CML-d2

Background: Asymmetric dimethylarginine (ADMA) and ε-N-carboxymethyl-L-lysine (CML) are microvascular risk factors and potential biomarkers of diabetic microvascular complication. Results: Sample preparation was achieved using acetonitrile for protein precipitation step. ADMA, CML and IS CML-d2 were separated with gradient on a Welch Ultimate(®) XB- NH2 column. The assays were validated according to current bioanalytical guidelines with respect to specificity, linearity (20-1000 ng/ml for ADMA in human plasma, 50-2000 ng/ml in urine, 10-500 ng/ml for CML in human plasma and urine), accuracy and precision, extraction recovery, matrix effect and stability. Conclusion: The LC-MS/MS method was successfully applied to quantification of ADMA and CML in plasma and urine samples from healthy individuals and patients with diabetic nephropathy.

ɛ-N-carboxymethyl-L-lysine (CML) is a stable chemical modification of protein lysine residues resulting from glycation and oxidation reactions and a potential biomarker of oxidative stress caused by sugar and lipid oxidation. In this study, a rapid, simple and sensitive method based on liquid chromatography-tandem spectrometry (LC-MS/MS) for the determination of CML in human plasma has been developed and validated. Sample preparation involved protein precipitation using trichloroacetic acid after addition of deuterated CML as internal standard. Chromatography was performed on an amino column by gradient-elution with a mobile phase containing acetonitrile:ultrapure water (80:20, v/v). CML and CML-d2 were detected by multiple reaction monitoring mode with ion pairs 205.0/130.1 and 207.2/84.1 respectively. The assay was linear in the range 10-1000 ng/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL and recovery >90%. Assay validation showed that inter- and intra-day precision and accuracy were satisfactory. The method was applied to compare plasma CML levels in healthy Chinese subjects and patients with diabetes and uremia. In healthy subjects CML concentration (mean±SD) was 16.6±7.8 ng/mL. CML level in diabetic patients was not significantly different from healthy subjects whereas the level in patients with uremia was significantly higher than both healthy subjects and diabetic patients (P<0.001). The method will be useful to assess the value of CML as a biomarker of diabetic vascular complications resulting from elevated oxidative stress.