Analysis of 3'-azido-3'-deoxythymidine levels in tissues and milk by isocratic high-performance liquid chromatography

A sensitive high-performance liquid chromatographic (HPLC) assay was established to analyze levels of the antiretroviral agent 3'-azido-3'-deoxythymidine (AZT, zidovudine) in serum, milk and tissue extracts. After methanol precipitation, serum samples could be injected directly into the HPLC apparatus, whereas tissue extracts required further clarification. Recovery of AZT was virtually complete. Isocratic elution with a mobile phase consisting of 6% acetonitrile and 0.1 M ammonium acetate, pH adjusted to 4.5 with glacial acetic acid, resulted in good resolution of AZT and its metabolites; retention times for AZT and the internal standard, p-nitrophenol, were 20 and 37 min, respectively. Using this method, we have demonstrated that AZT crosses both the blood-brain and placental barriers and is excreted into milk at high levels.