Rapid simultaneous determination of arginine and methylated arginines in human urine by high-performance liquid chromatography–mass spectrometry

ArticleinAnalytica Chimica Acta 487(2):145-153 · July 2003with6 Reads
DOI: 10.1016/S0003-2670(03)00554-3
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.
  • [Show abstract] [Hide abstract] ABSTRACT: N(G)-Monomethyl-L-arginine (L-NMMA), N(G),N(G)-dimethyl-L-arginine (ADMA), and N(G),N(G)'-dimethyl-L-arginine (SDMA) are emerging cardiovascular risk factors. A high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of L-NMMA, ADMA and SDMA is described. The assay employed 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a fluorescent derivatization reagent. After solid phase extraction with cation-exchange column, the methylated arginines were converted to fluorescent derivatives with NBD-F, and the derivatives were separated within 32 min on a reversed-phase column. Nomega-Propyl-L-arginine was Used as an internal standard. Extrapolated detection limits were 12 nM (12 fmol per injection) for L-NMMA and 20 nM (20 fmol per injection) for ADMA and SDMA, respectively, with a signal-to-noise ratio of 3. The calibration curves for L-NMMA, ADMA and SDMA were linear within the range of 50-5000 fmol. The method was applied to the quantitative determination of L-NMMA, ADMA and SDMA in 200 microl of rat plasma. The concentrations of L-NMMA, ADMA and SDMA in rat plasma were 0.16 +/- 0.03, 0.80 +/- 0.25 and 0.40 +/- 0.21 microM, respectively (n = 5).
    Full-text · Article · Mar 2005
  • [Show abstract] [Hide abstract] ABSTRACT: A column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method for the determination of three methylated arginines, NG-monomethyl-L-arginine (L-NMMA), NG,NG-dimethyl-L-arginine (asymmetric dimethyl-L-arginine, ADMA), and NG,NG′-dimethyl-L-arginine (symmetric dimethyl-L-arginine, SDMA), which are endogenous nitric oxide synthase inhibitors, was developed. After fluorescence derivatization of plasma samples with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized methylated arginines were trapped on a cation exchange column with filter to remove proteins, separated within 42 min on a reversed-phase column, and detected at an emission wavelength of 530 nm with excitation at 470 nm. The detection limits were 10 fmol for L-NMMA and 20 fmol for ADMA and SDMA with a signal-to-noise ratio of 3. A good linearity for calibration curves for each methylated arginine was observed within the range of 50–5000 fmol using homoarginine as an internal standard. The proposed method was applied to the quantitative determination of L-NMMA, ADMA and SDMA in rat plasma. The concentrations of L-NMMA, ADMA and SDMA in rat plasma were 0.16 ± 0.01, 0.73 ± 0.02 and 0.41 ± 0.05 µmol l−1, respectively (n = 5).
    Article · Nov 2005
  • [Show abstract] [Hide abstract] ABSTRACT: A fast, simple and sensitive column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was developed on a monolithic silica column for the determination of N(G),N(G)-dimethyl-L-arginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. After fluorescence derivatization of plasma samples or homogenized tissues with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized ADMA was trapped on a cation-exchange column and separated within 15 min on a monolithic silica column. The detection limit for ADMA was 36 nM (250 fmol per injection) when the signal-to-noise ratio was 3. A good linearity for calibration curve for ADMA was observed within the range of 140 nM (1.0 pmol per injection) - 140 microM (1.0 nmol per injection) using N(G)-monomethyl-L-arginine (L-NMMA) as an internal standard. The proposed method was used for the quantitative determination of ADMA in rat plasma. The concentrations of ADMA in rat plasma were 0.82+/-0.05 microM (n=4). Furthermore, the method developed was applied to determine dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity in rat kidney, which was assayed by measuring the amount of ADMA metabolized by the enzyme.
    Article · Dec 2006
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