Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration

Abstract

N(G) ,N(G) -dimethyl-l-arginine (asymmetric dimethylarginine, ADMA),N(G) -monomethyl-l-arginine (l-NMMA) and N(G) ,N(G') -dimethyl-l-arginine (symmetric dimethylarginine, SDMA) are released during hydrolysis of proteins containing methylated arginine residues. ADMA and l-NMMA inhibit nitric oxide synthase by competing with l-arginine substrate. All three methylarginine derivatives also inhibit arginine transport. To enable investigation of methylarginines in diseases involving impaired nitric oxide synthesis, we developed a high-performance liquid chromatography (HPLC) assay to simultaneously quantify arginine, ADMA, l-NMMA and SDMA. Our assay requires 12 μL of plasma and is ideal for applications where sample availability is limited. We extracted arginine and methylarginines with mixed-mode cation-exchange columns, using synthetic monoethyl-l-arginine as an internal standard. Metabolites were derivatized with ortho-phthaldialdeyhde and 3-mercaptopropionic acid, separated by reverse-phase HPLC and quantified with fluorescence detection. Standard curve linearity was ≥0.9995 for all metabolites. Inter-day coefficient of variation (CV) values were ≤5% for arginine, ADMA and SDMA in human plasma and for arginine and ADMA in mouse plasma. The CV value for l-NMMA was higher in human (10.4%) and mouse (15.8%) plasma because concentrations were substantially lower than ADMA and SDMA. This assay provides unique advantages of small sample volume requirements, excellent separation of target metabolites from contaminants and validation for both human and mouse plasma samples.

Keywords: ADMA; Arginine; L-NMMA; Nitric Oxide Synthase; SDMA; Vascular Homeostasis.

Figure 1

Resolution and identification of analytes. Arginine (50 µm),N^G‐monomethyl‐l‐arginine (l‐NMMA; 0.5 µm), homoarginine (0.5 µm), asymmetric dimethylarginine (ADMA; 0.5 µm), symmetric dimethylarginine (SDMA; 0.5 µm) and monoethyl‐l‐arginine (MEA; 50 µm) were analyzed individually to determine retention times. Peak identities: 1, arginine; 2, l‐NMMA; 3, homoarginine; 4, ADMA; 5, SDMA; and 6, monoethyl‐Larginine (MEA, internal standard). Concentrations in human plasma (middle panel) were 89.7 µm arginine, 0.09 µm l‐NMMA, 0.35 µm ADMA and 0.28 µm SDMA. Concentrations in mouse plasma (bottom panel) were 83.7 µm arginine, 0.14 µm l‐NMMA, 0.51 µm ADMA and 0.13 µm SDMA. Arrows at 20 and 37 min indicate switch to increased sensitivity (PMT gain 16) and back to normal sensitivity (PMT gain 11), respectively.

Figure 2

Survey of human and mouse plasma for potential contaminants. Human and mouse plasma were processed with protein precipitation [no solid‐phase extraction (SPE)] or with solid phase extraction using mixed‐mode cation exchange columns (+SPE). For these analyses, plasma samples were not spiked with MEA (internal standard) and photomultiplier tube (PMT) gain remained constant (11) for the duration of the run. Plasma samples were compared with a combined standard (top panel) to identify potential contaminants that may co‐elute with target analytes if not fully eliminated by SPE. Arrows at 20 and 37 min indicate switch to increased sensitivity (PMT gain 16) and back to normal sensitivity (PMT gain 11), respectively. Sensitivity was not increased in the bottom two panels and traces were artificially shifted up (no SPE) and down (+SPE) to aid visualization. Peak identities: 1, arginine; 2, l‐NMMA; 3, homoarginine; 4, ADMA; 5, SDMA; 6, MEA; 7, alanine; and 8, taurine.

Figure 3

Plot of standard curves. 9‐point dilutions of a combined external standard were prepared and assayed in triplicate. Combined standards contained arginine, l‐NMMA, ADMA, SDMA and MEA (internal standard). The fluorescence detector photomultiplier tube gain was set to 11 for peaks detected at normal sensitivity curves (A) and to 16 for peaks detected with increased sensitivity (B). In each dilution, arginine and MEA were 100× more concentrated than l‐NMMA, ADMA and SDMA. R ² values were 0.9998 for arginine, 0.9998 for MEA, 0.9995 for l‐NMMA, 0.9996 for ADMA and 0.9995 for SDMA.