Analysis of Drugs and Metabolites in Blood and Urine using Automated Disposable Pipette Extraction

Significance of the Topic

Reliable and rapid determination of drugs and metabolites in blood and urine plays a key role in clinical diagnostics, forensic investigations, anti-doping control and toxicology studies. Traditional solid phase extraction workflows are time-consuming, labor-intensive and require large solvent volumes, creating a bottleneck in high-throughput laboratories. The development of an automated, disposable pipette extraction (DPX) approach addresses these challenges by streamlining sample preparation, reducing manual handling, and delivering consistent, sensitive results.

Objectives and Overview of the Study

This work aimed to implement and validate an automated DPX procedure on the GERSTEL MPS 2 autosampler under MAESTRO control for comprehensive analysis of acidic, basic and neutral drugs in small volumes of blood or urine. Key goals included: ultrafast sample cleanup, integration of large volume injection for solvent elimination and analyte concentration, just-in-time preparation via the MAESTRO PrepAhead function, and demonstration of performance comparable to or exceeding typical LC-MS/MS applications.

Methodology and Instrumentation

Disposable Pipette Extraction tips packed with mixed-mode cation exchange sorbent (DPX-CX) were used to capture a broad range of analytes without the need for prior sorbent conditioning. The general workflow was:

• Spike samples with deuterated internal standards.
• Protein precipitate blood with acetonitrile; hydrolyze urine with β-glucuronidase in 0.1 M phosphate buffer at 55 °C, then precipitate enzyme.
• Dispense 250 μL 30 % acetonitrile onto DPX sorbent, aspirate and mix the sample, discard to waste after 30 s.
• Wash sorbent with 0.5 mL 30 % and 0.5 mL 100 % acetonitrile; discard washes.
• Elute analytes with 0.7 mL 2 % NH₄OH–78 % CH₂Cl₂–20 % isopropanol into GC vials.
• Perform large volume injection (50 μL) in a cooled injection system (CIS 4) to evaporate solvent online.
• Derivatize benzodiazepines via sandwich injection of MTBSTFA or BSTFA and eluent in the CIS or in-vial, followed by GC–MS analysis in full-scan or SIM mode.

Used Instrumentation

• Agilent 6890N GC with 5975 inert XL MSD
• Gerstel CIS 4 PTV inlet for large volume injection and inlet derivatization
• Gerstel MPS 2 PrepStation dual-rail autosampler with DPX and Automated Liner EXchange (ALEX) options
• DPX-CX 1 mL tips and analytical grade solvents and reagents

Main Results and Discussion

Automated DPX enabled a complete extraction cycle in approximately 6 minutes per sample, synchronized with GC–MS runs using MAESTRO PrepAhead. Key findings:

• Basic drugs (TCAs, PCP, methadone, meperidine, cocaine) in 0.25 mL whole blood achieved LODs <0.1 ppm in full-scan mode, with recoveries of 67–111 % and RSDs <10 %.
• Benzodiazepines in 0.2 mL hydrolyzed urine reached LODs around 1 ng/mL; most compounds yielded recoveries of 50–100 % after MTBSTFA derivatization in the CIS.
• Opiates in blood (codeine, morphine, 6-MAM, oxycodone) achieved LODs <1 ng/mL with automated in-vial BSTFA derivatization and large volume injection.
• Sandwich injection in the CIS effectively removed solvent, enabled on-inlet derivatization and concentration, maintaining liner performance over >50 injections without manual intervention.

Benefits and Practical Applications of the Method

This automated DPX workflow offers:

• High throughput “just-in-time” preparation synchronized with analysis, maximizing instrument utilization and reducing wait times.
• Minimal manual labor and solvent consumption compared to traditional SPE.
• Flexible derivatization strategies for GC–MS target classes.
• Robust reproducibility and sensitivity suitable for clinical, forensic and QA/QC laboratories.

Future Trends and Opportunities

Ongoing developments may include:

• Extension of DPX sorbent chemistries for emerging drug classes and metabolites.
• Integration with LC–MS/MS platforms for complementary workflows.
• Further miniaturization and online coupling of extraction and analysis systems.
• Application of artificial intelligence for automated method optimization and real-time data interpretation.

Conclusion

The combination of automated disposable pipette extraction, large volume injection in a cooled inlet and MAESTRO-controlled autosampling delivers a rapid, sensitive and reproducible approach for drug and metabolite analysis in small blood and urine samples. This workflow addresses key bottlenecks in sample preparation, enabling high-throughput, consistent results with significant reductions in labor and solvent use.

Reference

• S. T. Ellison, W. E. Brewer and S. L. Morgan, “Comprehensive Analysis of Drugs and Metabolites in Urine with Disposable Pipette Extraction”, PittCon 2008, New Orleans, LA.