Description
A specific and sensitive method was developed for the analysis of creatinine in urine. This method utilizes liquid-liquid extraction, isotope dilution mass spectrometry (IDMS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). A mixture of creatinine-d3 internal standard and a urine sample was first dried and then extracted with 1-octanol to dissolve the creatinine. The creatinine dissolved in the 1-octanol was free of salts and was extracted back into water for analysis by hydrophilic interaction liquid chromatography (HILIC) and MS/MS with positive ion electrospray ionization (ESI). Creatinine is quantified using the ion transitions of m/z 114 to m/z 44 for creatinine-h3 and m/z 117 to m/z 47 for creatinine-d3. The specificity of the method was established by running samples in the MS/MS mode, and was indicated by no interferences observed in the eluting region of creatinine when creatinine-free sample solutions were analyzed. The method is linear, with regression coefficient (R2) of 0.9997, in the creatinine concentration range from 0.010 mg/mL to 12 mg/mL (1.0 to 1200 mg/dL). The limit of detection, established by the signalto- noise ratio (S/N) of three was 0.003 mg/mL, and the limit of quantitation at S/N of ten was 0.01 mg/mL. The precision of the method determined by replicate analyses of a sample was 3.37% RSD (n=6, at 2.0 mg/mL). The day-to-day precision, determined by analyzing a urine sample in six replicates each on two different days, was 3.65% RSD (n=12, at 2.0 mg/mL). The average accuracy of the method, determined by analyzing nine creatinine-free synthetic urine samples spiked with known amounts of creatinine at 0.05, 0.5, and 6 mg/mL, in triplicates at each level, was 106%. The recovery was in the range 100-110% at concentrations higher than 0.5 mg/mL, and averaged 111% at 0.05 mg/mL. The results from 23 urine samples generated by this method and an established method currently used in our lab were compatible.
