![]() ![]() The method was validated to meet the College of American Pathology (CAP), Clinical Laboratory Improvement Amendments (CLIA) and New York Department of Health (NYDOH) protocols. Samples underwent solid-phase extraction prior to LC–MS-MS analysis. The positive cutoff was 200 ng/mL for all of the drugs. The set of 100 urine specimens positive by EMIT II (false-positive specimens) was confirmed to be negative by an LC–MS-MS method developed in-house that quantitates AMP, methamphetamine, ethylenedioxyethylamphetamine (MDE, MDEA, Eve), methylenedioxymethamphetamine (MDMA, Ecstasy, XTC) and methylenedioxyamphetamine (MDA). Standards were purchased from Cerilliant (Round Rock, TX, USA). Furthermore, we wanted to investigate whether the false-positive specimens were caused by psychoactive drugs such as synthetic cathinones. TOF technology could enable the identification of unknown compounds by acquiring exact mass and retention time in full-scan mode, which was an added benefit in comparison with LC–MS-MS. We ran this set of 100 false-positive specimens by TOF using a targeted database to identify potential compounds that may have caused the false-positive IA results. ![]() We then evaluated a separate set of 100 specimens that screened positive by IA for AMP and/or MDMA, but did not confirm by LC–MS-MS. We retrospectively evaluated 3,571 specimens screened by the AMP and MDMA EMIT II tests that were reflexed to confirmation testing by LC–MS-MS to determine positivity rates and the percentage of false positives. The aim of this study was to combine accurate mass data from TOF with structural data derived from the literature and CAS to determine whether compounds yet unknown to cross-react with ecstacy (MDMA) or AMP are present in urine samples that were positive by AMP or MDMA IAs, but did not confirm by LC–MS-MS. Large compound databases, such as Chemical Abstracts (CAS), can be searched to associate compounds with similar structural and physical properties with known molecular formulas. While HRMS provides accurate chemical formulas, it provides limited information of the chemical structure for analytes with unknown retention times. Accurate mass data are used to assign a molecular formula to a compound, and analyte retention time helps to correlate molecular formula with known compounds. High-resolution mass spectrometry (HRMS) techniques, such as liquid chromatography time-of-flight (TOF) mass spectrometry, provide accurate mass measurements for identification of compounds present in a specimen. Specifically, amphetamine (AMP) IAs demonstrate broad antibody cross-reactivity with non-targeted compounds that are structurally or physically related to phenethylamines ( 1, 2) ( 12). However, IA drug screens have limited sensitivity and specificity due in part to capture antibody cross-reactivity with structurally similar interfering compounds, producing false-positive results. Urine drug testing is frequently performed by immunoassay (IA) due to ease of use, rapid turn-around time and the ability to screen multiple drug classes. Structural data of targeted antigens can be utilized to correlate HRMS-derived chemical formulas with structural analogs. Urine analysis by HRMS correlates accurate mass with chemical formulae, but provides little information regarding compound structure. The chemical formulas and exact masses of 145 structures (of 20 chemical formulas) were compared against masses identified by TOF. Compounds known to have cross-reactivity with the IAs were identified in the structure-based search. Chemical formulas and exact masses of 145 structures were then compared against masses identified by TOF. Separately, SciFinder (Chemical Abstracts) was used as an in silico structure search to generate a library of compounds that are known to cross-react with AMP/MDMA IAs. Hundred false-positive IA specimens for AMP and/or MDMA were analyzed by an Agilent 6230 time-of-flight (TOF) mass spectrometer. We employed two techniques, high-resolution mass spectrometry (HRMS) and an in silico structure search, to identify compounds likely to cause false-positive results. Some amphetamine (AMP) and ecstacy (MDMA) urine immunoassay (IA) kits are prone to false-positive results due to poor specificity of the antibody. ![]()
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