Development and validation of two LC-MS/MS methods for the detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine using turbulent flow chromatography

Abstract

In the context of driving ability diagnostics in Germany, administrative cutoffs for various drugs and pharmaceuticals in urine have been established. Two liquid chromatography–tandem mass spectrometry methods for simultaneous detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine were developed and validated. A 500-μL aliquot of urine was diluted and fortified with an internal standard solution. After enzymatic cleavage, online extraction was performed by an ion-exchange/reversed-phase turbulent flow column. Separation was achieved by using a reversed-phase column and gradient elution. For detection, a Thermo Fisher TSQ Quantum Ultra Accurate Mass tandem mass spectrometer with positive electrospray ionization was used, and the analytes were measured in multiple-reaction monitoring mode detecting two transitions per precursor ion. The total run time for both methods was about 15 min. Validation was performed according to the guidelines of the Society of Toxicological and Forensic Chemistry. The results of matrix effect determination were between 78 % and 116 %. The limits of detection and quantification for all drugs, except zopiclone, were less than10 ng/mL and less than 25 ng/mL, respectively. Calibration curves ranged from 25 to 200 ng/mL for amphetamines, designer amphetamines, and benzoylecgonine, from 25 to 250 ng/mL for benzodiazepines, from 12.5 to 100 ng/mL for morphine, codeine, and dihydrocodeine, and from 5 to 50 ng/mL for buprenorphine and norbuprenorphine. Intraday and interday precision values were lower than 15 %, and bias values within ± 15 % were achieved. Turbulent flow chromatography needs no laborious sample preparation, so the workup is less time-consuming compared with gas chromatography–mass spectrometry methods. The methods are suitable for quantification of multiple analytes at the cutoff concentrations required for driving ability diagnostics in Germany.