Assessment of false positive rates of immunoassay kits for ecstasy compared to chromatographic method

Background & Aims: In Turkey, enzyme multiplied immunoassay technique (EMIT) is widely used for urine drugscreening analysis. Although positive interferences are expected in immunochemical analysis, in Manisa MentalHealth and Diseases Hospital, a significant number of samples tested positive for ecstasy using the EMIT-based kit (SIEMENS) were found to be false positive when re-evaluated by high resolution liquid chromatography-tandemmass spectrometry (UHPLC-MS/MS-ORBITRAP).Ecstasy (MDMA) and its metabolite (MDA) have structural similarities with numerous substances such as amphetamine-type stimulants, ranitidine, ephedrine, pseudoephedrine, cathinones, etc. Because ecstasy and/or itsmetabolite can cross-react with antibodies in the amphetamine screening test kit, urine specimens with true ecstasy positivity are also positive for amphetamines. Therefore, the possibility of "false ecstasy positivity" is stronglyconsidered in specimens in which amphetamine positivity does not accompany by ecstasy positivity. This study aimed to evaluate the analytical performance of the three immunoassay methods by re-analyzing samples predicted as possible "false positives" at EMIT with biochip array technology (BAT) (Randox) and DRI (ThermoFisher) compared to LC-MS/MS confirmation.Methods: The urine sample results of donors aged 18-65 years, admitted to the Toxicology Laboratory of Manisa Mental Health and Diseases Hospital for forensic or clinical drug analysis between December 2023- February2024 (n=2618) were retrospectively reviewed. These samples were analyzed using EMIT-based Siemens brand drugscreening kits on the Siemens ADVIA-1800 autoanalyzer. Among the samples positive for ecstasy (n=349, 13.3%),only samples negative for amphetamines were selected (n=100, 3.8%). One aliquot of these samples was reanalyzed with Thermo Fisher DRI kit on Siemens ADVIA-1800 and Randox BATkit on Randox Multistat. Indeed, confirmatory analyses of these samples were performed on a Sciex QTRAP 5500Plus LC-MS/MS device.A cut-off value of 500 mg/dL was used for immunochemical methods and LOQ for LC-MS/MS (2.89 ng/mL for ecstasy, MDMA; 2.82 for ecstasy metabolite, MDA). Based on the LC-MS/MS results, the results of the three immunoassay methods were classified as true positive (TP), true negative (TN), false positive (FP) and false negative (FN).Results & Discussion: Fifty six percent of samples included into the study (n=100) were under judicial supervisionand probation, 36% were from the Addiction Detoxification Center, 9% were from the psychiatry clinic. The confirmatory analysis with LC-MS/MS revealed that 96% of samples were false positives and only four sampleswere true positives (three of which were among the six samples detected >1000 ng/mL by EMIT). DRI found only8% positive, of which three were true positives (75% of true positives) and five were false positives. BAT detectedone of four true positives (25% of true positives), while no false positives were detected in negative samples. TwoLC-MS/MS techniques ORBITRAP and SCIEX QTRAP 5500 plus provided consistent results in 4 true positive samples. One true positive sample was positive by EMIT, but negative by the other two immunochemical methods.In summary, among the ecstasy-positive samples (n=100), 96% (n=96) were false positives with EMIT. Among these, 5.2% (n=5) were false positives with DRI, while no false positives were identified with BAT. Among the four truepositive samples, 75% (n=3) tested positive with DRI, and 25% (n=1) tested positive with BAT.Based on these findings, the false positive rate of the EMIT-based Siemens kit seems to be relatively high. BAT,which doesn’t yield false negatives, appears to be highly specific (100%). DRI had 94.8% specifity. However, dueto the limited number of true positive samples evaluated, no conclusions can be drawn regarding sensitivity. Conclusion: The EMIT-based ecstasy kit from Siemens has a significant false positive rate, which increases theneed for confirmation and imposes a financial burden. Since our study is based on possible false positive samples with EMIT, it includes a limited number of cases in terms of true positivity. Our investigation will continue byexpanding the sample size to include more true positives.