Determination of drugs of abuse in biological samples by accelerated solvent extraction and LC-MS/MS

Significance of the topic

Determination of drugs of abuse in biological specimens is critical in forensic toxicology. Accurate identification and quantification support legal investigations, clinical toxicology, and public health monitoring. Traditional sample preparation methods like solid-phase extraction (SPE) deliver reliable results but involve manual steps, high solvent use, and extended processing times. Accelerated solvent extraction (ASE) emerges as an automated alternative that can enhance throughput, reduce operator bias, and lower solvent consumption while maintaining high analytical performance.

Objectives and study overview

This study aimed to evaluate the feasibility of ASE combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantifying 36 target analytes in human blood and urine. These compounds span psychoactive drugs, antagonists, medications, and anesthetics. Samples from postmortem cases were split and processed by both ASE and conventional SPE. Comparative data on recovery, sensitivity, precision, and matrix effects were generated to assess ASE as a routine forensic extraction method.

Methodology

Biological samples (blood and urine) were collected from ten human cadavers and stored at –20 °C. Each sample was divided for parallel SPE and ASE workflows. SPE involved cartridge conditioning, sample loading at pH 6.88, sequential elutions, vacuum drying, and reconstitution in methanol. ASE was performed in 10 mL stainless steel cells loaded with diatomaceous earth and sample spiked with internal standard (proadifen, SKF-525A). Extraction parameters were: n-hexane:acetone (4:1) at 80 °C, 1500 psi, 5 min static time, one cycle, 60% rinse, 100 s purge.

After evaporation, extracts were reconstituted in 100 µL methanol and analyzed by LC-MS/MS. Chromatographic separation used a C18 column (50×2.1 mm, 1.9 µm) at 35 °C with a gradient of 20 mM ammonium formate/0.1% formic acid and methanol at 0.4 mL/min. Detection employed a triple-quadrupole mass spectrometer with heated electrospray ionization, combined full-scan and data-independent acquisition (DIA).

Instrumentation used

• Thermo Scientific Dionex ASE 350 Accelerated Solvent Extractor
• Thermo Scientific TSQ Fortis Triple-Quadrupole Mass Spectrometer
• Thermo Scientific Surveyor MS Quaternary Pump with Degasser
• Surveyor AS Auto-Sampler with Rheodyne valve
• Thermo Scientific HyperSep Verify CX SPE cartridges

Main results and discussion

Both extraction methods delivered acceptable recoveries (60–140%) and precision (%CV ≤15%). ASE offered comparable or improved recoveries for most classes; benzodiazepines showed notably higher recoveries (e.g., diazepam: 92.7% ASE vs. 85.2% SPE in blood). Some analytes (e.g., LSD) favored SPE, highlighting the need for method tuning per compound. ASE extracts exhibited lower background signals, improved peak resolution, and reduced matrix effects (±15% normalized). Limits of detection and quantification met forensic criteria (LOD S/N ≥3, LLOQ S/N ≥10, bias ≤20%). Carry-over was minimal (<20% of LLOQ). Stability, selectivity, and calibration performance complied with SWGTOX guidelines.

Benefits and practical applications

• Higher throughput: 12 min per ASE extraction vs. manual SPE steps.
• Reduced solvent use: 30 mL/sample vs. larger volumes in SPE.
• Automation: minimization of operator-dependent variability.
• Versatility: adjustable temperature, pressure, and solvents for diverse analytes.
• Applicability: suitable for routine forensic and clinical toxicology laboratories.

Future trends and opportunities

Advances may include integration with ultra-high-performance liquid chromatography (UHPLC) systems, on-line coupling for direct transfer of ASE extracts to LC-MS/MS, and miniaturized or green extraction solvents. Expansion to other matrices (hair, bile, bone) and emerging psychoactive substances will further enhance forensic capabilities. Data-independent acquisition strategies and high-resolution mass spectrometry could refine confirmatory analyses.

Conclusion

This work demonstrates that accelerated solvent extraction is a robust, efficient, and high-throughput alternative to conventional SPE for forensic analysis of drugs of abuse. ASE reduces solvent consumption and labor, delivers reliable recoveries across multiple drug classes, and streamlines sample preparation without compromising analytical quality.

