Navigation Links
A simple and rapid LC-MS/MS method for the simultaneous determination of nine antiretroviral drugs commonly used in Europe (protease Inhibitors and non-nucleoside reverse transcriptase inhibitors)

Purpose

In clinical research, the monitoring of protease inhibitor (PI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) antiretroviral drugs is of significant interest due to the importance of assessing inter-individual and intra-individual metabolism variability. To be of practical use, it is essential that the analytical method used to monitor these drugs is fast and easy to perform, with minimal sample preparation, and without compromising precision and accuracy.

Compared with other methods, liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) has the potential for simultaneous determination of antiretroviral drugs with greater specificity, lower detection limits and a broader dynamic range. Several approaches have been proposed for a fast and stable LCMS/ MS platform for antiretroviral drug monitoring (1, 2). So far, however, these methods have not adequately addressed crucial LC/MS/MS configuration and sample preparation parameters required to ensure robustness, cost-effectiveness, and long-term viability.

Overview

This application note describes a LC/MS/MS procedure for the simultaneous determination of nine PI and NNRTI antiretroviral drugs that are commonly used in Europe. Using the procedure presented here, these drugs were determined quickly and reproducibly, with minimal sample preparation, operator intervention, and solvent consumption.

Key features

Sensitive and selective Multiple Reaction Monitoring (MRM) scan function for linear quantitative analysis with wide dynamic range

Patented LINAC collision cell for multi-component analysis at reduced MRM dwell times maintaining sensitivity and preventing crosstalk

Rugged and reliable triple quadrupole MS system for maximum uptime

Analyst software control of all peripheral devices for reduced solvent consumption, high reproducibility, and ease of use

Experimental conditions

Samples were prepared by adding 200 μL of a diluting solution to 100 μL of either blood serum, calibrators, or controls, and vortex mixing the sample for 30 seconds. After centrifugation at 13,000 g x 10 min., 200 μL of supernatant were transferred to either an autosampler vial or a microtiter plate.

The hardware configuration included an Applied Biosystems/MDS Sciex API 3000 Triple Quadrupole Mass Spectrometer equipped with TurboIonSpray source. The source operates in positive ion mode at a voltage of + 4500 volts and with a turbo gas flow of 8 L/min of air heated at 350C (nominal heating-gun temperature).

MRM measurements were made using declustering potential (DP) and collision energy (CE) values, which were automatically optimized by the software for each of the analytes. (DP values ranged between 40 and 70 V; CE values were between 19 and 45 eV).

Two-dimension chromatography was performed through a split arrangement of the single heads of an Agilent 1100 Binary Pump and a computer-controlled Valco Valve (10-port, 2 positions), plumbed as depicted in Figure 1. First dimension chromatography was accomplished via an Applied Biosystems POROS R2/20, 2.1 x 30 mm perfusion-column. Second dimension chromatography was performed by a Phenomenex Luna 5 μm Phenyl- Hexyl, 2 x 50 mm column (p.n. 00BA 4257-B0) housed in an Agilent Column Oven kept at 60oC. 10 μL samples were injected into the column via an Agilent 1100 Wellplate Autosampler, plumbed as shown in Figure 1.

All hardware set-up details, solution and reagent requirements, and operating parameters were stored on CD for quick transfer of the proposed methodology.

* Diluting solution consisted of acetonitrile containing 50 ng/mL of DESRapamycin as an internal standard.

Results and discussion

Results obtained using the proposed hardware configuration are summarized below.

Figure 2 shows the structure of some of the drugs tested in this study. Nevirapine is a NNRTI drug while the others belong to the PI group. MRM transitions for each of the measured analytes are listed in Figure 3.

Figure 4 shows a typical trace obtained on a serum sample spiked with 1 μg/mL of each drug. In these optimized conditions, the total run time was 6 minutes per sample.

Figure 5 illustrates the linearity obtained on calibrator samples with concentrations spanning from 0.1 to 5 μg/mL for each drug.

Figure 6 documents a reproducibility test made over a Serum sample spiked with 0.5 μg/mL of each drug.

Figure 7 shows the trace obtained on a serum sample from a patient treated with a drug cocktail containing Amprenavir and Ritonavir.

Conclusion

The following conclusions were drawn from these results:

The proposed method is fast. The sample-to-sample cycle time is only 6 minutes.

The proposed method is robust. More than 500 sample injections were made with no evidence of column saturation.

