Context matters: selecting LC-MS sample preparation methods for LC-MS/MS in clinical research and toxicology applications

Importance of the Topic

Liquid chromatography–mass spectrometry (LC-MS) has become essential in clinical and toxicological analysis due to its broad screening capacity, high sensitivity, and specificity compared with immunoassays. Proper sample preparation is critical to maintain instrument performance, extend maintenance intervals, and ensure accurate, robust results in complex biological matrices.

Aims and Overview of the Study

This white paper by Professor Alain Verstraete at Ghent University Hospital evaluates three LC-MS sample preparation methods—protein precipitation, liquid–liquid extraction (LLE), and solid-phase extraction (SPE)—for detecting therapeutic and narcotic drugs in small serum and urine volumes. It compares sensitivity, matrix depletion, throughput, and suitability for targeted and untargeted toxicology workflows.

Methodology and Instrumentation

• Sample matrices: low-volume serum (50–250 µL) and urine, covering a broad drug panel including amphetamines, opioids, benzodiazepines, barbiturates, cannabinoids, LSD, and designer drugs.
• Preparation techniques: dilution, protein precipitation, phospholipid removal, LLE, supported liquid extraction (SLE), offline SPE, and online SPE to balance simplicity, analyte concentration, and matrix cleanup.
• Hydrolysis: enzymatic and chemical cleavage of phase II metabolites in urine to improve detection of glucuronides and sulfates.
• Instrument platforms: Thermo Scientific Orbitrap Exploris 120 for high-resolution accurate-mass (HRAM) screening with polarity switching; TSQ Quantis Plus triple quadrupole for targeted quantitation; Thermo Scientific TraceFinder software with Tox Explorer Collection database.
• Acquisition parameters: full-scan resolution 35,000 with MS/MS at 17,500, 2.0 m/z isolation window, consistent retention time inclusion lists.

Main Results and Discussion

• Detection sensitivity: SPE and LLE outperformed precipitation, detecting 85–89 vs. 77 out of 102 analytes at low-to-medium concentrations. Precipitation’s lower sample volume limited sensitivity despite its simplicity.
• Matrix effects: Precipitation and dilution reduce background but do not concentrate analytes. Phospholipid removal improved selectivity but added cost. LLE, SLE, and SPE offered superior analyte enrichment at the expense of complexity and time.
• Throughput and automation: Automated SPE workflows on robotic platforms enabled preparation of 96 samples in under three hours, supporting high-throughput clinical settings.
• Robustness: Internal standard coefficients of variation (CV) were within 10%, and over 75% of analytes achieved CV ≤ 30%, satisfying clinical and forensic reproducibility guidelines.
• Tox Explorer Collection: Provided standardized methods, columns, calibrators, and HRAM spectral library for streamlined implementation and consistent performance across laboratories.

Benefits and Practical Applications

• Enhanced assay flexibility: A single LC-MS platform accommodates untargeted toxicology, therapeutic drug monitoring, hormone analysis, and vitamin profiling.
• Improved data quality: Efficient sample cleanup minimizes instrument fouling, reduces downtime, and lowers maintenance costs.
• Resource optimization: Choice of preparation method can be tailored to lab workload, staffing, and required sensitivity, balancing cost and performance.
• Futureproofing clinical services: Adoption of LC-MS expands test menus beyond immunoassays, meeting evolving diagnostic demands.

Future Trends and Opportunities

• Advances in automated sample prep platforms promise higher throughput with minimal hands-on time.
• Integration of AI-driven data processing and spectral matching will enhance untargeted screening capabilities.
• Development of robust multi-analyte calibrators and quality controls will simplify method validation and improve inter-lab harmonization.
• Emerging applications include quantification of therapeutic antibodies, coagulation factors, and novel psychoactive substances.

Conclusion

Selecting an optimal LC-MS sample preparation method requires balancing simplicity, cost, sensitivity, and throughput in line with laboratory context and regulatory requirements. While precipitation and dilution offer rapid, low-cost options, SPE and LLE deliver superior analyte enrichment for demanding toxicology assays. Automated workflows and comprehensive solution packages like the Tox Explorer Collection facilitate adoption of LC-MS in clinical settings, enabling robust, high-quality testing across diverse analyte classes.

References

1. Koal T et al., J. Steroid Biochem. Mol Biol. 2012, 129:129–138.
2. Yuan TF et al., J. Pharm. Biomed. Anal. 2019, 162:34–40.
3. Li H et al., Anal. Chem. 2012, 84:1267–1273.
4. Fresnais M et al., ACS Omega 2020, 5:24329–24339.
5. McDonald MG et al., J. Lipid Res. 2019, 60:892–899.
6. Jenkinson C et al., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2016, 1014:56–63.
7. Nakano Y et al., Methods Mol. Biol. 2019, 2030:253–261.
8. Onozato M et al., J. Chromatogr. Sci. 2020, 58:636–640.
9. Donners AAMT et al., Int. J. Lab. Hematol. 2020, 42:819–826.
10. El Amrani M et al., J. Pharm. Biomed. Anal. 2019, 175:112781.
11. Tong XS et al., J. Pharm. Biomed. Anal. 2004, 35:867–877.
12. Bjørk MK et al., Anal. Bioanal. Chem. 2013, 405:2607–2617.
13. Johnson-Davis KL, J. Appl. Lab. Med. 2018, 2:564–572.
14. Remane D et al., Clin. Biochem. 2016, 49:1051–1071.
15. Van Natta KL et al., Thermo Fisher Sci. Tech. Note 000794.
16. Thompson JP et al., Ann. Clin. Biochem. 2014, 51:312–325.
17. ANSI/ASB Standard 036, 2019.
18. GTFCh guidelines 2018.
19. ANSI/ASB Standard 121, 2021.
20. ANSI/ASB Standard 120, 2021.
21. ANSI/ASB Standard 119, 2021.