matrix effects in quantitative pesticide analysis using liquid chromatography–mass ... ·...

Guest Editor: Yolanda Pico

MATRIX EFFECTS IN QUANTITATIVE PESTICIDE ANALYSISUSING LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY

W.M.A. Niessen,1,2* P. Manini,3 and R. Andreoli31hyphen MassSpec, de Wetstraat 8, 2332 XT Leiden, the Netherlands2Department of Analytical Chemistry and Applied Spectroscopy,Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands3Laboratory of Industrial Toxicology, Department of Clinical Medicine,Nephrology and Health Sciences, University of Parma, via Gramsci 14,43100 Parma, Italy

Received 17 September 2005; received (revised) 27 November 2005; accepted 6 December 2005

Published online 16 June 2006 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20097

Combined liquid chromatography–mass spectrometry usingelectrospray or atmospheric-pressure chemical ionization hasbecome an important tool in the quantitative analysis of pesti-cide residues in various matrices in relation to environmentalanalysis, food safety, and biological exposure monitoring. Oneof the major problems in the quantitative analysis using LC–MS is that compound and matrix-dependent response suppres-sion or enhancement may occur, the so-called matrix effect.This article reviews issues related to matrix effects, focusing onquantitative pesticide analysis, but also paying attention toexpertise with respect to matrix effects acquired in otherapplication areas of LC–MS, especially quantitative bioanalysisin the course of drug development. # 2006 Wiley Periodicals,Inc., Mass Spec Rev 25:881–899, 2006Keywords: matrix effects; ion suppression; quantitativeanalysis; pesticide analysis; multiresidue analysis; electrosprayionization; atmospheric-pressure chemical ionization; LC–MS

I. INTRODUCTION

From the early days of the development of combined liquidchromatography–mass spectrometry (LC–MS) onwards, pesti-cide analysis has been an important application area. Whileinitially the research aimed at analyzing pesticides in environ-mental samples, especially water, more recently applicationswith respect to food safety and biological exposure monitoringbecome increasingly important. Quantitative analysis of pesti-cides and their transformation products are of special interest.This application has gained significant impetus from theintroduction of interfacing and ionization strategies based onatmospheric-pressure ionization, that is, electrospray ionization(ESI) and atmospheric-pressure chemical ionization (APCI).

An important issue in the method development of quanti-tative analysis using LC–MS via ESI or APCI is the possibleoccurrence of matrix effects. In most cases, a matrix effect isconsidered to be an (unexpected) suppression or enhancement ofthe analyte response due to co-eluting matrix constituents. It canbe easily detected by comparing the response obtained from astandard solution and that from a spiked pre-treated sample (post-extraction spike). An example of matrix suppression is illustratedfor the pesticide triflumisol in different matrices (Fig. 1).Triflumisol peaks acquired by LC-ESI-MS in selected reactionmonitoring (SRM) mode in solvent or post-extraction spikedextracts of eggplant, lettuce, and pepper are compared. Co-eluting matrix components in the pepper extracts almostcompletely suppress the analyte response, while for eggplantand lettuce extracts less, but still significant suppression isobserved (Aguera et al., 2004).

However, detailed studies on matrix effects in quantitativebioanalysis revealed that the ion suppression or enhancement isfrequently accompanied by significant deterioration of theprecision of the analytical method (Matuszewski, Constanzer,& Chavez-Eng, 1998, 2003). If post-extraction spiked samplesproduced from different lots of the same matrix, for example,‘‘plasma,’’ were analyzed, different results were obtained. Thisissue is nicely pictured in Figure 2. The precision (%RSD) uponrepetitive injection of post-extraction spiked plasma samples isplotted as a function of the analyte concentration for a single lotand for five different lots of plasma (Matuszewski, Constanzer, &Chavez-Eng, 2003). While for the single plasma lot the precisionis acceptable, it is not when the five different plasma lots are takeninto account. In practice, samples from different sources must beanalyzed. In a discussion on matrix effects, it can be useful todiscriminate between ion suppression (or enhancement) by thematrix at one hand, and different matrix effects exerted bydifferent sample lots at the other hand. A useful nomenclaturewas suggested and is adopted in this article: the difference inresponse between the solvent sample and the post-extractionspiked sample is called the ‘‘absolute matrix effect,’’ while thedifference in response between various lots of post-extraction

Mass Spectrometry Reviews, 2006, 25, 881– 899# 2006 by Wiley Periodicals, Inc.

————*Correspondence to: W.M.A. Niessen, hyphen MassSpec Consultancy,

de Wetstraat 8, 2332 XT Leiden, the Netherlands.

E-mail: [email protected]

spiked samples is called the ‘‘relative matrix effect’’ (Matus-zewski, Constanzer, & Chavez-Eng, 2003). If no counteraction istaken, an absolute matrix effect will primarily affect the accuracyof the method, while a relative matrix effect will primarily affectthe precision of the method.

A repeatable and reproducible ion suppression (or enhance-ment) in a quantitative analytical method should not posesignificant problems, at least when analyte standards areavailable. Such problems can be eliminated by the use ofmatrix-matched standards or other means, as outlined in moredetail below. However, if post-extraction spiked samples ofdifferent lots of the same matrix after sample pre-treatment and

LC give different and/or (strongly) varying analyte responses, thematrix effect has a much greater impact on the reliability of theanalytical data (Matuszewski, Constanzer, & Chavez-Eng, 1998,2003; Bogialli et al., 2004).

Some researchers believe that the use of tandem massspectrometry (MS–MS) or SRM eliminates or reduces matrixeffects. However, this is a misconception. The matrix effects aregenerally due to the influence of co-eluting compounds onthe actual analyte ionization process, that is, well before theanalyte ions enter the high vacuum of the mass analyzer.Therefore, matrix effects must be solved prior to or during theanalyte ionization by ESI or APCI.

FIGURE 1. Matrix suppression in the LC-ESI-MS analysis of triflumisol in different matrices. The

compound was isolated by liquid extraction with ethyl acetate, evaporation to dryness, and reconstitution in

methanol. Reprinted from Aguera et al. (2004) with permission. �2004, Elsevier B.V.

FIGURE 2. Precision (n¼ 5, %CV) of a bioanalytical method at various analyte concentrations,

determined in either a single plasma lot or in five different plasma lots. While for the single plasma lot

the precision is acceptable, it is not one five different plasma lots are taken into account. Reprinted from

Matuszewski, Constanzer, & Chavez-Eng (2003) with permission. �2003, American Chemical Society.

& NIESSEN, MANINI, AND ANDREOLI

882 Mass Spectrometry Reviews DOI 10.1002/mas

Response suppression or enhancement effects may beexerted by any co-eluting component entering the atmospheric-pressure ionization source via the liquid stream. Some mobile-phase additives are also known to suppress or enhance analyteresponse. Although such effects are sometimes called matrixeffects, it appears useful to discriminate between effects due tothe analytical system, for example, mobile-phase composition,source parameters, and effects due to the actual analyte matrix.

Although one may erroneously get this impression, matrixeffects are certainly not only observed with ESI-MS. Already in1988, sample effects were observed in the thermospray LC–MSanalysis of labeled serotonin and some catecholamines (Gelpı,Abian, & Artigas, 1988). More recently, matrix effect in theultratrace analysis of pesticide residues in food and bioticmatrices by GC–MS or LC–MS were extensively reviewed(Hajslova and Zrostlıkova, 2003). The consequences of ionsuppression in LC–MS in residue analysis were discussed(Antignac et al., 2005).

