Simple, rapid and micro high-pressure liquid chromatographic determination of endogenous "true" creatinine in plasma, serum, and urine.

Previously published methods for endogenous creatinine levels in plasma, serum, or urine lack specificity or are subject to interferences from endogenous or exogenous substances. The developed simple, rapid, and specific high-pressure liquid chromatographic method includes the novel deproteinization and extraction of 1 volume of plasma or serum with 2.5 volumes of acetonitrile and also of 1 volume of urine with 40 volumes of a 20% water-80% acetonitrile solution. An aliquot of the supernate is then injected directly into the chromatograph. A cation-exchange column and acidified (0.02% of 85% phosphoric acid) 0.1 M ammonium phosphate solution as the mobile phase, with a flow rate of 2 ml/min, were used. Creatinine, with a retention time of 3.8 min, was monitored via its UV absorption at 215 nm. Both peak heigh and integrated area methods of quantitation yielded the same results. Several methods were employed to show that the "suspected" creatinine peak from plasma samples was due entirely to the "true" creatinine. No interference was found in samples obtained from normal and renal patients. The day-to-day variation in the detector response was small. Each assay requires only about 5 min for completion. Ten microliters of plasma or serum or 1 microliter of urine is sufficient for analysis.