High-performance liquid chromatographic assay of asymmetric dimethylarginine, symmetric dimethylarginine, and arginine in human plasma by derivatization with naphthalene-2,3-dicarboxaldehyde

Asymmetric dimethylarginine (ADMA) is an emerging cardiovascular risk factor. Its increased levels have been hypothesized to be a cause of endothelial dysfunction in pathological conditions such as hypertension, dyslipidemia, renal failure, hyperglycemia, and hyperhomocysteinemia. It acts as a potent competitive inhibitor of nitric oxide synthase. Methods using ortho-phthaldialdehyde (OPA) as derivatization reagent are widely performed in HPLC determination of ADMA, but they produce derivatives whose fluorescence rapidly decreases during time. Moreover, these methods do not allow a clear separation of ADMA from its stereoisomer symmetric dimethylarginine (SDMA). Our work describes a new method to determine ADMA, SDMA, and arginine that uses, as derivatizing reagent, naphthalene-2,3-dicarboxaldehyde (NDA). Chromatograms with low background, showing a complete separation of ADMA and SDMA, are obtained: NDA derivatives are considerably more stable than the OPA derivatives. The calibration curves of ADMA and SDMA are linear within the range of 0.01-16.0 muM. Coefficients of variation are less than 1.7% for within day and less then 2.3% for day to day. Absolute mean recoveries from supplemented samples are between 100 and 104%. These characteristics make this method reliable and easily manageable for large routine analyses. (C) 2003 Elsevier Science (USA). All rights reserved.