Confirmatory method

Blank values must be reported they should not be higher than 30% of the LOQ. Confirmatory method/technique must be described if appropriate... 

Confirmatory techniques must be submitted if the analytical method is not highly specific. A confirmatory method will not be required if the original method uses GC/MS, provided that at least three fragment ions with an m z ratio of >100 are used for identification/quantitation. The rationale for the selection of the ions monitored should also be provided. When a confirmatory method/technique is required to demonstrate specificity, the properties of the analyte should be considered when deciding on an appropriate method/technique. In SANCO/825/00 acceptable confirmatory techniques are specified as follows ... 

The extent of validation of confirmatory techniques is currently under consideration. Qne approach is that the extent of validation may be smaller than for the enforcement method. In principle, validation in triplicate at the relevant concentration level (LOQ or MRL) is sufficient. In the case where an MRL is set for multiple crops, a single validation in all representative crop groups is sufficient. A confirmatory method for residues in air is not required if a corresponding method was submitted for the other sample matrices. This approach is realized in Germany. ... 

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 use of standards prepared in control matrices is typically not allowed for determinative procedures because control tissues are not routinely available to regulatory laboratories. When a matrix effect alters the spectrum or chromatography of an analyte relative to the pure standard, so that confirmatory criteria cannot be met, a control extract containing standard may be substituted for pure standard. Justification, with CVM concurrence, should be provided for confirmatory methods that use fortified control extracts. 

The method trial process for NADA methods is different to the process for non-NADA methods. However, the validation protocol followed by the participating laboratories and the requirements for acceptance of the method are the same. The trial process also differs for determinative procedures and confirmatory procedures. Determinative procedures are evaluated using the multiple laboratory process, whereas the confirmatory method needs to be evaluated only in a single government laboratory. 

Another difference between determinative and confirmatory method trial procedures is the way in which sample extracts are prepared for analysis. Most current methods submitted for review use the same sample extraction technique for both the determinative and confirmatory procedures. In those cases where the same extraction technique is used, the sponsor may provide the prepared extract to the FDA laboratory for analysis. Any problems with the extraction procedure will have been corrected during the determinative method trial. 

For confirmatory methods, the confirmatory procedure criteria described previously should be met. All negative control samples should fail to meet the confirmation standard established in the procedure. All samples fortified at or above the tolerance and all incurred residue samples at or above the tolerance should meet the confirmation standard (to confirm) described in the SOP. It has been argued that it is not necessary for incurred samples containing the marker residue at a concentration below the tolerance to meet established confirmatory criteria. However, failure to confirm the marker residue in these samples may indicate a lack of robusmess of the procedure. Any procedure that had this problem would be closely examined to ensure that the method would meet the needs of the Agency. 

To demonstrate the validity of an analytical method, data regarding working range/ calibration, recovery, repeatability, specificity and LOQ have to be provided for each relevant sample matrix. Most often these data have to be collected from several studies, e.g., from several validation reports of the developer of the method, the independent laboratory validation or the confirmatory method trials. If the intended use of a pesticide is not restricted to one matrix type and if residues are transferred via feedstuffs to animals and finally to foodstuffs of animal origin, up to 30 sets of the quality parameters described above are necessary for each analyte of the residue definition. Table 2 can be used as a checklist to monitor the completeness of required data. 

Origin of sample Matrix type Main study Independent lab. validation" Confirmatory method"... 

Another equally important consideration before development of a determinative or confirmatory method is an understanding of the chemical properties of the analyte. Such an understanding becomes the cornerstone of a successful method since the unique chemical properties of each analyte provide the basis for isolation and detection schemes. Table 1 lists some of the important chemical properties that could be considered. For example, knowing the or p/fb of an analyte could influence the choice of a liquid-liquid extraction scheme, solid-phase extraction (SPE) cartridge, mobile phase pH, or mass spectrometric ionization. Knowing the overall polarity of the analyte can be very helpful in the evaluation of an extraction or separation. Currently, computational methods are available to obtain an estimate of the logP... 

This selected ion monitoring (SIM) approach typically has greater applicability in cases where sensitivity is more of a concern. Kiehl and Kennington developed a swine liver confirmatory method for tilmicosin that confirmed structure based upon monitoring a parent ion and two additional structural fragment ions. A discussion of the validation requirements for confirmatory methods is provided in Section 6. 

Once the determinative or confirmatory method has been developed to take full advantage of the chemical properties of the analyte molecule, a study is necessary to prove that the method is valid. Criteria for method validation are outlined in guidelines from the US FDA, US EPA, and EU. A summary of the differences in regulatory requirements for method validation is provided in Table 3. The parameters addressed by all of the regulatory guidelines include accuracy, precision, sensitivity, specificity, and practicability. 

Confirmatory methods Minimum of 3 ions Relative abundance 10% external standard (or full spectra) Ions characteristic of molecule None Points system based on technique used... 

Confirmatory methods must be sufficiently robust to accurately verity the structure of the analyte. For the US FDA, the validation procedure for confirmatory methods is currently defined by Sphon. Validation criteria include reproducible chromatographic separation, ions chosen to be characteristic of the molecule. 

The method using GC/MS with selected ion monitoring (SIM) in the electron ionization (El) mode can determine concentrations of alachlor, acetochlor, and metolachlor and other major corn herbicides in raw and finished surface water and groundwater samples. This GC/MS method eliminates interferences and provides similar sensitivity and superior specificity compared with conventional methods such as GC/ECD or GC/NPD, eliminating the need for a confirmatory method by collection of data on numerous ions simultaneously. If there are interferences with the quantitation ion, a confirmation ion is substituted for quantitation purposes. Deuterated analogs of each analyte may be used as internal standards, which compensate for matrix effects and allow for the correction of losses that occur during the analytical procedure. A known amount of the deuterium-labeled compound, which is an ideal internal standard because its chemical and physical properties are essentially identical with those of the unlabeled compound, is carried through the analytical procedure. SPE is required to concentrate the water samples before analysis to determine concentrations reliably at or below 0.05 qg (ppb) and to recover/extract the various analytes from the water samples into a suitable solvent for GC analysis. 

