Bethany V. Pond, Jason S. Wood, and August Specht Varian, Inc.
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.
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).
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 internal standard (10 pg/μL of Alprazolam-d5) and the analytes in the same concentrations as used in the no matrix preparation
Pre-Protein Crash Matrix Preparations:
0.3 mM BSA in PBS was spiked with the internal standard (10 pg/μL of Alprazolam-d5) and the analytes in the same concentrations as the no matrix preparations. An equal volume of the 80:20 acetonitrile:methanol solution containing 0.1% acetic acid was added to the spiked BSA solutions. The mixtures were centrifuged for 10 minutes at 12000 rpm and then refrigerated for 1.5 hours. The supernatant was removed and subsequently analyzed.
The samples were run in triplicate for calibration curve purposes on a 500-MS Ion Trap Mass Spectrometer.
Each analyte was run individually with the internal standard in an MRM experiment for a total run time of 5 minutes. For the no matrix run, the analytes and internal standard were run individually in a MS/MS experiment with the product scan centered around the specific product ions.
Results and Discussion
Calibration curves were run without the matrix, with theafter crash spiked matrix, and the before crash spiked matrix with good linearity observed for each analyte. The extracted ion chromatogram of 500 fg (on column) of Temazepam (m/z = 255.2) in the before crash matrix is shown in the top panel of Figure 1, while the bottom panel of Figure 1 is the extracted ion chromatogram of the internal standard, Alprazolam-d5 (50 pg on column). The Signal-to-Noise (S/N P-P) ratio of temazepam in the spiked matrix was determined to be 16:1 at 500 fg on column.
The LOD (limit of detection) values for the three different types of matrix environments are summarized in Table 1. A calibration curve of Alprazolam in a before crash spiked matrix of these compounds is shown in Figure 2. An R2 value > 0.99 was observed for both compounds in all of the matrices studied, over the calibration range of 0.2 pg/μL 1000 pg/μL (1 pg 5000 pg on column). An LOQ = 0.2 pg/μL (1 pg on column) was determined for all three matrix conditions for both Alprazolam and Temazepam.
Even with the addition of the matrix, the range of quantitation did not change. Figure 3 shows the extracted ion chromatogram (m/z = 255.2) of Temazepam in all three of the matrix conditions.
It is evident from Figure 3 that the 500-MS Ion Trap is capable of handling a biological matrix and thus is still able to reach a low level of detection under the conditions described. These results show that the 500-MS Ion Trap is a sensitive and quantitative tool for the detection of target analytes in biological matrices.
The measurements taken with Alprazolam and Temazepam spiked into bovine plasma matrix illustrate that it is possible to reach low levels of quantitation (low- to sub-pg level) using the 500-MS Ion Trap Mass Spectrometer. Quantitative analysis of Temazepam and Alprazolam in a biological matrix has been demonstrated in the 500-MS instrument illustrating its excellent performance in both quantitative and qualitative analysis.