Multi-residue

We can list the following areas as prime targets essential oil and natural product analysis, chiral analysis (e.g. of fragrances), trace multi-residue analysis, pesticide monitoring, and further petroleum products applications, in fact any separation where simply greater resolution and sensitivity is demanded-which means probably almost... 

J. Cai and J. Henion, Quantitative multi-residue determination of /3-agonists in bovine urine using on-line immunoaffinity extr action-coupled column packed capillary liquid cliromatogr aphy-tandem mass spectr ometry , 7. Chromatogr. 691 357-370 (1997). 

Kasprzyk-Hordern B, Dlnsdale RM, Guwy AJ (2007) Multi-residue method for the determination of basic/neutral pharmaceuticals and illicit dmgs in surface water by solid-phase extraction and ultra performance liquid chromatography-positive electrospray ionisation tandem mass spectrometry. J Chromatogr A 1161 132-145... 

Togola A, Budzinski H (2008) Multi-residue analysis of pharmaceutical compounds in aqueous samples. J Chromatogr A 1117 150-158... 

Gros M, Petrovic M, Barcelo D (2006) Development of a multi-residue analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS-MS) for screening and trace level determination of pharmaceuticals in surface and wastewaters. Talanta 70 (4) 678-690... 

As far as is practicable, the methods proposed must employ the simplest approach and commonly available equipment. If possible, standard multi-residue methods should be used. Descriptions of methods must be provided, including all necessary details. 

Figure 1 Development/vaUdation approach for multi-residue methods (hterature references in brackets)...

Owing to the complexity of multi-residue methods for products of animal origin, it is not possible to outline a simple scheme however, readers should refer to methods described in two references for detailed guidance (Analytical Methods for Pesticides in Foodstuffs, Dutch method collection and European Norm EN 1528. ) There is no multi-method specifically designed for body fluids and tissues. The latter matrix can be partly covered by methods for products of animal origin. However, an approach published by Frenzel et al may be helpful (method principle whole blood is hemolyzed and then deproteinized. After extraction of the supernatant, the a.i. is determined by GC/MS. The LOQ is in the range 30-200 ag depending on the a.i.). 

Moreover, new technologies such as LC/MS/MS should be considered and their potential should be recognized in the future. Currently food control laboratories monitor only a part of the pesticides used in their routine work. They prefer active ingredients that can be analyzed by multi-methods or some group-specific methods, because resources to check all relevant pesticides are normally not available. Therefore, many a.i. are monitored only on a case-by-case basis or not at all. An LC/MS multi-residue method, which may be developed in the future, could cover this gap to a large extent. 

Other considerations could include availability of reagent(s) or equipment, method for routine analyses vs limited samples, and confirmatory method vs multi-residues. Plan for method validation and/or analytical quality control. 

The multi-residue method DFG was intended to be used in state enforcement laboratories or in private contract or food industry laboratories. It was aimed initially only at plant materials and water and included a relatively large number of pesticides which are amenable to GC. 

The scope of the multi-residue method is extended permanently by testing and then including further active substances that can be determined by GC. Acidic analytes (such as phenoxyacetic acids or RCOOH metabolites) are included into the homogeneous partitioning by acidifying the raw extracts to a pH below the pKs value of the carboxylic acids. To include these analytes in the GC determination scheme they have to be derivatized with diazomethane, diazoethane, trimethylsilyldiazomethane, acidic esterification or benzylation, or by silanizing the COOH moiety. 

Once several target methods employing, e.g., LC/MS/MS techniques have been combined, a multi-residue method will evolve which includes the DEC S19 extraction procedures in combination with the generally applicable GPC cleanup and requires automatic multiple injections to circumvent the limitations of the limited HPLC peak capacity and the target-specific MS/MS methods. 

Non-NADA methods may be designed to detect multiple residues and they may be designed for use in multiple species. In order to validate these multi-residue methods, modifications to the validation protocol relative to single analyte methods are made. Additional laboratories will participate in the method trial, but the number of samples... 