The method for chloroacetanilide soil metabolites in water determines concentrations of ethanesulfonic acid (ESA) and oxanilic acid (OXA) metabolites of alachlor, acetochlor, and metolachlor in surface water and groundwater samples by direct aqueous injection LC/MS/MS. After injection, compounds are separated by reversed-phase HPLC and introduced into the mass spectrometer with a TurboIonSpray atmospheric pressure ionization (API) interface. Using direct aqueous injection without prior SPE and/or concentration minimizes losses and greatly simplifies the analytical procedure. Standard addition experiments can be used to check for matrix effects. With multiple-reaction monitoring in the negative electrospray ionization mode, LC/MS/MS provides superior specificity and sensitivity compared with conventional liquid chromatography/mass spectrometry (LC/MS) or liquid chromatography/ultraviolet detection (LC/UV), and the need for a confirmatory method is eliminated. In summary,... 

Full acceptance of HPLC/MS methods by the US EPA OPP as enforcement methods occurred between 1998 and 2001. For example, in 1998, the EPA OPP accepted HPLC/MS (without MS/MS) methods as primary enforcement methods, and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) only was suitable for confirmatory methods. However, in 2001, HPLC/MS/MS methods also became acceptable for primary enforcement. Table 4 summarizes the types of methods that were validated by the EPA OPP method validation program, for both food tolerance enforcement methods and environmental chemistry methods. 

Zitko [20] has described a confirmatory method in which the chloroparaffins in sediments are reduced to normal hydrocarbons which are then analysed by gas chromatography. This method lacks sufficient sensitivity for trace (sub-ppm) analysis and the confirmatory method may be difficult to apply. Friedman and Lombardo [21] have described a gas chromatographic method applicable to chloroparaffins that are slightly volatile the method is based on microcoulometric detection and photochemical elimination of chlorinated aromatic compounds that otherwise interfere. 

Data from a confirmatory method comparison should be provided if such method(s) is applicable to the same matrix(es) and concentration range(s). 

Elemental composition H 1.56%, Te 98.44%. The gas is identified by its physical properties and measured by chemical analysis. Two most confirmatory methods recommended here are (1) GC/MS, the characteristic mass ions should be in the range 126 to 132, and (2) furnace-AA or ICP emission spectroscopic analysis for metalic tellurium. For the AA analysis, hydrogen telluride gas should be passed through water and the solution acidified and analyzed for tellurim. Hydrogen may be measured by the classical combustion method involving oxidation to form water, followed by gravimetry. 

Kale, endive, carrots, lettuce, apples, potatoes, strawberries Extraction of crops with ethyl acetate and granular sodium sulfate, filtration, concentration with K-D. Sweep codistillation cleanup for GC. Florisil partition chromatography for polarographic confirmation. GC/KCI thermionic detector or GC/FPD polarographic confirmatory method No data for GC polarographic 0.2 ppm based on 1 g crop in 1 mL cell No data AOAC 1990... 

LC-PB-MS has been investigated as a potential confirmatory method for the determination of malachite green in incurred catfish tissue (81) and of cephapirin, furosemide, and methylene blue in milk, kidney, and muscle tissue, respectively (82). Results showed that the mobile-phase composition, nebulization-de-solvation, and source temperature all play an important role in the sensitivity of the method. The sensitivity increases with decreasing heat capacity of the mobile phase in the order methanol acetonitrile isopropanol water and with decreasing flow rate. A comparison of the PB with the thermospray interface showed that less structural information was provided by the latter, whereas the sensitivity was generally lower with the thermospray interface. 

Confirmatory methods based on LC-TSP-MS have further been developed for the determination of various penicillin derivatives, penicillin G, and cephapirin and its metabolite in milk. In the assay for penicillin derivatives (97), ammonium acetate buffer replaced the ion-pair reagents used in the mobile phase, whereas the positive-ion TSP mass spectra of penicillins displayed both MU and MNa ions, which provided unequivocal proof of the suspected drug residue. The detection limits in this assay were estimated to be in the range of 100-200 ppb. A detection limit of 100 ppb was also observed in one of the assays of penicillin G in milk (98), although another assay (99) offered a detection limit of only 3 ppb. Tills was probably due to the fact that only ultrafiltration was employed for milk cleanup in the former assay, while both protein precipitation and solid-phase cleanup were used in the latter. In the case of the cephapirin analysis (100), the principal metabolite in milk was identified as deacetylcephapirin by both LC-PDA detection and LC-TSP-MS. In the LC-MS method, the detection limits for cephapirin and deacetylcephapirin were 100 and 50 ppb, respectively. 

TLC-bioautography has been used in Canada since 1984 for the confirmation of tetracycline-positive in plant tests (65). However, TLC-bioautography is not quantitative and only gives direction to the analyst as to what confirmatory method of analysis should be used. Therefore, presumptive positives must be confirmed by physicochemical techniques that have been validated in terms of detection limit, precision, and accuracy. 

Another characteristic example of analytical strategy is that followed in the United Kingdom for the analysis of tranquilizers and -blockers (78). A total of 180 samples distributed over a whole year (15 samples per month) should be analyzed within a turnaround time of 28 days from receipt of sample. In that case, the expense of developing a two-tier analytical strategy was not justified by the sample throughput. Thus, the selection was direct application of a multiresidue LC/PDA confirmatory method (80). 

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