Preprogrammed control and operation of the LC peripheral units using the Analyst acquisition software were effective in reducing solvent consumption by precisely controlling the timing of the high-flow rate regimen during the sample clean up phase of the first dimension LC.

This method is fast and easy to perform, with minimal sample preparation without compromising reproducibility, precision and accuracy.

References

1. Volosov A, Alexander C, Ting L, Soldin SJ. Simple rapid method for quantification of antiretrovirals by liquid chromatography-tandem mass-spectrometry. Clinical Biochemistry 2002; 35:99-103.

2. Ghoshal AK, Soldin SJ. Improved Method for Concurrent Quantification of Antiretrovirals by LC-MSMS. Therapeutic Drug Monitoring 2003; 25:541-543.

3. Koal T, Dieters M, Casetta B, Kaever V. Simultaneous determination of 4 immunosuppressants by means of high speed and robust on-line SPEHPLC- MSMS. Journal of Chromatography 2004; B, 805:215-222.

Acknowledgements

Applied Biosystems/MDS SCIEX acknowledges Hlne Belva-Besnet and Bruno Casetta, and our collaborators Maryse Gravier, Yves Tavet and Didier Olichon from Laboratoire Pasteur Cerba, Cergy Pontoise, France, for providing the method, samples and data for this application note.


'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. New gravity-flow column gives faster and simpler purification of histidine-tagged proteins
2. A simple method to sequence from bacterial colonies using [a-33P] radiolabeled ddNTPs and Thermo Sequenase
3. Quantification of the Purinergic Receptor P2X3 using ICAT and Orthogonal 2D LC-MS/MS with an Ion Trap Mass Spectrometer
4. Determination of Acepromazine and its Major Metabolite in Equine Serum by LC-MS/MS using the Finnigan LCQ Deca XP Plus Ion Trap Mass Spectrometer
5. Comparison of different protein quantitation methods
6. Advancing the quality control methodology to assess isolated total RNA and generated fragmented cRNA
7. Sensitive detection of tumor cells in peripheral blood of carcinoma patients by a reverse transcription PCR method
8. Kinetic determination of vitamin B1
9. Fluorescence assay for determination of protease activity
10. Microdetermination of Phosphorus Using the SpectraMax Plus Microplate Spectrophotometer: Choice of Microplate, Cuvette, or Test Tube Assay Formats (MaxLine Application Note #24)
11. High-throughput gene expression analysis to screen for anti-asthma drugs
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/9/2016)... ... 2016 , ... PharmApprove announced today the hiring ... for Rare Disorders (NORD). Dorman will lead PharmApprove efforts to work with patients, ... drug regulatory review process. , “Adding Diane Dorman is just the latest in ...
(Date:2/9/2016)... Three-Year Initiative Supports Next Generation of Medical Geneticists and  ... Experiences SHPG ) ... of children born with rare diseases, as well as the future ... a new initiative designed to positively affect the lives of children ... disease care. --> To mark the company,s founding 30 ...
(Date:2/8/2016)... 8, 2016 Should antibiotic bone cement products ... to prevent infection after standard total hip or knee ... ECRI Institute have been fielding a lot lately. ... Bottom Line?" --> "Antibiotic Bone ... --> While there isn,t a simple ...
(Date:2/8/2016)... ... February 08, 2016 , ... Thomas J. Todorow has joined ... President for Corporate Services and the Chief Financial Officer at The Children’s Hospital ... Treasury, Managed Care Contracting, Supply Chain, and Investments. , Prior to joining CHOP ...
Breaking Biology Technology:
(Date:1/20/2016)... Jan. 20, 2016   MedNet Solutions , an ... spectrum of clinical research, is pleased to announce the ... achievements are the result of the company,s laser focus ... eClinical , it,s comprehensive, easy-to-use and highly affordable ... --> Key MedNet growth achievements in 2015 include: ...
(Date:1/18/2016)... , Jan. 18, 2016  Extenua Inc., ... that simplifies the use and access of ubiquitous ... go-to-market partnership with American Cyber.  ... extensive experience leading transformational C4ISR and Cyber initiatives ... integrating the latest proven technology solutions," said ...
(Date:1/13/2016)... ALBANY, New York , January 13, 2016 /PRNewswire/ ... Transparency Market Research has published a new market report ... Share, Growth, Trends, and Forecast, 2015 - 2023. According to ... mn in 2014 and is anticipated to reach US$1,625.8 ... from 2015 to 2023. In terms of volume, the ...
Breaking Biology News(10 mins):