The Guidance Document on Residue Analytical Methods requests the applicant to assess a standard multi-residue method by using standard steps. These steps are extraction with acetone or ethyl acetate, cleanup by gel permeation chromatography (GPC) and/or silica gel chromatography and final determination by GC. 

The best way to test the practicability of the multi-residue approach is to start with the GC determination step. Most often the inability to vaporize the intact compound means that it is not possible to include a new pesticide in the multi-residue scheme. In the case of common moiety methods, a decomposition step is needed to produce the common analyte. Often for that step, modification of the reaction conditions (such as pH and temperature) are necessary, which would lead to a significant deviation from standard multi-residue procedures. 

The elements of the multi-residue method should be used as needed. There is no requirement, for example, to test the full version of the German method DEG 19 without any deviation. This full method combines GPC and silica gel cleanup. A poor recovery of compounds from the silica gel is not a reason to reject the multiresidue approach, provided that the chromatograms of GPC eluates are free from interference. 

Occasionally, an additional derivatization step would allow the application of a multi-residue approach. Provided that this derivatization can be done after the standard cleanup, applicants are invited to present those methods. In most cases, for monitoring purposes a supplementary derivatization will be much simpler than a completely separate single-residue method. 

The enforcement methods provided by the applicants give basic information about appropriate cleanup steps and specific determination procedures. Typically, direct use of this developmental work occurred when a GC multi-residue method was found appropriate. Owing to the recent developments in the field of MS/MS with atmospheric pressure ionization, an alternative approach for those compounds that can be analyzed by liquid chromatography (LC) will soon be possible. It is important that some fundamental considerations for such method(s) should be agreed at the outset. Considerations include the most suitable extraction solvents and cleanup steps and some standard HPLC conditions. 

Both multi-residue methods are presented in several parts, which separate general considerations from procedures for extraction, cleanup and determination/ confirmation. Whereas in EN 12393 several extraction and cleanup steps cannot be combined arbitrarily, the modular concept is utilized to a greater extent in EN 1528. In the latter standard, there is no limitation to the combination of several extraction procedures, mostly designed for different commodities, e.g., milk, butter, cheese, meat or fish, with different cleanup steps. Both standards, EN 1528 and EN 12393, do not specify fixed GC conditions for the determination and confirmation. All types of GC instruments and columns, temperature programs and detectors can be used, if suitable. 

The other two CEN standards, for the determination of dithiocarbamate/thiuram disulfide residues and for the quantitation of bromide, are also separated into parts, but, in contrast to the multi-residue methods, complete methods are presented in each different part. Owing to this different approach and the reduced number of analytes, it was possible to validate these methods fully. 

CEN requirements for widely accepted multi-matrix/multi-residue methods... 

For multi-analyte and/or multi-matrix methods, it is not possible to validate a method for all combinations of analyte, concentration and type of sample matrix that may be encountered in subsequent use of the method. On the other hand, the standards EN1528 andEN 12393 consist of a range of old multi-residue methods. The working principles of these methods are accepted not only in Europe, but all over the world. Most often these methods are based on extractions with acetone, acetonitrile, ethyl acetate or n-hexane. Subsequent cleanup steps are based on solvent partition steps and size exclusion or adsorption chromatography on Florisil, silica gel or alumina. Each solvent and each cleanup step has been successfully applied to hundreds of pesticides and tested in countless method validation studies. The selectivity and sensitivity of GC combined with electron capture, nitrogen-phosphorus, flame photometric or mass spectrometric detectors for a large number of pesticides are acceptable. 

Many experts in Europe have tested the methods of both standards with various pesticide-matrix combinations in their own laboratories. Consequently, the responsible working groups of CEN TC 275 concluded that these are the best methods available. Nevertheless, there is no complete validation of all possible pesticide-matrix combinations. However, for most multi-residue methods within the standards all those pesticides which had been successfully tested in method validation trials and/or proficiency tests are listed. Also, matrices which had been examined in ring tests are listed. 

