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Liquid chromatography--tandem mass spectrometry method for the analysis of asymmetric dimethylarginine in human plasma.

Nitric oxide (NO) is essential in numerous physiologic processes and may be involved in related pathologic processes. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of isoforms of NO synthase in humans (1). ADMA originates from protein arginine methylation by protein arginine methyltransferases after protein hydrolysis (2). Enzymatic hydrolysis by dimethylarginine dimethylaminohydrolases to dimethylamine and citrulline is the major pathway for elimination of ADMA (3). Circulating ADMA is altered in patients with cardiovascular and neurologic diseases, erectile dysfunction, and many other disorders (4-6), and increased circulating ADMA independently predicts future cardiovascular events and mortality (7, 8). Short-time infusion of ADMA affects hemodynamics and cardiac function in humans (3, 9).

Analytical methods for the measurement of ADMA include HPLC, capillary electrophoresis, ELISA, and mass spectrometry (MS). The commonly used HPLC methods with fluorescence detection (10) measure o-phthaldialdehyde derivatives of ADMA. These derivatives are not stable and must be analyzed on-line. Moreover, ADMA must be separated by chromatographic means from its biologically inactive isomer, symmetric dimethylamine (SDMA). Thus, these HPLC methods have long analysis times of up to 45 min. Alternatively, ADMA and SDMA can be analyzed by gas chromatography--mass spectrometry (11,12), which shows different fragmentation patterns for the respective ions. These methods include several extraction and derivatization steps and require considerable analysis times. Recently, Kirchherr and Kuhn-Velten (13) developed a mass spectrometric method that omits derivatization procedures; however, chromatographic separation of ADMA from SDMA is still required for this method. Here we report a simple and rapid liquid chromatography---tandem MS (LC-MS/MS)-based method with 1 derivatization step and sample pretreatment consisting of protein precipitation. This is the first LC-MS--based method that separates methylated arginine derivatives by specific fragmentation patterns instead of by chromatographic separation.

Analyses were performed on a Varian 1200L Triple Quadrupole MS equipped with 2 Varian ProStar model 210 HPLC pumps and a Varian analytical column [50 x 2.0 mm (i.d.)] packed with Polaris [C.sub.18]-Ether (3 [micro]m bead size). The mobile phases consisted of methanol containing 1 mL/L formic acid (mobile phase A) and water containing 1 mL/L formic acid (mobile phase B). Chromatography was performed at 25[degrees]C with a flow rate of 0.4 mL/min. The gradient started with 2% A for 0.5 min and increased linearly to 50% A over 1.5 min, with subsequent reequilibration with 2% A for 2 min. Nitrogen was used as the nebulizing and drying gas (380[degrees]C) at 90 and 180 L/h, respectively.

For ionization in the positive electrospray ionization mode (ESI+), the needle and shield voltages were set at 5850 and 400 V, respectively. The following transitions were observed after fragmentation with argon (1.5 mTorr): m/z 231 [right arrow] 70 [collision energy (CE), -22 eV] for L-arginine (USP Reference Standard); m/z 238 [right arrow] 77 (CE, -24 eV) for L-[[sup.2.][H.sub.7]]-arginine (98 atom% [sup.2]H isotopic purity; 2,3,3,4,4,5,5-t-[[sup.2][H.sub.7]]-arginine; Euriso-top); m/z 259.3 [right arrow] 214 (CE, -16 eV) for ADMA (>99% purity; Sigma-Aldrich); m/z 259.3 [right arrow] 228 (CE, -14 eV) for SDMA (>99% purity; Sigma-Aldrich); and m/z 265.3 [right arrow] 220 (CE, -16 eV) for [[sup.2][H.sub.6]]-ADMA (98 atom% [sup.2]H isotopic purity; 3,3,4,4,5,5-[[sup.2][H.sub.6]]-ADMA). The synthesis and characterization of [[sup.2][H.sub.6]]-ADMA were performed as described previously (12). All compounds were analyzed as their butyl ester derivatives (see below). All parent ions (MS) represented the molecular protonated canons, i.e., [[M+H].sup.+]. The major daughter ions formed, i.e., m/z 70 and m/z 77, for L-arginine and L-[[sup.2][H.sub.7]]-arginine, respectively, correspond to a pyrrolinium ion as a cyclization product (14). Major daughter ions of m/z 259.3 were m/z 214 [[M+H - NHC[H.sub.3]C[H.sub.3]].sup.+] and m/z 228 [[M+H - N[H.sub.2]C[H.sub.3]].sup.+] for authentic ADMA and SDMA, respectively. The m/z 228 and m/z 214 ions were absent in the product ion mass spectra of ADMA and SDMA, respectively. We checked ion suppression by comparing peak areas of aqueous calibrator solutions without matrix and calibrator added to plasma matrix (15). Ion suppression was <10% for 5 different specimens.

