Whole blood continues to be a valuable tool in forensic toxicology for the immediate and near-term detection of illicit drugs and in cases where no other sample is available. Screening drugs of abuse can be complicated due to the wide variation of functional groups associated with different analyte classes. Most extraction techniques cannot extract all analytes using a single procedure without using non-optimal extraction protocols resulting in compromised extract cleanliness. Supported liquid extraction allows for the simultaneous analysis of cross functional analytes in a single extraction protocol without forfeiting extract cleanliness. The objective was to develop an automatable, common extraction procedure for multiple drug panels from whole blood matrix using supported liquid extraction (SLE) prior to UPLC-MS/MS analysis. The drug suites included amphetamines, benzodiazepines, cocaine, opiates, ketamine, buprenorphine, Z-drugs and THC-COOH.

Blank whole blood was spiked with drugs from various panels at 13ng/mL for the analytes and appropriate deuterated internal standards. Whole blood extraction was performed using ISOLUTE SLE+ 1mL capacity columns evaluating pH control and optimum extraction solvent combinations. The resultant extracts were evaporated to dryness and reconstituted in mobile phase for subsequent UPLC-MS/MS analysis. Samples were analyzed using a Waters ACQUITY UPLC coupled to a Quattro Premier XE triple quadrupole mass spectrometer (Waters). Positive ions were acquired using electrospray ionisation in the MRM mode.

Initial investigations focused on sample treatment. Extraction conditions were evaluated using spiked whole blood pre-treated 1:1 (v/v) with various ammonium hydroxide (aq) concentrations from 0.1 % up to 2 %. 0.1 % NH₄OH (aq) was selected as the pre-treatment as many of the drugs are basic in nature, however an environment at this pH value also allowed concomitant extraction of THC-COOH. Loading 1mL of pre-treated whole blood led to the extraction of matrix components in the form of red blood cells. Evaluation of various loading volumes from 500 to 900μL resulted in selection of 750μL (1:1, v:v) as the optimal loading volume for extract cleanliness. Dichoromethane, dichloromethane/isopropanol acid and methyl tert-butyl ether combinations with various modifiers were evaluated for extraction efficiency. Benzoylecgonine could only be extracted efficiently when using dichloromethane as the extraction solvent. The optimum extraction protocol provided recoveries greater than 70 % for nearly all drugs spiked into whole blood. Benzoylecgonine and buprenorphine recovered 47 % and 52 % respectively. RSD values for all drugs were under 10 %. Lower recoveries for benzoylecgonine and buprenorphine did not prevent quantitation at sub-ng/mL concentrations. Evaporative effects were eliminated using 0.05M HCl in methanol prior to evaporation. Calibration curves constructed from 1–500ng/mL demonstrated excellent linearity and r²>0.99 for all analytes. LLOQs were determined to be sub-ng/mL for each analyte when extracting 375μL of whole blood, with the exception of THC-COOH (5ng/mL). MRM analysis for common phospholipid molecules demonstrated the optimum method yields extremely clean extracts. This method was transferred to the Extrahera sample automation platform for repeat investigations into recovery, reproducibility and linearity and in nearly all analytes, the automation approach improves upon the manual approach for acquired data while also saving operator time.

This poster describes the suitability of ISOLUTE SLE+ for the rapid and reliable extraction of multiple analyte panels from whole blood in a single, fully-automatable assay on the Extrahera platform, prior to UPLC-MS/MS analysis.