Matrix effects are important in any type of quantitativeanalysis of real-life samples using LC–MS, but also in screeningstudies. This article reviews the current understanding and state-of-the-art in relation to matrix effects in quantitative analysisusing LC–MS, with a special focus on pesticide analysis.Since the mechanisms of matrix effects have been studied inconsiderable detail in relation to quantitative bioanalysis inpharmaceutical drug development, some of the data obtainedin that area are discussed as well.

II. INITIAL STUDIES INQUANTITATIVE BIOANALYSIS

Buhrman et al. were the first to report matrix-related ionsuppression in LC-ESI-MS in their comparison of three differentsample pre-treatment methods in the analysis of the platelet-activating factor receptor antagonist SR27417 in human plasma(Buhrman, Price, & Rudewicz, 1996). They evaluated theirresults in terms of extraction efficiency, ion suppression, andoverall process efficiency. With hexane liquid–liquid extraction(LLE), ethyl acetate back-extraction and solid-phase extraction(SPE), they achieved extraction efficiencies between 73% and78%. Significant ion suppression was observed in SPE andhexane LLE. They emphasized the importance of the sample pre-treatment as a tool to avoid or minimize matrix effects.

Subsequently, the group of Matuszewski discussed matrixeffects in more detail, that is, in the bioanalysis of indinavir andfinasteride in human plasma (Fu, Woolf, & Matuszewski, 1998;Matuszewski, Constanzer, & Chavez-Eng, 1998).

In the method development for the analysis of indinavir inhuman plasma, comparison was made between two sample pre-treatment methods, that is, dilution of urine and LLE with methyltert-butyl ether, two chromatographic systems, that is, with lowand high capacity factor, and the use of either isotopically labeledor analog internal standards (Fu, Woolf, & Matuszewski, 1998).They concluded that the potential effect of co-eluting ‘unseen’endogenous species on the analyte response should be evaluatedduring method development and validation of bioanalytical LC–MS methods.

Method development in the quantitative bioanalysis offinasteride was initially directed at the use of ESI-MS(Matuszewski, Constanzer, & Chavez-Eng, 1998). An analoginternal standard (IS) was used. The precision in the finasteridepeak area upon injection of standard solutions was better than5%RSD, while a precision ranging between 17%RSD and43%RSD was obtained with extracts of five different lots ofcontrol human plasma. This indicates a significant relative matrixeffect. Subsequently, the response of the internal standard in post-extraction spiked plasma samples was evaluated for fivedifferent plasma extracts under two different sample pre-treatment and two different chromatographic conditions.Although the enhanced selectivity of the extraction andefficiency of the LC separation showed some improvement, theprecision and accuracy of the ESI-MS method remainedinadequate to support clinical studies. Therefore, an LC–MSmethod based on the use of APCI was successfully developedand validated.

III. MATRIX EFFECTS IN PESTICIDE ANALYSIS

Pesticides and related compounds are analyzed in three differentmatrices. Initially, LC–MS was applied in the determination ofpesticides in environmental samples, especially different watercompartments, but also soil, sludge, and other environmentallyrelevant matrices (Hogenboom, Niessen, & Brinkman, 2001;Geerdink, Niessen, & Brinkman, 2002; Pico, Blasco, & Font,2004). More recently, there is a growing interest in the use of LC–MS in the determination of pesticide residues in food, mainlyfruits and vegetables (Pico, Blasco, & Font, 2004). In addition,there is significant interest in the determination of pesticides inbiological fluids and tissues, especially with respect to biologicalexposure monitoring (Barr & Needham, 2002) and/or (forensic)toxicology (Lacassie et al., 2001). Given the difference inmatrices, these application areas are treated separately in thefollowing sections.

A. Pesticides in Water and OtherEnvironmental Matrices

Environmental analysis of pesticides is primarily aimed at waterused for human consumption and the production thereof from itsprimary sources, surface water and ground water. Early warning,monitoring, and surveillance programs have been established(Hogenboom, Niessen, & Brinkman, 2001; Geerdink, Niessen, &Brinkman, 2002; Pico, Blasco, & Font, 2004). Europeandirectives for drinking water (98/83/EC) and similar directivesfrom regulatory bodies in other countries are relevant withrespect to the concentration levels that need to be analyzed. TheEuropean directive states that the concentration of pesticides andits related products should not exceed the level of 0.1 mg/L forindividual compounds and 0.5 mg/L for the total pesticideconcentration. Monitoring for compliance to such regulationsinvolves: (i) multiresidue screening of target compounds, (ii)quantitative analysis and confirmation of identity of anypesticide at or above the threshold level, (iii) collection ofinformation on non-target compounds that may be considered

MATRIX EFFECTS IN LC–MS &

Mass Spectrometry Reviews DOI 10.1002/mas 883

harmful in a similar way as pesticides, for example, pesticidetransformation products.

In the more recent studies, often criteria established byregulatory bodies for residues of veterinary medicine in food, forexample, 2002/657/EC within the European Union, are appliedin quantitation and confirmation of identity (Hernandez et al.,2004a). In addition, monitoring of other environmental compart-ments is performed, for instance to acquire knowledge on the useand proliferation of pesticides, their persistency in the environ-ments, and their possible toxic effect on plants, animals and otherbiota. Some examples of studies where special attention was paidto matrix effects are reviewed below.

Although some early studies indicated response suppressionby matrix constituents (Honing et al., 1996), the interest in matrixeffects followed the growing awareness of this phenomenon inquantitative applications of LC–MS in the drug discoverydevelopment studies. Early studies on multiresidue analysis ofacidic pesticides, organophosphorus pesticides, and imidazoli-none herbicides in estuarine water, ground water, and soil do notmention any problems with ion suppression due to matrix effects(Chiron et al., 1995; Lacorte and Barcelo, 1996; Lagana, Fago, &Marino, 1998). In general, analyte recoveries, usually better than80%, were determined without paying attention to the fact thatthe values obtained might result from a combination of poorextraction recovery and losses due to ion suppression. Despite theoften high background levels and even, in some cases, theobservation of a humic acid hump in the total-ion chromato-grams, no questions were raised concerning the potentialinfluence of co-eluting compounds on the ionization efficiency.The success in such quantitative studies should perhaps beattributed to the relatively high chromatographic resolutionachieved on the 100–250 mm long LC columns used, showingsignificant retention of analytes relative to the solvent front in thegradient elution runs, and the relatively elaborate sample pre-treatment performed.

Problems with matrix effects were reported for desmethyl-metoxuron in a single short-column LC–MS study (Hogenboomet al., 1997). Poor accuracy and precision were achieved in LC-APCI-MS for this polar transformation product due to co-elutionwith the solvent front, which resulted from the poor chromato-graphic resolution inherent to the 10-mm long columnsused. However, these data were not interpreted in terms ofmatrix effects.

Steen et al. investigated the influence of different amounts of(co-extracted) humic acids on the signal intensity for atrazineand three of its transformation products, diuron and two of itstransformation products, chloridazon, metolachlor, and AIPA(Steen et al., 1999). Direct infusion of surface water sampleswithout sample pre-treatment resulted in more than 90%suppression, relative to standard solutions (Fig. 3). Muchbetter results were achieved when LC was applied in combinationwith various SPE strategies (SPE at either pH 3 or pH 7, ordual-SPE approach), although for some compounds still morethan 25% suppression was observed. It was also found that thesignal suppression depends on the origin of the sample. Inthis particular study, surface water from the Amsterdam-Rhine Canal showed more pronounced suppression than thelow-salinity water from Western Scheldt estuaries (Steenet al., 1999).

