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Detection of Alprazolam and Temazepam in a Bovine Plasma Matrix Using the Varian 500-MS Ion Trap Mass Spectrometer

Bethany V. Pond, Jason S. Wood, and August Specht Varian, Inc.

Introduction

Alprazolam (Xanax) and Temazepam (Restoril) were analyzed in a Bovine Serum Albumin (BSA) matrix, both before and after protein precipitation using Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC/ESI/MS/MS). The ability of the Varian 500-MS IT Mass Spectrometer to detect and accurately quantitate these drugs in the BSA matrix is investigated here.

Instrumentation

Varian ProStar 210 Solvent Delivery Module (2)

Varian 500-MS Ion Trap Mass Spectrometer equipped with an ESI source

Varian Prostar 420 Autosampler

Materials and Reagents

All solvents (reagent or HPLC Grade) and the Bovine Serum Albumin (Part # A4503-10G) were purchased from Sigma-Aldrich Corporation (St. Louis, MO). The analytes, Temazepam (Part # T-907) , Alprazolam (Part # A-903), and Alprazolam-d5 (Internal Standard, Part #A-910), were purchased from Cerilliant Corporation (Round Rock, TX).

Sample Preparation

Stock solutions were purchased at a concentration of 1 mg/mL in methanol. Further dilutions of the stock solutions were carried out in a 1:1 mixture of 2mM ammonium acetate in 0.1 % aqueous formic acid: 0.1% formic acid in acetonitrile.

No Matrix Sample Preparations:

Solutions ranging in concentration from 0.1-1000 pg/μL of alprazolam and temazepam were made from the stock solution.

Post-Protein Crash Matrix Preparation:

Equal Volumes of 0.3 mM Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS) and an 80:20 Acetonitrile:Methanol solution containing 0.1% Acetic Acid were centrifuged for 5 minutes at 12000 rpm and refrigerated for 1.5 hours. The supernatant was removed and spiked with the int
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