Analysis of Psychotropic Drugs in Whole Blood Utilizing Simultaneous Scan/MRM Measurements (1)

Significance of the Topic

Whole blood contains high levels of endogenous lipids, such as cholesterol and fatty acids, which can coelute with target analytes and hinder accurate detection of psychotropic benzodiazepines. Reliable separation and identification of compounds like triazolam and etizolam are critical in forensic toxicology and clinical analysis to ensure precise screening and quantification in complex biological matrices.

Objectives and Study Overview

This study demonstrates a simultaneous scan and multiple reaction monitoring (MRM) approach using GC-MS/MS to differentiate triazolam and etizolam from overlapping cholesterol signals in whole blood. By combining full scan data for broad screening with targeted MRM transitions, the method aims to achieve both comprehensive detection of medicinal toxicants and selective quantification of the two benzodiazepines.

Used Instrumentation

• Gas Chromatograph–Triple Quadrupole Mass Spectrometer: GCMS-TQ8030 (Shimadzu)
• Capillary Column: Rxi-5Sil MS, 30 m × 0.25 mm i.d., 0.25 µm film thickness
• Autosampler: AOC-20i+s with solvent flush mode

Methodology and Instrumentation

Sample pretreatment employed liquid–liquid extraction on EXtrelut NT3 cartridges. Whole blood aliquots were split into acidic (pH 5) and basic (pH 9) fractions, loaded, and eluted with a 3:1 chloroform/isopropanol mixture. The combined eluates were dried over silica gel under nitrogen and reconstituted in 200 µL of the same solvent mixture. An internal standard containing deuterated PAH isomers (1 µg/mL) was co-injected.
Chromatographic separation used a splitless injection at 260 °C, with an oven program from 60 °C (2 min) to 320 °C at 10 °C/min. Helium carrier gas was controlled for linear velocity of 45.6 cm/s. Mass spectrometric detection combined full scan (m/z 45–700) at 5 000 u/s with MRM events targeting transitions for triazolam (m/z 313→277, 313→278, 313→242) and etizolam (m/z 342→272, 342→245, 342→266).

Main Results and Discussion

Full scan chromatograms revealed that cholesterol elutes at the same retention time as triazolam and etizolam, making identification ambiguous. MRM chromatograms, however, selectively monitored specific precursor-product ion pairs, effectively separating the benzodiazepines from the cholesterol background. At a spiked concentration of 500 ng/mL in whole blood, both compounds were clearly detected without mutual interference.

Benefits and Practical Applications

• Enhanced selectivity: Two-stage mass separation eliminates lipid interference.
• Dual-purpose data: Combined scan for broad toxicant screening and MRM for targeted confirmation.
• Robust quantification: Reliable detection of triazolam and etizolam in forensic and clinical samples.

Future Trends and Potential Uses

Advancements may include expanding MRM libraries for additional drug classes, integrating high-resolution MS to further reduce background noise, and automating sample preparation. Adaptation to other challenging matrices (e.g., oral fluid, tissue) could broaden forensic and clinical applications.

Conclusion

The simultaneous scan/MRM GC-MS/MS approach successfully overcomes cholesterol interference, enabling selective and sensitive detection of triazolam and etizolam in whole blood. This dual-mode method streamlines forensic toxicological workflows by providing both comprehensive screening and precise quantification.

References

No external literature list provided within the original document.