Hogenboom et al. compared overall recoveries of polar andacidic pesticides, that is, without discriminating betweenextraction recovery and signal suppression, in three differentSPE-LC–MS-MS setups, including two dual-column SPEprocedures (Hogenboom et al., 2000). The overall recovery fromtap water, showing less matrix contamination, was always betterthan that from surface water, while the dual-SPE-columnapproach showed improvement compared to the single-SPE-column one. In another study, slightly lower overall recoverieswere also reported for surface water compared to drinking waterfor phenoxyacid herbicides (Marchese et al., 2002).

Dijkman et al. systematically studied the effects of matrixinterferences in the LC–MS analysis of acidic pesticides(Dijkman et al., 2001). Three different LC systems werecompared: (i) a single column LC, (ii) on-line SPE–LC, and(iii) coupled-column LC–LC. Different column combinationsfor LC–LC were evaluated as well. Both ESI and APCI in thenegative-ion mode were applied for analyte ionization prior toMS–MS in SRM mode. Effects of salinity and dissolved organiccarbon (DOC) were investigated. Milli-Q and tap water werefortified with 12 mg/L DOC. They conclude that saline samplescan only be analyzed after column-switching, either SPE–LC orLC–LC because otherwise the response of early elutingcompounds is severely influenced, and that the LC–LC approachwith Supelco ABZþ material in the second column is the mostversatile setup to exclude matrix effects (Dijkman et al., 2001).

The limited interest in matrix effects in subsequent yearsshould perhaps be understood as an indication that no seriousproblems with matrix effects are observed in this applicationarea. Researchers seem to be satisfied with determining overallrecoveries better than 80% and %RSD better than 10%–15%.

In recent years, environmental water analysis has broadenedits focus from pesticide analysis towards a wider variety ofcompounds, especially endocrine disrupters, including surfac-tants and their transformation products, pharmaceuticals such asestrogens and antibiotics, marine toxins like microcystinhepatotoxins (Petrovic et al., 2002; Zwiener & Frimmel,2004a,b). In this respect, matrix effects were evaluated bycomparison of response in solvent standards and post-extractionspiked samples for six different water compartments, that is,surface water, rain water, ground water, channel water, waste-water treatment plant effluents, and industrial effluents (Benijtset al., 2004). In addition, the influence of various mobile-phase additives on the observed matrix effect was evaluated(see below).

B. Pesticide Residue Analysis in Food

Pesticides are widely used in food production in either field orpost-harvest protection of crops. Monitoring is aimed at avoidingrisks with these toxic compounds to consumers. Maximumresidue levels (MRL) have been established by regulatory bodies,for example, 2003/13/EC, to ensure food safety in this respect.Typical MRL are in the range of 0.01 and 5 mg/kg, depending onthe pesticide. Monitoring for adherence to regulations involves:(i) screening for target compounds, which are known to beapplied on certain crops, (ii) quantitative analysis and confirma-tion of identity, and (iii) more general screening programs. In



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these studies, often criteria established by regulatory bodies forresidues of veterinary medicine in food, for example, 2002/657/EC within the European Union, are applied in quantitation andconfirmation of identity.

Food extracts are obviously far more complex matrices thanenvironmental water. Therefore, considerable more attention ispaid to matrix effects, especially absolute matrix effects, in thedevelopment of quantitative methods for pesticides in food.

Response enhancement due to matrix constituents wasreported by Barnes et al. for the LC-APCI-MS analysis ofdiflubenzuron and clofentezine in fruit drinks based on plums,strawberries, and blackcurrants (Barnes et al., 1995). Subse-quently, the same group showed that different calibration curveswere obtained in LC-APCI-MS for the pesticide fenbutatin oxideusing matrix-matched standards of tomatoes, cucumbers, andbananas. Minor enhancement was found for tomatoes andcucumbers, while suppression was observed for bananas (Barneset al., 1997b). These and other data from the same group (Barneset al., 1997a) indicate the importance of the use of matrix-matched standards in pesticide analysis in food. On the otherhand, no influences on responses of 12 carbamates were foundfrom 10 vegetable and fruit samples (Di Corcia et al., 1996).

Matrix effects are known to be both compound and matrixdependent. In this respect, interesting results were reported forthe analysis of chlormequat in different matrices. While onegroup prefers the use of matrix-matched standards in the analysisof chlormequat in pears (Startin et al., 1999), another found nostatistically significant difference between solvent standards andmatrix-matched standards in the analysis of chlormequat intomato products (Careri et al., 2002). In both cases, ion-exchangeLC in combination with ESI-MS was applied. In the lattermethod, which is in agreement with the recommended methodwithin EU (CEN, 2002), a (D4)-isotopically labeled internalstandard was used, which provides adequate correction formatrix effects (see below).

Despite the fact that matrix-matched standards were used inthe analysis of carbendazine, thiabendazole, and six phenylureaherbicides in orange extracts, significant matrix effects wereobserved (Bester et al., 2001). The application of an additionalclean-up step using size-exclusion chromatography solved thisproblem.

In developing a multiresidue method for the determinationof 74 pesticides in fruit and vegetables, the absolute matrixeffects in 13 representative matrices were studied: lemon,grape, strawberry, apple, nectarine, tomato, carrot, green salad,cucumber, eggplant, spinach, potato, and pepper (Ortelli, Edder,& Corvi, 2004). Comparison of the response between standardsolutions and matrix extracts fortified at 0.1 mg/kg was made.The results were reported, averaged per pesticide, as an accuracyand %RSD (n¼ 13). The mean accuracy value was 92% withvalues ranging from 63% to 133% and with %RSD ranging from10% to 40%. The %RSD values clearly indicate that matrix-matched standards must be used because matrix effects aresignificantly different for different matrices (and differentanalytes). Similar data were reported for 57 pesticides andmetabolites in fruits and vegetables in 14 different commoditygroups by others (Jansson et al., 2004). Commodity groupsare fruits or vegetables which share common features, forexample, high water content, high fat content, high acid content

(SANCO/825/00). Matrix effects in the multiresidue analysisof 20 pesticides in 8 different vegetables, that is, cucumber,tomato, pepper, green bean, eggplant, zucchini, melon, andwatermelon, were investigated to select one representativematrix for calibration purposes in the analysis of samples fromall 8 different matrices (Martınez Vidal et al., 2005). A cucumberblank extract was selected for this purpose. It thus may beconsidered as a representative matrix for vegetables in thiscommodity group (fruiting vegetables). For some pesticides andmatrices, differences in the calculated concentrations higherthan 10% were obtained in comparing calibration using therepresentative cucumber matrix and calibration using a matrix-matched standard.

The group of Pico compared various extraction proceduresin the LC–APCI-MS analysis of pesticides in fruits by LC-MS,for example, SPE, LLE, solid-liquid extraction (SLE), stir-barsorptive extraction (SBSE), matrix solid-phase dispersion(MSPD), solid-phase microextraction (SPME) (Blasco, Font, &Pico, 2002, 2004; Juan-Garcıa et al., 2004; Soler, Manes, & Pico,2004, 2005). In these studies, matrix effects were primarilyinvestigated regarding their influence on the extraction recovery,that is, in term of overall recoveries. In a comparison betweenSPE and SBSE for six fungicides in grapes, no difference inresponse was observed for SBSE between standards in methanoland in matrix, while in SPE a 0%–15% response enhancementwas observed (Juan-Garcıa et al., 2004). In a comparison betweenMSPD and SLE in the LC–ESI-MS analysis of pesticides inoranges and strawberries, it was shown that the matrixsuppression was lower in MSPD extracts for propanil, cyproco-nazole, pyrifenox, and tebufenpyrad, while it was lower in SLEextracts for kresoxim methyl, 8-cyhalotrin, acrinatrin, andcarbosulfan. For pyriproxifen, more than 30% signal enhance-ment was observed in both MSPD and SLE extracts (Soler,Manes, & Pico, 2004). A similar comparison was made for othercompounds and other matrices (Soler, Manes, & Pico, 2005). Inanother study, SLE was applied to extract imidacloprid,carbendazim, thiabendazole, methiocarb, imazalil, and hexythia-nox from oranges prior to LC–APCI-MS analysis (Blasco, Font,& Pico, 2004). Matrix enhancement was observed for the firstthree, and suppression for the latter three pesticides. The use ofmultiple stages of MS–MS in the ion-trap instrument resulted ina reduced matrix effect, that is, both less enhancement or lesssuppression. No clear explanation could be given for this effectbecause the matrix effect occurs in the ion source prior to anymass analysis.