The sensitivity achieved (LOD) is not normally presented. It is recognized that different laboratories determine dissimilar values for this parameter and even within a laboratory the repeatability of the LOD is low. Most often, the lowest validated concentration gives an impression about the lowest levels that can be analyzed generally with acceptable results. A measure of selectivity is the intensity of blank results. This intensity is discussed by the participants of inter-laboratory validation studies. However, results are not reported and limits are not defined by CEN TC 275. The results of method validations of the several multi-residue/multi-matrix methods are not reported in the same way, but newer methods with limited scope generate analogous tables with validation results (as an example, see Table 7). 

Each individual method collection comprises a large number of methods, which often have different validation statuses. For instance, the most important Swedish multi-residue method (based on ethyl acetate extraction, GPC and GC) is validated for many pesticides by four laboratories, but other methods are presented with singlelaboratory validation data. Some methods in the Dutch and German manuals were tested in inter-laboratory method validation studies, but others by an independent laboratory or in a single laboratory only. 

To reduce the effort, another validation procedure is used for extension of the German multi-residue method to a new analyte. Actually, more than 200 pesticides can be analyzed officially with this method, which is the up-to-date version of the better known method DFG SI9. A typical validation is performed by at least three laboratories, which conduct fortification experiments at the same three levels with at least four representative matrices. These representative matrices are commodities with high water content (e.g., tomato), fruits with high acid content (e.g., lemon), dry crops (e.g., cereals) and commodities with high fat content (e.g., avocado). 

Calibration data (e.g., linearity or sensitivity) are not discussed in detail between laboratories, but a typical calibration starts with 50% of the lowest fortification level and requires at least three additional calibration levels. Another point of calibration is the use of appropriate standards. In 1999 a collaborative study tested the effect of matrix residues in final extracts on the GC response of several pesticides.Five sample extracts (prepared for all participants in one laboratory using the German multi-residue procedure) and pure ethyl acetate were fortified with several pesticides. The GC response of all pesticides in all extracts was determined and compared with the response in the pure solvent. In total, 20 laboratories using 47 GC instruments... 

If analytical methods are validated in inter-laboratory validation studies, documentation should follow the requirements of the harmonized protocol of lUPAC. " However, multi-matrix/multi-residue methods are applicable to hundreds of pesticides in dozens of commodities and have to be validated at several concentration levels. Any complete documentation of validation results is impossible in that case. Some performance characteristics, e.g., the specificity of analyte detection, an appropriate calibration range and sufficient detection sensitivity, are prerequisites for the determination of acceptable trueness and precision and their publication is less important. The LOD and LOQ depend on special instmmentation, analysts involved, time, batches of chemicals, etc., and cannot easily be reproduced. Therefore, these characteristics are less important. A practical, frequently applied alternative is the publication only of trueness (most often in terms of recovery) and precision for each analyte at each level. No consensus seems to exist as to whether these analyte-parameter sets should be documented, e.g., separately for each commodity or accumulated for all experiments done with the same analyte. In the latter case, the applicability of methods with regard to commodities can be documented in separate tables without performance characteristics. 

Obviously, a best or generally accepted documentation of performance data of validated multi-residue methods does not exist. Too many data are collected and then-detailed presentation may be confusing and impractical. Additionally, the validation of multi-residue methods is a continuous on-going process which started for many pesticides 20 years ago, when less comprehensive method requirements had to be fulfilled. For this reason, a complete and homogeneous documentation of method validation data cannot be achieved. 

S.L. Reynolds, R. Fussel, M. Caldow, R. James, S. Nawaz, C. Ebden, D. Pendhngton, T. Stijve, and H. Desirens, Intercomparison Study of Two Multi-residue Methods for the Enforcement of EU MRLs for Pesticides in Fruit, Vegetables and Grain, European Commission, BCR Information, Chemical Analysis Contract No. SMT4-CT-95-2030 Reports EUR 17870EN (1997), EUR 18639 EN (1998), EUR 19306 EN (2000) and EUR 19443 EN, European Commission, Brussels (2001). 

Analytical methods for representative anilides are reported in this article. In addition, they are also applicable as multi-residue methods. 

A multi-residue method based on SPE cleanup and gas chromatography/ion trap mass spectrometry (GC/ITMS) was developed for the determination of 120 pesticides and related metabolites in two soils with organic matter contents of 4.0-5.2%. 

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