field, the latter is morecommonly encountered, as its lower LODs permit com-pliance with the strict MRLs set by governing authori-ties. Aspects regarding unequivocal confirmation of toxiccompounds must also be mentioned. In ion trap, forexample, product-ion scan permits confirmation ofanalytes, while in triple-quadruple at least two precursorion-product ion transitions must be acquired. For multi-residue analysis, a greater number of compounds can bemonitored using a triple-quadrupole instrumentalthough increases in LODs are frequently observed. Anadditional advantage of ion-trap instruments is theirhigh selectivity, utilizing multi-step MS, which can avoidinterferences.For quantitative purposes, isotopic dilution is used in

those instances where a small number of compoundshave to be determined. For multi-residue analysis, thematrix-matched method is recommended, since nolabeled standards for all analytes are currently available.External calibration is rarely used and then is only pro-posed for pesticides in simple matrices, such as vege-tables and fruits, and always after demonstrating that nomatrix effect occurs.For routine analysis of food-product compounds, only

well-established tandem techniques, such as triplequadrupoles and MS/MS in ion traps, are in commonuse. More recent approaches in LC–MS/MS, includinglinear traps, new-generation triple quadrupoles, andhybrid instruments, Q-TOF and Q-linear traps that aregaining widespread acceptance in other fields, have not



yet been used in food analysis. These instruments offeradvantages, such as high scanning speeds, accuratemass measurement (TOF), and increased sensitivity(linear traps and new-generation triple quadrupoles). Nodoubt their applicability to this field will be demonstratedin the near future.

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LC–MS/MS analysis of organic toxics in food

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