Quantitation of 122 Drugs in Urine by Triple Quadrupole Mass Spectrometry

Significance of the Topic

Forensic toxicologists increasingly face the challenge of quantifying a broad and evolving panel of drugs and metabolites in urine samples. Conventional immunoassays, GC-MS or LC-UV methods require separate workflows for different compound classes, increasing time and cost. A single LC-MS/MS method capable of analyzing polar and non-polar, positively and negatively ionizing analytes in one run offers significant practical advantages for forensic, clinical, and workplace testing laboratories.

Objectives and Study Overview

The primary goal was to develop and validate a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method on a Thermo Scientific™ TSQ Endura™ triple quadrupole instrument. This approach targets the simultaneous quantitation of 122 drugs and metabolites in human urine, covering opiates, opioids, benzodiazepines, barbiturates, amphetamines, tricyclic antidepressants, designer drugs, and more.

Methodology and Instrumentation

• Sample Preparation: Urine aliquots were spiked with stable isotope-labeled internal standards and subjected to ß-glucuronidase hydrolysis. The deconjugated mixture was extracted via liquid-liquid extraction (Amtox A™ tubes), dried, and reconstituted for analysis.
• Liquid Chromatography: Gradient elution using 10 mM ammonium acetate in water (solvent A) and methanol (solvent B) on a Thermo Scientific™ Accucore™ PFP fused-core column (2.6 µm, 100 × 2.1 mm). The gradient ramped from 2 % to 100 % B over 10 min at 0.5 mL/min; total run time was 15 min with a 10 µL injection volume.
• Mass Spectrometry: A TSQ Endura triple quadrupole equipped with Ion Max™ NG heated electrospray source performed fast SRM acquisition (up to 500 SRM/s). Two transitions per analyte provided ion ratio confirmation; a single transition was monitored for each of 84 isotope-labeled internal standards, totaling 328 SRM transitions in positive and negative modes.
• Data Processing: Thermo Scientific™ TraceFinder™ software was used for peak integration, calibration curve fitting, and ion ratio confirmation.

Main Results and Discussion

Limits of quantitation (LOQs) ranged from 0.5 to 50 ng/mL for most analytes, with back-calculated concentrations within 20 % of nominal and ion ratios meeting defined tolerances. Intra-assay precision across quality control levels (1, 10, 100 ng/mL) was generally below 10 % RSD and always within 17 %. Matrix effect evaluation, using 12 lots of blank urine, showed <2 % failure for compounds with isotope-labeled analogs versus 21 % for those without. Representative chromatograms and calibration curves demonstrated consistent sensitivity and linearity (R2 > 0.9) across the panel.

Benefits and Practical Applications

• Single-run analysis of 122 diverse compounds reduces instrument time and sample handling compared to multiple targeted assays.
• Fast polarity switching and high SRM acquisition speed maintain sensitivity and quantitative performance even with narrow chromatographic peaks.
• Stable isotope-labeled internal standards effectively mitigate matrix effects, ensuring reliable quantitation across varied urine samples.
• Applicable to forensic and clinical toxicology, workplace drug testing, and research laboratories requiring high-throughput, robust drug screening.

Future Trends and Potential Applications

Advancements may include expanding the analyte panel to encompass newly emerging designer drugs, integrating high-resolution mass spectrometry for unknown screening, and implementing automated sample preparation systems to increase throughput. Data analytics and machine learning could further enhance compound identification, peak integration, and data review processes. The flexibility of LC-MS/MS methods positions them as a central platform for evolving toxicology and pharmacokinetic studies.

Conclusion

The developed HPLC-MS/MS method on the TSQ Endura instrument successfully quantifies 122 drugs and metabolites in urine with high sensitivity, precision, and throughput. A unified sample preparation procedure, combined with fast SRM acquisition and stable isotope-labeled internal standards, meets or exceeds forensic toxicology requirements, offering a streamlined workflow for comprehensive urine drug analysis.

Reference

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