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fundamentally different analytical principle, to reduce the potential impact of interfering species (such as cross-reactants in immunoassays). The rst assay must be highly sensitive to identify presumptive positive samples and eliminate false negatives, while the second, quantitative method must be highly speci c to eliminate false positive results. In this example, urine specimens from six healthy male subjects with a history of marijuana use, who were undergoing medical treatment, were initially tested using eight semiquantitative cannabinoid immunoassays (Table 16.7). In these assays, the metabolite 11-nor-9-carboxy- 9-tetrahydrocannabinol (THCCOOH) was detected. The effect of two different cutoff values (100- and 50-mg/L THCCOOH) were investigated with respect to sensitivity, speci city and ef ciency, as de ned previously in Eq. 16.31 16.33. In this study, all the specimens were also assayed by GC MS, as a quantitative con rmatory method, using a cutoff value of 15 mg/L; this value was used to de ne positive and negative test results (!15 and <15 mg/ L, respectively). True positive specimens were de ned as having immunoassay results equal to or greater than the speci ed cutoff concentration (100 or 50 mg/L) and !15 mg/L THCCOOH by GC MS. True negative specimens had results less than the cutoff concentrations for the immunoassays and the GC MS test. False positive samples had immunoassay results greater than or equal to the immunoassay cutoff concentration and <15 mg/L THCCOH by GC MS. False negative samples had immunoassay results lower than the speci ed immunoassay cutoff concentration and !15 mg/L THCCOOH by GC MS. In this study, from 957 specimens analyzed, GC MS results indicate 151 positive and 806 negative results. These are compared with results of the immunoassay methods in Table 16.7. This study concluded that lowering the cannabinoid cutoff concentration from 100 to 50 mg/L THCCOOH increased the percent correct identi cation of true-positive specimens by all of the commercial immunoassays tested. Therefore, the sensitivities of all of the immunoassays were enhanced, resulting in improved ef ciency for these assays. As expected, the speci city decreased slightly when the lower (50 mg/L) cutoff value was used. It is also evident from the results shown in Table 16.7 that there are discrepancies between the results of the eight commercial immunoassays. Preliminary tests suggest that these discrepancies can be attributed to differences in antibody selectivities, since different antibodies are used in the different assays; these antibodies may be expected to show different cross-reactivities with other cannabinoid metabolites, as well as other interfering species. SUGGESTED REFERENCES
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J. C. Miller and J. N. Miller, Statistics for Analytical Chemistry, Elis Horwood PTR Prentice Hall, New York, 1993. G. T. Wernimont, Use of Statistics to Develop and Evaluate Analytical Methods, W. Spendley Ed., Association of Of cial Analytical Chemists, Arlington, VA, 1985.
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R. Caulcutt and R. Boddy, Statistics for Analytical Chemists, Chapman and Hall, London, 1983. A. Fajgelj and A. Ambrus, Eds., Principles and Practices of Method Validation, The Royal Society of Chemistry, Cambridge, UK, 2000. M. L. Bishop, J. L. Duben-Engelkirk, and E. P. Fody, Clinical Chemistry, Lippincott Williams & Wilkins, Philadelphia, 2000. D. C. Harris, Quantitative Chemical Analysis, W. H. Freeman and Co., New York, 2002.
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1. V. P. Shah et al., Pharmaceut. Res. 17, 2000, 1551 1557. 2. W. Horwitz, Anal. Chem. 54, 1982, 67A 76A. 3. P. Willetts and R. Wood, in Principles and Practices of Method Validation, A. Fajgelj and A. Ambrus, Eds., Royal society of Chemistry, Cambridge, UK, 2000, pp. 271 288 4. B. Nix and D. Wild, in The Immunoassay Handbook, D. Wild, Ed., 2nd Ed., Nature Publishing Group, New York, 2001, pp. 198 210. 5. P. Ertl and S. R. Mikkelsen, U.S. Pat. 6,391,577 BI, granted May 21, 2002, and P. Ertl, E. Robello, F. Battaglini, and S. R. Mikkelsen, Anal. Chem. 72, 2000, 4957 4964. 6. M. T. Shipchandler and E. G. More, Clin. Chem. 41, 1995, 991 994. 7. A. J. Engelen and P. H. G. Randsdorp, J. AOAC Inter. 82, 1999, 112 118. 8. M. A. Huestis, J. M. Mitchell, and E. J. Cone, Clin. Chem. 40, 1994, 729 733.
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