Simultaneous quantitative determination of N(G),N(G)-dimethyl-L-arginine or asymmetric dimethylarginine and related pathway's metabolites in biological fluids by ultrahigh-performance liquid chromatography/electrospray ionization-tandem mass spectrometry.
BACKGROUND
Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) formation inhibitor, has emerged as a promising biomarker of NO-associated endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. The interest in potentially fundamental role of this metabolite, in basic and clinical research, led to the development of numerous analytical methods for the quantitative determination of ADMA and dimethylarginines in biological systems, notably plasma, serum and urine.


OBJECTIVE
The aim of this work was to present a simple, fast and accurate UPLC-tandem-MS-based method for the simultaneous determination and quantification of arginine, ADMA, SDMA, NMMA, homo-arginine and citrulline. This method is designed for high sample throughput of only 10 μL of human plasma, serum or urine.


METHODS
The analysis time is reduced to 1.9 min by an ultrahigh-performance liquid chromatography run coupled with electrospray ionization (ESI) in the positive mode tandem mass spectrometry detection.


RESULTS
The method was validated in plasma, serum and urine. Correlation coefficients (r(2)) of the calibration curves in all matrices considered ranged from 0.9810 to 0.9993. Inter- and intra-assay precision, accuracy, recovery and carry-over were evaluated for validation. The LOD was 0.01 μM for all compounds in water, plasma and serum and 0.1 μM in urine. The LOQ was 0.05 μM for ADMA, SDMA, NMMA and H-Arg and 0.5 μM for Arg and Cit in water, plasma and serum; while in urine was 0.1 μM for ADMA, SDMA, NMMA and H-Arg and 0.5 μM for Arg and Cit. The precision was ranged from 1% to 15% expressed as CV% and the accuracy (bias %) was <±7% for all added concentrations with the exception of NMMA (-10%). ADMA mean plasma levels, measured in healthy adults and newborns, were in accord with literature data published: (M±SD) 0.56±0.10 μM and 0.84±0.21 μM, respectively, showing that ADMA levels in plasma decreased with age. In serum we have similar data (0.54±0.18 μM and 1.14±0.36 μM), while in neonatal urine ADMA was 11.98±7.13 μmol mmol(-1) creatinine.


CONCLUSIONS
Data from calibration curves and method validation reveal that the method is accurate and precise. The fast run time, the feasibility of high sample throughput and the small amount of sample required make this method very suitable for routine analysis in the clinical setting.
Di Gangi
Iole Maria
IM
Department of Pediatrics Salus Pueri, University of Padua, Via Giustiniani, 3 I-35128 Padua, Veneto, Italy.
Chiandetti
Lino
L
Gucciardi
Antonina
A
Moret
Vittoria
V
Naturale
Mauro
M
Giordano
Giuseppe
G
D000328:Adult; D001120:Arginine / Q000031:analogs & derivatives / Q000032:analysis* / Q000097:blood / Q000652:urine; D001826:Body Fluids / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002956:Citrulline / Q000032:analysis / Q000097:blood / Q000652:urine; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000097:blood / Q000652:urine; D006801:Humans; D007231:Infant, Newborn; D008875:Middle Aged; D009569:Nitric Oxide / Q000037:antagonists & inhibitors; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004791:Enzyme Inhibitors; D002956:Citrulline; D009569:Nitric Oxide; C018524:N,N-dimethylarginine; D001120:Arginine