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

ernal 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
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