The role of GC-MS / MS with triple quadrupole in pesticide residue analysis in food and the environment Autores /

Gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) using a triple quadrupole (QqQ) analyzer has in the last few years become a powerful technique for the determination of pesticide residues due to its robustness, and excellent sensitivity and selectivity. This review gives an overview of currently published applications of GCMS/MS with a QqQ analyzer for pesticide residue analysis of different food and environmental sample matrices. This technique allows the reliable quantification and identification of low pesticide concentrations for non-polar (semi) volatile compounds belonging to different chemical families. It has allowed a notable improvement of methods performance in comparison with the traditional GC methods with single stage quadrupole MS.


Introduction
Pesticide residue analysis (PRA) of food and environmental materials has become an important specialized field of modern analytical chemistry.The necessity of advanced analytical methods for its application in monitoring programs that ensure food-safety and environmentally responsible agricultural practices has been frequently highlighted.
Reliable and sensitive analytical methods able to reach the low limits of quantification (LOQ) required by the legislation are needed.In most cases, LOQs lower than 0.01 mg kg −1 in food and lower than 0.1 μg L −1 in water are needed for monitoring purposes, where the reliable identification and quantification of hundreds of pesticide residues in many different matrices is normally pursued.In recent years, new developments in sample preparation and instrumentation, especially dealing with chromatographic techniques coupled to mass spectrometry (MS) or tandem MS, have allowed the high quality standards required from a qualitative and quantitative point of view in PRA to be achieved.
In the past decades, gas chromatography coupled to mass spectrometry (GC-MS) methods have been mostly based on selected ion monitoring (SIM) or full scan modes, evolving from single quadrupole (Q) to ion trap (IT) analysers.The first papers dealing with PRA by GC-MS can be traced back to 1970's when the determination of a reduced number of pesticides was carried out using packed column GC systems coupled to mass spectrometers with single quadrupole analyzers.In a recent review, 1 it has been reported that the single quadrupole is still the most used analyzer in combination with GC.
Similarly, Botitsi et al. 2 showed that during the period of 2006 to 2009, 26 out of the 47 reviewed papers that employed GC-MS for the determination of pesticides in food and water were based on single quadrupole analysis.According to data reported, only 9 papers dealt with the use of triple quadrupole (QqQ), the rest being methods based on IT and time-of-flight (TOF) analyzers.In the review article from Andreu and Picó 1 on PRA in biota, 18 out of 24 papers reviewed dealt with the use of single quadrupole GC-MS.
Despite the wide existing applications, methods based on the use of single quadrupole instruments suffer from low sensitivity when working in the full scan mode.The sensitivity can be improved by working in the SIM mode, but the identification potential and the non-target/retrospective analysis capabilities are sacrificed.After development of the single quadrupole, the next step in the evolution of mass analyzers in pesticide residues analysis (without eliminating the use of the single quadrupole) was the increased use of ion trap mass spectrometers that allowed full spectra based methods to be developed with suitable sensitivity (similar to that obtained by a quadrupole in the SIM mode) in a single run.A number of papers have been published demonstrating the capability of ion trap analyzers for carrying out tandem MS experiments, improving sensitivity and selectivity, but losing the non-target capabilities.The fact that the MS/MS working mode of an ion trap is a product ion scan results in the co-elution of several analytes, or sample matrix components and notably reduces sensitivity and the number of points across the chromatographic peak.In the late 90s, the introduction of GC-TOF MS resulted in an improvement of full scan based methods and a step forward in non-target analysis.TOF MS is able to provide full spectrum acquisition data at high sensitivity.
High-speed (HS) TOF MS, with a fast data acquisition rate (up to 500 spectra per second), is an excellent technique for GC × GC MS detection, for which the data acquisition speed is the most limiting factor.On the contrary, mainly coupled to 1D-GC and with lower data acquisition rates (20-25 Hz) and a narrower dynamic range, highresolution (HR) TOF MS provides sensitive full spectrum data with high mass accuracy, allowing the resolution of peaks from closely related interfering matrix components.In the last years, many papers can be found dealing with the determination of pesticides in food and environmental samples by GC × GC-(HS) TOF MS 3,4 but much less related with GC (HR) TOF MS. 5,6 The most important limitation of TOF MS is related to its low sensitivity which can make it troublesome to reach the required limits of detection (LOD)   and, in the case of the HR TOF, also the peak saturation problems that occur in short dynamic ranges.
