Liquid chromatography

One of the disadvantages of GC has always been, and in fact still is, the requirement that the compound to be determined has to be thermally stable and should have a sufficient volatility. In GC analysis, these compounds either show sign of thermal decomposition or fail to elute to column. In recent years many new pesticides have been developed that are not very well amenable to GC. Among the analytical approaches, LC is the preferred separation technique for the most polar and thermally labile pesticides. There are some classes of pesticides for which HPLC is superior to GC, such as carbamates, urea herbicides, benzoylurea insecticides and benzimidazole fungicides.

HPLC methods for the determination of pesticides could employ reversed-phase. As most pesticides are low-polarity compounds, they often are analysed by reversed-phase chromatography with C18 or C8 columns and aqueous mobile phase, followed by UV adsorption, UV diode array, fluorescence or mass spectrometric detection.

In the determination of phenylureas, which are used extensively to protect a large number of crops against weeds, LC methods have been used in various matrices. UV detection used in many methods [73,74]) is sensitive enough in most cases but lacks selectivity, especially when trace levels in complex samples need to be assayed. Effective clean-up before LC/UV determination could be sufficient to isolate phenylureas from matrix interferences well enough to allow determination at low levels required by current regulator laws for baby and organic foods (p 0.010 mg/kg).

Sannino [75] described a method for quantitative determination of nine phenylurea herbicides in potatoes, carrots and mixed vegetables. Samples are extracted with acetone, partitioned with ethyl acetate-cyclohexane (50 + 50, v/v) and cleaned up by GPC with ethyl acetate-cyclohexane (50 + 50, v/v) as eluent. A further purification on Florisil cartridge was necessary to obtain an extract free from interferences when detected by LC with UV at 254 nm.

The fluorescence detector has been in use for a number of years on a more or less routine basis. Fluorimetry is much more selective and in many cases much more sensitive than UV absorbance spectrometry. The better selectivity is because two wavelengths, the excitation and the emission wavelength, are involved, and by a careful selection of these two wavelengths a very selective detection is possible. The very high selectivity of fluorimetry is of course also a disadvantage because there are only a rather limited number of compounds that can be detected with good sensitivity. Some classes of compounds show a strong native fluorescence, but there are not many pesticides that show a native fluorescence.

An example of LC method with fluorescence detector is the determination of TBZ and carbendazim, two compounds that belong to the benzimidazole class. They are systemic fungicides used as either pre-harvest or post-harvest treatment for the control of a wide range of fruit and vegetable pathogens. Carbendazim is the major metabolite and fungitoxic principle of benomyl and thiophanate methyl (TM). Sannino [76] used a reversed-phase liquid chromatographic method for determining carbendazim, TBZ and TM in fruit products (nectars, purees, concentrates and jams). The extraction and clean-up method, based on the procedure developed by Bicchi et al. [77], is schematized in Figure 6. The fungicides were separated and quantified by using an ion-pairing mobile phase with UV and fluorescence detectors in tandem, following the procedure of Gilvydis and Walters [78]. UV and fluorimetric detectors connected in series allowed the simultaneous determination of the non-fluorescent TM and fluorescent carbendazim and TBZ. Figure 7 shows the chromatograms of apricot puree extract and the sample spiked with 0.1 mg/kg of carbendazim and TBZ and 0.01 mg/kg of TBZ.

For those compounds which do not possess appreciable native fluorescence, a derivatization technique can be employed.

In pesticide residue analysis the main application of derivatization is the determination of NMCs. They comprise an important class of insecticides, widely used for crop protection with some of the most common ones being carbaryl, carbofuran, aldicarb, methomyl and oxamyl. Carbaryl has been included in the final list of compounds to be considered for periodic re-evaluations by the 2001 Joint FAO/WHO Meeting on the Pesticide Residues [79]. NMCs can be determined by UV detector [80].

However, the sensitivity and selectivity offered by UV detection is very poor, because the carbamates present their absorption maximum at about

Figure 6 Scheme for simultaneous determination of benzimidazole fungicides employed by Sannino [76].

