Quantitative determination of seven synthetic cathinones (stimulants) from stabilized human urine by UHPLC-MS/MS for forensic toxicology

Significance of the Topic

The emergence of new psychoactive substances such as synthetic cathinones poses a challenge for forensic and clinical laboratories. Rapid identification and quantitation of these stimulants in biological matrices is critical for public health monitoring, legal cases and quality control. Conventional methods often require derivatisation or suffer from matrix effects, limiting throughput and reliability.

Study Objectives and Overview

This study aimed to develop and validate a quantitative UHPLC MS/MS method for seven synthetic cathinones in human urine stabilized with ascorbic acid. The approach integrates mixed mode solid phase extraction with hydrophilic interaction liquid chromatography on a Syncronis HILIC column. Performance parameters including linearity, sensitivity, precision, accuracy, recovery and matrix effects were evaluated.

Methodology

Sample preparation relies on a simple protein precipitation and mixed mode SPE cleanup. Urine samples (100 µL) acidified with phosphoric acid are loaded onto SOLA SCX 10 mg 96 well plates. After washing steps with acidified water and methanol, analytes are eluted with ammonia in acetonitrile, evaporated and reconstituted for analysis. The 6 minute isocratic UHPLC separation uses acetonitrile and 10 mM ammonium formate on a 100 × 2.1 mm Syncronis HILIC 1.7 µm column at 30 °C.

Instrumentation Used

• Vanquish Horizon UHPLC system with binary pump, split sampler and active preheater
• TSQ Quantiva triple stage quadrupole mass spectrometer with HESI ion source
• SOLA SCX SPE 10 mg 96 well plates
• Chromeleon CDS software for data acquisition and processing

Main Results and Discussion

The method exhibited linear calibration from 0.1 to 100 ng/mL with a 1/x² weighted regression. The lower limit of quantification was 0.1 ng/mL for all analytes. Intra batch accuracy bias remained within ±9.3 % and precision below 8 % CV across LLOQ, low, medium and high QC levels. Recoveries ranged from 85.9 % to 94.1 %. Matrix factors normalized to a deuterated internal standard fell between 0.897 and 1.01 with CV ≤9.5 %, indicating minimal ion suppression. No significant carryover or interferences were observed in blank urine from multiple sources.

Benefits and Practical Applications

• Rapid 6 minute analysis per injection with minimal sample consumption
• Sensitive quantitation without derivatisation steps
• Robust orthogonal cleanup to mitigate matrix effects
• Suitable for high throughput forensic toxicology and clinical screening

Future Trends and Potential Applications

Expanding the method to cover additional emerging cathinones and other novel psychoactive substances will support laboratories facing evolving drug trends. Integration with high throughput automation and advanced data processing may further boost productivity. Continued development of specialized HILIC and mixed mode phases will enhance selectivity and robustness for complex biological samples.

Conclusion

The presented UHPLC MS/MS workflow delivers a fast, reliable and sensitive solution for quantifying synthetic cathinones in stabilized human urine. Its simplicity, automation compatibility and strong analytical performance make it well suited for routine forensic toxicology, clinical research and quality control applications.