One of the major developments in the field of PRA has been the commercialization of liquid chromatography (LC) coupled to QqQ MS systems, which has benefited greatly from the high sensitivity and selectivity of tandem MS in the selected reaction monitoring (SRM) mode.Thus, QqQ has been the analyzer most used in LC-based methods in the 2006-2009 period, 2 although this fact was not such evident for GC-based methods, which have suffered a notable delay in the wide acceptance of this analyzer in comparison to LC-MS/MS.In fact, LC-QqQ MS/MS started to be applied in PRA in the early 90's, while GC-QqQ MS/MS was applied around 15 years later.The first publications, between 2003 and 2005, reported the use of GC-QqQ MS/MS in food matrices like tobacco, oil, baby food or cucumber [7][8][9][10] and in human fat. 11veral papers have been published on the comparison of different analyzers in GC-MS.Mezcua et al. 12 compared GC-Q MS and GC-IT MS(/MS) for the determination of insecticides in vegetables indicating that no significant differences were found in terms of sensitivity, although IT under MS/MS conditions was superior to GC-Q MS in the identification capability.Garrido et al. 13 made an interesting comparison between two MS/MS systems, QqQ and IT, concluding that intraday-precision was similar for both, but interday-precision was found to be worst in the case of QqQ.In contrast, better linearity ranges were achieved for QqQ together with lower matrix effects especially for dirty samples e.g.fat containing samples.Additionally, a larger number of compounds can be included in a single injection in QqQ (SRM mode), although regarding identification capabilities IT in the tandem MS mode gives more information for a better confirmation of positive findings due to the MS n possibilities.
Several ionization techniques have been used in GC-MS over time, 14,15 electron ionization (EI) being the most popular and widely used.EI offers valuable information on the molecule structure (several fragment ions and, in some cases, molecular ions).It is a robust and universal ionization source that generates highly reproducible spectra that can be searched in available commercial spectral libraries for the identification of non-target compounds.However, EI generates highly extensive fragmentation.In some cases, it leads to mass spectra without abundant/intense characteristic peaks (e.g.molecular ion), and the sensitivity obtained can be poor.In those cases, alternative approaches, such as chemical ionization (CI), which can be applied in both the positive or negative mode allow mass spectra to be obtained with a predominant molecular ion peak and low fragmentation.CI has been applied in PRA, especially for the determination of organohalogenated pesticides due to its better sensitivity and selectivity for some of these compounds. 16,17However, there is a low number of applications compared to EI, and it is not posible to carry out spectral library searching as it is not commercially available for CI.
A promising source is atmospheric pressure chemical ionization (APCI), which opens a new perspective in the development of GC-MS/MS methods.This source has been recently tested in PRA and offers very attractive features for compounds that suffer extensive fragmentation in the EI mode. 18,19

GC-MS/MS applications in food analysis
Nowadays, the control of pesticide residues in food commodities has become a requirement for compliance with the legislation, ensuring safety of the population and international and national trade.The determination of GC-amenable pesticides in food samples by using tandem MS with a QqQ analyzer has emerged in the last decade as a valuable approach, which allows higher selectivity and sensitivity and minimizes or even removes most chromatographic interferences.This section reviews the papers published in the last ten years related to the determination of pesticide residues in food samples by GC-QqQ MS/MS (Table 1).The most relevant aspects related to the studied pesticides, types of matrices, sample preparation procedures and analytical measurements are discussed in the present review.