190 nm. Post-column hydrolysis and derivatization coupled with fluorescence detection has been accepted as a standard protocol by several official organizations (US-FDA, EN 14185-2) [81,82]. The derivatization is specific for primary amines and thus adds a high degree of selectivity to the detection compared to direct UV absorbance detection of the parent compounds. The carbaryl metabolite, 1-naphthol, which is not carbamate, is unaffected by this reaction sequence, but exhibits an equally strong native fluorescence. Thus, on the basis of the pioneering work by Moye et al. [83] which was refined by Krause [84] various sensitive and selective LC methods including post-column derivatization for simultaneously determining NMCs in complex matrices such as vegetables [85,86] and meat [87] as well as water [88] have been proposed. The typical method is as follows: the pesticides are extracted from food with organic solvents, the extract is cleaned up by a method based on liquid-liquid or solid-liquid partition, and is analysed by LC with post-column derivatiza-tion. Podhorniak [89] uses an acetone extraction, followed by an aminopropyl SPE. A method is presented for the determination of 24 compounds (13 parent NMC pesticides and their metabolites, and piperonyl butoxide) in selected fruit and vegetables. The Comite Europeen de Normalisation (CEN; European Committee for Standardization) Technical Committee CEN/TC 275 ''Food Analysis-Horizontal Methods'' has prepared the European Standard EN 14185-2 [82] method for the determination of NMC in fruit and vegetables based on post-column derivatization. The extract is purified on a diatomaceous earth column.

A disadvantage of the post-column technique is that special equipment is needed, such as a mixer chamber, a reactor and an extra pump. In addition, coexisting substances having fluorescence from food, especially from citrus fruits, frequently interfere with the determination of the pesticides.

5.2.1 Liquid chromatography-mass spectrometry

The number of compounds that cannot be determined by GC because of their poor volatility, high polarity and thermal instability has grown dramatically in the last few years. Agrochemicals belonging to carboxamide, quinazolin, phenoxypyrazol, strobilurin, pyrimidine, triazol, carbamate, neonicotinoid, morpholine classes are representative of the newly introduced molecules. The identification of pesticides in complex samples can be a problem for LC with traditional detection methods such as UV or even diode array detection (DAD), because the latter technique may not be specific enough for spectral differences that are too small. A fluorescence detector can offer a greater selectivity compared to UV detection, but if there are many different compounds to be analysed, this technique will not provide adequate results [90].

Nowadays, liquid chromatography coupled with mass spectrometry (LC/MS) is becoming one of most powerful techniques for the residue analysis of polar, ionic or low volatility pesticides in fruit and vegetables. Compared to traditional detectors electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources in combination with MS instruments have increased the sensitivity of LC detection by several orders of magnitude. Single quadrupole l.O

UV (28Onm)

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was the first mass spectrometer introduced in the market for combination with LC by means of atmospheric pressure ionization (API) sources. Those interfaces have the characteristic of being designed to provide a soft ionization process that lead to a mass spectrum with only few ions (commonly only the molecular ion), without the required specific structure diagnostic ions. The poor fragmentation of molecules is translated in deficient specificity because isobaric interferences (compounds with the same m/z ratio) or multiple component spectra are frequently observed in extracts of complex matrices [91]. Then, in contrast to GC/MS, single quadrupole mass spectrometers are not used in the majority of recent studies dealing with LC/MS.

A disadvantage of single quadruple instruments is the high intensity of background signals derived from sample matrix and HPLC solvent clusters. Owing to this chemical noise in real samples, very low LODs cannot be achieved even if the sensitivity of these instruments is high.

The chemical background can be reduced significantly if MS-MS in combination with SRM is applied. Reaction-monitoring modes enhance the detection limits in analytical procedures. In a QqQ, Q1 and Q3 quadrupoles are both fixed at a single mass. This technique is applied when the precursor and the product ions are known before analysis. Even if the co-extracted matrix component has the same molecular mass of a pesticide it can be separated in SRM experiments because their fragmentation in the collision cell most often results in different product ions. Therefore, MS-MS offer excellent sensitivity and unsurpassed selectivity. For this reason, QqQ analysers have been the most widely used technique for the determination of pesticides in food until now. In the last 4 years, many LC-MS-MS methods have been developed for multiclass pesticides in fruit and vegetables and water [92-101]. Nowadays, a modern commercial QqQ mass spectrometer is suitable to detect approximately 100 analytes simultaneously with sensitivity sufficient for residue determination at the 0.010mg/kg level [92]. In the case of sufficiently high concentrations, the simultaneous observations of approximately 200 SRM transitions are feasible. The use of time window programs (periods) is not necessary unless the number of analytes to be analysed within one run is significantly increased, or pesticides with very low response are determined. Because of the high sensitivity achieved by MS-MS, gradient elution on a small reversed-phase column is usually used. However for confirmation purposes at least two transitions must be recorded.