We used 3 different calibrators, L-arginine, ADMA, and SDMA, at 6 different concentrations (n = 5 each): 0, 25, 50, 100, 250, and 500 [micro]mol/L for L-arginine and 0, 0.25, 0.5,1, 2, and 4 [micro]mol/L for ADMA and SDMA, respectively. Aqueous stock solutions of L-arginine, ADMA, and SDMA were made by weighing authentic material supplied by the manufacturer. The calibrators were treated exactly the same as patient plasma samples. L-[[sup.2][H.sub.7]]-Arginine and [[sup.2][H.sub.6]]-ADMA were added as internal standards at concentrations of 50 and 2 [micro]mol/L, respectively. [[sup.2][H.sub.6]]-ADMA was used as internal standard for ADMA and SDMA. Quantitative determinations in biological samples (50 [micro]L) were performed by dilution of 5 [micro]L of aqueous internal standards (500 [micro]mol/L L-[[sup.2][H.sub.7]]-arginine and 20 [micro]mol/L [[sup.2][H.sub.6]]-ADMA) corresponding to concentrations of 50 and 2 [micro]mol/L, respectively, in 50 [micro]L of biological sample. Subsequently, proteins were precipitated with 100 [micro]L of acetone, and dried supernatants were derivatived. Compounds were derivatived with 100 [micro]L of 1 mol/L HCl in 1-butanol for 17 min at 65[degrees]C. After evaporation, samples were reconstituted in 1 mL of water; 20 [micro]L of the water extract was injected on the column. The mean (SD) retention times of the butyl ester derivatives of L-arginine, ADMA, and SDMA, respectively, were 1.03 (0.04) min (CV = 3.9%; n = 6), 1.84 (0.02) min (CV = 1.3%), and 2.03 (0.02) min (CV = 0.8%). Differences in retention times for unlabeled and labeled compounds were not statistically significant.

[FIGURE 1 OMITTED]

The linear regression equations for peak-area ratio (LC-MS/M5; y) and ratio of injected calibrators (x) were as follows: y = 0.94x - 0.001 ([r.sup.2] = 0.999) for L-arginine; y = 0.98x + 0.02 ([r.sup.2] = 0.999) for ADMA; and y = 2.05x + 0.01 ([r.sup.2] = 0.999) for SDMA. Ideally, regression analysis of peak-area ratios should give a slope of 1; however, the intensities of the daughter ions obtained for labeled and unlabeled compounds were not the same. Thus, only under MS conditions was a slope of 1 obtained: The linear regression equations for peak-area ratio (LC-M5; y) and ratio of injected calibrators (x) were as follows: y = 1.003x - 0.02 ([r.sup.2] = 0.999) for L-arginine; y = 1.001x + 0.01 ([r.sup.2] = 0.999) for ADMA; and y = 1.002x + 0.03 ([r.sup.2] = 0.998) for SDMA. Nevertheless, calibration curves were calculated to account for these differences, and the slopes of the calibration curves were included in all plasma concentration calculations. The lower limits of detection, defined as a signal-to-noise ratio of 3, were 45 nmol/L for L-arginine, 3 nmol/L for ADMA, and 2 nmol/L for SDMA.

