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 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.

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:
(Date:6/26/2015)... ... June 26, 2015 , ... ReliantHeart, Inc. , an innovative ... the first implant of the HeartAssist5® LVAD (HA5) to the 68th consecutive implant, there ... Thrombus that originates in an LVAD is referred to as de novo thrombus. De ...
(Date:6/25/2015)... 2015 Current asthma medications, which work ... by dilating the airways, can stop working over ... and Women,s Hospital and Harvard Medical School supports ... certain sensory nerve endings in the lungs that ... in the journal Neuron on June ...
(Date:6/25/2015)... 2015  BioMed Realty Trust, Inc. (NYSE: BMR ... ), a world leader in genomics focused on improving ... has signed a 20-year lease for a 155,000 square ... Cambridge, England . The project will provide ... skilled workforce in the dynamic research cluster anchored by ...
(Date:6/25/2015)... Israel , June 25, 2015 ... investors to co-invest with Startup Nation,s leading angels and ... appointment of Darya Fuks as Portfolio Manager.  ... Investment Bank, will lead iAngels, deal flow and due ... development efforts for all iAngels portfolio companies. An investment ...
Breaking Biology Technology:HeartAssist5® Remote Monitoring has alerted no patterns of de novo thrombus in 68 consecutive implants 2HeartAssist5® Remote Monitoring has alerted no patterns of de novo thrombus in 68 consecutive implants 3Targeting Nerve Endings To Curb Allergic Asthma 2Targeting Nerve Endings To Curb Allergic Asthma 3Targeting Nerve Endings To Curb Allergic Asthma 4Targeting Nerve Endings To Curb Allergic Asthma 5BioMed Realty And Illumina Announce New Build-To-Suit Laboratory Building In Cambridge's Growing Life Science Cluster 2BioMed Realty And Illumina Announce New Build-To-Suit Laboratory Building In Cambridge's Growing Life Science Cluster 3Former J.P. Morgan Vice President Darya Fuks Joins iAngels 2Former J.P. Morgan Vice President Darya Fuks Joins iAngels 3
... Dendreon Corporation (Nasdaq: DNDN ) management will host a conference call ... second quarter 2009 financial results. Access to the discussion may be obtained as ... 11:30 AM ET /8:30 AM PT, Date: ... (domestic) or +1-719-325-4873 (international), Webcast: www.dendreon.com (homepage and investor ...
... , , , PITTSBURGH, July 30 ... the three and six months ended June 30, 2009. , , ... Adjusted diluted EPS, which excludes the impact of certain purchase accounting items ... $0.65 for the three and six months ended June 30, 2009, compared to $0.20 ...
... a weakened strain of the malaria parasite that will be ... developed in collaboration with researchers from the US, Japan and ... Malaria kills more than one million people each year and ... of at least 35 million years of healthy, productive human ...
Cached Biology Technology:Dendreon to Hold Conference Call on Tuesday, August 11, 2009 at 11:30 AM ET 2Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 2Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 3Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 4Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 5Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 6Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 7Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 8Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 9Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 10Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 11Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 12Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 13Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 14Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 15Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 16Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 17Mylan Reports Adjusted Diluted EPS of $0.32 for the Quarter Ended June 30, 2009 18First genetically engineered malaria vaccine to enter human trials 2
(Date:6/17/2015)... SAN JOSE, Calif. , June 17, 2015 ... leader in human interface solutions, today announced that ... has adopted the Synaptics ® ClearPad ® ... family of display driver integrated circuits (DDICs) for ... Note Pro. By leveraging ClearPad for full in-cell ...
(Date:6/16/2015)... 16, 2015 Fingerprint Cards (FPC) raises ... it will amount to at least 340 MSEK to that ... rapid increase in market growth and orders received the revenue ... that revenue for 2015 will exceed 1,500 MSEK to an ... 2,200 MSEK. Due to receipt of orders of ...
(Date:6/11/2015)... June 11, 2015 Daon, a global ... that its IdentityX Mobile Authentication Platform v4.0 has ... Alliance is an industry consortium launched in 2013 ... simpler, stronger authentication.  In order to receive certification, ... rigorous series of tests that measure compliance and ...
Breaking Biology News(10 mins):Synaptics Touchscreen and Display Driver IC Solutions Power Xiaomi's Latest Flagship Smartphones 2Synaptics Touchscreen and Display Driver IC Solutions Power Xiaomi's Latest Flagship Smartphones 3Fingerprint Cards Raises the Revenue Guidance for Q2 2015 and for the Full Year 2015 2Fingerprint Cards Raises the Revenue Guidance for Q2 2015 and for the Full Year 2015 3Daon's IdentityX Mobile Authentication Platform One of First Products to be Certified by FIDO Alliance 2
... protective delivery vehicle that shuttles friendly bacteria safely through ... boost for the probiotics industry. The new technology ... drugs and even increase calcium absorption, according to research ... at the University of York this week. The ...
... coli strain that hit Germany in May 2011 ... of collaboration across the scientific community should give insight ... the Society for General Microbiology,s Autumn Conference 2011. ... BGI, China. Their sequence was provided in draft form ...
... this math more evident than in vertebrates, which are programmed ... do not. Snakes, of course, have no digits, and birds ... handle on how these developmental changes are orchestrated in the ... Which digits are they: a thumb with index and middle ...
Cached Biology News:New polymer research could boost probiotics industry 2Crowd-sourcing the E. coli O104:H4 outbreak 2
... 8 micro-bore tubes to 1 outflow ... tips, Rapid (several millisecond) solution change ... prevention , Solid construction 360, 250 ... For mounting on any micromanipulator Available ...
... (BTI-TN-5B1-4) derived from Trichoplusia ni egg ... be capable of expressing significantly higher ... to other insect cells. High Five ... time as adherent cultures- Quick adaptation into ...
... Racks are ideal for storage and transport ... polypropylene racks withstand extreme temperatures, making them ... may also be autoclaved for use in ... side accommodates 0.5ml microcentrifuge tubes, the other ...
... Signet offers the most comprehensive, ... requirements. We have incorporated unprecedented quality ... capabilities. This includes proven detection chemistries ... to the TechMate protocol. It also ...
Biology Products: