Analysis of Antiretrovirals Used in Combination HIV Therapy

By: Jacinth A. M. McKenzie, Carmen T. Santasania, David S. Bell, Reporter EU Volume 18

Jacinth A. M. McKenzie, Carmen T. Santasania, and David S. Bell


The simultaneous determination of antiretrovirals from three therapeutic classes is demonstrated using the Supelco AscentisTM RP-Amide column.

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Antiretroviral agents are used in the treatment of human immunodeficiency virus (HIV) and acquired immuno deficiency syndrome (AIDS). To date, five therapeutic classes have been developed: nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs), protease inhibitors (PIs) and fusion inhibitors (1). Effective treatment is usually accomplished using at least three drugs from more than one therapeutic class. For example, recommendations for initial treatment include the combination of two NRTIs or NtRTIs with either an NNRTI or a PI (2).

In the study of drug efficacy, pharmacokinetics, prevention and management of adverse reactions, therapeutic drug monitoring of antiretrovirals is performed. This is generally achieved by HPLC coupled with UV-diode array or mass spectrometry detection following solid phase extraction (3,4). Drawbacks of previous HPLC methods include the use of ion-pair reagents (5) and limited specificity for a single drug class (6). This report describes a simple HPLC method for the simultaneous determination of antiretrovirals from three drug classes using a new proprietary surface optimized embedded polar group (EPG) stationary phase, Ascentis RP-Amide. The Supelco Ascentis RP-Amide phase demonstrates advantages in polar analyte retention as well as improved selectivity when compared to traditional C18 stationary phases.

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Experimental Approach

The USP assay method for nevirapine was modified by applying a gradient to include assay of other antiretrovirals. Alternately, if a MS detector is desired, minor adjustments to the mobile phase can be made. All chemicals utilized were obtained through Sigma-Aldrich. Analytes were obtained from USP or other sources.

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Representative structures from three drug classes and an optimized chromatogram obtained on the Ascentis RP-Amide EPG phase for the simultaneous analysis of seven antiretrovirals are provided in Figures 1 and 2A, respectively. When zalcitabine is added to the analyte mixture (Figure 2B), a small interfering peak appears as a shoulder on peak 8. The shoulder appears only after zalcitabine is analyzed in combination with one or more of the protease inhibitors and thus appears to be a reaction product of the compounds.

Figure 1. Representative structures of an NRTI (Stavudine), an NNRTI (Nevirapine) and a PI (Saquinavir)p>

Figure 2A. Separation of Seven Antiretrovirals Using the Supelco Ascentis RP-Amide

For compatibility with mass spectrometric detection, the phosphate buffer initially employed was replaced with 0.1% ammonium acetate in water. Using this mobile phase in combination with acetonitrile, the elution of all 7 drugs was completed in 15 minutes (Figure 3).

Figure 3. Reconstructed Ion Chromatogram of Seven Antiretrovirals Using Supelco Ascentis RP-Amide and MS Detection

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Simultaneous analysis of an NNRTI, nevirapine, NRTIs (lamivudine, zidovudine, stavudine), and PIs (ritonavir, lopinavir, saquinavir) was achieved in a single run without the use of ionpair reagents utilizing a new EPG column, Ascentis RP-Amide. In comparison, alkyl type phases (C18 and C8) often require the use of ion pair reagents to achieve simultaneous monitoring of different therapeutic classes. Simultaneous monitoring of drugs used in combination therapy using the proprietary, surface optimized Ascentis RP-Amide stationary phase is advantageous in detecting interactions that can lead to clinical failures or shortfalls encountered in HIV treatment.

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  1. C.P.W.G.M. Verweij-van Wissen, R.E. Aarnoutse and D.M. Burger, J. Chromatogr. B. 816 121-129 (2005).
  2. Panel on Clinical Practices for Treatment of HIV Infection, Guidelines for the use of antiretroviral agents,
  3. M. Jenal, S. Rao, M. Gatz, D. Whigan, J. Chromatogr. B. 795 273-289 (2003).
  4. B. Fan, J.T. Stewart, J. Liq. Chromatog. Relat. Technolog. 24 3017 (2001).
  5. G. Aymard, m. Legrand, T. Trichereau, B. Diquet, J. Chromatogr. B. 744 227-240 (2000).
  6. V.A. Simon, M.D. Thiam, L.C. Lipford, J. Chromatogr. A. 913 447-453 (2001).

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