We validated our LC-MS/MS method by adding different concentrations of L-arginine, ADMA, and SDMA in quintuplicate to samples. L-arginine was added at 0, 0.5, 1, 25, 50, 100, and 250 [micro]mol/L, and ADMA and SDMA were added at 0, 0.05, 0.1, 0.5, 1, 2, and 4 [micro]mol/L. Linear regression analysis between measured (y) and added (x) concentrations yielded the following slopes and y-intercepts: 1.01 and 70.3 [micro]mol/L ([r.sup.2] = 0.999) for L-arginine; 1.00 and 0.45 [micro]mol/L ([r.sup.2] = 0.999) for ADMA; and 1.01 and 0.42 [micro]mol/L ([r.sup.2] = 0.999) for SDMA. Data from these validation experiments are listed in Table 1. The observed recoveries of the added analytes were 97.7%-111% for L-arginine, 96.9%-105% for ADMA, and 90.8%-109% for SDMA. All CVs were <6% for the imprecision and <20% for recovery except for the addition of 0.5 [micro]mol/L L-arginine. Thus, the analytical range of the method was 1-250 [micro]mol / L for L-arginine and 50 nmol / L to 4 [micro]mol / L for ADMA and SDMA, with 1 [micro]mol/L and 50 nmol/L representing the lower limits of quantification for L-arginine and ADMA and SDMA, respectively. The addition of L-arginine, ADMA, or SDMA did not influence the measurement of the other analytes. We also determined within- and between-run imprecision (CVs; n = 10). The within-run CVs were 3.3% for L-arginine, 2.6% for ADMA, and 2.5% for SDMA. Between-run CVs were 4.7% for L-arginine, 4.1% for ADMA, and 3.9% for SDMA

We also measured ADMA in plasma samples from healthy human volunteers (n = 22) by this LC-MS/MS method. Shown in Fig. 1 is a typical chromatogram of a human plasma sample. Samples were aliquoted (50 [micro]L) and frozen at -20[degrees]C before analysis. The mean (SD) concentrations of L-arginine, ADMA, and SDMA were 65.6 (23.4), 0.55 (0.14), and 0.69 (0.23) [micro]mol/L, respectively. The mean (SD) molar ratio of L-arginine to ADMA was 132 (55).

Our LC-MS/MS-based method allows for the simultaneous determination of ADMA, SDMA, and L-arginine and has been validated for the measurement of these 3 analytes in human plasma. Values obtained for L-arginine, ADMA, and SDMA in human plasma are within previously reported ranges (11-13,16). Major advantages over previous reported methods for the measurement of ADMA include a minimized sample volume and reduction in laboratory work. In particular, in comparison with other LC-MS-based methods (13,14,16,17), sample run time has been reduced to 4 min including reequilibration. Chromatographic separation required only slightly more than 2 min. This considerable reduction in analysis time was achieved by use of the specific fragmentation patterns of the ester derivatives of ADMA and SDMA. Identical MS spectra were obtained from the ester derivatives of ADMA and SDMA, but the major fragments in the daughter ion spectra for ADMA and SDMA were at m/z 214 and m/z 228, respectively. These ions were specific for ADMA and SDMA: they were absent in the corresponding spectra of SDMA and ADMA, respectively. In contrast to previous reports, the proposed method allows, for the first time, spectrometric instead of chromatographic separation of these methylated arginines (see the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/ vol51 /issue7/).

References

(1.) Tsikas D, Boger RH, Sandmann J, Bode-Boger SM, Frolich JC. Endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox. FEBS Lett 2000;478:1-3.

(2.) McBride AE, Silver PA. State of the Arg: protein methylation at arginine comes of age. Cell 2001;106:5-8.

(3.) Achan V, Broadhead M, Malaki M, Whitley G, Leiper J, MacAllister R, et al. Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase. Arterioscler Thromb Vasc Biol 2003;23:1455-9.

(4.) Boger RH, Bode-Boger SM, Szuba A, Tsao PS, Chan JR, Tangphao O, et al. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation 1998;98:1842-7.

(5.) Selley ML. Increased concentrations of homocysteine and asymmetric dimethylarginine and decreased concentrations of nitric oxide in the plasma of patients with Alzheimer's disease. Neurobiol Aging 2003;24:903-7.

(6.) Maas R, Zabel M, Wenske S, Ventura R, Schwedhelm E, Steenpass A, et al. Erectile dysfunction in patients with cardiovascular disease and diabetes: evidence for disturbed relation of L-arginine and the endogenous inhibitor of NO-synthase asymmetrical dimethylarginine (ADMA). Z Kardiol 2004; 93(Suppl 3):V1235.

(7.) Zoccali C, Bode-Boger S, Mallamaci F, Benedetto F, Tripepi G, Malatino L, et al. Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study. Lancet 2001; 358:2113-7.

(8.) Boger RH, Lenzen H, Hanefeld C, Bartling A, Osterziel KJ, Kusus M, et al. Asymmetric dimethylarginine: an endogenous inhibitor of NO synthase is a predictor of the risk for coronary heart disease-results of the multicenter CARDIAC study. Circulation 2003;108(Suppl IV):256.