The different physicochemical properties of the pesticide chemical classes increase the difficulty when developing a simultaneous analytical method for multiresidue analysis of food commodities.Thus, analytical methodologies based on GC-QqQ MS/MS for the determination of pesticides from the same family are quite common, e.g. for the determination of organochlorine (OC), 8,20,21 organophosphorus (OP) pesticides, [22][23][24] or of pyrethroids 19,25 in food commodities.Some particular examples are the GC-QqQ MS/MS method developed by Le Faouder et al. 26 just for fipronil, or by Peruga et al. for chlorothalonil. 279][30][31][32] However, the majority of applications (around 70%) deal with multiresidue methods for multiclass pesticides in food samples, 7,9,10,[16][17][18] the most adequate strategy available for monitoring purposes that minimizes time, costs, reagents, labors and hazards in order to obtain rapid analytical results in response to urgent demands. Moreoer, most multiclass methods published include more than one hundred pesticides in their target list, among insecticides, herbicides, acaricides, fungicides, etc. 10,33,35- 38,40,41,43,44,46-52,57 In this respect, remarkable papers are those published by Okihashi et al. 35 and by Banerjee et al., 57 who have developed analytical methodology for the determination of up to 260 and 349 pesticides in fruits and vegetables, respectively.In PRA, the term Food includes a wide range of treated products, fruits, vegetables, grains and other commodities.Even after being washed, stored, processed and prepared, some pesticide residues may remain in both fresh products and processed foods.From the overview of the applications shown in Table 1, it can be seen that fruits and vegetables are the most frequent samples analyzed.10,[16][17][18][19]24,27,[32][33][34][35][36][37]39,41,44,46,[48][49][50]52,54,55,57,59 Other matrices analyzed are oils and fats, 8,22,23,38,40 cereals, 38,39,43,59 muscles and livers, 28- 30,58 tobacco, 7,42 ginseng, 47,56 animal feeds, 20,26 milk, 25,26 eggs, 31 wine, 51 mussels, 21 baby food 9 and fruit-based soft drinks.53 GC-MS/MS methods for pesticide residues include the extraction of the analytes from the matrix, appropriate cleanup of the raw extracts and final measurement.The most used approach for extraction of pesticides from food samples is nowadays the QuEChERS procedure (Quick, Easy, Cheap, Effective, Rugged and Safe), which has been widely reported in the literature.60 Some variations of the original method have led to two modified methods: the acetate buffered method (AOAC official Method 2007.1)61 and the citrate buffered method (CEN Standard Method EN 15662).39 The QuEChERS procedure in combination with GC-QqQ MS/MS is one of the preferred approaches at present for residue determination of GC-amenable pesticides.Among the methods reported, it can be mentioned that those of Leandro et al., 9 Plaza Bolaños et al. 36 and Wong et al., 46 used the original method without modifications.In contrast, other authors obtained good results using the acetate buffered version, [17][18][19]41,52 although citrate modification seems to be more used in this field.In fact, the citrate buffered version has become the most applied extraction method in pesticide residue analysis in food by GC-QqQ MS/MS.32,38,39,42-44,49- 51,55 In these methods, a subsequent cleanup step is applied based on dispersive-solid phase extraction (d-SPE) using different sorbents, such as primary secondary amine (PSA), C 18 , Z-Sep Plus and/or graphitized carbon black (GCB), depending on the complexity of the matrix.
After sample preparation, the final extract is commonly injected into the inlet system with a classical split/splitless injector.In some cases, the final extracting solvent is not appropriate for injection into the split/splitless system, due to the high volume-expansion coefficient during vaporization.Solvent exchange prior to chromatographic injection to an adequate solvent such as toluene, 18,19,38,43,44,46,47,50,51,55 acetone 32,35 or hexane 24 could be a good choice to solve this problem.][50][51][52]57,58,62 Apart from PTV, other large volume injection (LVI) systems are on-column injection or concurrent solvent recondensation (CRS) injection. 55garding to the GC-QqQ MS/MS measurement (Table 1), EI is the most used ionization mode for the determination of pesticides in food, although the use of negative CI (NCI) has also been reported. 7,16,17,25For some compounds, with highly electronegative elements, such as halogen, oxygen, etc., the use of the NCI mode usually provides better sensitivity and selectivity.The use of the QqQ allows selected reaction monitoring (SRM) to be applied, one of the most selective and sensitive approaches for simultaneous quantification and confirmation in PRA, when adequate precursor and product ions are selected.In this way, most matrix interferences are minimized, or even eliminated, improving the selectivity and the sensitivity, and reaching low detections limits due to the lower chemical noise in the chromatograms.
