Improved method for the measurement of cimetidine in human serum by reverse-phase high-pressure liquid chromatography

An improved method for the measurement of cimetidine in human serum by reverse-phase HPLC has been developed. The assay involves the addition of the following to 1.0 ml serum: 5 ml of ethyl acetate/isopropanol (96:4 by volume); 0.1 ml of 5N NaOH; and 0.1 ml of the internal standard, N-cyano-N'-methyl-N"-[3-(4-imidazolyl)propyl]-guanidine, which is a close structural analog of cimetidine. The extracted cimetidine is quantitated with high-pressure liquid chromatograph containing a reverse-phase column and a variable-wavelength UV detector (228 nm). The mobile phase consists of methanol in 5 mM K2HPO4 (adjusted to pH 2.8) as a 10:90 mixture by volume. At a flow rate of 2 ml/min, the retention times for the internal standard and cimetidine are 2.8 and 6.2 min, respectively. The standard curve for cimetidine is linear from 0.1 at least 4.0 micrograms/ml in serum. The CV of this assay for cimetidine, obtained from analysis of six replicate samples of a 1.0 microgram/ml serum pool, is 2%. The CV of this method obtained from the daily analysis (N = 12) of the 1.0 microgram/ml cimetidine standard, is 3%, and that from the 0.5 microgram/ml standard is 5%. Cimetidine was found to be stable in refrigerated or frozen serum for 1 month and in whole blood for 24 hr either at room temperature or refrigerated. In the serum from 13 patients receiving cimetidine therapy, the trough cimetidine levels varied from less than 0.1 to 2.7 mg/ml.