HPLC analysis of asymmetric dimethylarginine (ADMA) and related arginine metabolites in human plasma using a novel non-endogenous internal standard.
BACKGROUND
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthesis which has been implicated in the endothelial dysfunction. Methods for ADMA measurement often yield widely differing results, and few methods simultaneously offer satisfactory accuracy and precision. We describe a fully validated HPLC method for analysis of arginine and its methylated derivatives in human plasma using a novel internal standard.


METHODS
Arginine and related metabolites are extracted from plasma by solid phase extraction (SPE), derivatised with ortho-phthaldialdehyde and separated by isocratic reverse phase chromatography. Monoethylarginine (MEA), which is not endogenously present in human plasma was used as internal standard. SPE and chromatographic procedures are optimised and recovery, precision, linearity and sensitivity of the assay established. The suitability and performance of MEA is compared with that of monomethylarginine (MMA), the internal standard most commonly used in HPLC methods.


RESULTS
SPE yields high and reproducible recoveries (>90%). The analytes of interest are chromatographically well resolved. The method has high sensitivity (LOD, 0.01 micromol/L for arginine and 0.001 micromol/L for ADMA, SDMA and homoarginine) and good precision (CV, 2.5% for ADMA). The data obtained with the internal standards MEA and MMA is comparable in terms of assay precision and population reference intervals.


CONCLUSIONS
We describe an optimised isocratic HPLC method for the simultaneous measurement of arginine and related metabolites in plasma which exhibits satisfactory precision and is suitable for routine use. Its main advantage over other published HPLC methods is the use of the novel internal standard, MEA, which unlike other commonly used internal standards is not inherent in human plasma.
Blackwell
Scott
S
Department of Clinical Biochemistry, Scottish Trace Element and Micronutrient Reference Laboratory, Royal Infirmary, Glasgow, UK.
O'Reilly
Denis St J
DS
Talwar
Dinesh K
DK
D001120:Arginine / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism / Q000592:standards; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D012015:Reference Standards; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods*
C018524:N,N-dimethylarginine; D001120:Arginine