The effect of the solvent choice for the liquid extraction oforganophosphorus pesticides from fruits and vegetables on thematrix suppression was evaluated for cabbage and grapes (Mol,van Dam, & Steijger, 2003). Dispersion of the fruit or vegetablein acetone and subsequent extraction by dichloromethane/petroleum ether did not result in any matrix-induced suppressionor enhancement. With ethyl acetate extraction, the matrix effectwas very limited (<10%), while significant effects were observedwith aqueous acetic acid, methanol, or acetone as extractionsolvent.

Severe absolute matrix effects were reported in the analysisof triflumisol in pepper (Fig. 1), as the signal was suppressedalmost completely. Signal enhancements were observed for othercompounds, for example, for dimethomorf and azoxystrobin in



pepper and lufenuron and flufenoxuron in eggplant (Aguera et al.,2004). The results of a study on the absolute matrix effects in thedetermination of 24 new pesticides in processed fruits andvegetables are shown in Figure 4 (Sannino, Bolzoni, & Bandini,2004). The responses of solvent standards and matrix-matchedstandards of apple puree, concentrated lemon juice, andtomato puree were compared. While the matrix effects haveonly a limited effect for most compounds, strong signalenhancement was observed for indoxacarb in all three matricesand fenpyroximate in apple and lemon samples, while theresponse of etofenprox was significantly suppressed in all threematrices. Similar results were reported by others for otherpesticides and matrices (Granby, Andersen, & Christensen,2004)

A comparison of the slopes of calibration curves of matrix-matched standards of 15 pesticides in 7 different matrices, thatis, pepper, tomato, apple, broccoli, lemon, orange, and melonshowed that some compounds experienced negligible matrixeffects in these matrices, for example, carbendazim andthiabendazole (Ferrer, Garcıa-Reyes, & Fernandez-Alba, 2005;Ferrer et al., 2005; Ferrer, Thurman, & Fernandez-Alba, 2005).For other compounds, for example, triflumizol, the responsestrongly depended on the matrix, although the pepper matrixshowed little influence in this case (in contrast to Fig. 1). Forbroccoli, the last four eluting pesticides showed higher matrixsuppression (Ferrer, Garcıa-Reyes, & Fernandez-Alba, 2005).

From these as well as other published studies, it may beconcluded that matrix effects can play a very important role in theresidue analysis of pesticides in food. Most researchers recognizethe importance of these effects to achieve reliable results. Theseeffects must be studied for each individual pesticide and in eachindividual matrix, although different commodities have beendefined recently (SANCO/825/00). In general, matrix-matchedstandards are considered the most useful approach to eliminatethe consequences of absolute matrix effects on the reliability(accuracy and precision) of the data. However, systematic studiesshowing that matrix effects for a particular pesticide and aparticular matrix are similar for fruits or vegetables from, forinstance, different regions have not yet been reported. Matrix-matched standards are frequently made using extracts ofbiologically grown fruits and vegetables.

C. Pesticides in Biological Fluids and Tissues

To monitor biological exposure effects of pesticides, thecompounds as well as their metabolites are analyzed in biologicalfluids and tissues. In general, the analysis is concentrated atcarefully selected biomarkers of exposure, for example, atrazinemercapturate as the major human metabolite of the herbicideatrazine (Beeson, Driskell, & Barr, 1999). Obviously, theanalysis of urine samples is favored because of the non-invasivesampling, especially for the more polar pesticides or theirbiomarkers, although analysis in serum, plasma, breast milk, andtissues has been performed as well. Reviews on analyticalmethods for exposure monitoring (Barr & Needham, 2002) andthe application of LC–MS in this area (Hernandez, Sancho, &Pozo, 2005) were published. With respect to matrix effects, tosome extent advantage can be taken from the wide experience in

dealing with matrix effects in especially plasma analysis,acquired in quantitative bioanalysis for (pre-)clinical studiesfor drug development.

Contrary to pesticide analysis in water and food, wheremultiresidue analysis is pursued, the number of target com-pounds in exposure monitoring is generally very limited.Therefore, isotopically labeled internal standards can be applied,which correct for matrix effects. This is demonstrated inmonitoring urinary metabolites of atrazine, malathion, and 2,4-dichlorophenoxyacetic acid (Beeson, Driskell, & Barr, 1999).Matrix-matched standards are applied as much as possible,although in some cases finding appropriate blanks can be dif-ficult in human exposure studies (Manini, Andreoli, & Niessen,2004).

Coupled-column chromatography, involving the heart-cutof a fraction from the chromatogram of the first column andtransfer and separation onto a second column, in combinationwith ESI-MS, was applied to reduce matrix effects in thedetermination of chlorpyrifos and its main metabolite 3,5,6-trichloro-2-pyridinol in human serum and urine (Sancho, Pozo, &Hernandez, 2000). The same approach was later on used in thedetermination of 4-nitrophenol and 3-methyl-4-nitrophenol,metabolites of the organophosphorus pesticides parathion andfenitrothion, respectively, in human urine (Sancho et al., 2002;Hernandez, Sancho, & Pozo, 2004b).

In the method development for the urine analysis of 10biomarkers of exposure to a variety of organophosphoruspesticides, it was demonstrated that matrix-induced ion suppres-sion is especially important for early eluting compounds, whilelater eluting compounds are not affected (Olsson et al., 2003).The response of all analytes in fortified urine samples wasevaluated as a function of the injection volume. For the twoearly eluting compounds, a non-linear increase of the responsewas observed at injection volumes higher than 5 mL, whilewith later eluting compounds 10 mL could be injectedwithout any problem. For two other compounds, acephate andmethamidophos, an additional clean-up step was necessary toeliminate matrix effects. Unfortunately, the clean-up step withsolid-supported LLE showed a poor recovery for these twocompounds.

In a more elaborate study, involving a wider variety of targetmolecules, that is, metabolites from organophosphorus pesti-cides, synthetic pyrethroids, selected herbicides and DEET, thesame group followed a different approach to evaluate matrixeffects (Olsson et al., 2004). The analytes are both carboxylicacids, which are partly analyzed using APCI-MS, partly usingESI-MS, and non-carboxylic acids, which are analyzed usingAPCI-MS. After the sample was applied to the SPE cartridge, thelatter was washed with 0.1% acetic acid and subsequently driedby vacuum to remove the water, which may contain salts andpolar biomolecules. To evaluate matrix effects, the methanoleluate from the SPE cartridge was collected in five fractions,which were subsequently analyzed by LC–MS. As expected, thefirst methanol fraction showed more suppression in ESI-MS thanlater fractions. Unexpectedly, the same effect was observed fortwo compounds in APCI-MS. In addition, most carboxylic acidsshowed reduced response in the later eluted fractions, both inESI-MS and APCI-MS. The latter observation could not readilybe explained (Olsson et al., 2004).



