SPECIFIC HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY WITH ULTRAVIOLET DETECTION FOR THE DETERMINATION OF 1-(2-CHLOROETHYL)-3-SARCOSINAMIDE-1-NITROSOUREA IN PLASMA

A facile, sensitive and highly specific HPLC method for assaying 1-(2- chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) in plasma has been developed. The drug was efficiently isolated from plasma by extractio n with tert,-butyl methyl ether. A structurally related compound with similar physicochemical properties served as the internal standard (I. S.). Following evaporation-of the organic solvent, the extract was rec onstituted with 0.05 M ammonium acetate buffer, pH 5.0, and loaded ont o a 4 mu m Nova-Pak C-18 column (15 cm X 3.9 mm), which was preceded b y a 7 mu m Brownlee RP-18 precolumn (1.5 cm X 3.2 mm). Chromatography was performed at ambient temperature using a mobile phase of methanol- 0.1 M ammonium formate buffer, pH 3.7 (25:75, v/v). UV absorbance of t he effluent was monitored at 240 nm. A flow-rate of 1.0 ml/min was use d for analyzing mouse and dog plasma extracts. Under these conditions, the drug eluted at 4.0 min and was followed by the I.S. at 6.1 min. A n automatic switching valve was employed to allow the precolumn to be flushed 1.5 min into the run, without interrupting the flow of the mob ile phase to the analytical column, thereby preventing the apparent bu ild-up of extractable, strongly retained, UV-absorbing components pres ent in mouse and dog plasma. Operating in this manner, more than 100 s amples could be analyzed during a day using a refrigerated autosampler for overnight injection. The method was readily adapted to the determ ination of SarCNU in human plasma by simply decreasing the eluent flow -rate to 0.6 ml/min, whereby SarCNU and the I.S. eluted at approximate ly 5.8 and 9.1 min, respectively. Furthermore, the switching valve was not necessary for the analysis of human plasma samples. With a 50-mu l sample volume, the lowest concentration of SarCNU included in the pl asma standard curves, 0.10 mu g/ml, was quantified with a 7.8% R.S.D. (n=27) over a 2 month period. Plasma standards, with concentrations Of 0.26 to 5.1 mu g/ml, exhibited R.S.D. values ranging from 1.3 to 4.7% . Thermospray-ionization MS detection was used to definitively establi sh the specificity of the method. The sensitivity of the assay was sho wn by application to be more than adequate for characterizing the plas ma pharmacokinetics of SarCNU in mice.