- Title
- Gradient high performance liquid chromatographic method for the simultaneous analysis of efavirenz, emtricitabine and tenofovir
- Creator
- Koekemoer, Sonya Mariana
- Date Issued
- 2016
- Date
- 2016
- Type
- text
- Type
- Thesis
- Type
- Masters
- Type
- MPharm
- Identifier
- http://hdl.handle.net/10962/54679
- Identifier
- vital:26599
- Description
- In 2014, approximately 6.8 million people in South Africa were HIV-positive, and the majority of those affected are aged 15 or older. A fixed-dose combination (FDC) antiretroviral (ARV) dosage form containing one non-nucleotide reverse transcriptase inhibitor (efavirenz) and two nucleotide reverse transcriptase inhibitors (emtricitabine and tenofovir) was licensed in South Africa in April 2013. New consolidated guidelines for HIV management and prevention of mother to child transmission (PMTCT) were published by the South African Department of Health in December 2014 and the FDC is now the recommended first-line treatment for HIV-positive patients. According to these guidelines all such people aged 15 and older, and weighing more than 40 kg, with a CD4 count of ≤ 500/ μl will be eligible for antiretroviral therapy (ART) using the FDC. In addition every pregnant and breastfeeding woman is eligible for lifelong ART regardless of CD4 count and EFV can be used as first-line treatment for pregnant women regardless of the length of gestation state of the pregnancy at that time. The use of this simplified regime is likely to promote much needed and improved adherence to therapy. An investigation into the development of a stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method for the simultaneous quantitation of EFV, FTC and TNF was undertaken. Isocratic HPLC analysis was found to be unsuitable due to the highly polar FTC molecule eluting in the void. Therefore a gradient HPLC method was developed and validated. The method was validated according to the International Conference on Harmonisation, now known as International Council for Harmonization (ICH). Correlation coefficients > 0.999 were obtained for each assessment of linearity and FTC, TNF and EFV are linear in the range 0.4-40 μg/ml, 0.6-60 μg/ml and 1.2-120 μg/ml. The equation of the best-fit least squares regression lines for FTC, TNF and EFV were y = 0.0191x+0.0007, y = 0.0163x+0.0116 and y = 0.01x+0.016, respectively. The method is accurate as the y-intercept was < 2% of the detector response for all ARV, and the method is precise in terms of intra- and inter-assay precision as all % RSD < 2%. The stability-indicating nature of the method was demonstrated under acidic, alkaline and oxidative stress in addition to UV exposure and elevated temperatures, and the individual chromatograms were overlaid using Empower® 3 Software to establish whether there was interference with the peaks of interest. The forced degradation studies demonstrated the selectivity of the method for the ARV compounds. The method was applied to assay and in vitro dissolution studies of commercially available tablets. The amount of each active ingredient released from Atripla® was determined and compared to the amount of each drug released from Aspen Efavirenz® and Truvada® (a combination of FTC and TNF). The percent FTC released from Atripla® and Truvada® was similar based on the acceptance criteria for immediate-release BCS class 1 compounds. Statistical analysis was undertaken to compare the dissolution profiles of FTC, TNF and EFV. The percent of these compounds released in these studies indicate that bioequivalence testing would be required to declare these products interchangeable. The validated RP-HPLC and in vitro dissolution test method are suitable for routine quality control testing of solid oral dosage forms containing EFV, FTC and TNF, and as the dissolution method can discriminate between different formulations of the same molecule, these tools can also be used for analysis during formulation development studies. The method is not suitable for the analysis of the ARV plasma due to lack of sensitivity and an inability to quantitate the compounds at the required concentration levels. The use of HPLC with mass spectroscopy for quantitation would enhance the sensitivity of the method and may eliminate the quantitation of the molecules in the presence of interference that was observed when using UV detection. Fixed dose combination tablets are convenient for patient therapy and it is likely that in the future more molecules will be formulated into such dosage forms. However formulations such as these can pose significant difficulties when developing and using analytical methods for the quantitation of all compounds in the dosage form at the same time, in particular when the compounds have vastly different physico-chemical properties that impact the quality of a separation and therefore the analysis. Therefore when embarking on the development of FDC product cognisance of the difficulties of developing single methods for the analyses is required and approaches to overcome these difficulties should be considered.
- Format
- Format
- 130 leaves
- Publisher
- Rhodes University
- Publisher
- Faculty of Pharmacy, Pharmacy
- Language
- English
- Rights
- Koekemoer, Sonya Mariana
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Collections
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