Analytical methods pesticide confirmation

The reliability of the analytical method was confirmed by analyzing insecticide-fortified extracts of untreated soil. An average recovery of 85% was achieved with DDT. The gas chromatograph was standardized by injecting known amounts of pesticides before and after every two or three unknowns. 

The development of a robust analytical method is a complex issue. The residue analyst has available a vast array of techniques to assist in this task, but there are a number of basic rules that should be followed to produce a reliable method. The intention of this article is to provide the analyst with ideas from which a method can be constructed by considering each major component of the analytical method (sample preparation, extraction, sample cleanup, and the determinative step), and to suggest modern techniques that can be used to develop an effective and efficient overall approach. The latter portion emphasizes mass spectrometry (MS) since the current trend for pesticide residue methods is leading to MS becoming the method of choice for simultaneous quantitation and confirmation. This article also serves to update previous publications on similar topics by the authors. ... 

The most widely regarded approach to accomplish the determination of as many pesticides as possible in as few steps as possible is to use MS detection. MS is considered a universally selective detection method because MS detects all compounds independently of elemental composition and further separates the signal into mass spectral scans to provide a high degree of selectivity. Unlike GC with selective detectors, or even atomic emission detection (AED), GC/MS may provide acceptable confirmation of the identity of analytes without the need for further information. This reduces the need to re-inject a sample into a separate GC system (usually GC/MS) for pesticide confirmation. Through the use of selected ion monitoring (SIM), efficient ion-trap or quadrupole devices, and/or tandem mass spectrometry (MS/MS), modern GC/MS instruments provide LODs similar to or lower than those of selective detectors, depending on the analytes, methods, and detectors. 

In food analysis, sensitivity is not the only requirement for analytical method development. Besides confirmation of the identity of pesticides, the identification of nontarget analytes is also important. One powerful tool is LC/MS, especially when it is combined with appropiate sample-treatment procedures it allows one to obtain detection limits adequate for trace-level analysis. Liquid chromatography-MS has demonstrated that it is an effective way to obtain both qualitative and quantitative information. 

In both these areas, chemical derivatisation has traditionally played a role and with the advent of gas chromatography an even more important role. The reasons for preparing a derivative suitable for GC analysis are many and varied and have been discussed thoroughly in a number of books and reviews (l- >). For convenience they are summarised in Table I. As can be seen, two different types of chemical derivatisation techniques are mentioned under Item 4 of Criteria, There is the chemical derivatisation of a pesticide as a pre-requisite of the method of analysis, e.g. esterification of the chlorophenoxy acids, as well as derivatisation as a method for confirmation of identity. The former must meet all the requirements associated with a practical, viable analytical procedure while for the latter the emphasis is on speed, ease of operation and reproducibility. 

In connection with the development of an analytical method (13) for the determination of organophosphorus pesticides in human blood and urine, mass spectral confirmation of a series of methylated and ethylated derivatives of the hydrolytic and metabolic products of these insecticides was required. The urine of an individual occupationally exposed to parathion was extracted with a 1 1 (v/v) solvent mixture of acetonitrile and diethyl ether. Simultaneously, the intact organophosphorus insecticides were hydrolyzed by adding a portion of 5N hydrochloric acid to... 

Of all the systems which have been utilized for the analysis of pesticide residues, combined gas chromatography-mass spectrometry afiFords a particularly useful approach because positive identification of the components of a mixture can be made without prior separation at sensitivities compatible with the limited quantities of residues generally available. From the results of recent studies involving the application of this technique, it has been demonstrated that available residue analytical methods provide efficient isolation and adequate cleanup of extracts of human, animal, and environmental media in most cases to permit gas chromatographic-mass spectrometric analyses with maximum confidence. Additionally, it has been shown that this combined technique will conveniently provide definitive and conclusive confirmation of residue identity as well as characterization of residues and their metabolites of unknown structure. 

More attention should be paid to the needs of the residue analyst working at the bench. Multidetection analytical methods are an enormous gain, but we need to beware of constantly adding new pesticides or breakdown products to an already-existing multidetection scheme. To do this will eventually make confirmation of identity an impossible job. For... 

Of the more-or-less pure organic constituents detectable in water as analytical methods improve, the insecticides, fungicides, and weedkillers probably have received the most attention. Although inland surface waters have been monitored for about a decade, essentially nothing is known about pesticide levels in the ocean, and existing information comes from indicator organisms. Even current quantitative data from water analyses stretch the limits of accuracy and confirmation of identity (23). 

An overview of several recent applications of UHPLC-MS(/MS) methods for the multi-residue analysis of pesticides in food products has been presented. In order to cope with the necessity of high throughput and fast analysis of pesticide residues in food, all aspects of analytical method development, that is, sample extraction and clean-up, chromatographic analysis, and quantitation and confirmation aspects, must be taken into account. 

