Method trials

The US Food and Drug Administration (FDA) evaluates methods to be used in government regulatory laboratories for the determination and confirmation of drug residues in food derived from animal products. The FDA Center for Veterinary Medicine (CVM) oversees the validation (i.e., demonstration that the method is suitable for use) via a protocol known as a method trial. CVM ensures that the appropriate government laboratories have the tools needed to monitor the Nation s food supply. 

The FDA requires [FDC Act, Section 512 (b)(1)(G)] that methods used for the detection and confirmation of drug residues in animal products be practicable. Overseeing the reliability of these methods is the responsibility of the FDA CVM. The methods are corroborated using an interlaboratory evaluation of the method known as a method trial. The method trial is used to demonstrate that the method is suitable for use to detect and confirm drug residues and can be performed by a trained analytical chemist. 

The NADA method approval process consists of three phases (1) method development by the sponsor and generation of information to establish that the method satisfies acceptability criteria (2) FDA review of the sponsor s data to determine suitability of the method and (3) the method trial , an inter-laboratory study, which determine whether the method meets performance criteria when used in multiple laboratories. The inter-laboratory method trial procedure provides an indication of a method s ability to be used as a practicable and reliable regulatory tool. Sponsors are urged to develop methods that are mgged and exceed rather than meet the minimal standards of acceptability. Those methods that appear marginally acceptable after review often do not pass the inter-laboratory method trial. 

The non-NADA method trial process mirrors the NADA process. Methods are developed, reviewed for scientific and technical soundness, and validated in multiple laboratories, and the data generated are analyzed to determine if the method is suitable for its intended use. 

Prior to a method trial, the FDA strongly recommends that a second analyst or independent laboratory perform the method. The independent analyst is asked follow the method SOP as written. This analyst should not have been involved in developing the method or be familiar with it in any way. The purpose of the independent analysis is to determine if a qualified chemist can perform the method described without input other than that provided in the written instmctions. This trial mn will typically identify problems with the SOP that are not apparent to the method developer. Although not required by the FDA, the independent assessment can identify potential problems with the method SOP prior to the lengthy and costly method trial. A trial mn offers the method developer an opportunity to correct problems and to increase the probability that subsequent method trials will be successful. Finally, the method developer should realize that the variability achieved in his/her laboratory is often less than that realized by less experienced analysts. If a method cannot achieve a suitable degree of repeatability in the developer s laboratory, it should not be expected to do any better in other laboratories. 

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. 

The evaluation of all NADA analytical methods was previously conducted exclusively by the CVM. Since 1995, the CVM has offered sponsors of NADA residue methods the option of conducting the method trial through a Sponsor Monitored Method Trial (SMMT) process. The SMMT is conducted according to CVM specifications with CVM oversight. The resultant performance data must be reviewed and judged acceptable by CVM before the method is approved. 

In the SMMT process, draft protocols are reviewed, and guidance provided to the sponsor to help ensure that the format and specifications are adequate. The protocol should be approved by CVM prior to the initiation of the method trial. Once the protocol and method description are acceptable to CVM, the methods are sent to the participating laboratories for review, and a method demonstration is scheduled. The method demonstration, attended by all participating laboratory analysts, involves review of the study protocol and method SOP and a laboratory demonstration of the method. Ideally, all revisions are completed by the end of the demonstration and the study protocol is signed. 

At a minimum, the method will be tested in one FDA laboratory and two contract laboratories selected by the sponsor. If the method is for a new animal drug in tissue regulated by the United States Department of Agriculture (USDA) as part of the meat inspection program, a Food Safety and Inspection Service (FSIS)/USDA laboratory will be included if sufficient resources are available. The method trial will be conducted using control and incurred target tissues that are supplied by the sponsor. The sponsor may, on request, supply new or unusual reagents or standards. 

Following the completion of the trial, each participating contract laboratory provides a report of their results to the method trial Study Director. The government laboratory(ies) provide their results to the CVM method trial coordinator. The sponsor compiles the final results from participating laboratories into a summary report. A final version of the SOP is also provided that includes any revisions made because of observations made during the trial. The summary report, electronic and hard copies of all laboratory results, work sheets, and reports from each of the participating laboratories are sent to CVM for final review and acceptance. This should include electronic copies of all information necessary to verify all of the results. 

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. 

The final difference is that the FDA analyst alone makes the recommendation based on the data for the acceptance of the confirmatory procedure. The conclusion of the analyst stating the suitability of the procedure for confirming the presence of the marker residue is sent directly to the CVM method trial coordinator in the Office of New Animal Drug Evaluation (ONADE) and not back to the sponsor as with the determinative procedure. 

The FDA coordinates the method trial process for non-NADA methods. The sample requirements are the same as for the NADA trials. Non-Federal laboratories such as contract laboratories and State laboratories can participate in the process. For a single-residue method, the minimum numbers of samples and laboratories are the same as for NADA method trials. 

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... 

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. 

In 1994, only 15% of EPA method validations (tolerance method validation and environmental chemistry method validations) that involved GC were carried out using GC/MS. In 2002, this number is reversed in that 85% of the GC methods that were validated by both programs used GC/MS. Many of the compounds investigated in these method trials were polar compounds, and hence these compounds required derivatization in order to be amenable to GC. One common methylating agent is (trimethylsilyl)diazomethane, which is used, for example, to methylate the sulfonamide flumetsulam. As opposed to HPLC/MS, where derivatization is often not necessary, the GC/MS procedure involves an extra step to methylate this compound, under dry conditions, prior to determination by GC/MS. 

The optimum value of the relaxation factor k can be obtained from the largest eigenvalue of s. This eigenvalue may, in turn, be obtained approximately from the relative magnitude of vectors oik) and oik+1). Improvements discussed in the next section, and amplified in the next chapter, significantly alter the method. Trial-and-error choice of k is therefore preferable and probably necessary. 

Maruo K, Oota T, Tsurugi M, Nakagawa T, Arimoto H, Hayakawa M, Tamura M, Ozaki Y, Yamada Y. Noninvasive near-infrared blood glucose monitoring using a calibration model built by a numerical simulation method trial application to patients in an intensive care unit. Applied Spectroscopy 2006, 60, 1423-1431. 

Separation Method. Trial experiments with small portions of the coked catalyst in progressively more dense mixtures of carbon tetrachloride (d - 1.594 g/cc) and 1,1,2,2- tetrabromoethane (d - 2.967 g/cc) showed that the lightest catalyst fractions exhibited a density of about 2.3 g/cc. A solvent mixture with d - 2.33 g/cc was prepared from a mixture of 46.4 ml of carbon tetrachloride and 53.6 ml of tetrabromoethane. A 20 g portion of coked catalyst was placed in a 120 ml quantity of this solvent mixture in a 250 ml Teflon centrifuge bottle. The mixture was thoroughly agitated for several minutes and then placed in a centrifuge at 3,600 rpm for 30 minutes. Upon removal from the centrifuge, the mixture was allowed to stand for several hours in order to obtain well-defined float and sink fractions. 

J, Jacobian of partial derivatives in Newton-Raphson method, trial ft. 

Change in the independent variable to move it toward solution in a Newton-Raphson method, trial k, Sec. 4.2.6. 

Method Trial Reaction Time Residues 14C Reacted A406... 

---