Fortified sample

Determine the amount of residue found in each of the fortified samples. 

For confirmatory procedures, the fortified sample sets at half and twice the tolerance... 

On the other hand, single-residue methods developed by the applicants give basic information about appropriate cleanup steps and specific determination procedures. In addition, not many laboratories other than those from the applicants are able to test the real solvent extraction efficiency. The reason is that extraction studies need radio-labeled incurred residues instead of fortified samples. Hence enforcement methods provided by the manufacturers accelerate the development of methods which meet the needs of (official) food control laboratories. 

Enforcement methods provided by the manufacturer are not generally tested in the laboratories of the European regulatory authorities. Very often, proposed methods are evaluated by assessing the logic of proposed procedures and only for the completeness of validation data. For this theoretical review process, as much information as possible should be available. Recovery data from many validation experiments with different kinds of matrices and the resulting chromatograms of control and fortified samples provide the confidence needed by the referee. In the following sections, the most important aspects of this evaluation will be considered. 

Sensitivity At appropriate concentrations All available detectors should be tested with all fortified samples in >3 laboratories No criteria necessary Comparison of results in different laboratories... 

The HPLC elution pattern is affected to some extent by the pH of the mobile phase. Moderate pH adjustment to optimize the resolution between EMA and MEMA may be performed. Retention time can be affected greatly by the history of the HPLC column and also the buffer/methanol ratio. The mobile phase ratio should be adjusted to provide adequate separation and retention. Control and fortified samples should be run in the same analytical set with treated samples. 

In order to estimate the analytical accuracy of the method with a given set of water samples, a certain number of control water samples should be fortified with a known amount of each herbicide. Control water samples are fortified at different analyte levels across the range of anticipated concentrations. For example, 0.010g of analyte is necessary for a 0.05 agL fortification of a 200-mL sample. This would be accomplished by adding 1.0 mL of a 0.010 o.gmL solution to the sample. The deuterated standards are not incorporated in the fortification solutions but may be added to all control and fortified samples for internal correction of recovery. The following solutions are used to fortify control water samples ... 

Fortification of control water samples must be made at this point by adding the correct volume of the appropriate fortification solution. Optima Grade bottled water is used as the matrix for the controls and the laboratory-fortified samples for all water types. Add 1.0 mL of the 0.10 qg mL mixed deuterated herbicide internal standard solution to all samples including control and fortified water samples. A 200-mL volume is sufficient to quantitate to levels of 0.05 M-gL ... 

At least one control water sample must be analyzed concurrently with the water samples to determine the presence of matrix interferences and/or background levels of the metabolites. Optima-grade bottled water is used as the matrix for the controls and the fortified samples for all wafer fypes, because obtaining ground and surface water specimens that are completely free of the metabolites is difficult. Our analyses of ground and surface waters have demonstrated the presence of low-level interferences in these matrices. Interferences from other pesticides are unknown, because none have been examined. However, none are expected due to the high level of specificity of the LC/MS/MS analysis. 

The minimum detectable level is estimated with the dinifroaniline signal-to-noise ratios (S/N). With fortification levels between 0.2 and 0.5mgkg the recovery of trifluralin from plant matrices is 70-99% with the LOD/LOQ being 0.005 mg kg according to the analytical method of the Ministry of the Environment, Japan. In multiresidue analysis by GC/NPD, the percent recoveries of pendimethalin from each crop with a fortification level of 0.25 mg kg were brown rice 70, potato 70, cabbage 80, letmce 89, carrot 84, cucumber 64, shiitake 74, apple 76, strawberry 99, and banana 99%. The LOD for each sample was 0.01 mg kg for pendimethalin. In residue analysis by GC/ECD, recoveries of the majority of dinifroaniline herbicides from fortified samples of carrot, melon, and tomato at fortification levels of 0.04—0.10 mg kg ranged from 79 to 92%. The LODs were benfluralin 0.001, pendimethalin 0.002 and trifluralin 0.001 mg kg for the GC/ECD method. ... 