Elucidation of how the general principles underlying the concept of validation should be expressed in practice is an evolving process, as exemplified by the ongoing evolution of validation requirements for bioanalytical assays in the pharmaceutical industry (Shah 1992, 2000 FDA 2001 Viswanathan 2007). The complementary principle of fitness for purpose (Section 9.2) applies not only to the assay method but also to the validation process itself. Procedures that are considered to be fit for purpose in validation of an analytical method to be used in drug development, for example, need not necessarily apply to, e.g., methods used to screen pesticide residues in foodstuffs. As noted in Section 9.2, this point of view appears to be consistent with the definition of validation applied to all measurements (ISO 1994) Validation Confirmation by examination and provision of objective evidence that the particular requirements for a specified intended use are fulfilled. Of course, some basic principles are common to all validation schemes. 

Clearly, different compromises must be applied compared with those for analytical methods that target only one or two specific compounds, with respect to analysis time, degree and level of validation criteria including accuracy and precision, recovery, confirmation of identity etc. The broader the class of compounds to be analyzed, the lower the level of validation that is possible, unless it is accepted that large amounts of time and resources are available. Usually broad class multi-residue analyses are designed as semi-quantitative screening methods usually followed by identity confirmation. This section describes some examples of such methods developed for pharmaceutical and pesticide residues in water. 

In some cases, confirming identification of components obtained from soil, such as pesticides, is essential. Thus, the uncertainty in some analyses needs to be addressed. This can be accomplished by identifying the components using two entirely different methods such as IR spectroscopy and MS. Although GC-IR-MS methods can positively identify separated components, the IR component of the system is not nearly as sensitive as are the GC and MS components. This detracts from the usefulness of this method. However, in cases where the level of analyte is not limiting, which frequently occurs in soil extracts, this can be an excellent method to use. Also, with modern concentration techniques, it is neither difficult nor time-consuming to concentrate analytes to a level that is identifiable by IR spectroscopy [17,18],... 

Second column confirmation must be used in pesticide, PCBs, and chlorinated herbicide analyses by EPA Methods 8081, 8082, and 8151, respectively. In these methods, two columns with dissimilar polarities and two ECDs provide compound identification and quantitation. This technique produces a lower rate of false positive results, but does not eliminate them completely. This is particularly true for low concentrations of pesticides and herbicides, where non-target compounds, such as constituents of the sample matrix or laboratory artifacts listed in Table 4.3, produce chromatographic peaks on both columns. These interference peaks cannot be distinguished from the target analytes based on retention time only and cause false positive results. 

A variety of methods have been developed for sampling pesticides in air. Suitable procedures must deal with difficulties posed by the uncertainty regarding the physical state (aerosols, solid particles, vapors) of airborne residues, their relatively low concentrations (less than 1 mg/m, ca 80 ppb), fluctuations in pesticide concentrations and the levels of potential interferences with time, potential reactivity during the sampling process, and limited availability of sampling devices. These are in addition to the problems of cleanup, recovery, quantitation, and confirmation which are common to trace analytical processes once the sample has been collected and brought to the laboratory for determination. 

The modem GC data system will produce a report of peaks detected with the retention time, peak area, and peak height. In order to identify the analytes of interest and quantify the data, a series of calibration standards are required to be analyzed followed by samples. The calibration standards will identify retention times for analytes, surrogates, and internal standards. With the exception of MS analysis, compounds are identified in chromatograms based solely on their retention time. Positive confirmation can be done by analyzing the same sample extract on a different type (polarity) of GC column. If the compound is detected at the same concentration from both GC columns, then the data can be reported (e.g., US EPA Method 8081—OC Pesticides—requires analysis on a DB-5 column with confirmatory analysis on a DB-17 column). For MS analysis, multiple ion chromatograms... 

Ultraviolet spectrophotometry is considered a valuable tool as an aid for confirming the identification of pesticide residues. A correlation between the UV spectrum and the structure of several pesticides is discussed. Knowledge of such correlation may provide clues about the general type of chromophore present and may help the analyst to design analytical procedures. The transparency of many groups in the near UV imposes a limitation on interpretations of the absorption bands in this region. However, when taken in conjunction with the information obtained by IR, NMR, and mass spectroscopy, UV spectra may lead to structural proposals of value to the pesticide analyst. A discussion of the methods that have been utilized for the analysis of pesticides on the submicrogram level is also presented. 

The superior selectivity and high-speed acquisition rate of the GC-MS/MS technique allows interference-free quantification, even with peak co-elution and provides positive confirmation in a single analytical run. To accurately monitor pesticide residues, a high throughput multi-residue method that can quantitate a large number of pesticide residues during a single analysis is described. 

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