(9.) Kielstein J, Impraim B, Simmel S, Bode-Boger SM, Tsikas D, Frolich JC, et al. Cardiovascular effects of systemic nitric oxide synthase inhibition with asymmetrical dimethylarginine in humans. Circulation 2004;109:172-7.

(10.) Tsikas D, Junker W, Frolich JC. Determination of dimethylated arginines in human plasma by high-performance liquid chromatography. J Chromatogr B 1998; 705:174-6.

(11.) Tsikas D, Schubert B, Gutzki FM, Sandmann J, Frolich JC. Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography--tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative. J Chromatogr B 2003; 798:87-99.

(12.) AlbsmeierJ, Schwedhelm E, Schulze F, Kastner M, Boger RH. Determination of [N.sup.G],[N.sup.G]-dimethy-L-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry. J Chromatogr B 2004;809:59-65.

(13.) Kirchherr H, Kuhn-Velten WN. HPLC-tandem mass spectrometric method for rapid quantification of dimethylarginines in human plasma. Clin Chem 2005;51:249-52.

(14.) Vishwanathan K, Tackett RL, Stewart JT, Bartlett MG. Determination of arginine and methylated arginines in human plasma by liquid chromatography--tandem mass spectrometry. J Chromatogr B 2000;748:157-66.

(15.) Annesley TM. Ion suppression in mass spectrometry. Clin Chem 2003;49: 1041-4.

(16.) Martens-Lobenhoffer J, Bode-Boger SM. Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectrometry. J Chromatogr B 2003;798:231-9.

(17.) Huang LF, Guo FQ, Liang YZ, Li BY, Cheng BM. Simultaneous determination of L-arginine and its mono- and dimethylated metabolites in human plasma by high-performance liquid chromatography--mass spectrometry. Anal Bioanal Chem 2004;380:643-9.

Previously published online at DOI: 10.1373/clinchem.2004.046037

Edzard Schwedhelm, [1] * Jing Tan-Andresen, [1] Renke Maas, [1] Ulrich Riederer, [2] Friedrich Schulze, [1] and Rainer H. Boger [1] ([1] Institute of Experimental and Clinical Pharmacology, University Hospital Hamburg-Eppendorf, and [2] Institute of Pharmacy, University of Hamburg, Hamburg, Germany; * address correspondence to this author at: Institute of Experimental and Clinical Pharmacology, University Hospital Hamburg-Eppendorf, 20246 Hamburg, Germany; fax 49-40-428039757, e-mail schwedhelm@uke.uni-hamburg. de)
Table 1. Precision of the LC-MS/MS method for the
measurement of L-arginine, ADMA, and SDMA in
human plasma.

 Concentration Mean (SD) Mean (SD)
 added, measured recovery of
 [micro]mol/L concentration, added
 Analyte (n = 5) [micro]mol/L analyte, % CV, %

L-Arginine 0 69.4 (1.0) NA (a) 1.4
 0.5 70.0 (0.6) 111 (22) 0.9
 1 70.4 (0.4) 97.7 (19) 0.6
 25 95.5 (1.0) 104 (5.5) 3.2
 50 122 (2.6) 105 (2.3) 2.1
 100 173 (5.5) 104 (2.5) 3.2
 250 320 (8.6) 100 (1.5) 2.7

ADMA 0 0.448 (0.007) NA 1.5
 0.05 0.496 (0.011) 96.9 (10.1) 2.3
 0.1 0.553 (0.004) 105 (3.9) 0.8
 0.5 0.949 (0.012) 100 (1.1) 1.3
 1 1.464 (0.022) 102 (1.0) 1.5
 2 2.422 (0.030) 98.7 (1.4) 1.3
 4 4.443 (0.104) 99.9 (1.2) 2.3

SDMA 0 0.424 (0.024) NA 5.6
 0.05 0.479 (0.008) 109 (14.6) 1.7
 0.1 0.515 (0.013) 90.8 (11.4) 2.5
 0.5 0.924 (0.043) 100 (3.9) 4.7
 1 1.449 (0.041) 103 (3.7) 2.9
 2 2.441 (0.070) 101 (3.1) 2.9
 4 4.480 (0.109) 101 (2.4) 2.4

(a) NA, not applicable.
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Title Annotation:Technical Briefs
Author:Schwedhelm, Edzard; Tan-Andresen, Jing; Maas, Renke; Riederer, Ulrich; Schulze, Friedrich; Boger, Ra
Publication:Clinical Chemistry
Date:Jul 1, 2005
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