LC-MS-MS Measurements of Urinary Creatinine and the Application of Creatinine Normalization Technique on Cotinine in Smokers' 24 Hour Urine

A simple and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS-MS) method was developed and validated for the quantification of creatinine in human urine. The analysis was carried out on an Agilent Zorbax Eclipse XDB-C18 column (2.1 × 150 mm, 3.5 μm). The mobile phase was 0.1% formic acid in water and 0.1% formic acid in acetonitrile (50/50, v/v). Linear calibration curves were obtained in the concentration range of 1–2000.0 ng/mL, with a lower limit of quantification of 0.99 ng/mL. The intra- and interday precision (RSD) values were below 3%. The method was successfully applied to a bioequivalence study of creatinine in Chinese smokers and nonsmokers. The total cotinine in 24 h urine and cotinine : creatinine ratio were also positively associated (Pearson R = 0.942, P < 0.0001). However, cotinine : creatinine ratio varied significantly across smoking groups for the difference of individual. 24 h urinary cotinine was more appropriate for expressing correlation with tar than cotinine : creatinine ratio.