Fast and Easy Separation of 23 Drugs of Abuse Including High, Stable Resolution of Isobaric Opioids from Human Urine by UHPLC-MS/MS

Importance of the Topic

Screening of drugs of abuse in human urine is critical for clinical research, toxicology and forensic applications. Laboratories require rapid, high-throughput methods that can resolve structurally similar compounds, maintain consistent performance over hundreds of injections and minimize downtime due to column degradation.

Study Objectives and Overview

This study aimed to develop and demonstrate a fast, reliable UHPLC-MS/MS workflow for the separation and quantification of 23 common drugs of abuse in diluted human urine. Particular focus was placed on achieving baseline resolution of key isobaric opioid pairs (morphine/hydromorphone and codeine/hydrocodone) and assessing long-term stability over 1000 replicate injections.

Methodology and Used Instrumentation

Sample preparation consisted of a simple dilute-and-shoot protocol: 100 µL of spiked urine mixed with 900 µL of 0.1 % formic acid in water. Chromatographic separation employed a Thermo Scientific Vanquish Horizon UHPLC system with an Accucore Biphenyl 2.6 µm, 50 × 2.1 mm column at 45 °C. A ballistic gradient from 95% aqueous to 100% methanol (both containing 0.1 % formic acid) was executed over 4 minutes, with a total cycle time of 5.5 minutes at 0.75 mL/min and 2 µL injection volume. Detection used a Thermo Scientific TSQ Quantiva triple quadrupole MS with HESI-II Ion Max source, operating in positive ionization MRM mode. Data were processed using Chromeleon 7.2.8 CDS.

Main Results and Discussion

All 23 analytes exhibited sharp, symmetrical peaks and full separation of critical isobaric opioids. Resolution remained unchanged through 1000 injections, with retention time %CV of 2.49 % for morphine, 0.07 % for alprazolam and 0.14 % for 11-hydroxy-THC. Key chromatographic metrics such as peak width at half height and asymmetry showed negligible drift, underscoring the column’s robustness under high-throughput conditions.

Benefits and Practical Applications

• Fast cycle time (5.5 minutes) enables high sample throughput for clinical and forensic screening.
• Excellent and stable resolution of isobaric opioids reduces false positives and enhances quantitation accuracy.
• Simple dilute-and-shoot sample prep streamlines workflow and is amenable to automation.
• Robust performance over at least 1000 injections minimizes maintenance and downtime.

Future Trends and Potential Uses

Future developments may extend this approach to emerging novel psychoactive substances, incorporate more advanced ion mobility separations, and integrate automated sample handling. Advances in stationary phase chemistries and MS instrumentation will continue to improve sensitivity, selectivity and throughput in drug screening workflows.

Conclusion

The combination of a high-efficiency biphenyl column, rapid gradient UHPLC and targeted MS/MS detection provides a powerful, reliable solution for high-throughput screening of drugs of abuse in urine. The method delivers excellent resolution, consistent retention stability and simplified sample preparation, meeting the demands of modern clinical and forensic laboratories.

References

• Find out more at www.thermofisher.com/appslab
• Find out more at www.thermofisher.com/biphenyl