In general, the criteria used for confirmation/identification of positive samples are not treated in detail in most of the papers published.Some authors do not mention this issue, and only acquire one SRM transition for each analyte without mentioning any confirmation criteria. 7,37,425][56][57][58][59] Some authors 8,17,22,23,25 have used the mere presence of a second SRM transition as confirmation criteria for positives in the samples.However, the European Commission Decision 2002/657/EC 63 implements the concept of identification points (IPs).In the case of MS/MS determination, 1 identification point is earned from a precursor ion, and 1.5 identification points are earned from a resulting product ion.For the unequivocal confirmation of the identity of compounds at least 3 and 4 identification points are required for legal and banned substances, respectively. 43This has been applied by several authors such as Garrido Frenich et al., 30 Plaza Bolaños et al., 31,36 Walorcyzk 38,43 and Fernández Moreno et al. 41 in their work.Nowadays, the most widely accepted approach is based on the presence of chromatographic peaks at the two (or three 53 ) transitions acquired, together with agreement of the R t and the evaluation of the intensity ratio between the quantification (Q) and the confirmation (q) transition, and comparison with those of the reference standard within the maximum tolerances established by the European Commission Decision 2002/657/EC 21,28,40,44,[46][47][48][49][50] and the SANCO 64 guidelines. 16,27,39,53dern QqQ instruments allow the simultaneous acquisition of two or more transitions in just a single GC analysis.However, some publications reported 10,33,37 performed a sequential approach with two sample injections: the first for rapid screening, with acquisition of only one transition, and the second for the confirmation and quantification of the compounds previously detected in positive samples, with acquisition of 2 or 3 transitions.As a particular example, Fuentes et al. 22,23 determined OP pesticides in olive oil by GC with flame photometric detection (GC-FPD) and the identity of residues in positive samples was confirmed through GC-QqQ MS/MS analysis by acquiring two transitions.
Once the identity of the analyte in the sample has been confirmed, quantification of pesticide residues is normally the next objective.Different approaches have been reported for quantification of pesticide residues in food, commonly considered as a complex matrix.1]57,59 Cervera et al. 48studied the matrix effect of several food matrices (orange, nectarine, spinach, raisin, paprika, cabbage, pear, rice, legume and gherkin) comparing the response of reference standards prepared in solvent with the response of matrix-matched standards.Most of the pesticides showed an evident signal enhancement in the presence of matrix, and matrix-matched calibration using relative responses to an internal standard was required for the correct quantification of compounds.On the contrary, Nardelli et al. 20 performed the quantification of OC pesticides in fish feed by using standard solutions in solvent and in the matrix as calibration curves, no differences were observed between the results obtained using the different sets of standards.Other applications have been reported in the literature in which standard solutions in solvent were used for analysis of different matrices such as cabbage, 32 milk, 25 mussel 21 and ginseng. 56As occurs with other analyzers used in GC-MS, the triple quadrupole analyser, even working in the SRM mode, is affected by coextracted matrix components that may lead to an enhancement of the chromatographic signal or to a reduction of the analyte response in comparison to the signal in pure solvent, as these effects are normally a consequence of problems coming from the GC system.Thus, when coextracted matrix components compete with the target pesticides to access the active sites of the GC system and/or when they are protected from decomposition in the hot injector, a matrix-induced response enhancement is observed.Conversely, accumulation of non-volatile coextracted matrix components in the GC system helps to generate new active sites, and matrix-induced response diminishment occurs. 45nsitivity is an important parameter to measure the potential of a method in PRA.
