(M1030-01-08) Quantitation of Diazepam and Its Metabolites Using High-Throughput Rapidfire Mass Spectrometry

Purpose: The analysis of diazepam and its metabolites (benzodiazepines) in urine and plasma is a need for monitoring and identification of drug abuse in clinics and forensic technology. Moreover, quantitation of benzodiazepines in serum or plasma may help to determine optimum chronic dosing for the patients. In this work, we have developed and optimized a method for analyzing diazepam and its metabolites using high-throughput triple quadrupole mass spectrometry system. Although several liquid and gas chromatographic method has been reported for analyzing benzodiazepines, using rapidfire/MS/MS method accelerates quantitation speed to almost 15 times faster than LC/MS/MS. In this work, the glucuronide metabolites were analyzed as well to see the possibility of diazepam/ glucuronide ratio calculation as a biomarker of the time elapsed after diazepam administration¹.

Methods: The RapidFire RF400 system (Agilent Technologies [Biocius], Woburn, MA) was interfaced with an Agilent G6495 TQ mass spectrometer. Samples were aspirated directly from 96-well plates into a 10-µL sample loop and passed through an SPE cartridge with solvent A at a flow rate of 1.5 mL/min for 3 s. After the desalting step, analyte retained on the cartridge was eluted with solvent B to the mass spectrometer at a flow rate of 1.25 mL/min for 5 s, and another 1.5 s from the re-equilibration step., the entire sampling cycle was 12 s per well, which reduced the sample analysis time by at least 15-fold compared with the standard LC-MS analysis. C4, C8, C18, and phenyl SPE cartridges were applied for this study with the different solvents (pH from 2.7 to 4.7 by formic acid and ammonium formate combination). The results were evaluated by the LC-MS analysis using the G6495 TQ mass spectrometer coupled to an Agilent 1290 Infinity II LC System. Analytical samples were separated using an Agilent Zorbax SB-Phenyl 4.6x50mm 3.5uM column at a flow rate of 0.7 mL/min with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the solvent A and B respectively. Elution conditions were the following: 0 to 0.3 min, 10% solvent B; 0.3 to 1.7 min, a linear gradient change from 10% B to 98% B; 2 to 2.3min, 98% B; and 2.3 to 3.5 min, 10% B. The peak integration was performed using the Agilent MassHunter Quantitative analysis software.

Results: The optimum method was achieved with a C4 SPE cartridge and 0.1% formic acid in water and 0.1% formic acid in acetonitrile as solvent A and B&C respectively. Pooled standard curve prepared by spiking diazepam and its metabolites into pre-crashed drug-free human urine and plasm in the range of 0.005 nM-200 nM (12 samples). The samples were analyzed by monitoring MRM transitions corresponding for each compound reported in Table1. The limits of detection were determined for all the metabolites (reported in Table 1). The analytes had excellent linearity within the measured ranges with R² values greater than 0.99. Figure 1 shows representative standard curves for diazepam, oxazepam and oxazepam glucuronide in plasma. The accuracy of the measurements was evaluated by six quality control samples (triplicate). The accuracy of calculated concentrations is ±20% for QC samples. The results of RF/MS/MS were also consistent with the results of the LC/MS/MS and were correlated by R²>0.99.

Conclusion: This SPE/MS/MS methodology using High-Throughput RapidFire Mass Spectrometry provides comparable results to LC/MS/MS. Diazepam and its metabolites were accurately and simultaneously analyzed at 12 seconds per sample ( >250 samples per hour).

References: 1. Wang R, Wang X, Liang C, Ni C, Xiong L, Rao Y, Zhang Y. Direct determination of diazepam and its glucuronide metabolites in human whole blood by μElution solid-phase extraction and liquid chromatography-tandem mass spectrometry. Forensic Sci Int. 2013;233(1-3):304-11.


Table 1: MRM transitions and the limits of detection (LOD) for diazepam and its metabolites


Figure 1: Representative standard curves for diazepam and its metabolites in plasma. The average of three measurements for each concentration and standard deviation are plotted.