Biomarker Quantification: Development of Fit for purpose LC-MS/MS Method for Determination of Methyl guanidine in Mice Urine

Abstract:

Introduction: Accurate quantitation of biomarkers is always challenging, it becomes really tedious when biomarker has poor retention on chromatographic column and possess a chemical structure resistant for derivatisation. Methyl guanidine is product of protein catabolism, normally gets excreted in urine. Endogenous methyl guanidine concentrations in urine increases if there is reduce urine production or conversion of creatinine to methyl guanidine as proposed in patients with chronic renal failure. Increased level of methyl guanidine promotes apoptosis of renal proximal tubular cells in vitro, which in-turn could result in renal failure. Therefore Methyl guanidine can be considered as putative biomarker for renal failure studies. Method: Artificial urine was used as surrogate matrix for preparation of calibration standards, while quality control standards were prepared in authentic mice urine diluted 50 fold with artificial urine prior to extraction. For determination of basal levels of endogenous methyl guanidine urine samples from naïve mice were quantified. Moreover 50 fold dilution of quality control standards and study samples with artificial urine makes test matrix almost similar to that of calibration standards. Result: Developed method was found to linear 2ng/ml to 1000 ng/ml, with R2 more than 0.98.Basing on the mean endogenous basal levels of methyl guanidine determined in un-treated C57BL/6J mice urine, developed method can accurately quantify up to 10 fold up regulation and up to 20 fold down regulation of methyl guanidine concentrations. Conclusion: A fast, robust and cost effective LC-MS/ MS method was developed for determination of MG in mice urine. This is the first LCMS/ MS assay for direct quantitation of MG in mice urine samples. Approach followed for quantitation of MG is in-expensive over procurement of stable labeled standards, moreover 50 fold dilution of quality control standards and study samples with synthetic urine makes test matrix almost similar to that of calibration standards.

Key words: Methyl guanidine, Biomarker Quantification, Mice urine, Surrogate matrix, Synthetic urine, LC-MS/MS method.