Investigations of CE and HPLC methodologies for the analysis of anti-retroviral drugs
Auberger, Ann Marie
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Two of the most widely used analytical separation techniques are high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). HPLC utilizes pressure to drive a mobile phase through a stationary bed, which interacts with solute particles in order to achieve separation. In CE, an applied voltage is employed to separate species in accordance to their size-to-charge ratios. Both techniques can be coupled with a variety of detection schemes, such as ultra-violet (UV), fluorescence, and mass spectrometry (MS), making them useful for the separation of a wide variety of species, from small ions to macromolecules. Due to this versatility, both separation techniques have played significant roles in biological separations. Nucleoside Reverse Transcriptase Inhibitors (NRTIs) are a class of antiretroviral drugs used to treat patients infected with the human immunodeficiency virus (HIV). Once incorporated into the body, NRTIs are metabolized into their active triphosphate (TP) forms. Structural similarities to naturally occurring deoxyribonucleotides enable the NRTI-TPs to act as competitive inhibitors of the viral enzyme, reverse transcriptase, and as chain terminators for the growing viral DNA strand. To date, a standard, direct method for analysis of multiple NRTI-TPs has not been reported. Such a method can significantly simplify chemical analysis of the NRTI drugs often used in HIV treatment. Separation and quantification of multiple triphosphorylated drugs could accelerate the assessment of patient response to the drugs, resulting in establishment of appropriate dosage regimes. The research presented in this dissertation focuses on studies that can facilitate the analysis of five NRTI-TPs, zidovudine triphosphate (ZDV-TP), 2’3’-dideoxyadenosine triphosphate (ddA-TP), zalcitabine thiotriphosphate (ddC-TP), lamivudine triphosphate (3TC-TP) and stavudine triphosphate (D4T-TP), and adenosine triphosphate (ATP). For their direct analysis, three different approaches were examined: anion-exchange HPLC, capillary electrophoresis with field-amplified sample stacking (CE-FASS), and lanthanide enhancement. The anion-exchange HPLC research focused on making an existing method MS compatible, since the original method, which utilized UV detection, gave detection limits that were not appropriate for the analysis of the NRTI-TPs in clinical applications. The effect of removing mobile phase components (e.g., sodium chloride) on the analytical separation was studied; more MS compatible additives were introduced (e.g., ammonium acetate). The substitution had little impact on the quality of the separation; however, the quantity of salt in the mobile phase remained high. Implementation with MS detection is still limited by the lack of technology for online desalting. Due to their long fluorescence lifetimes, lanthanide sensitizers are commonly used as fluorescent tags for the analysis of biomolecules in time-resolved studies. We explored the application of a terbium (Tb) based chelating sensitizer for the detection of the NRTI-TPs. This chelating sensitizer utilized 7-amino-4-methyl-2(1 H )-quinolinone (carbostyril 124, cs124) as the sensitizer and diethylenetriaminepentaacetic acid (DTPA) as a chelating ligand. The sensitizer absorbs UV light and transfers it to the lanthanide, whereas the chelator holds the sensitizer in close proximity to the lanthanide so that energy transfer from the sensitizer to the lanthanide can occur efficiently. When cs124-DTPA was added to Tb 3+ solutions, a significant enhancement of lanthanide’s fluorescence signal was observed. Preliminary studies in which ATP was added to the Tb-cs124-DTPA moiety showed an increase in the fluorescence signal, indicating that this lanthanide sensitizer could be useful for fluorescent labeling of the NRTI-TPs. CE-FASS utilized online sample preconcentration, which enabled the NRTI-TPs to be focused into narrow bands. Thus, concentration sensitivity, which is generally a limitation in traditional CE methods, was significantly improved. We studied the effect of several parameters (e.g., pH and injection lengths) on preconcentration enhancement to achieve the desired enhancement effect. After optimization, we demonstrated that detectability of the NRTI-TPs can be increased 10,000-fold with regards to traditional CE methods.