INTRODUCTION The Pesticide Manual lists 860 pesticides, most of which are still sold worldwide. 1 A

3.1 INTRODUCTION The Pesticide Manual lists 860 pesticides, most of which are still sold worldwide. 1 A

considerable volume of registration data is submitted by the applicant for statutory approval, which relies on the determination of the concentration of the pesticide and associated metabolites and degradation products in a wide variety of matrices and their structural characterization. Another driver for the analysis of pesticide residues in food is to generate the monitoring data needed to back up the statutory approval process. Checks are carried out to ensure that no unexpected residues are occurring in crops and that residues do not exceed the statutory maximum residue levels (MRLs). Such surveillance is carried out as part of national and international programs and also by the food industry and their suppliers to demonstrate ‘‘due diligence’’ under food safety legislation. Following notification of an MRL violation, brand owners may choose to sample and analyze foodstuffs on a ‘‘positive release’’ basis to ensure that the materials are compliant before distribution. While laborator- ies undertaking the pesticide residue analysis for a survey might have up to 1 month to report their findings, a much more rapid approach to analysis is required for positive release situations (e.g., 24 h).

Residues of pesticides used for crop protection, on animals, for public hygiene use, in industry and in the home or garden are found in rivers and groundwater. In the United Kingdom, requirements for water analysis vary depending on whether it is

for monitoring trends, 2 Environmental Quality Standards (EQSs), 3 tailored to local pesticide usage patterns, 4 or drinking water. 5 Analysis is also needed to investigate the concentration of pesticides and their metabolites in samples from humans; both by the chronic exposure of the general population to pesticides 6 and by occupational exposure for those working with pesticides. 7 Biological monitoring of exposure involves the measurement of a biomarker (normally the pesticide or its metabolite)

in biological fluids. 8 Pesticides can poison wildlife, including beneficial insects and some pets. Cases relate to abuse of a product where the pesticide is used to

deliberately and illegally poison animals. 9 Relevant tissues from casualties, includ- ing whole bees, are analyzed to help assess the probable cause of the incident and

whether any pesticide residues found contributed to the death or illness of the animal. Analyses must prove reliable, be capable of residue measurement at very low levels (sub ppb), and also provide unambiguous evidence of the identity and magnitude of any residues detected. More recently, additional emphasis has been on shortening analysis times to deal with high sample throughput. Depending on the purpose of the analysis, determination of pesticide residues may be termed target (compound) analysis or nontarget analysis. Checking food or surface and ground- water has been typically achieved by target compound analysis as the relevant

Analysis of Pesticides by Chromatographic Techniques 61 analytes are fixed by the residue definition given in the various regulations, which

may include significant metabolites or degradation products. In general, MRLs are in the range of 0.01

–10 mg=kg but can be lower for infant food where there is no approved use (Limit of Determination MRL, typically between 0.01 and

0.05 mg=kg). Although EU regulation of residues in drinking water does not contain detailed residue definitions, the high sensitivity of target compound analysis has been employed as a practical compromise to meet the 0.01 mg=L limit. For target compound analysis, characteristic ions for the analyte are selected before starting the analysis. An unexpected compound cannot be detected if its relevant ions are not selected and will be missed if present in the sample. Pesticide misuse can be missed due to incomplete target compound lists and strategic data regarding changing patterns in both legitimate and illegal use of pesticides cannot be captured. A nontarget analytical approach provides rapid and accurate screening of unknown substances in food and water and also when determining whether pesticide abuse caused the death of an animal. For nontarget screening, instruments must be able to generate sufficient information for elucidation of residues by providing either mass spectra for interpretation or accurate mass information from which empirical formu- lae can be deduced. This information must be generated while maintaining the high sensitivity required, for example, to detect violations of limit of detection (LOD)- based limits in food or water. When dealing with unknowns there is often a lack of reference standards, used in target compound analysis for unequivocal identification through the standard’s characteristic chromatographic behavior and mass spectrum.