Pizzutti et al. [101] developed and validated a method for the analysis of 169 <-

Figure 7 Chromatograms of (a) apricot puree free from carbendazim, TM and TBZ, and (b) apricot puree spiked with 0.1 mg/kg of carbendazim and TM and 0.0010 mg/kg of TBZ obtained by UV (at 280 and 305 nm), and (c) fluorescence detection at excitation/emission wavelengths of 280/310 nm, immediately following carbendazim elution at 305/345 nm. Column: 5 mm Supelcosil LC-18-DB; injection volume, 25 mL; mobile phase; 35% methanol in ion pairing solution. 1 g sodium decanesulfonate dissolved in a mixture of 200 mL of water, 7mL of phosphoric acid and 10 mL of triethylamine and diluted to 1 L with water); column temperature: 40°C and flow rate: 1.5 mL/min. Reproduced from Ref. [76] with permission from Elsevier, Copyright 1995.

pesticides in soya, without clean-up, by LC-MS-MS using positive and negative ESI. In total, 155 pesticides were analysed in the positive mode and 14 pesticides in the negative mode, each mode in one single chromatographic run.

ITDs may also operate in the MS-MS mode, which reduces the background to a level known from MS-MS. When performing MS-MS, quadrupole ion trap (QIT) instruments are generally more difficult to handle than QqQ analysers but they have the advantage of working in product-ion-scan without loss of sensitivity. Moreover, QIT offers the possibility of performing multiple-stage fragmentation (MSn). However, ion collection, fragmentation, and mass analysis of fragments in a step-by-step process in traps require much more time than in QqQ instruments, which do this in parallel. Furthermore, ion traps suffer from a limited dynamic range, a smaller potential to fragment very stable ions and the inefficiency to trap low-mass fragments.

Soler et al. [102] compared QITand QqQ for determining pesticides in orange samples. The results indicated that TQ provides higher precision, better linearity, it is more robust and when the purpose of the analysis is quantitative determination, it is preferable over QIT. However, the LOQs were almost the same for both instruments.

All LC-MS instruments can be equipped with at least three types of soft ionization sources, which are ESI, APCI and photoionization. Up to now, applications of photoionization to the determination of pesticides have been rarely published. ESI and APCI are used more often. The ESI coupled with MS-MS is supposed to have a high sensitivity and selectivity for a wider range of pesticides in food. There are several instrumental parameters that have drastic influence on the ionization efficiency. The instrumental optimization includes the adjustment of typical interface parameters such as the ionization voltage in ES, the pressure of the spraying/nebulizing gases, the interface temperature and the clustering potential.

The mobile phase is important to obtain a good chromatographic separation, but it also affects the analyte ionization and the sensitivity of the mass spectrometry. Usually, the use of MS-MS does not require any chromatographic separation between analytes, because it is very rare to find molecules that share the same unique transition. However, the simultaneous analysis of a high number of compounds by MS-MS requires at least a sufficient chromatographic separation, in order to reduce the matrix effect. Methanol and/or acetonitrile are usually used as organic modifiers. LC separations are sometimes improved at acidic pH, using acetic acid or formic acid, as such or in combination with ammonium acetate or ammonium formate. The intensity of signal can be increased in the positive ionization (PI) mode; however the presence of H+ ions inhibited the negative ionization.

A relatively new and valuable technique in the field of pesticide analysis is the TOF-MS. The accurate mass measurements of the TOF-MS (typical mass error < 2 mDa) ensure an equal selectivity as that obtained with the two type of MS-MS. The high degree of selectivity is obtained by removing matrix interferences at the same nominal mass measurement (using the exact mass chromatograms). The main advantage of this type of instrument is the identification of non-target pesticides and unknown peaks in a sample even if analytical standards are not available. Thurman et al. [103] developed an identification scheme using a combination of LC/TOF-MS (accurate mass) to generate elemental composition of ions and LC/MS ITMS (MS-MS) providing complementary structural information, which is useful for the elucidation of unknown organic compounds at trace levels in citrus fruit extracts. This scheme has been applied to identify two post-harvest fungicides (imazalil and prochloraz), the main degradation product of imazalil and a non-previously reported prochloraz degradation product.

The high speed and acceptable sensitivity have made TOF an attractive alternative to quadrupole or QqQ. Recently, LC/TOF-MS has been proven to be a sensitive and selective method for the determination and confirmation of pesticide residues in vegetables and fruit [104] obtaining LODs in compliance with established MRLs. Gilbert-Lopez et al. [105] developed a method based on LC/ESI-TOF-MS for the determination of 12 pesticides (MCB, TBZ, imazalil, tridemorph, triadimefon, bitertanol, prochloraz, flutriafol, myclobutanil, ipro-dione, diphenylamine and procymidone) in fruit-based baby foods. The confirmation of the target pesticides was based on accurate mass measurements of protonated molecules (M + H)+ and fragment ions. LODs were between 0.1 and 10 mg/kg depending on pesticide studied.

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