UPLC-MS/MS Analytical Method for the Simultaneous Quantification of Diazepam, Nordazepam, and Oxazepam in Patients With Alcohol Dependence.

BACKGROUND

To establish a method for the simultaneous quantification of diazepam (DIA) and its active metabolites, nordazepam (NorD) and oxazepam (OXAZ), and provide a reference range for therapeutic concentrations in patients with alcohol dependence.

METHODS

Simple and direct protein precipitation was used to extract the biological samples. Subsequent separation was performed on an Agilent XDB-C18 column (50 mm × 4.6 mm, 1.8 μm) with a column temperature maintained at 35 °C and a flow rate of 0.5 mL/min via ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The mobile phase consisted of methanol-water containing 5 mM ammonium formate (75:25, v/v). Detection was conducted using electrospray ionization in multiple reaction monitoring modes: m/z 284.6→193.2 for DIA, m/z 270.5→140.1 for NorD, m/z 286.9→241.1 for OXAZ, m/z 289.6→198.2 for DIA-D5, m/z 275.5→140.0 for NorD-D5, and m/z 291.9→246.1 for OXAZ-D5. The linear response range for DIA, NorD, and OXAZ was 1-1500 ng/mL.

RESULTS

The key parameters of the bioanalytical method were validated: the average extraction recovery was 95%-101% (CV <6%); calibration curves exhibited good linearity over the concentration range (R2 ≥0.99 for all analytes); accuracy was within 85%-115%; and intra-day and inter-day precision were satisfactory (CVs <15%). The concentrations of analytes in 26 routine therapeutic drug monitoring (TDM) samples from patients with alcohol dependence were determined.

CONCLUSIONS

We developed and validated a rapid, simple, and economic UPLC-MS/MS method for the quantification of DIA, NorD, and OXAZ in human serum. The method is well-suited for the determination of serum levels of DIA and its active metabolites in patients with alcohol dependence, and could be further applied to TDM and subsequent studies.