Over the past decades, approaches to trace level determination of pesticides have changed considerably, moving away from the use of GC with selective detectors to the sensitivity and selectivity offered by GC-MS. The commercialization of atmos-

pheric pressure ionization with tandem mass spectrometers 11 enabled the determin- ation of pesticides and their degradation products that are polar, relatively nonvolatile, and=or thermally labile, and, therefore not amenable to GC analysis. 12 Further developments in both detection and column technology enabled the scope for LC to be significantly enlarged and now LC-MS offers a similar breath of analysis to

GC-MS (e.g., 171 pesticides and=or metabolites). 13 The use of alternative mass analyzers to the single quadrupole (Q) (i.e., various types of ion trap, triple quadru- poles, and time-of-flight [TOF]) and their various combinations (e.g., QTOF) has improved the capabilities of the instruments available. Table 3.1 provides an over- view of the advantages and disadvantages of each analyzer for both GC-MS and LC-MS. The vast majority of pesticides sought is amenable to multiresidue approaches and can now be thoroughly isolated from water and complex food matrices without the large amounts of natural material coextracted with the pesticides interfering with the analysis. For example, out of ~400 pesticides routinely targeted

using the QuEChERS method, 14 217 are analyzed employing LC-MS=MS and 187 employing GC-MS and GC-TOF MS techniques. In a fascinating recent review, 15

Alder compared the scope and sensitivity of GC coupled with EI and single quadru- pole MS with LC combined with tandem mass spectrometry for the analysis of 500 high-priority pesticides concluding that both techniques are still needed to cover the wide range of pesticides to be monitored. A number of compounds are not amenable to multiresidue analysis and so require separate, so-called, single residue methods.

62 Analysis of Pesticides in Food and Environmental Samples

TABLE 3.1 Capabilities of the Different Analyzers for Pesticide Residue Analysis

Analyzer

Disadvantages Quadrupole (Q):

Advantages

High sensitivity in SIM mode Poor sensitivity in scan mode GC-MS, LC-MS

(0.1 –1 pg), good dynamic range (50 –500 pg), low selectivity for (five orders of magnitude), good

complex matrices, SIM needs selectivity in CI, low cost

preselection, unit mass resolution Quadrupole ion trap

High=medium sensitivity in scan and Low selectivity for complex matrices (QIT): GC-MS,

product ion scan modes (0.1 –10 pg), in MS mode, limit on number of GC-MS=MS,

library-searchable EI and product ions that can be determined LC-MS, LC-MS=MS

ion spectra, good selectivity in CI, simultaneously, limited dynamic MS n , fast acquisition rate, low cost

range (3 –4 orders of magnitude), limited mass range in MS=MS, unit mass resolution

Triple Quadrupole Excellent sensitivity (10 –100 fg) The number of MRM channels that (QqQ): GC-MS=MS,

and selectivity in MRM mode, can be monitored at any one time is LC-MS=MS

good dynamic range (five orders limited, MRM needs preselection, of magnitude), concurrent

unit mass resolution, high cost monitoring of many channels High-speed time-of-

High sensitivity (0.1 –1 pg), Low selectivity, limited dynamic flight (TOF): GC-MS

library-searchable EI spectra, range (four orders of magnitude), very fast acquisition rate

unit mass resolution, high cost Enhanced resolution

High sensitivity (0.1 –1 pg), good Limited dynamic range (four orders TOF: GC-MS,

selectivity, accurate mass, fast of magnitude), not true ‘‘high LC-MS

acquisition rate resolution,’’ high cost Qq-linearIT (QqLIT):

Excellent sensitivity (10 –100 fg) Unit mass resolution, high cost LC-MS=MS

and selectivity in MRM mode, high sensitivity in product ion scan mode, MS n

QTOF: LC-MS=MS High sensitivity (0.1 –1 pg), good Limited dynamic range (four orders selectivity, accurate mass of both

of magnitude), not true high precursor and product ions, fast

resolution, high cost acquisition rate

In the next sections, the use of GC and LC with selective detectors will be briefly discussed followed by an exploration of the three stages key to the successful application of both GC-MS and LC-MS: sample introduction, chromatography and subsequent ionization, and mass analysis (mass spectrometry).