Automated Extraction of a Drugs of Abuse Panel from Human Urine Using Biotage® Extrahera™ LV-200 and Microelution SPE Prior to UPLC-MS/MS Analysis

Significance of the Topic

The reliable detection of drugs of abuse in urine is critical for clinical diagnostics, forensic investigations and workplace compliance testing. Automated, high-throughput sample preparation coupled with sensitive UPLC-MS/MS analysis enhances laboratory efficiency, reduces operator error and delivers robust quantitative data across a broad multi-class panel of analytes.

Objectives and Overview of the Study

This study evaluates an automated microelution solid-phase extraction (SPE) workflow using Biotage Extrahera LV-200 and Biotage Mikro CX 96-well plates to isolate over 50 common drugs of abuse and metabolites from hydrolyzed human urine prior to UPLC-MS/MS analysis. The goals were to:

• Develop a mixed-mode SPE protocol delivering clean extracts with high recovery and reproducibility.
• Compare manual versus automated processing in terms of recovery, precision and throughput.
• Demonstrate linearity and limits of quantitation (LOQs) suitable for low-pg/mL detection.

Methodology and Instrumentation

Sample Preparation:

• 100 µL internal standard solution spiked into 1 mL urine, equilibrated 1 hour.
• Dilution with 950 µL 100 mM ammonium acetate pH 5 and addition of β-glucuronidase, incubated at 60 °C for 2 hours.
• SPE using 2 mg Biotage Mikro CX plates (mixed reversed-phase/strong cation exchange); conditioning with methanol, equilibration with 4% phosphoric acid, sample loading, sequential washes, and microelution with DCM/MeOH/NH4OH (78:20:2, v/v).
• Evaporation under nitrogen (TurboVap 96 Dual), reconstitution in 90:10 water/MeOH with 0.1% formic acid.

UPLC-MS/MS Analysis:

• UHPLC: Shimadzu Nexera with Restek Raptor Biphenyl column (100×2.1 mm, 2.7 µm); gradient from 80:20 to 0:100 aqueous/organic mobile phases containing 2 mM ammonium formate and 0.1% formic acid; flow rate 0.4 mL/min; injection 5 µL; column at 30 °C.
• Mass Spectrometry: Shimadzu 8060 triple quadrupole with ESI+; optimized MRM transitions for >50 analytes; interface at 400 °C; gas flows set for robust desolvation.

Main Results and Discussion

Recovery and Precision:

• Average recoveries >60% for most analytes with RSDs <5%.
• Comparable performance between manual and automated SPE, with slightly improved reproducibility on the Extrahera.

Linearity and LOQ:

• Calibration range 1–1000 pg/mL delivered r2 >0.999 for all analytes.
• LOQs ranged from 1 pg/mL (e.g., methamphetamine, EDDP) up to several hundred pg/mL for challenging compounds.

Chromatography:
Representative UPLC-MS/MS traces demonstrated baseline resolution across multiple drug classes (cocaine, opioids, benzodiazepines, amphetamines), confirming method specificity.

Benefits and Practical Applications

• High-throughput capacity: ~25 minutes to process 96 samples automatically (excluding hydrolysis and evaporation).
• Minimal solvent and sample consumption owing to microelution format.
• Integration with automated platforms reduces manual handling and potential variability.
• Suitable for clinical, forensic and workplace drug monitoring laboratories requiring multi-class analysis.

Future Trends and Possibilities

Emerging developments may include:

• Direct-injection compatible elution solvents to eliminate evaporation steps.
• Broader panels incorporating novel psychoactive substances.
• Integration with laboratory information management systems (LIMS) for seamless data flow.
• Green SPE chemistries and miniaturization to further reduce solvent usage.

Conclusion

An automated mixed-mode microelution SPE workflow using Biotage Extrahera LV-200 and Mikro CX plates provides robust, reproducible extraction of a comprehensive drugs-of-abuse panel from human urine. Coupled with UPLC-MS/MS, the method achieves excellent recoveries, precision, linearity and low LOQs, supporting high-throughput toxicology testing.

References

• Biotage Application Note AN964.v1, Automated Extraction of Drugs of Abuse from Urine Using Biotage Extrahera LV-200 and Microelution SPE, 2022.