Rapid and sensitive determination of zidovudine and zidovudine glucuronide in human plasma by ion-pair high-performance liquid chromatography

A rapid, simple and sensitive method is described for the simultaneous determination in human plasma of the well known antiviral agent zidovudine (AZT) and its main metabolite, zidovudine-5'-O-glucuronide (G-AZT), using a solid-phase extraction method for sample preparation and a rapid ion-pair HPLC separation method with diode-array ultraviolet detection. The method overcomes the problems experienced in other procedures because of the large difference in polarity of the two compounds and the pH-sensitive retention of G-AZT by using n-octylamine to increase the retention of the glucuronide and improve the overall chromatographic behaviour. AZT and G-AZT are eluted at 3.6 and 5 min, respectively, with 7-ethyltheophylline used as internal standard eluting at 4.2 min. Caffeine, a good marker for the elution of related compounds present in plasma, appears well before, at 2.6 min, Quantification limits were established at 0.025 and 0.050 mu g/ml for AZT and G-AZT, respectively. The improvement in method reproducibility due to the late elution of G-AZT could be observed even at the quantification limit at which an inter-assay relative standard deviation of only 6.4% was found after 3 months of routine use of the method.