Rapid simultaneous determination of arginine and methylated arginines in human urine by high-performance liquid chromatography–mass spectrometry
ABSTRACT A simple, sensitive and fast method using reversed-phase high-performance liquid chromatography (HPLC)–mass spectrometry (MS) coupling with an atmospheric pressure chemical ionization (APCI) interface was developed for simultaneous separation and determination of l-arginine (ARG), NG,NG-dimethylarginine (ADMA) and NG,N′G-dimethylarginine (SDMA) in human urine. This method involved the use of the [M+H]+ ions of ARG, ADMA and SDMA at m/z 175, 203 and 203 in the selective ion monitoring (SIM) mode. Satisfactory separation was achieved on a mm Shimadzu VP-ODS column by using the mobile phase consisting of water (95%), acetonitrile (5%) and trifluoroacetic acid (TFA, 0.4%). l-Homoarginine was used as the internal standard for the assay. With an isocratic HPLC, the total LC–APCI–MS analysis time was less than 5 min, making the method the fastest and most specific method reported to date. The limits of quantification (LOQ) were found to be 0.2 μmol l−1 for ARG, ADMA and SDMA. The inter-assay precision and accuracy were in the range of 2.1–6.8 and −4.4–5.4%, respectively. The intra-assay precision and accuracy were in the order of 1.6–4.1 and −1.8–3.2%, respectively. The recoveries were between 98.2 and 103.2%. With the help of the proposed method, the levels of ARG, ADMA and SDMA in human urine were determined.
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ABSTRACT: 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. 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. 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. 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. 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.Analytica chimica acta 09/2010; 677(2):140-8. · 4.31 Impact Factor
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ABSTRACT: L-Arginine (ARG) is converted to nitric oxide (NO) and L-citrulline (CIT) by endothelial nitric oxide synthase which is competitively inhibited by asymmetric dimethylarginine (ADMA). We have developed a liquid chromatography-mass spectrometric method for the simultaneous determination of endogenous ARG, labeled ARG (¹⁵N₄-ARG), CIT, ADMA, and its inactive isomer, symmetric dimethylarginine (SDMA) in biological samples. Concentrations of unlabeled ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA in EA.hy926 human endothelial cell lysate, cell incubation media, rat plasma or rat urine were measured by hydrophilic-interaction liquid chromatography electrospray tandem mass spectrometry. ¹³C₆-ARG, D₄-CIT and D₇-ADMA were used as internal standards for ARG and ¹⁵N₄-ARG, CIT, and dimethylarginines, respectively. The calibration curves of ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA were linear and independent of several sample matrices. Intra- and inter-day variabilities for the quantification of all the compounds were below 15% in quality control samples. Application of this method to determine the uptake as well as efflux of these compounds was illustrated through in vitro cell study by exposing human endothelial cells to ¹⁵N₄-ARG, which allowed the observation of generation of ¹⁵N₃-CIT and ¹⁵N₃-ARG in the cell lyate. Use of these isotopes adds insights into the cellular handling of endogenous vs. exogenous ARG. Application of this method for rat plasma and rat urine assays was demonstrated after ARG oral supplementation in rats. An LC-MS/MS method was developed to quantify 6 ARG-related compounds simultaneously, utilizing 3 separate internal standards. This assay allows concurrent monitoring of uptake, efflux and metabolic processes when isotope-labeled ARG and CIT are measured, and can be applied for determination of these compounds in rat plasma and rat urine.Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 03/2011; 879(7-8):467-74. · 2.78 Impact Factor
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ABSTRACT: A method for the simultaneous analysis of asymmetric dimethylarginine, symmetric dimethylarginine, monomethylarginine and arginine in human plasma and urine, with short analysis time and isotopic internal standardisation for each analyte is described. The method requires neither sample derivatisation nor the need for chromatographic separation of analytes. The method described shows good precision and accuracy and is suited for both research purposes and implementation in the busy, routine clinical laboratory. In addition the synthesis and utilisation of isotopically labelled symmetric dimethylarginine and monomethylarginine is described for the first time, avoiding the use of surrogates such as homoarginine for internal standardisation.Journal of Chromatography B 09/2008; 874(1-2):27-32. · 2.69 Impact Factor