References

• 1. Abend AM, Chung L, McCollum DG, Wuelfing WP. Automated accelerated solvent extraction and HPLC analysis of ivermectin in meat-based formulations. J Pharm Biomed Anal. 2003;31:1177–1183.
• 2. Ottonello G, Ferrari A, Magi E. Determination of PCBs in fish: ASE and GC-MS. Food Chem. 2014;142:327–333.
• 3. Sun H, Zuo Y, Qi H, Lv Y. ASE with capillary electrophoresis for fluoroquinolones and sulfonamides in aquatic products. Anal Methods. 2012;4:670–675.
• 4. Tao Y, Chen D, Wei H, et al. ASE-derivatization LC-MS/MS of nitrofurans in freshwater fish. Food Addit Contam A. 2012;29:736–745.
• 5. Wang B, Xie X, Zhao X, et al. ASE method for thiamphenicol, florfenicol and amine in poultry eggs. Molecules. 2019;24(9).
• 6. Wang B, Pang M, Zhao X, et al. Comparison of LLE and ASE for chloramphenicol, thiamphenicol, florfenicol, and amine in eggs. J Mass Spectrom. 2019;54:488–494.
• 7. Wu JJ, Mak YL, Murphy MB, et al. Validation of ASE LC-MS/MS for ciguatoxin-1 in fish. Anal Bioanal Chem. 2011;400:3165–3175.
• 8. Chuang YH, Zhang Y, Zhang W, et al. Comparison of ASE and QuEChERS for pharmaceuticals in vegetables. J Chromatogr A. 2015;1404:1–9.
• 9. Marchese S, Perret D, Gentili A, et al. ASE and LC-MS determination of arylphenoxypropionic herbicides in soil. Rapid Commun Mass Spectrom. 2001;15:393–400.
• 10. Popp P, Keil P, Möder M, et al. ASE followed by GC, HPLC, and GC-MS for PAHs, pesticides, and PCBs. J Chromatogr A. 1997;774:203–211.
• 11. Conte E, Milani R, Morali G, Abballe F. Comparison of ASE and traditional methods for diflufenican in soil. J Chromatogr A. 1997;121–125.
• 12. Pallaroni L, Björklund E, Von Holst C. Microwave-assisted and ASE of zearalenone. Mycotoxin Res. 2002;18:74–77.
• 13. Ge X, Wu X, Liang S, Sun H. ASE cleanup and HPLC for cyromazine and melamine in plants. J Chromatogr Sci. 2014;52:751–757.
• 14. Yang G, Sun Q, Hu Z, et al. ASE-DLLME and GC-MS of essential oil from Ligusticum. J Sep Sci. 2015;38:3588–3598.
• 15. Zhang Y, Yang J, Shi R, et al. ASE-SPE GC-ECD analysis of OCPs in crops. Chromatographia. 2011;73:385–391.
• 16. Zhang YN, Yang XL, Bian YR, et al. ASE-SPE-HPLC of PAHs in soil and earthworms. Chin J Anal Chem. 2016;44:1514–1520.
• 17. Zhu QZ, Sun Q, Su ZG, et al. ASE for stable isotope analysis in soil water. Chin J Anal Chem. 2014;42:1270–1275.
• 18. Kellogg JJ, Wallace ED, Graf TN, et al. Conventional and ASE of green tea for metabolomics. J Pharm Biomed Anal. 2017;145:604–610.
• 19. Sant’Anna SG, Oliveira CDR, Diniz EM, Yonamine M. ASE GC analysis of nicotine in meconium. J Anal Toxicol. 2012;36:19–24.
• 20. Mantovani CC, Lima MB, Oliveira CDR, et al. ASE for cocaine biomarkers in meconium. J Chromatogr B. 2014;957:14–23.
• 21. Franceschetti L, Di Candia D, Giordano G, et al. Drugs in bone: skeletal remains study. Int J Legal Med. 2021;135(2):457–463.
• 22. Giordano G, Biehler-Gomez L, Seneci P, et al. Detecting drugs in dry bone over 23 years. Int J Legal Med. 2021;135(2):457–463.
• 23. Iida T, Todaka T. Dioxin measurement in human blood: improved method. Ind Health. 2003;41:197–204.
• 24. Kuriki K, Tajima K, Tokudome S. ASE for fatty acids in plasma and erythrocytes. Lipids. 2006;41:605–614.
• 25. SWGTOX. Standard practices for method validation in forensic toxicology. J Anal Toxicol. 2013;37:452–474.