Extraction of a Drugs of Abuse Panel from Whole Blood Using ISOLUTE® SLE+ Prior to UPLC-MS/MS Analysis

Significance of the topic

The accurate extraction and quantitation of drugs of abuse in whole blood is critical for forensic toxicology, clinical diagnostics, and workplace screening. Traditional liquid–liquid extraction methods can suffer from emulsion formation, poor reproducibility, and labor‐intensive workflows. Supported liquid extraction (SLE) using ISOLUTE SLE+ columns addresses these limitations by enabling high recoveries, minimal matrix effects, and compatibility with high‐throughput automation.

Objectives and overview of the study

This application note describes a robust protocol for extracting 49 analytes spanning cocaine, opioids, benzodiazepines, amphetamines, cannabinoids, and other drug classes from 500 µL whole blood samples. The goal was to streamline sample preparation prior to UPLC–MS/MS analysis, achieve low limits of quantitation (LLOQs as low as 0.007 ng/mL), and demonstrate reproducible performance suitable for automation.

Methodology and instrumentation

• Sample pretreatment: 500 µL whole blood was spiked with stable‐isotope internal standards, equilibrated, and basified with 0.1% ammonium hydroxide aqueous solution.
• Supported liquid extraction: ISOLUTE SLE+ 1 mL columns were loaded with 750 µL pretreated blood, allowed to absorb, and sequentially eluted with dichloromethane/isopropanol (95:5 v/v) and methyl tert‐butyl ether (MTBE) in three 2.5 mL aliquots, with vacuum assists to maximize recovery.
• Post‐elution processing: Eluates were collected into tubes containing 100 µL of 50 mM HCl in methanol to stabilize free‐base analytes, evaporated to dryness under air or nitrogen using a TurboVap LV, and reconstituted in 100 µL methanolic mobile phase B followed by 400 µL aqueous mobile phase A.
• LC conditions: Waters ACQUITY UPLC with a Restek Raptor Biphenyl 2.7 µm column (100 × 2.1 mm) and biphenyl guard, gradient from 5% to 100% organic over 13.5 min at 0.4 mL/min, column at 40 °C.
• MS/MS detection: Waters Premier XE triple quadrupole with electrospray ionization in positive MRM mode, source temperatures 150 °C (ion source) and 450 °C (desolvation), optimized cone voltages and collision energies for each analyte.

Main results and discussion

• Analyte recoveries: Typical recoveries exceeded 80% across most drug classes when spiked at 13 ng/mL, with relative standard deviations (RSDs) below 10%.
• Calibration and linearity: Quadratic fitting over 1–500 ng/mL yielded coefficients of determination (r2) above 0.99 for all compounds, with representative curves for morphine, amphetamine, benzoylecgonine, and diazepam demonstrating excellent sensitivity.
• Lower limits of quantitation: LLOQs ranged from 0.007 ng/mL (midazolam, hydromorphone) to 5 ng/mL (THC-COOH), enabling detection of trace levels.
• Extract cleanliness: Total ion chromatograms of phospholipid transitions showed negligible residual matrix interferences compared to acetonitrile-precipitated blood, indicating superior cleanup.

Benefits and practical applications

• Streamlined workflow: Eliminates emulsion issues and reduces manual handling compared to traditional LLE.
• Automation compatibility: Method successfully automated on Biotage Extrahera™, processing 24 samples in under 41 minutes with comparable or improved recoveries and precision.
• Broad analyte coverage: Suitable for panels including stimulants, opioids, benzodiazepines, cannabinoids, and designer drugs.
• Regulatory compliance: Low LLOQs and robust linearity support forensic and clinical applications requiring stringent validation criteria.

Future trends and potential applications

• Integration with high-resolution mass spectrometry to expand screening capabilities for novel psychoactive substances.
• Miniaturization of sample volumes and further reduction of solvent consumption for greener workflows.
• Application to alternative matrices such as plasma, oral fluid, or dried blood spots for decentralized testing.
• Real-time data processing and AI-driven decision support integrated into automated platforms.

Conclusion

The presented ISOLUTE SLE+ protocol offers a rapid, reliable, and automatable approach for extracting a comprehensive drugs of abuse panel from whole blood. High recoveries, low matrix effects, and sensitive MS/MS detection enable confident quantitation across a wide concentration range, making this method well suited for routine toxicology, forensic, and clinical laboratories.