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)


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.


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.


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.


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.


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.



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:

(Date:11/24/2015)... Switzerland (PRWEB) , ... November 24, 2015 , ... ... plant and the environment are paramount. Insertion points for in-line sensors can represent ... has developed the InTrac 781/784 series of retractable sensor housings , which ...
(Date:11/24/2015)... 24, 2015 /CNW Telbec/ - ProMetic Life Sciences Inc. (TSX: ... Mr. Pierre Laurin , President and Chief Executive Officer ... upcoming Piper Jaffray 27 th Annual Healthcare Conference to ... 1-2, 2015. st , at 8.50am (ET) ... throughout the day. The presentation will be available live via ...
(Date:11/24/2015)... 2015  PDL BioPharma, Inc. (PDL) (NASDAQ: PDLI ) ... president and chief executive officer, will present at the 27 ... New York City . The presentation will ... 2015 at 9:30 a.m. EST. and ... at least 15 minutes prior to the presentation to allow ...
(Date:11/24/2015)... Nov. 24, 2015 According to two new studies, ... This is something that many doctors, scientists, and public health ... remains: with fewer PSA tests being done, will there be ... Dr. David Samadi, "Despite the efforts made in ... the second leading cancer cause of death in men, killing ...
Breaking Biology Technology:
(Date:11/16/2015)... , Nov 16, 2015  Synaptics Inc. ... human interface solutions, today announced expansion of its ... ™ touch controller and display driver integration ... of smartphones. These new TDDI products add to ... (HD resolution), TD4302 (WQHD resolution), and TD4322 (FHD ...
(Date:11/10/2015)... Nov. 10, 2015  In this report, ... basis of product, type, application, disease indication, ... this report are consumables, services, software. The ... safety biomarkers, efficacy biomarkers, and validation biomarkers. ... are diagnostics development, drug discovery and development, ...
(Date:11/2/2015)... Nov. 2, 2015  SRI International has been awarded ... preclinical development services to the National Cancer Institute (NCI) ... provide scientific expertise, modern testing and support facilities, and ... pharmacology and toxicology studies to evaluate potential cancer prevention ... The PREVENT Cancer Drug Development Program is an NCI-supported ...
Breaking Biology News(10 mins):