B-389 A High-Throughput, Fully Automated Magnetic Bead-Based Extraction for Multi-Class Drug-of-Abuse Analysis in Oral Fluid

Abstract

The increasing prevalence of polysubstance use and the drawbacks of urine have driven interest in oral fluid (OF) for drugs-of-abuse (DOA) testing. However, OF brings its own analytical challenges including generally lower collection volumes and analyte concentrations, matrix effects and interferents, and the need for more robust and sensitive sample preparation procedures. Additionally, reliance on the Quantisal™ collection device, the only 510(k)-approved OF storage and transport device on the market, introduces dilution and buffer components that complicate downstream LC-MS/MS analysis. Conventional extraction techniques lack sensitivity and/or don’t afford automation at scale, limiting broader adoption of OF. We present the first magnetic bead-based extraction (mSPE) method for small molecule analysis in Quantisal™ for drug abuse testing. A 12-drug panel spanning cationic, zwitterionic, and hydrophobic analytes was extracted using a mixed mode (MCX) sulfonic acid functionalized Fe3O4 sorbent and is automated on a ThermoFisher KingFisher™ Flex platform in a 96-well plate format. The panel comprised buprenorphine, fentanyl, mitragynine, hydrocodone, codeine, oxycodone, pregabalin, gabapentin, alprazolam, oxazepam, temazepam and THC. Sorbent specific surface area, pore size and diameter were optimized for the MCX sorbent. Extraction was performed using 800 µL of OF sample in Quantisal™, the KingFisher™ Flex system with a 30-minute automated protocol involving binding, 3 wash steps and elution. Sample was subsequently dried and reconstituted. A 4 µL of reconstituted extract was analyzed on a Shimadzu LCMS-8060 triple quadrupole LC-MS/MS system with a Shimadzu prominence HPLC system using a 6.6 min method with Raptor Biphenyl 2.7 µm, 50 x 3.0 mm HPLC column. Precision and accuracy were evaluated using quality control (QC) materials. Analytical measuring range (AMR) was established. Matrix effect and recovery were quantified via the Matuszewski approach. The MCX microspheres demonstrated an approximately spherical morphology with particle diameters ranging from 15–50 µm, a specific surface area of 600–800 m²/g, and an average pore size of 60–80 angstroms. Excellent linearity was achieved for all analytes across the clinically relevant AMR, with R² values greater than 0.996. Within-run and between-run precision were within15% at concentrations slightly below the limit of quantification (LOQ) and less than 9% within the AMR. Bias was within 15% below the LOQ and less than 12% within the AMR. Matrix effects were within13% relative to neat solution responses. Extraction recovery ranged from 34.49% to 92.08%, providing adequate signal at LOQ. We developed and validated a highly automated magnetic bead–based extraction (mSPE) method for the quantitative analysis of 12 clinically relevant drugs of abuse in oral fluid collected using Quantisal™. To our knowledge, this is the first mSPE method specifically designed for small-molecule analysis in this collection matrix. Comprehensive validation demonstrated analytical performance suitable for clinical laboratory implementation. The workflow is completed within 30 minutes with minimal hands-on time and integrates seamlessly into automated platforms, enabling faster and more efficient quantification compared with conventional extraction approaches. The MCX Fe3O4 sorbent supports broad analyte coverage across diverse physicochemical properties, providing flexibility to incorporate emerging targets in an evolving toxicology landscape.