Weigh 2.5 or 5 g of crop matrix into a blending vessel. Fortify samples at this point with the appropriate analytical standards. Allow the solvent to evaporate. Add 100 mL of acetone-water (4 1, v/v) and blend the mixture using an Omni mixer equipped with a macro generator for 5 min at 6000-7000 rpm. Filter the sample through a Whatman 934 AFI glass-fiber filter paper on a Buchner funnel/vacuum flask setup. Rinse the blending cup and filter cake with 100 mL of acetone. Transfer the filtrate into a 200-mL TurboVap vessel. 

The injection standards of carfentrazone-ethyl must be in acetonitrile. Other solvents (e.g., ethyl acetate) lead to poor chromatography following injection of matrix samples. This can lead to apparent enhanced recoveries of analyte in the fortified samples. 

An immunoassay was developed to determine the penicillinase stable isoxazolyl penicillins cloxacillin and dicloxacillin in milk by Usleber et alJ The assay detected lOpgkg" of cloxacillin and 30pgkg of dicloxacillin with recoveries of 102% and 84%, respectively. The calibration curve was prepared by fortifying skimmed milk powder (lOOgL ) with standards. Fortified samples were prepared in pasteurized milk and analyzed directly after decreaming by centrifugation. This immunoassay was performed with minimal sample preparation, probably because the extensive water solubility of the penicillins prevents problems associated with more lipid-soluble analytes. 

Untreated (control) soil is collected to determine the presence of substances that may interfere with the measurement of target analytes. Control soil is also necessary for analytical recovery determinations made using laboratory-fortified samples. Thus, basic field study design divides the test area into one or more treated plots and an untreated control plot. Unlike the treated plots, the untreated control is typically not replicated but must be sufficiently large to provide soil for characterization, analytical method validation, and quality control. To prevent spray drift on to the control area and other potential forms of contamination, the control area is positioned > 15 m away and upwind of the treated plot, relative to prevailing wind patterns. 

An example of adequate sample homogenization is given in Table 4. The experiment was conducted with two replicate treated soil samples. Each replicate was analyzed in duplicate. Three different sample aliquots (2, 5 and 10 g) were used from each replicate. Analyses of controls and fortified samples were also conducted concurrently with treated samples to evaluate method performance (i.e., extraction recoveries). These results show that residue values are the same regardless of sample size. Thus, thorough homogenization of soil samples coupled with mgged analytical methodology provides for satisfactory residue analysis. 

Each sample was fortified with chlorpyrifos, as a reference standard, to determine the recovery during each extraction. Three portions of solvent were used, and the combined extract for each sample was dried with sodium sulfate. Analyses employed gas chromatography/flame photometric detection. Limits of detection for vegetation and animal tissues were 0.2 and 0.007 pg respectively. Recoveries from fortified samples were 82%. Diazoxon occurrence was infrequenf and at trace concentrations. 

Once method validation has been completed, the treated samples may be analyzed. The method should be under control so that no additional changes will be necessary. Analysis of laboratory-fortified samples and control samples will be used to monitor the quality of the study. The purpose of laboratory-fortified samples is confirmation of the recovery efficiency of residues from the sample matrix. A minimum of two laboratory recovery samples need to run with each set. Recoveries should average 70-120%. 

Another important quality measure is the analysis of the field-fortified samples. The field-fortified samples have been handled, shipped and stored in the same manner as the treated samples. Any loss of analyte in the field-fortified samples will be used to correct residue levels of the field treated samples. It is best practice to keep the storage interval as short as possible to minimize losses. The acceptable storage time will vary according to the stability of each compound in aqueous solution. A good guideline is to analyze the samples within 30 days of sampling. 

Control urine should be collected from individuals who have no apparent past history of exposure to the active ingredient. This control urine must be stored frozen until used for field fortification purposes. The urine is then thawed, shaken well, and a certain amount should be aliquoted into a small jar/bottle to use for field fortification. The active ingredient is then added to the urine using a 1-mL volumetric pipet, the solution is shaken well, and the sample is immediately frozen. Occasionally, the fortified sample can be left at room temperature or at some lower temperature in a liquid state to simulate field storage during collection of the urine sample. After leaving the sample at such temperatures for the prescribed length of time, the sample is immediately stored frozen. 

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