LOD and LOQ are usually calculated for this purpose, although their estimation is a controversial issue, due to the different ways of calculation.This makes it troublesome to perform a realistic comparison of the values reported in the literature.In order to compare the sensitivity of the applications reviewed, the lowest concentration level validated was used as an indicator of the sensitivity of GC-MS/MS methods.Most of the publications (Table 1) used a lowest level validated in the range of 5-10 μg kg −1 . 8,10,17,24,27,37- 39,43,44,46,48-52,54,57,59The use of a large injection volume resulted in an increase in the sensitivity, and lowered the method validation concentration down to 1 μg kg −1 .Thus, Leandro et al., 9 Belmonte Valles et al. 16 and Sapozhnikova and Lehotay 58 validated their procedure at the lowest level of 1 μg kg −1 in baby foods, fruits and vegetables, or in catfish muscle, using large volume injections of 8, 2 and 5 μL, respectively.

GC-MS/MS applications in the analysis of environmental samples
The extensive use of pesticides in agriculture and their industrial applications in the last decades, together with the persistence of some of these compounds, has led to their wide presence in the different compartments of the environment.Consequently, there is a need to know the concentration of these contaminants in the aquatic environment, although they normally are found at the μg L −1 level or below.To this aim, strict regulations and environmental monitoring programs have to be adopted to accurately determine the concentration levels of pesticides.GC-QqQ MS/MS is an attractive technique with strong potential in the determination of low levels of pesticides in environmental samples, as occurs in food analysis.
The number of papers published until now related to environmental applications of GC-QqQ MS/MS in pesticide residue analysis is not as large as for food (Table 2).6][67][68][69][70][71][72][73][74][75] Only two articles deal with around hundred target analytes: Barco-Bonilla et al. 67 included 139 analytes in waste water samples analysis and Martínez Vidal et al. 69 included 98 pesticides in soil analysis.
or stir-bar sorption extraction (SBSE). 70Airborne particulate matter has been another environmental matrix analyzed by GC-QqQ MS/MS, 71 using microwave-assisted extraction (MAE) followed by GPC as a cleanup step.As shown in Table 2, water has been the most common matrix studied in the environmental field.6][67][68] Some exceptions applied to pyrethroids pesticides that were extracted using ultrasound-assisted emulsification extraction (UAEE) with chloroform, 25 and SBSE which was used for the analysis of pesticides in river water 73,77 or in drinking water. 74,78Garrido Frenich et al. 72 compared both SPME and hollow fiber liquid phase microextraction (HF-LPME) for the extraction of pesticides in drinking water, concluding that SPME and GC-MS/MS offered the best compromise in terms of quality, speed and reliability.
After extraction (and occasionally cleanup), GC-amenable pesticides described in Table 2 were determined by GC-QqQ MS/MS in the SRM mode.In all the publications, at least two transitions were acquired and most used the EI mode.Feo et al. 25 concluded that the best selectivity and sensitivity for the determination of pyrethroids in water and sediment was obtained by using GC-MS/MS in the NCI mode.
