DETERMINATION OF 2'-DEOXY-3'-THIACYTIDINE (3TC) IN HUMAN URINE BY LIQUID-CHROMATOGRAPHY - DIRECT-INJECTION WITH COLUMN-SWITCHING

3TC (GR109714X) is a cytidine dideoxynucleoside analogue which has bee n shown to have in vitro activity against a variety of strains of HIV- 1 and is currently being investigated in clinical trials as a treatmen t for HIV infection. An HPLC method for the determination of 3TC in hu man urine has been developed and validated. The method allows direct i njection Of urine (10 mul) using HPLC column switching followed by UV detection. On-line extraction is performed using a Spherisorb-SCX (5 m um, 20 x 4.0 mm) eluted with deionized water at 1 ml min-1. 3TC is ret ained while the bulk of urine constituents are eluted to waste. The SC X column is then backflushed to a BDS-Hypersil-C18 (5 mum, 250 x 4.6 m m) and eluted with 100 mM acetate pH 4.5-methanol (95:5, v/v) for fina l separation. 3TC is detected by UV absorbance at lambda = 285. The qu antitation range of the assay was 0.5-500 mug ml-1. The method has dem onstrated sufficient ruggedness to be used in support of 3TC clinical trials. Application to other cytidine analogues including DDC has been demonstrated.