Navigation Links
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 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 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.

Conclusion

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.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
2. A New PCR-based Mycoplasma Detection Method
3. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
4. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
5. Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes
6. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
7. The DIG System Nonradioactive and Highly Sensitive Detection of Nucleic Acids
8. Quantitative Measurement of Cell Proliferation Using the BrdU ELISA: A Comparison Between Colorimetric and Chemiluminescent Detection
9. Quantification of Nucleosomes in Serum by the Cell Death Detection ELISAplus
10. A Further Step in Understanding Apoptosis Direct Detection of PARP Cleavage
11. In Situ Cell Death Detection Kit
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/23/2016)... , June 23, 2016   Boston Biomedical ... novel compounds designed to target cancer stemness pathways, ... been granted Orphan Drug Designation from the U.S. ... of gastric cancer, including gastroesophageal junction (GEJ) cancer. ... designed to inhibit cancer stemness pathways by targeting ...
(Date:6/23/2016)... , June 23, 2016 A person commits a ... crime scene to track the criminal down. An ... Food and Drug Administration (FDA) uses DNA evidence to track ... Sound far-fetched? It,s not. The FDA has increasingly ... support investigations of foodborne illnesses. Put as simply as possible, ...
(Date:6/23/2016)... ... June 23, 2016 , ... ... and Mold) microbial test has received AOAC Research Institute approval 061601. , “This ... introduced last year,” stated Bob Salter, Vice President of Regulatory and Industrial Affairs. ...
(Date:6/23/2016)... YORK , June, 23, 2016  The Biodesign ... to envision new ways to harness living systems and ... Modern Art (MoMA) in New York City ... than 130 participating students, showcased projects at MoMA,s Celeste ... Paola Antonelli , MoMA,s senior curator of architecture ...
Breaking Biology Technology:
(Date:3/31/2016)... 2016   LegacyXChange, ... "Company") LegacyXChange is excited to release its ... to be launched online site for trading 100% guaranteed ... will also provide potential shareholders a sense of the ... an industry that is notorious for fraud. The video ...
(Date:3/29/2016)... March 29, 2016 LegacyXChange, Inc. ... "LEGX" and SelectaDNA/CSI Protect are pleased to announce our ... in a variety of writing instruments, ensuring athletes signatures ... created collectibles from athletes on LegacyXChange will be assured ... the DNA. Bill Bollander , CEO ...
(Date:3/22/2016)... , March 22, 2016 ... report "Electronic Sensors Market for Consumer Industry by Type ... Others), Application (Communication & IT, Entertainment, Home ... Global Forecast to 2022", published by MarketsandMarkets, ... expected to reach USD 26.76 Billion by ...
Breaking Biology News(10 mins):