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IV. MECHANISMS OF MATRIX EFFECTS

To obtain a better understanding of matrix effects in quantitativebioanalysis, the group of King studied mechanistic aspects of ionsuppression by a number of experiments (King et al., 2000). Bythe use of a dual ESI-APCI source and a dual-sprayer ESI source,they demonstrated that the matrix effect primarily is a liquid-phase and not a gas-phase process. By collection of residues fromthe curtain plate in a Sciex API source and re-injection, theydemonstrated that an increase of the amount of analyte found atthe curtain plate was observed when non-volatiles are present inthe liquid flow, originating from either non-volatile mobile-phaseadditives or from non-volatile sample constituents. These non-volatiles prevent the preformed analyte ion to escape from thedroplet into the gas phase. This indicates at least one of the causesof ion suppression in ESI-MS (King et al., 2000).

Essentially similar results were reported by others in a studyon matrix effects in the analysis of the insecticides G-, methoxy-,and hydroxy-fenozide in wheat forage extracts (Choi, Hercules,& Gusev, 2001a,b). A twofold reduction in response wasobserved between injections of 5 or 100 mL extract. They alsoshowed that by collection of the relevant chromatographic peakand re-injection in the same chromatographic system, the matrixsuppression was significantly reduced. Some mobile-phaseadditives were found to reduce the matrix suppression, asdemonstrated in Figure 5 (Choi, Hercules, & Gusev, 2001b). The

effect of mobile-phase composition on the extent of matrixeffects was also studied by others (Benijts et al., 2004).

By comparison of the results in ESI-MS and APCI-MS, thegroup of King also concluded that the formation of solid analyte-inclusion particles is a possible cause of ion suppression in APCI-MS. This study emphasizes the general importance to removenon-volatile sample constituents during sample pre-treatment(King et al., 2000). It was also concluded that APCI-MS is lessprone to matrix effects than ESI-MS, which is also confirmedby others (Barnes et al., 1997a; Chin, Zhang, & Karnes, 2004;Souverain, Rudaz, & Veuthey, 2004). However, one shouldcertainly not exclude the occurrence of matrix effects in APCI-MS, as demonstrated in some bioanalytical studies (Mei et al.,2003; Sangster et al., 2004).

Although the presence of non-volatiles is certainly impor-tant with respect to matrix effects, it does not tell the completestory. In early mechanistic studies, for example, (Kebarle &Tang, 1993), it was demonstrated that compounds with highproton affinities may also suppress the analyte response in ESI-MS, potentially also via gas-phase processes. Other mechanisticstudies indicate the importance of surface activity in the ESIprocess (Cech and Enke, 2000; Zhou and Cook, 2001). Matrixcomponents with higher surface activity may suppress theionization of the analyte. In addition, matrix constituents (ormobile-phase additives) that may form strong ion-pairs with thepre-formed analyte ions are known to suppress the response of

FIGURE 5. Effect of the mobile-phase composition on the extent of matrix suppression in the LC–MS

analysis of: (1) G-fenozide, (2) hydroxy-fenozide, and (3) methoxy-fenozide in wheat forage extracts

(——— standard sample, � � � � � � � matrix extract sample obtained by liquid–liquid extraction). Solvent is

acetonitrile:water (A) without additive, (B) with 1 mM ammonium formate, (C) with 0.1% (v/v) formic acid,

and (D) with 0.01% (v/v) NH4OH. Reprinted from Choi, Hercules, & Gusev (2001b) with permission.

�2001, Springer Verlag.



these analytes. In a recent study on matrix effects in the analysisof pharmaceuticals in wastewater, it was found that surprisinglythe majority of matrix effects was due to low molecular weightcompounds (<1 kDa) (Kloepfer, Quintana, & Reemtsma, 2005).

Finally, the extent of matrix effects in the bioanalysis of aparticular compound also depends on the source design of theLC–MS system utilized (Mei et al., 2003). Apart from changingthe ionization techniques, using an LC–MS instrument fromanother manufacturer may in some cases solve problems withmatrix effects as well.

V. DETECTION OF MATRIX EFFECTS

There are several ways to detect the presence of a matrix effect.Obviously, different procedures are required for absolute matrixeffects and for relative matrix effects. The most straightforwardway to evaluate an absolute matrix effect is to compare theresponse of an analyte in a standard solution and in a post-extraction spiked sample (matrix-matched standard) (Matus-zewski, Constanzer, & Chavez-Eng, 2003). Difference inresponse indicates ion suppression or ion enhancement. Thisapproach is widely applied, as shown by the examples onpesticide analysis quoted above (see also Figs. 1, 3, and 4).

In a detailed study on matrix effects in quantitativebioanalysis, Matuszewski et al. discussed various methods toadequately evaluate matrix effects (Matuszewski, Constanzer, &Chavez-Eng, 2003). A fairly complete description of the absolutematrix effects can be achieved by acquiring calibration plots withthree sample sets. The first sample set consists of the analyte andthe IS in mobile phase (standard solution), the second sample setconsists of post-extraction spiked plasma samples (matrix-matched standard), and the third set of pre-extraction spikedplasma samples (fortified real sample). From the peak areasacquired for these calibration plots one can calculate thepercentage matrix effect (%ME) from:

%ME ¼ Area of post-extraction spike

Area of standard� 100 ð1Þ

the recovery (%RE) of the sample pre-treatment method from:

%RE ¼ Area of pre-extraction spike

Area of post-extraction spike� 100 ð2Þ

and finally the overall process efficiency (%PE) from:

%PE ¼ Area of pre-extraction spike

Area of standard� 100 ¼ ð%ME �%REÞ

100

ð3Þ

This approach also accounts for possible influences of the analyteconcentration relative to that of the matrix. Comparison ofcalibration plots of standard solutions and matrix-matchedstandards has been reported in several pesticide-related studiesas well, for example (Barnes et al., 1995, 1997a,b; Startin et al.,1999; Careri et al., 2002; Ferrer, Garcıa-Reyes, & Fernandez-Alba, 2005; Ferrer et al., 2005; Ferrer, Thurman, & Fernandez-Alba, 2005). The terms proposed here differ somewhat from theterms introduced earlier (Buhrman, Price, & Rudewicz, 1996).

By the definition give here, ion enhancement can be accountedfor as well.

It is important to appreciate the difference between therecovery and the overall process efficiency. The recoverymeasures the efficiency of the analyte extraction process duringsample pre-treatment. However, it can only be determinedadequately by means of a method free from matrix effects.Otherwise, the overall process efficiency rather than theextraction recovery is determined. In fact, because in manystudies LC–MS was used to determine the recovery, quite oftenthe overall process efficiency is reported as the ‘‘recovery’’instead of the actual extraction recovery. The extraction recoveryshould be independent of the LC–MS interface and ionizationmethod used. When a recovery statistically significantly higherthan 100% is reported, one can be sure that the overall processefficiency is given, and not the extraction recovery. In such a case,the matrix effect provides signal enhancement.

In practice, the absolute %ME has limited relevance becauseit is generally more important to demonstrate the absence ofrelative matrix effects. For quantitative bioanalysis, the relativematrix effect has been evaluated by comparing the analyteresponse in extracts of plasma from five different sources(Matuszewski, Constanzer, & Chavez-Eng, 2003). This can beperformed by comparison of the slopes of the calibration plots (orthe %RSD in these slopes) using these five plasma lots. Thiswould imply that for the determination of all three parameters%ME, %RE, and %PE a total of 105 analyses would be needed,assuming five plasma lots and a 7-point calibration. Alternatively,relative matrix effects may be evaluated by a comparison of theprecision expressed as %RSD in repetitive injection of standardsand post-extraction spiked samples derived from the variousplasma lots. This would imply a total of 30 analyses, assuming5 injections of the standard and each of the 5 plasma lots at oneconcentration. If significant differences in these %RSD valuesexist, a relative matrix effect is present between the differentsample batches (Avery, 2003; Matuszewski, Constanzer, &Chavez-Eng, 2003).

