Automated Hydrolysis, Extraction and Analysis of Synthetic Cathinones in Urine using a Robotic Autosampler and LC-MS/MS Platform

Significance of the Topic

Synthetic cathinones known as bath salts represent a challenging class of novel psychoactive substances. They are unregulated stimulants that can pose significant health and legal risks. Rapid and reliable detection of these compounds in urine is essential for forensic investigation drug monitoring and clinical toxicology. Automation of sample preparation and analysis improves throughput consistency and data quality

Objectives and Overview of the Study

The study aimed to develop a fully automated workflow integrating enzymatic hydrolysis dispersive solid phase extraction and LC-MS/MS analysis for synthetic cathinones in urine. The approach utilized a GERSTEL MPS robotic autosampler to perform controlled hydrolysis and cleanup followed by quantitation on an Agilent 1260 HPLC coupled to an Ultivo triple quadrupole mass spectrometer

Methodology and Instrumentation

• Sample pretreatment using 100 µl urine spiked with deuterated internal standards
• Enzymatic hydrolysis at room temperature using recombinant beta glucuronidase B-One for 15 minutes with controlled pH and mixing
• Automated dispersive solid phase extraction with acetonitrile precipitation and formic acid wash using DPX tips
• LC conditions employing a Poroshell 120 EC-C18 column at 60 °C with a 0.1% formic acid in water and acetonitrile gradient over a 10 minute run
• Mass spectrometry in positive electrospray mode with scheduled multiple reaction monitoring transitions for target analytes

Used Instrumentation

• GERSTEL MPS roboticPRO sampler with DPX option and agitator module
• Agilent 1260 HPLC system with Poroshell 120 EC-C18 column 3.0 x 50 mm 2.7 µm
• Agilent Ultivo triple quadrupole mass spectrometer with Jet Stream electrospray source
• Cheminert C2V six port injection valve with 2 µl stainless steel loop

Main Results and Discussion

Automated hydrolysis achieved complete deconjugation of morphine within 15 minutes as demonstrated by plateaued signal response. Calibration curves for mephedrone and MDPV exhibited linearity with correlation coefficients greater than 0.99. Limits of quantitation were at 2.5 ng/mL. Accuracy across low middle and high quality control levels averaged 99.4% precision ranged from 2.6 to 19.3% coefficient of variation

Benefits and Practical Applications of the Method

• High throughput processing minimizes manual intervention and reduces turnaround time
• Automated workflow enhances reproducibility and lowers risk of human error
• Dispersive SPE effectively reduces matrix interference from urine samples
• Approach is applicable for routine forensic toxicology and clinical monitoring of novel stimulants

Future Trends and Potential Applications

Expansion of the automated platform may include additional drug classes and integration with high resolution mass spectrometry for broader screening capabilities. Advances in robotics and software could enable real time data feedback and decision support. Adaptation to other biological matrices such as blood or oral fluid can further extend its utility in various analytical settings

Conclusion

The fully automated protocol combining rapid enzymatic hydrolysis dispersive clean up and LC-MS/MS analysis delivers accurate precise and high throughput quantitation of synthetic cathinones in urine. Implementation on a robotic autosampler with DPX extraction substantially reduces hands on time and improves laboratory efficiency

Reference

• Technical Data Sheet product B-One Kura Biotech Inc January 2020