Pitarch et al. 65 developed the first GC-MS/MS methodology for priority organic pollutants in water, including several pesticides.Although the EI mode was used for the general method, a supplementary methodology based on GC-(NCI) MS using the selected ion recording (SIR) mode was proposed for quantification and confirmation of OC pesticides as it allowed notable sensitivity improvement for these compounds.As regards confirmation identity, it is based on the presence of at least two SRM transitions 25,71,72,76,77,79 and R t agreement, although several authors also took into account the experimental relative intensity ratio of the sample and the theoretical ratio of the reference standard, using the maximum deviations established in the European Commission Decision 2002/657/EC, 21,65,68,75,79,80 or based on other defined tolerances. 66,70,73,74,78antification for water samples has been mostly based on calibration in solvent. 25,65,66,68,72For more complex matrices, matrix-matched calibration provided better results, as in the case of soil samples, 69,76 marine sediments 21,70 or even airborne particulate matter. 71e GC-MS/MS methods applied in the analysis of environmental samples presented excellent sensitivity.In the case of soils and sediments, the lowest level validated was as low as 1 ng g −176 or 5 ng g −1 . 25,69The purity of the air was evaluated by analyzing the airborne particulate matter performing the validation at the lowest level of 10 ng mL −1 . 71As regards water samples, the lowest level validated is reported to be 0.14 ng L −1 for a variety of priority organic pollutants. 73As expected, the lowest level validated in waste water was much higher, as a consequence of the higher matrix complexity. 67,75,77

Trends and perspectives
After its first use for PRA around 10 years ago, GC-QqQ MS/MS has been consolidated in most laboratories.This technique has the degree of robustness required to be widely applied at present, and the improvements offered as regards method sensitivity and selectivity are widely recognized.The determination of pesticide residues by GC-MS is commonly based on the use of relatively long capillary columns (25-30 m) with internal diameters of 0.25-0.32mm by using typically low polarity stationary phases (from 100% methyl silicone to 5% phenyl methyl silicone in most cases) leading to chromatograms of tenths of minutes.Fast GC coupled to MS has been shown to be an interesting alternative that, through different instrumental approaches, allows increased sample throughput by reducing the analysis time. 81,82In this way, low-pressure GC-QqQ MS/MS has been applied to pesticide residue analysis, with an important increase in sensitivity, shorter run time, higher sample loading and increased ruggedness. 81,83Another approach is based on the use of narrower columns with internal diameters of 0.1 mm i.d.combined with fast column temperature programming, resulting in an increase in sensitivity, a reduction in the analysis time and peak width and thus an increase in resolution, thereby making it feasible to determine even more than one hundred pesticides in analysis times lower than 10 minutes. 82,84e use of APCI as an ionization source for GC-MS methods, is a major advance that will greatly improve pesticide residue determination (and other GC amenable compounds) due to its soft ionization behaviour in comparison with that obtained by EI.
Portolés et al. 18,19 have demonstrated the capabilities of this soft ionization source for producing spectra with much lower fragmentation than that obtained by EI, where the molecular ion is commonly absent (or with low abundance).In these cases, when using EI it is necessary to select a fragment ion as a precursor ion in the MS/MS method and, consequently, not only the sensitivity but also the specificity of the method can be affected.With APCI, M + or [M + H] + is the base peak of the spectra in most cases.Under these conditions, precursor-ion selection would no longer require a compromise between selectivity and sensitivity, allowing more specific MS/MS experiments.This approach has not yet been exploited in pesticide residues analysis but it will surely be a major advance in this field in the near future.

A
combination of GC-MS/MS and LC-MS/MS, both with a triple quadrupole analyser, is one of the most current powerful approaches in PRA.They are complementary techniques that allow the determination of pesticides and metabolites within the whole range of physico-chemical properties, such as volatility, polarity and thermal stability.The combined use of both techniques allows the monitoring of hundreds of compounds that are GC or LC amenable.The present trend in multiresidue analysis is the application of generic sample preparation leading to sample extracts that are analysed by both LC-MS/MS and GC-MS/MS, this being nowadays one of the most "universal" approach in PRA.2013, 758, 80-92.84.R. Húšková, E. Matisová, S. Hrouzková and L. Švorc, Analysis of pesticide residues by fast gas chromatography in combination with negative chemical ionization mass spectrometry, J. Chromatogr., A, 2009, 1216(35), 6326-6334 .

Table 2 (
76ntinued)Depending on the complexity of the matrix, different techniques have been used to extract the analytes.For example, soil generally requires the use of stronger techniques capable of extracting potentially bound residues.Only two applications dealt with soil analysis: Martínez Vidal et al.69who used a pressurized liquid extraction (PLE) technique with a mixture of ethyl acetate and methanol for investigation of multiclass pesticides, and Rashid et al.76who developed a methodology based on acetate buffering QuEChERS with a posterior liquid-liquid partition (LLP) cleanup for the determination of OC pesticides.In the case of sediments, analytical methodologies are based on extraction with different solvents followed by an additional cleanup or concentration step using SPE