To the best of our knowledge, studies on relative matrixeffects in the analysis of pesticides in fruits and vegetables havenot yet been reported. An evaluation of the matrix effects indifferent environmental water compartments, that is, surfacewater, rain water, ground water, channel water, wastewatertreatment plant effluents, and industrial effluents has beenreported. In this study, the responses of solvent standards andpost-extraction spiked samples were compared for six differentwater compartments (Benijts et al., 2004). Given the differentsample origin, these studies are best considered as evaluation ofthe absolute matrix effects in various sample types rather thanthe study of a relative matrix effect. Milk from six differentsources was applied in developing residue analysis of carbamatesin milk. Three compounds, that is, carbaryl, pirimicarb, andaldicarb showed significant matrix suppression. For pirimicarband aldicarb, the effect could be diminished by improved LCseparation (less steep gradient, resulting in doubling of theanalysis time). The slower gradient did not result in animprovement for carbaryl. Comparison of the results fromdifferent milk samples showed the absence of a relative matrixeffect, as the %RSD in the mean carbaryl concentration wasbetter than 7% (Bogialli et al., 2004).



The group of King proposed a post-column infusion system,which enables the evaluation of the absolute matrix effectsof different sample pre-treatment procedures (Bonfiglio et al.,1999; King et al., 2000). In this system (see Fig. 6), continuouspost-column infusion of the analyte of interest is performed,while blank matrix extracts obtained with different sample

pre-treatment methods are injected onto the LC column. Thisenables evaluation of the absolute matrix effects on the analyte asa function of the chromatographic retention time. An example ofthe results is shown in Figure 7. Using this infusion method,matrix effects can be monitored even beyond the analytical runtime, which is especially important in high-throughput repetitive

FIGURE 6. Scheme of the post-column infusion system to evaluate sample pretreatment methods with

respect to matrix effects. Reprinted from King et al. (2000) with permission. �2000, American Society for

Mass Spectrometry.

FIGURE 7. Typical results of a post-column infusion experiment. In the top chromatogram the separation

of the parent drug, its metabolite and an analog internal standard is shown, indicating the expected retention

times of the compounds. The bottom chromatogram shows the matrix effect on the response of the parent

drug. The ion suppression and ion enhancement effects prevent the reliable determination of the target

compound. Reprinted from M.D. Nelson and J.W. Dolan, Ion suppression in LC–MS–MS, a case study,

LC �GC Eur., (February 2002) 73 with permission. �2002, Advanstar Communications Ltd.



injection of samples in an isocratic LC system. One can actuallyobserve long-term ion suppression effects exerted on subsequentinjections. From these results, accumulation of matrix effects in along sample series can be anticipated (Bonfiglio et al., 1999).

The post-column infusion system is an elegant approach tomonitor matrix effects in a pragmatic and phenomenologicalmanner, that is, without addressing questions related themolecular or mechanistic causes of the matrix effects. As such,it is widely applied in method development for quantitativebioanalysis, for example, in developing methods for thequantitative screening of drug discovery compounds by fast-gradient (1 min run time) LC–MS (Hsieh et al., 2001; Tiller andRomanyshyn, 2002; Mei et al., 2003), in the optimization of thesample pre-treatment in the analysis of methadone, comparingfour off-line and three on-line sample pre-treatment methods(Souverain, Rudaz, & Veuthey, 2004), and in evaluating variousprotein precipitating additives (Polson et al., 2003). One shouldkeep in mind that this elegant approach enables the evaluation ofan absolute matrix effect only. For assessing a relative matrixeffects, responses (and their %RSD) of extracts from differentsample lots must be evaluated.

The usefulness of the post-column infusion system wasquestioned, especially for cases where matrix-matched calibratorsmust be used (Kaufmann & Butcher, 2005). A high-frequencysegmented addition of analyte is proposed as an alternative to thecontinuous post-column addition. This causes a pulsation of thebaseline at a fixed frequency. The frequency should be chosen toprovide peak widths of the spike smaller than that of the analyte inthe LC run. Any change in peak height indicates suppression orenhancement. In this way, the peak areas of target compounds maybe corrected for the suppression or enhancement effect. Theapproach was demonstrated for oxytetracycline, which shows atwofold signal enhancement in honey. The most importantproblem of the approach is in signal deconvolution (Kaufmann& Butcher, 2005). Kaufmann and Butcher appear to assume thatthe post-column infusion system is actually applied to correct formatrix effects in real sample analysis. At least, that is what they areaiming at with their alternative method. In practice, the post-column infusion method is used differently, that is, mainly to getinsight in the occurrence of matrix effects and to optimize samplepre-treatment in terms of removing matrix constituents. One mayquestion whether the segmented post-column analyte addition is auseful tool in the analysis of large series of real samples.

Obviously, the use of the post-column infusion system islimited to target compound analysis with only a limited number oftarget compounds. The effort involved in developing a multi-residue analysis of a wide variety of target compounds in this waywould be enormous. In principle, the post-column infusion systemaims at optimization of the sample pre-treatment method in termsof reducing the matrix suppression for a particular analyte. In amultiresidue study, one would have to strike too many compro-mises between optimum approaches for particular analytes.

VI. REDUCTION OR ELIMINATION OFMATRIX EFFECTS

Matrix effects are due to co-eluting matrix constituents. They arecompound and matrix dependent. It is important to state that the

components responsible for the matrix effects are often notionized by ESI-MS or APCI-MS, which means that they cannotbe detected by MS. Various compound classes have beenidentified as a potential influential matrix constituents suppres-sing or enhancing the analyte response, for example, non-volatilesample constituents, salts, compounds with high surface activityand/or ion-pairing properties, compounds with high protonaffinity (in positive-ion mode) or low gas-phase acidity (innegative-ion mode). However, it is not clear whether this list isconclusive. And often the exact composition of a complex matrixis unknown.

There are two general approaches to deal with matrix effectsin quantitative analysis. First, one may eliminate the sampleconstituents responsible for the matrix effects. This would implyimprovement of the sample pre-treatment and/or the chromato-graphic separation (efficiency and/or resolution). Although thiswould in principle be the best approach, it may be difficult toachieve, especially in multiresidue analysis, where a variety ofinterferences may be involved. Alternatively, one may reduce oreliminate the influence that matrix effects have on the accuracyand/or precision of the method. The latter may be achieved by oneor a number of the following measures: change to anotherionization methods, change the mobile-phase composition, and/or use adequate standardization. In many cases, both approachesmust be combined to achieve adequate quantitative results.

A. Remove Matrix Constituents:Improve Sample Pre-Treatment

Improvements in sample pre-treatment procedures aim atreducing the amount of matrix components that are introducedinto the analytical system. It may involve a more selective analyteextraction procedure or a more extensive sample clean-up prior toinjection into the LC–MS system. When a large number ofsamples have to be analyzed, improvement of the sample pre-treatment in most cases is the most effective method in reducingor eliminating matrix effects.

Improvements in sample pre-treatment procedures havereceived considerable attention in environmental water analysis,aiming at either improved extraction recovery for more polartarget compounds, reduced matrix interferences of especiallyhumic acids, or both. In this respect, approaches can bementioned involving dual-SPE-column procedures (Geerdinket al., 1998; Steen et al., 1999; Hogenboom et al., 2000), the useof selective SPE-packing materials such as restricted accessmaterial (RAM), molecularly imprinted polymers (MIP) (Koeberet al., 2001), and immunoaffinity materials (Hennion & Pichon,2003). Some of the advanced methods are more laborious andtime-consuming. With respect to food analysis, the comparisonof various sample pre-treatment methods by the group of Picomay serve as an example (Blasco, Font, & Pico, 2002, 2004;Juan-Garcıa et al., 2004; Soler, Manes, & Pico, 2004, 2005).

