Confirmatory assay

For confirmatory assay, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is becoming more frequently used in the analysis of OTC owing to its high sensitivity and ability. Electrospray ionization (ESI) [55-57] and atmospheric pressure chemical ionization (APCI) [41] methods combined with tandem mass spectrometry are favored because of their higher sensitivity and better reproducibility. Hamscher et al. [58] developed a method for the determination of persistent TC residues in soil fertilized with manure by HPLC tandem mass spectrometry, MS-MS, and confirmation by MS-MS-MS. Zhu et al. [59] developed an LC-tandem mass spectrometry for the analysis of common tetracyclines in water. The detection limit for oxytetracycline was 0.21 pg/L. Lykkeberg et al. [60] used LC-MS/MS for determination of oxytetracycline and its impurities EOTC, TC, ETC, ADOTC, oc-AOTC, and /i-AOTC. 

DNA arrays certainly offer an extremely high degree of multiplex capability for testing for multiple targets simultaneously in confirmatory assays. As the methods for detecting hybrids on the arrays improve, perhaps it will be possible to eliminate the requirement for PCR. PCR does amplify targets of interest to levels where detection is currently possible, but the... 

A positive result from a eukaryotic reporter assay would lower the ranking of a compound for development, alert lead optimization chemists and similarly trigger a confirmatory assay and alert safety assessment. 

WJ Blanchflower, RJ McCracken, AS Haggan, DG Kennedy. Confirmatory assay for the determination of tetracycline, oxytetracycline, chlortetracycline and its isomers in muscle and kidney using liquid chromatography-mass spectrometry. J Chromatogr B 692 351-360, 1997. 

WJ Blanchflower, SA Hewitt, DG Kennedy. Confirmatory assay for the simultaneous detection of 5 penicillins in muscle, kidney and milk using liquid chromatography-electrospray mass spectrometry. Analyst 119 2595-2601, 1994. 

Confirmatory assay Performed without S-9 activation and exposure period extended to 24 hours to confirm mutation assay results... 

The use of alkaloids as therapeutic drugs, drugs of abuse and deliberate poisons has required the development of screening and confirmatory assays for these... 

E. Daeseleire, H. De Ruyck, R. Van Renterghem, Confirmatory assay for the simultaneous detection of penicillins and cephalosporins in milk using LC—AIS—AIS, Rapid Commun. Mass Spectrom., 14 (2000) 1404. 

Ito, Y. Ikai, Y. Oka, H. Matsumoto, H. Miyazaki, Y. Takeha, K. Nagase, H. Application of ion-exchange cartridge clean-up in food analysis. IV. Confirmatory assay of benzylpenicilhn, phenoxymethylpeniciUin, oxacillin, cloxacillin, nafcillin and dicloxacilhn in bovine tissues by hquid chromatography-electrospray ionization tandem mass spectrometry. J. Chromatogr., A 2001, 911, 217-223. 

McKeon. M.L. and Pbil. M. (1991). Piperonyl Butoxide in the Assay for Unscheduled DNA Synthesis in Rat Liver Primary Cell Cultures with a Confirmatory Assay. Unpublished report no. 14413-0-447R from Hazleton Washington. Kensington, Maryland, ITS A. Undertaken for the PBO Task Force. Washington IX . USA. 

Recent advances in robotics, cheminformatics, and assay formats, including miniaturization, have made the generation of small-molecule hits by high-throughput screening (HTS) campaigns a more or less routine procedure in the pharmaceutical and increasingly the academic sectors see also Chapters 1 and 2). Active compounds identified from HTS are then validated in confirmatory assays and their respective activities characterized in more detail... 

Cifone MA, L5178Y TK+/- Mouse Lymphoma Forward Mutation assay with a Confirmatory Assay, Covance Laboratories Inc., Vienna, VA, Covance No. 22636-0-43lOECD, U.S. Army Center for Health Promotion and Preventive Medicine, Aberdeen Proving Ground, MD, 2002. 

Once an ADA-positive sample has been shown to be specific in the confirmatory assay, typically the next step is to determine the titer of the sample. Often, the titer is determined in the screening assay, using the same approach used initially to determine positive samples. Serial dilutions of the sample are made prior to analysis. 

The safe concentration of drug-related residue must be known in order to determine the withdrawal period for a veterinary product. Often the toxicity data is incomplete and an estimate must be made to progress with requisite residue studies. One approach is to conduct a total residue study with sufficiently widely-spaced sacrifice intervals to assess the rate of depletion of total residue over the projected range of probable safe concentrations. A zero-withdrawal sacrifice interval should be included. The target tissue and marker residue are identified and surveillance/confirmatory assays developed. If a major portion of residue is non-extractable (bound) and the marker is undetectable at times when total residue is still significant, a residue bioavailability study may be necessary. To complete the data package, final residue and comparative metabolism studies are conducted. Studies on the metabolism of flunixin in cattle will illustrate this approach. 

The confirmatory assay for flunixin in bovine liver utilized capillary gas chromatography/mass spectrometry to isolate and confirm the structure of the marker residue. The extraction procedure is similar to that for the surveillance assay, except that following extraction of the acidified aqueous layer, the organic layer is dried with sodium sulfate, concentrated to dryness under nitrogen, derivatized with trimethylsilane, and a portion of the derivatized extract analyzed by capillary gas... 

The mean concentration of flunixin in the liver was 389 ng/g after 12 hours, 53 ng/g after 24 hours, and 13 ng/g after 48 hours of withdrawal from treatment. At 48 hours, only two of the five animals treated had residues above the limit of quantitation (8 ng/g). No flunixin was detected in the 72-hour withdrawal liver samples. Confirmation analyses by GC-MS indicated that detected residues were flunixin. As shown in Figure 2, the flunixin concentrations detected in the livers of treated cattle at 12 and 24 hours post final dose in both the total and final residue depletion studies are similar. However, in both studies, the concentrations of detected flunixin are low with respect to total radiolabeled residue. These results suggest that a significant portion of the total residue may be bound and/or the existing surveillance and confirmatory assays do not adequately extract flunixin residues from the tissue of treated animals. The need for further work on the bound residues and the existing assays will depend on the final safe concentration assigned to the drug. 

Bogialli S, Curini R, Di Corcia A, Lagana A, Mele M, Nazzari M, Simple confirmatory assay for analyzing residues of aminoglycoside antibiotics in bovine milk Hot water extraction followed by liquid chromatography-tandem mass spectrometry, J. Chromatogr. A 2005 1067(l-2) 93-100. 

Daeseleire E, De Ruyck H, Van Renterghem R, Rapid confirmatory assay for the simultaneous detection of ronidazole, metronidazole and dimetridazole in eggs using liquid chromatography-tandem mass spectrometry. Analyst 2000 125 1533-1535. 

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