ANTIRETROVIRAL DRUG DETECTION IN URINE USING HIGH PERFORMANCELIQUID CHROMATOGRAPHY

Abstract

Human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) was first recognised in 1981. The explosive worldwide pandemic has become the leading cause of death in South Africa. An estimated 10.2% of the South African population is currently infected with HIV/AIDS and more than 2500 new infections occur on a daily basis, revealing South Africa as the country with the highest infection rate worldwide. A devastating fifteen percent of new infections worldwide occur in South Africa alone despite extensive roll out programs implemented by the department of health. Extensive research and drug development was conducted since its discovery. Many antiretroviral drugs (ARVs) have been developed and various treatment protocols have been implemented to combat the epidemic, but no cure has been discovered yet. When a patient is first diagnosed, an aggressive treatment regimen of 3 or more antiretrovirals is prescribed to reduce the viral load and increase the life expectancy of a positive patient. The ARVs are well absorbed with good bioavailability after oral administration. The drugs are metabolised by the liver and mainly excreted in the urine. Unfortunately all ARVs pose the threat of toxicity. The roll out program implemented by the department of health now reaches more infected patients than ever before. The increased availability of these drugs increases the likelihood of misuse or accidental toxicity. Treatment of such a patient can be problematic and very costly if a correct diagnosis cannot be made. Increased requests for therapeutic drug monitoring of the ARVs to establish patient compliance and to monitor pharmacokinetic interactions with other drugs was also a key motivator for the development of a method for ARV determination in our area. The current high performance liquid chromatography (HPLC) method developed by the Department of Pharmacology/Toxicology can identify the presence of lamivudine, abacavir, zidovudine, nevirapine and efavirenz in urine. Continued research in the field of drug development launches new drugs on the market on a regular basis. Treatment regimens change as the new drugs become available, and older line ARVs become outdated. This dynamic and TDM requests warrant the necessity to extend the application of the method to accommodate new ARVs. It is also important as part of method validation to analyse samples from patients using antiretroviral drugs to see if the method that was developed complies with the analytical criteria required. Methods: Lamivudine, abacavir, zidovudine, nevirapine, efavirenz, stavudine, lopinavir and tenofovir were extracted from urine using a liquid-liquid extraction and analysed with a Hewlett-Packard (HP) 1090 Liquid Chromatograph. The eluent was monitored and full spectra of each peak were recorded at 210 nanometre (nm) with an ultraviolet-visible spectrophotometry (UV/VIS) Diode Array detector. Results: Spectra recorded for each peak showed excellent reproducibility and agreement to database recorded spectra. The calibration curves of the ARVs were linear and reproducible. Recovery exceeded 74%. Validation data for control samples show inter – assay precision with a coefficient of variation (CV) of < 16.9%. Short-term stability testing indicated that concentrations remained stable at room temperature and refrigeration, but freezing of samples is not advised. Conclusion: A qualitative HPLC method for the identification of ARVs in urine was developed and validated. This analysis can be used as a component of an emergency toxicological screening in suspected overdoses. It requires minimal sample pre–treatment, small sample volume, minimal expertise, and provides results within 30 minutes.