While sample pre-treatment methods may reduce theconcentration of interfering components, there are two alternativeapproaches, that is, sample dilution and smaller injection volumes.In both cases, the amount of matrix components introduced intothe analytical system is reduced, resulting in reduced matrixsuppression (Choi, Hercules, & Gusev, 2001a,b; Dams et al.,



2003). Although this seems to be a very simple approach, it cannotalways be applied, because it inevitably increases the detectionlimits of the compounds of interest. As such, it may become aproblem to detect the compounds at the required MRL.

Results from various groups, for example (Olsson et al.,2003; Hogenboom et al., 1997), indicate that elimination ofmatrix components is especially important for early elutingcompounds. This probably explains why matrix effects were firstobserved in high-throughput quantitative bioanalysis, wherecolumn length and thus analyte separation from the solvent fronthas been minimized to reduce the analysis time.

B. Remove Matrix Constituents:Improve Chromatography

With respect to chromatographic resolution, the practice inmultiresidue analysis of pesticides is different from the bioana-lytical practice in drug development. In the latter case, one or onlya few target analytes must be determined. Given the large numberof samples, it has become common practice to significantly reducethe column length to 20–50 mm, with its negative effect onchromatographic resolution. However, in this type of bioanalysis,often only separation from the solvent front is pursued.

In multiresidue analysis, however, conventional columnlengths (100–250 mm) are applied, providing far betterseparation. As such, limited gain can be achieved in reducingmatrix suppression by improving the resolution in multiresiduepesticide analysis. Doubling the analysis time by using a lesssteep solvent strength gradient was successfully applied toeliminate severe matrix suppression of pirimicarb and aldicarb inthe analysis of carbamate insecticides in milk (Bogialli et al.,2004). Probably the most important issue is to achieve sufficientanalyte retention to move also the more polar compoundssufficiently away from the solvent front. In this respect, the use ofhydrophilic interaction chromatography (HILIC) might be auseful alternative in some cases. In HILIC, silica columns areapplied in combination with organic/aqueous mobile phases. Asa result, the retention order is reversed compared to reversed-phase LC. The method has been successfully applied inquantitative bioanalysis of some very polar drugs (Naidong,2003). Stationary phases especially designed for HILIC arecommercially available.

Coupled-column LC–LC approaches have been demon-strated to enable a reduction of matrix effects, allowing for directurine injection in biological exposure monitoring studies(Sancho, Pozo, & Hernandez, 2000; Sancho et al., 2002;Hernandez, Sancho, & Pozo, 2004b). This is especially true,when sufficient orthogonality of the phase systems can beachieved. However, the coupled-column approach is a typicaltarget-compound oriented method, which is not readily adaptedto multiresidue analysis. Comprehensive LC�LC, which wouldbe applicable to multiresidue studies, is still in its infancy.

C. Eliminate Effects on Accuracy and/orPrecision: Change Ionization Mode

A change in ionization mode, mostly from ESI-MS to APCI-MS,has been shown to be a successful way of eliminating matrix

effects (Matuszewski, Constanzer, & Chavez-Eng, 1998, 2003).Similarly, the use of the negative-ion mode instead of thepositive-ion mode, or vice versa, may provide reduction of matrixeffects. In practice, this will not be possible for all target analytesbecause they do not all give similar response in either ESI-MSor APCI-MS, or in either positive-ion or negative-ion mode, ashas been systematically investigated for over 75 pesticides andrelated compounds (Thurman, Ferrer, & Barcelo, 2001). Inthe analysis of pharmaceuticals in wastewater samples, it wasshown that a post-column splitter to reduce the LC flow-rateto 20–100 mL/min resulted in a 45%–60% reduction of thematrix effects (Kloepfer, Quintana, & Reemtsma, 2005).This is in agreement with observation of reduced matrix effectsin nanoelectrospray-MS experiments, for example (Ganglet al., 2001).

Atmospheric-pressure photoionization (APPI) has beenpromoted as an alternative ionization method next to ESI-MSand APCI-MS (Raffaelli and Saba, 2003). It should not onlyextend the applicability range towards less polar molecules, butalso reduce matrix effects in quantitative analysis (Hsieh et al.,2003; Hanold et al., 2004). APPI-MS has not yet been widelyapplied in pesticide analysis.

D. Eliminate Effects on Accuracy and/orPrecision: Mobile-Phase Composition

Because analyte ionization may be strongly influenced by themobile-phase composition in both ESI-MS and APCI-MS,different mobile-phase additives may influence the extent ofmatrix suppression in a particular method. An example of thiswas given in Figure 5, where the effect of three different mobile-phase additives, that is, 1 mmol/L ammonium formate, 0.1%formic acid, and 0.01% ammonium hydroxide, on the signalsuppression of three fenozides was investigated (Choi, Hercules,& Gusev, 2001b). In another study (Benijts et al., 2004), oppositeresults were achieved: mobile-phase additives led to an increaseof the signal suppression. In this study, the influence on the matrixeffects (%ME) was compared for formic and acetic acid at 0.01%and 0.1% concentrations (v/v) and for ammonium formate andacetate at 1 and 5 mM concentration. Next to various non-pesticide endocrine disruptors, such as bisphenol A, estradiol,ethinyl estradiol, and alkyl phenols, some carbamates andtriazine herbicides were studied as model compounds. The latterwere studied in the positive-ion mode. The addition of both acidsand buffers resulted in lower %ME for the pesticides in thepositive-ion mode as well as for most other model compoundsin the negative-ion mode, that is, in an increase of the signalsuppression.

E. Eliminate Effects on Accuracy and/orPrecision: Standardization

Probably the most effective way to eliminate the adverseinfluence of matrix effects on the accuracy and/or precision ofan analytical method is the application of the standard additionmethod (Stuber and Reemtsma, 2004). However, this method istime-consuming and laborious. Therefore, the standard additionmethod is not frequently applied (see below).



In multiresidue analysis of pesticides, generally no internalstandards are applied. This is due to the difficulty to selectappropriate standards for a wide variety of target compounds. Inprinciple, one would need to introduce several internal standards,ideally even one standard per target compound. This wouldseriously limit the sensitivity of the method as it doublesthe number of SRM transitions that have to be monitored. Ithas clearly been demonstrated above that matrix-matchedstandards must be and can be applied to achieve reliable andsufficient precise results. Obviously, the use of matrix-matchedstandards can pose an enormous amount of work when awide variety of pesticides are monitored in a wide variety ofmatrices, for example, in relation to pesticide analysis in fruitsand vegetables. In this respect, the studies on various commod-ities of fruits and vegetables (SANCO/825/00) and potentialrepresentative matrices for each of these commodities are ofgreat practical importance (Jansson et al., 2004; Martınez Vidalet al., 2005).

Although not widely applied in pesticide analysis, it seemsuseful to discuss some issues related to internal standards. Whenthe number of target compounds is limited, internal standardscan be applied, either analog or stable-isotopically labeledinternal standards. Because the matrix effect may be (strongly)compound dependent, the ionization of an analog internalstandard and the analyte may be differently affected by thematrix. It was shown that the precision of a method in which ananalog internal standard is used, can be significantly improved bymodifying the mobile-phase conditions in such a way that analyteand analog internal standard co-elute (Kitamura et al., 2001).The intra-day precision in the analysis of 2-C-ethynylcytidineas analyte and 30-C-ethylcitidine as analog internal standardimproved from 5.2–16.2%RSD with separation to 2.7–4.2%RSD without separation between analyte and internalstandard (Kitamura et al., 2001). Instead of modifying theseparation conditions, selection of another more appropriateanalog should be considered.

