A New Bioanalytical Method for the Determination of Alprazolam in Human Plasma

A new liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of alprazolam in human plasma, using lorazepam as internal standard. Detection was performed using positive ion electrospray tandem mass spectrometry on an Agilent 1200 Triple Ouad 6410A system. The mass transition ion-pair was followed as m/z 309→ 281 for alprazolam and m/z 321→ 275 for lorazepam, in multiple-reaction monitoring mode. The plasma samples were extracted with tert-buthyl methyl ether and separated by liquid chromatography. Alprazolam and the internal standard were separated on a Zorbax SB-CN column using a mobile phase consisting in 70:30 (v/v) mixture of acetonitrile and 0.5% formic acid aqueous solution, with a flow rate of 0.8 mL/min. The proposed method has been validated for the concentration range in between 0.1 and 50 ng/mL and a correlation coefficient of 0.9991. The precision and accuracy were within 12% for intra-day and inter-days assays. The overall recoveries for alprazolam and lorazepam were 76% and 88%, respectively. The overall time of one analysis was 4 minutes. The assay has proven to be sensitive, specific and reproducible, suitable for the quantitative determination of alprazolam in human plasma.