MSACL 2025 Abstract

Automated Microelution Plate Solid Phase Extraction for Quantitative LC-MS/MS Analysis of Fentanyl Analogs, Nitazenes, and Xylazine in Human Urine

Qi Huang, Heather Eastwood, Steven Alo, Karsten Liegmann, John D Laycock
Tecan, Baldwin Park, CA, United States

Qi Huang (Presenter) Tecan

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
The continued prevalence of fentanyl and its analogues, as well as nitazenes and xylazine, underscores the urgent need for reliable analytical tools in clinical and forensic toxicology, as these substances continue to drive overdose fatalities. Accurate detection of these substances in biological matrices is critical in clinical and forensic toxicology. This presentation evaluates the use of a 96-well microplate based SPE format offering microelution capabilities for the extraction of fentanyl analogs, nitazenes, and xylazine from human urine, using automated and semi-automated liquid handling and positive pressure instruments.

OBJECTIVES:
This study demonstrates the extraction of fentanyl analogs, nitazenes, and xylazine from human urine using a strong cation exchange SPE sorbent, packed into a microelution plate. The adoption of such plate enables streamlined automation, improved sample throughput, and significantly lower elution volumes compared to traditional SPE columns. This study explores a fully automated and a semi-automated workflow.


METHODS:
The sample panel consisting of 30 analytes was spiked into drug-free human urine (Utak) yielding a concentration of 1 ng/mL. Samples were loaded onto Tecan HPSCX 96-well microelution SPE plates. For each of the SPE workflows, both the Resolvex® A200 and i300 instruments were used for positive pressure loading and solvent dispensing, while the Resolvex® i300, integrated on a Fluent automated workstation, also provided drying of purified samples. After wash steps, a low elution volume totaling 50uL, delivered as two 25uL dispense and elute steps were used to elute the analytes. LC-MS/MS quantitation was performed on the eluates, which were either injected directly or dried and reconstituted, before injection. The Sciex 7600 ZenoTOF in HR-MRM mode with internal standards, was used for quantitation.

RESULTS:
The microelution plate SPE workflow was evaluated for extraction efficiency, reproducibility, and operational robustness. LC-MS/MS analysis on the Sciex 7600 of the extracted samples provided ample sensitivity for the majority of the compounds, at a concentration of 1ng/mL in urine. To assess the efficiency of the SPE extraction, the recovery of the samples was calculated using a post-extraction spike in. The sample recovery showed high extraction efficiency, with 90% of analytes achieving recoveries ≥80%. Technical replicate samples were processed to evaluate the reproducibility, demonstrating low CVs and high quantitative stability of the workflow. Two thirds of the samples analyzed resulted in CVs being lower than 10%.

CONCLUSION:
The SPE methods have been proven to effectively extract a panel of 30 compounds consisting of fentanyl and its analogues, as well as nitazenes and xylazines from human urine. By utilizing liquid handling and positive pressure instruments and the microelution plate based SPE workflow, the method was automated enabling low-volume elution, reduced hands-on time, and a simple, efficient protocol ideal for high-throughput toxicology assays and testing.