Methods of analysis
False positives and false negatives in meconium screening
False negatives It was reported that the method of isolating the drugs from the meconium substantially affects the outcome of the screen. When an essentially clean extract (i.e. drugs are isolated from the matrix using solvent or solid phase methods) is not used, a high rate of false negative results is observed. The immunoassay technique does not substantially affect the outcome of the analysis, but the sample preparation procedure does. False positives In a study conducted by Moore et al. (1995), 535 meconium samples were chosen which screened positively for at least one of the following drugs: cocaine metabolite, opiates, amfetamines or marijuana metabolite. The screening cut-off levels were 25 ng/g for all drugs except amfetamines (100 ng/g). Of these screen positive specimens, 285 (53.3%) were subsequently confirmed using GC-MS for one or more of the drugs at cut-off levels of 5 ng/g for all except marijuana metabolite (2 ng/g). Results of the study are presented in Table 8.9.
According to these results, immunoassay screening is falsely positive 46.7% of the time at the cut-off levels used, assuming that the correct drug metabolites are identified in the confirmatory procedure. It is possible that the immunoassay results are not in fact false positives but that there are drug metabolites present in meconium which are contributing to the immunoreactive response. These compounds are subsequently not determined in the confirmatory method, producing false negative results. Probably the most significant advance, to date, in the determination of drugs in meconium was reported by Steele et al. in 1993, who determined that for cocaine analysis there was a compound in meconium contributing to immunoreactive response which was not being confirmed by GC MS. The research group was unable to confirm a significant number of cocaine positive screens using a standard GC-MS assay which identified cocaine, cocaethylene, benzoylecgonine, ecgonine methyl ester and norcocaine. Subsequently, the authors determined that the significant contributor to the immunoassay was m-hydroxy benzoylecgonine, a previously unreported metabolite of cocaine in meconium. The authors noted some difficulties with the construction of a standard curve for m-hydroxy benzoylecgonine using meconium as the matrix. Following hydrolysis of meconium, the authors also concluded that m-hydroxy benzoylecgonine glucuronide has approximately the same immunoreactivity as unconjugated m-hydroxy benzoylecgonine.
The presence of this metabolite in significant concentrations in meconium demonstrated that the metabolic profile of newborns differs between urine and meconium, and therefore simple application of urine protocol to meconium specimens will cause false negative results. Ethically, it is mandatory to confirm all positives from the preliminary screening by GC-MS and screen-only meconium results should be interpreted with caution.
