Simultaneous Quantitation of 43 Drugs in Human Urine with a “Dilute-and-Shoot” LC-MS/MS Method

Significance of the Topic

This method addresses the critical need in forensic toxicology for a rapid and reliable approach to quantify multiple drugs of abuse and pain management compounds in urine. By combining dilute-and-shoot sample preparation with UHPLC MS MS, laboratories can achieve high throughput while maintaining sensitivity in complex biological matrices.

Objectives and Study Overview

The primary objective was to develop a streamlined LC MS MS protocol capable of simultaneously quantifying 43 analytes in human urine. Key aims included simplifying sample preparation, extending the linear range to 1000 ng/mL, minimizing matrix effects, and demonstrating method robustness for forensic applications.

Instrumentation

Chromatography and mass spectrometry were performed using:

• Thermo Scientific Accela 1250 pump with Open autosampler
• TSQ Quantum Access MAX triple quadrupole mass spectrometer
• Accucore PFP column (50×2.1 mm, 2.6 μm)
• Heated electrospray ionization source in positive mode

Methodology

Sample preparation involved:

• Spiking human urine with 43 drugs and internal standards at 250 ng/mL
• β-Glucuronidase hydrolysis at 60 °C
• Methanol addition and centrifugation at 17 000 g
• Twentyfold dilution and 50 μL injection

Chromatographic separation used a three-phase gradient of aqueous ammonium acetate formic acid, methanol ammonium acetate formic acid, and a high organic modifier at flow rates of 0.75 to 1.0 mL/min. SRM acquisition monitored two transitions per analyte, with quantifier and qualifier ions and ratio checks for confirmation. Calibration spanned 1 to 1000 ng/mL in blank urine.

Main Results and Discussion

Validation demonstrated:

• Absolute recoveries between 80 and 120 percent for 42 analytes; morphine recovery compensated by its deuterated internal standard
• Relative recovery CVs below 15 percent in nine individual urine samples
• Lower limits of quantitation of 2 to 20 ng/mL for 42 compounds and 50 ng/mL for tramadol
• Linear response with R2 greater than 0.99 up to 1000 ng/mL
• Accuracy at the LLOQ ranging from 89.9 to 118.4 percent
• Precision at the LLOQ with CVs up to 19.5 percent and at QC levels (20 and 200 ng/mL) CVs between 1.1 and 10.9 percent
• No detectable carryover

Benefits and Practical Applications

This dilute-and-shoot UHPLC MS MS method offers:

• Rapid turnaround with minimal sample handling
• High sensitivity for low-level detection
• Simultaneous quantitation of a broad panel of drugs and metabolites
• Robust performance in complex urine matrices

Its simplicity and throughput make it ideal for forensic, clinical, and workplace drug testing laboratories.

Future Trends and Possibilities

Emerging developments may include:

• Integration with high resolution mass spectrometry for broader screening
• Automation of sample preparation workflows
• Expansion to other biological matrices such as blood or saliva
• Application of data analytics and artificial intelligence for pattern recognition in toxicological profiles

Conclusion

The presented LC MS MS protocol provides a straightforward and efficient solution for the simultaneous quantitation of 43 drugs in human urine. It combines simple sample preparation, strong analytical performance, and minimal matrix interferences, meeting the demands of modern forensic toxicology.

References

No external literature was cited in the source document.