Controlling Crosstalk of Amphetamine and Methamphetamine in Urine Assays Using 96 Well Plates for LC-MS/MS Analysis

Importance of the topic

Urine assays for amphetamine and methamphetamine often face well‐to‐well evaporative crosstalk during extraction on 96-well plates, especially at high analyte concentrations. This phenomenon compromises assay accuracy in clinical, forensic, and toxicological applications, underlining the need for robust contamination control strategies.

Objectives and study overview

This study evaluates methods to mitigate evaporative crosstalk of volatile and non-volatile drugs in urine extractions. It investigates the effectiveness of the Biotage ACT Plate Adapter combined with hydrochloric acid in methanol at varying concentrations to control contamination for eight target analytes, with a focus on amphetamine and methamphetamine at clinically relevant thresholds.

Methodology and instrumentation

Samples of blank urine, calibrators, and high-level spiked specimens (50 000–100 000 ng/mL) were distributed across 96-well ISOLUTE SLE+ plates. After supported liquid extraction, eluates were evaporated at controlled temperatures and gas flows using a Biotage SPE Dry evaporator, with and without the ACT Plate Adapter. Hydrochloric acid in methanol (0.25 % and 0.5 %) was added prior to evaporation to promote salt formation.

The analytical system comprised a Shimadzu Nexera UPLC coupled to a Sciex 5500 triple quadrupole mass spectrometer.

Main results and discussion

Negligible crosstalk was observed for benzoylecgonine, morphine, and hydromorphone under all conditions. MDA, MDEA, and MDMA showed minor contamination (<5 ng/mL), which was largely prevented by the ACT Adapter. Amphetamine and methamphetamine exhibited significant crosstalk (2–200 ng/mL) without intervention; the ACT Adapter reduced this to 1–100 ng/mL. Optimal control (<30 ng/mL) was achieved using 10 μL of 0.5 % HCl in methanol with the ACT Adapter, satisfying SAMHSA confirmation cutoffs.

Benefits and practical applications

• The ACT Plate Adapter combined with optimized acid-in-solvent conditions offers a simple, effective contamination control strategy.
• Existing 96-well workflows in clinical, forensic, and QA/QC laboratories can incorporate these measures without major hardware changes.
• Reliable quantitation of amphetamine and methamphetamine supports regulatory compliance and data integrity.

Future trends and potential applications

Further developments may include reduced elution volumes, alternative plate formats, and automated integration to enhance throughput. Extension of salt-formation techniques to other volatile analytes and biofluids could broaden the impact across bioanalytical platforms.

Conclusion

Evaporative crosstalk in high-concentration urine assays can be effectively controlled by using the Biotage ACT Plate Adapter with 0.5 % HCl in methanol. This approach ensures accurate measurement of amphetamine and methamphetamine and aligns with forensic and clinical testing standards.

References

• Marin SJ, Merida III M, Smith J, Gairloch E. Controlling Crosstalk of Amphetamine and Methamphetamine in Urine Assays Using 96 Well Plates for LC-MS/MS Analysis. Biotage; 2014. Presented at ASMS 2020, Online. Part Number: P220.