Fast and easy separation of 23 drugs of abuse including high, stable resolution of isobaric opioids from human urine by UHPLC-MS/MS

Significance of the topic

The accurate and rapid determination of drugs of abuse in biological fluids is critical for clinical research, workplace testing, and forensic investigations. UHPLC-MS/MS methods that combine high-resolution separation with robust throughput can address the growing need to screen for both common and novel psychoactive substances.

Goals and study overview

This study presents a method for the fast and reliable separation of 23 drugs of abuse in human urine using a dilute-and-shoot sample preparation. Emphasis was placed on achieving excellent and stable resolution of isobaric opioids while maintaining high sample throughput and retention time reproducibility.

Methodology and instrumentation

• Sample preparation: Control human urine spiked at 500 ng/mL, diluted 1:9 with 0.1% formic acid in water (dilute and shoot).
• UHPLC: Thermo Scientific Vanquish Horizon system with Accucore Biphenyl 2.6 µm (50×2.1 mm) column.
• Chromatographic conditions: Gradient from 95% A (0.1% formic acid in water) to 100% B (0.1% formic acid in methanol) over 4 min; flow rate 0.75 mL/min; column temperature 45 °C; injection volume 2 µL; total cycle time 5.5 min.
• Mass spectrometry: Thermo Scientific TSQ Quantiva triple quadrupole; HESI-II probe in positive ionization; source temperature 440 °C; spray voltage 3200 V; MRM transitions optimized for each analyte.
• Data acquisition and processing: Chromeleon 7.2.8 CDS.

Main results and discussion

• Complete separation of 23 target analytes, including opioids, benzodiazepines, and cannabinoid metabolites, achieved in under 4 min.
• Critical isobaric pairs (morphine/hydromorphone and codeine/hydrocodone) resolved with resolution factors above 3.7, stable over 1000 injections.
• Retention time variability for morphine, alprazolam, and 11-hydroxy-THC remained low (CV < 2.5%) over 1000 replicates.
• Peak width at half height and peak asymmetry showed minimal change after 1000 injections, demonstrating column robustness without additional filtration or switching valves.

Benefits and practical applications

• High-throughput screening method suitable for routine clinical and forensic laboratories.
• Minimal sample preparation with a simple dilute-and-shoot approach, reducing processing time and risk of contamination.
• Robust and reproducible chromatographic performance supports reliable quantitation and reduces false positives in opioid analysis.
• Integration of UHPLC, solid-core biphenyl column, and triple quadrupole MS offers a streamlined workflow for drug-of-abuse panels.

Future trends and possibilities

• Expansion of analyte panels to include emerging novel psychoactive substances.
• Automation of sample preparation and data processing for increased laboratory efficiency.
• Application of the method to other biological matrices such as blood or oral fluid.
• Advancements in stationary phase chemistry and MS detectors could further enhance resolution and sensitivity.

Conclusion

The described UHPLC-MS/MS method provides a rapid, reliable, and high-resolution solution for the analysis of 23 drugs of abuse in human urine. The combination of a dilute-and-shoot protocol with the Accucore Biphenyl column and TSQ Quantiva MS system ensures robust performance over extensive use, making it ideal for high-throughput screening environments.

Reference

• Woodmansey K., Bardsley J., Tremintin S. Technical Note 21883. Thermo Fisher Scientific; 2018.