Automated High Throughput Sample Preparation and UPLC-MS/MS Analysis for the Quantification of EtG and EtS in Human Urine Using the Andrew+ Pipetting Robot

Importance of the Topic

Ethylglucuronide and Ethylsulfate are vital markers for monitoring alcohol consumption in clinical and forensic settings. These metabolites extend the detection window up to 80 hours after intake making them superior to direct ethanol measurement. Automated high throughput analysis enhances reliability and efficiency in laboratories dealing with large sample volumes.

Objectives and Study Overview

This work aimed to develop and validate an automated dilute and shoot workflow for the quantification of EtG and EtS in human urine. The study compares the performance of an Andrew+ Pipetting Robot based method to a traditional manual preparation, focusing on throughput, precision and recoverability.

Methodology

A two script approach was implemented on the Andrew+ pipetting platform to automate sample dilution and mixing steps in a 96 well format. One script used pipette based mixing while the other leveraged a Microplate Shaker plus. Internal standards were omitted in this demonstration and water was used for dilution. Method parameters such as mixing speed and pipetting settings were optimized via OneLab software for reproducible performance.

Used Instrumentation

• Andrew plus Pipetting Robot equipped with Bluetooth pipettes
• Microplate Shaker plus for automated mixing
• OneLab cloud native software for protocol creation
• ACQUITY UPLC I Class FTN system
• Xevo TQD triple quadrupole mass spectrometer
• ACQUITY UPLC CSH Phenyl Hexyl column

Main Results and Discussion

Both automated methods achieved comparable recoveries and precision to manual preparation workflows. Precision at two concentration levels for EtG and EtS met acceptance criteria with coefficients of variation below 20 percent. The recovery difference between automated and manual protocols was within two percent across quality control samples. The pipette mixing and shaker based workflows both delivered consistent results with minor improvements observed for EtS precision in the automated assays.

Benefits and Practical Applications

• Simple dilute and shoot approach adapted to automation
• High throughput processing using 96 well plates
• Reduction of human error through reproducible pipetting
• Better utilization of analyst time with minimal method training
• Easily transferable and modifiable methods via OneLab software

Future Trends and Potential Uses

Further integration of automated sample preparation with laboratory information management systems can streamline data flow and traceability. Expansion of similar automated workflows to additional biomarkers and biological matrices is feasible. Advanced robotics and AI driven method optimization could further enhance precision and throughput in clinical and forensic laboratories.

Conclusion

The automated dilute and shoot workflows on the Andrew plus platform provide accurate, precise and high throughput quantification of EtG and EtS in human urine. Both pipette based and shaker enabled mixing options yield results equivalent to manual protocols, offering flexible solutions for laboratories requiring efficient alcohol biomarker analysis.

Reference

Ohouo P Y Dixon N Lieu A Zavery Z Rosano T G UPLC MS MS Method for Quantitation of EtG and EtS in Human Urine Waters Application Note 720006273 May 2018