For these reasons, the use of an isotopically labeled internalstandard is preferred. In fact, in GC–MS and LC–MS, anisotopically labeled internal standard is considered to be the idealinternal standard, as it shows (almost) identical behavior to thetarget analyte in both sample pre-treatment, chromatography, andanalyte ionization. Unfortunately, isotopically labeled internalstandards are available for only a limited number of targetanalytes, they are expensive, and often difficult to obtain for othertarget compounds, especially with sufficient isotopic puritywith respect to the non-labeled variant. In addition, although it isgenerally believed that the use of an isotopically labeled internalstandard corrects for any matrix effects, data reported forthe bioanalysis of mevalonic acid indicate that this issue needsproper attention during method development and validation(Jemal, Schuster, & Whigan, 2003). In addition, mutualsuppression or enhancement of responses of an analyte and itsco-eluting isotopically labeled internal standard has occasionallybeen reported (Liang et al., 2003).

The EU standard method (CEN, 2002) for the LC–MSdetermination of chlormequat and mepiquat in fruit may serve asa good example of the use of isotopically labeled internalstandards. It was demonstrated, that by the use of a (D4)-labeledchlormequat as internal standard quantification of chlormequat in

tomato products could be evenly well achieved using solvent ormatrix-matched standards.

With both the analog and the isotopically labeled internalstandards, it is important to add these prior to sample pre-treatment. In that way, they can correct for analyte lossesduring sample pre-treatment as well as matrix-related responsesuppression or enhancement during analyte ionization.

The methods to reduce or eliminate matrix effects, discussedso far, strongly rely on the availability of an adequate blankmatrix to produce matrix-matched standards, and/or the avail-ability of appropriate compounds that may serve as analoginternal standards. When that is not the case, for example, in theanalysis of industrial effluents or the influents to a wastewatertreatment plant (Stuber & Reemtsma, 2004), dealing with matrixeffects is even more complicated. In the analysis of diarrheticshellfish poisoning toxins in scallops, various ways to correct formatrix effects were compared (Ito & Tsukada, 2001). Scallopextracts from different sources were found to show considerablerelative matrix effects. It was demonstrated that in this case thestandard addition method provided sufficiently accurate andprecise results.

An alternative approach with respect to internal standardi-zation is the use of the echo-peak technique (Zrostlıkova et al.,2002; Alder et al., 2004). The unknown sample and the standardsolution are injected within a short time period in each analysis(typically 30–50 sec between injections). As a result, thecompound in the unknown sample and its standard elute in closeproximity and are therefore expected to be affected by the matrixinterferences in the same way. The success of the echo-peaktechnique in the analysis of eight pesticides, spiked at 0.01mg/mLin an apple extract, in comparison to solvent and matrix-matchedstandards is shown in Figure 8. As shown, the echo-peaktechnique does not always adequately correct for matrix effects.This is for instance the case when the peak profiles of interferingmatrix constituents are apparently very sharp, as is the case forcarbaryl, teflubenzuron, and flufenoxuron, or when the analytegives a highly tailing peak, as is the case for imazalil (Zrostlıkovaet al., 2002). In a more elaborate study involving the analysis of70 pesticides in four different matrices, it was demonstrated thatthe echo-peak technique generated more accurate results for>70% of the cases than the use of solvent standards (Alderet al., 2004). The echo-peak technique appears to be especiallyattractive for screening purposes. When the echo-peak standardsare injected at MRL concentration, a too high level of one of thetarget compounds in the sample is readily recognized from theoccurrence of two peaks in the SRM trace.

VII. CONCLUSIONS AND PERSPECTIVES

Due to their unpredictable character, matrix effects in quantita-tive analysis using LC–ESI-MS or LC–APCI-MS are a seriousconcern, demanding sufficient attention during method develop-ment and validation. It is clear that the use of real sample extractsis necessary already at an early stage of method development, asthe matrix effect may have serious impact on the choice of themost appropriate sample pre-treatment method, ionizationmethod and mode, and even the most adequate mobile-phasecomposition. While advanced sample pre-treatment methods can



help in reducing or eliminating matrix effects, such procedurescome with additional difficulties when many compounds withwidely differing physicochemical properties must be analyzed.With respect to the development of multiresidue methods, thematrix effects are even more challenging to the analyticalresearch. Some attention should also be paid to the investigationof relative matrix effects in the analysis of pesticides in fruits andvegetables from different sources, for example, grown indifferent regions.

The best way to eliminate the influence of matrix effects onthe accuracy and precision of a quantitative method is the use ofisotopically labeled internal standards. Obviously, limitedavailability and high costs of such standards, as well as thedifficulties involved in using a large variety of standards inmultiresidue methods, to some extent excludes the wideapplication of such multiple isotopically labeled internalstandards. As a result, matrix effects continue to providechallenges in developing reliable quantitation in multiresiduemethods.

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Wilfried Niessen graduated in Chemistry at the Free University in Amsterdam,

Netherlands in 1981 and received his PhD at the University of Amsterdam in 1986 on a

thesis on open-tubular liquid chromatography–mass spectrometry. Since then, he has been

involved in research and education related to the application of hyphenated mass

spectrometry methods in pharmaceutical, environmental, biochemical, and other applica-

tion areas. Between 1986 and 1996, he was an assistant professor within the Leiden

Amsterdam Center for Drug Research in Leiden, Netherlands. From 1996 onwards, he

works in his company hyphen MassSpec, involved in consulting and courses in analytical

mass spectrometry and LC–MS. Since 2003, he combines this with a part-time position as

extraordinary professor in bioanalytical mass spectrometry within the Faculty of Sciences

at the Free University in Amsterdam. He is the author or editor of several books, including

Volume 8: Hyphenated Methods of Elsevier’sEncyclopedia ofMass Spectrometry, and he is

a member of the editorial board of Journal of Chromatography A. His main research

interests involve the principles, technological developments, and the application of LC–MS

and related techniques in biological, pharmaceutical, and food safety application areas.

PaolaManini graduated in Chemistry at the University of Parma, Italy in 1992 and received

her PhD in Chemical Sciences at the same University in 1997 presenting a dissertation

concerning the development of innovative analytical methods using LC-MS in several

application fields, including food analysis and the characterization of organometallic

compounds. During her PhD research, she spent 6 months at the Free University of

Amsterdam where she carried out research on LC–MS quantification of pesticides in water

and vegetable samples. As a post-doc at the Medical School of Parma, she is using her

experience on MS-hyphenated techniques in the field of environmental and occupational

toxicology. Her research activity is mainly devoted to the LC–MS characterization of the

metabolism of industrial chemicals (n-hexane, styrene, naphthalene) and environmental

pollutants (benzene, toluene) and to the development of new biomarkers of exposure. She



developed and validated analytical methods for the determination of volatile organic

compounds in biological samples by solid-phase microextraction GC–MS.

Roberta Andreoli graduated in Chemistry at the University of Parma, Italy in 1996 and

obtained a Masters in Occupational and Environmental Toxicology at the same University

during 1997–1998. As a research fellow at the Laboratory of Industrial Toxicology of the

Medical School of Parma, she gained experience in the use of GC–MS and LC–MS for the

determination of organic solvents and their metabolites in complex biological matrices

(blood, urine, exhaled breath condensate). At the moment, she is a PhD student in Health

Sciences, directing her studies to the use of mass spectrometry for the analysis of

biomarkers of oxidative stress in patients with respiratory disorders and in workers exposed

to pneumotoxic agents.



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