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 microM. 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.
Marra
Maurizio
M
Diabetology Unit, Department of Gerontological Research, INRCA, Via della Montagnola 81, I-60131 Ancona, Italy. m.marra@inrca.it
Bonfigli
Anna Rita
AR
Testa
Roberto
R
Testa
Ivano
I
Gambini
Anna
A
Coppa
Gilberto
G
D001120:Arginine / Q000031:analogs & derivatives* / Q000097:blood* / Q000737:chemistry; D002318:Cardiovascular Diseases / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D009281:Naphthalenes; D012015:Reference Standards; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism
D007202:Indicators and Reagents; D009281:Naphthalenes; C024917:symmetric dimethylarginine; C018524:N,N-dimethylarginine; C027717:2,3-naphthalenedicarboxaldehyde; D001120:Arginine