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Abstract INTRODUCTION:
In conventional forensics testing, toxicology urine screening typically employs Immunoassay-based (IA) tests which are prone to several limitations including false negatives and cross-talking, to name a few. Further, the frequent addition of new test targets results in slow test improvements and the IA-based assay industry cannot adequately respond to this fast-moving introduction of new substances. Herein, we report on a rapid, simple, and mass spectrometry-based urine screening method using DART-MS/MS for six common forensic compound groups, which vastly improves the acquisition of positive results without false-positive consequences, and one which is amenable to quick response for new drug targets.
OBJECTIVES:
Direct Analysis in Real Time-MS/MS (DART-MS/MS) is a fast and simple chromatography-free solution for wide-audience urine screening workflows. The primary goal of this method is to provide enhanced data quality by leveraging high resolution mass spectrometry with analysis times that are similar to immunoassays. The secondary goal is to simplify and improve the conventional urine-screening workflow by introducing high quality commercial kits to lower the threshold of entry into mass spectrometry-based screening.
METHODS:
The screening workflow includes the following compound groups: 1 Stimulants (amphetamine, methamphetamine, MDA), 2 Opiates (codeine, oxycodone, oxymorphone, tapentadol), 3 Benzodiazepines (alprazolam, diazepam, oxazepam, temazepam), 4 Benzodiazepine metabolites (7-aminoclonazepam, alpha-hydroxyalprazolam), 5 Illegals (benzoylecgonine, 6-MAM, THC-COOH), and 6 Opioids (fentanyl, acetylfentanyl, nor-fentanyl).
Development:
Analysis was performed using the newly introduced Bruker EVOQ DART-TQ+ platform. This platform utilizes a fully integrated DART ion source, an improved EVOQ triple quadrupole mass spectrometer, and single point of entry software to enable automatic acquisition and processing of a standard 96 sample set.
DART parameters, including temperature, scan type, and scan time, were optimized to maximize both sensitivity and precision. All drugs were tuned prior to analysis.
Calibration series and QCs were prepared in pooled human urine with deuterated internal standards. Liquid-liquid extraction was performed by concentrating the organic layer across a 2h preparation time. Aliquots of 2 µL were loaded onto a Bruker HTS-96 plate and ionized using the integrated DART-MS source for analysis amounting to 20-30 seconds per sample and a total run time < 1.5h.
Validation:
Regression curves were comprised of 5 calibrators, blanks, matrix blanks, and 3 QCs run in triplicate. Each set of samples was run multiple times.
RESULTS:
The drug groups 1-5 were analyzed by DART-MS/MS and the DART parameters were optimized for each group. Helium stream temperature was determined to be as follows for the six groups analyzed: (1) 200 C; (2) 350 C; (3) 350 C; (4) 350 C; (5) 350 C; (6) 350 C. All samples were run in scan mode with a 5-8 second duration. Data were processed against the internal standards using tqControl and the integrated MS quantitation features. All drugs investigated were analyzed via quadratic regression curves in the range tested, with R2 values of 0.985 or better for screening. Quadratic regression was selected to extend the screen range. Most drugs passed the validation protocol following traditional FDA requirements.
CONCLUSION:
This DART-MS/MS method describes a suitable chromatography-free approach for screening urine toxicology samples using mass spectrometry. The rapid analysis times, simplicity, and data quality were deemed to exceed current immunoassay times, complexity, and rate of false positives. Furthermore, this solvent-free approach reduces costs and environmental impact. NOTE: Analysis of a correlation set against a validated assay is pending. |
= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
