High-throughput CZE-UV determination of arginine and dimethylated arginines in human plasma

Experimental studies document that increased asymmetric dimethylarginine (ADMA) blood levels inhibit NOS significantly, reducing NO generation. ADMA measurement often needs sample cleanup by SPE prior to chromatography and precolumn derivatization that cannot be easily employed in a routine clinical setting. We set up a new reliable CE method to measure ADMA, symmetric dimethylarginine (SDMA), and arginine without sample extraction or precolumn derivatization in order to examine their concentrations in human plasma. Sample was concentrated prior to CE injection and analytes were monitored by UV detection. CE analysis was performed in an uncoated fused-silica capillary, 75 microm id and 60.2 cm length (50 cm to the detection window), injecting 1 s water plug (0.5 psi) followed by 10 s of the sample (0.5 psi). Separation was carried out in a 50 mmol/L Tris-phosphate run buffer at pH 2.30, 15 degrees C and 15 kV (75 microA) at normal polarity. Recovery of plasma ADMA was 101-104% and inter-day CV was less than 3%. Assay performance was evaluated measuring the levels of arginine and its dimethyl derivatives in 77 subjects. Passing-Bablok regression and Bland-Altman test for methods comparison suggest that the data obtained by our method and by a reference CE-LIF assay are similar.     

Improved method for plasma ADMA, SDMA, and arginine quantification by field-amplified sample injection capillary electrophoresis UV detection

Here, we describe an easy field-amplified sample injection capillary electrophoresis method with UV detection for the separation and detection of free plasma arginine and dimethylated arginines. The analytes were baseline-separated within 22?min by using 50?mmol/L Tris phosphate pH 2.3 as running buffer. The plasma samples were treated with acetonitrile/ammonia for protein elimination, the supernatants were dried, re-swollen in water and directly injected in the capillary without complex cleanup by solid phase extraction and/or tedious sample derivatization procedures. Due to the stacking effects of the electrokinetic injection, it was possible to operate a consistent on-line pre-concentration of the analytes before running the electrophoresis. This procedure allowed to reach a detection limit in the real sample of 10?nmol/L for dimethylated arginines and 20?nmol/L for arginine, thus improving about threefold our previous method, that required a more complicated pre-analytical procedure to concentrate samples. The recovery of plasma ADMA was 99-104% and inter-day CV was less than 3%. The assay performance was evaluated measuring the levels of arginine and its dimethyl derivatives in 50 subjects. The statistical tests for the methods comparison suggest that the data obtained by our new method and by our previous CE assay are similar.     

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

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. © 2015 The Authors Biomedical Chromatography published by John Wiley & Sons, Ltd.     

A novel mass spectrometry‐based method for simultaneous determination of asymmetric and symmetric dimethylarginine,l‐arginine and l‐citrulline optimized for LC‐MS‐TOF and LC‐MS/MS

Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by nitric oxide synthase by conversion of l‐ arginine to l‐ citrulline. l‐ Arginine methylated derivatives, asymmetric and symmetric dimethylarginines (asymmetric dimethylarginine, ADMA, and symmetric dimethylarginine, SDMA), regulate l‐ arginine availability and the activity of nitric oxide synthase. As such, they have been frequently investigated as potential biomarkers in pathologies associated with dysfunctions in NO synthesis. Here, we present a new multistep analytical methodology based on liquid chromatography combined with mass spectrometry for the accurate identification of l‐ arginine, l‐ citrulline, ADMA and SDMA. Compounds are measured as stable 2,3,4,5,6‐pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse‐phase column. Serum aliquots (100 μL) were spiked with isotope‐labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25°C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R ² = 0.9966–0.9986) for all derivatized compounds. Good accuracy (94.67–99.91%) and precision (1.92–11.8%) were observed for the quality control samples. The applicability of the method was evaluated in a cohort of angiological patients and healthy volunteers. The method discerned significantly lower l‐ arginine and l‐ citrulline in angiologic patients. This robust and fast LC‐ESI‐MS method may be a useful tool in quantitative analysis of l‐ arginine, ADMA, SDMA and l‐ citrulline.     

On-line electrophoretic sample clean-up for sensitive and reproducible μCE immunoassay

We report a novel on-line electrophoretic sample clean-up approach for highly sensitive and reproducible microchip electrophoretic (μCE) immunoassay of low-abundance proteins in human serum. The method takes advantage of the differential effect of field-amplified sample stacking on molecules with different electrophoretic mobility. Large interfering proteins are removed from the loading channel by simple voltage control, resulting in selective concentration and injection of smaller target analytes to the separation channel. As a proof of concept, an antibody-free injection mode was developed for direct μCE immunoassay of human insulin-like growth factor-I (IGF-I) in serum samples without any additional purification steps. Clear and sharp peaks were obtained for IGF-I with low background and excellent reproducibility. Besides, the assay sensitivity was further increased by addition of ethanol to the sample buffer at a concentration of 50% right before performing the μCE detection. The lower limit of detection of IGF-I achieved 0.68 ng mL(-1), with an overall signal enhancement factor of 2750. The established on-line electrophoretic sample clean-up approach may find wide applications in the development of other microchip-based high-throughput analytical platforms for clinical and biological use.     

Possible indirect detection of rHuEPO administration in human urine by high-performance liquid chromatography tandem mass spectrometry

The present study is based on the assumption that changes in an ADMA-DDAH-NOS (ADMA-asymmetrical dimethylarginine; DDAH-dimethyl-arginine dimethylaminohydrolase; NOS-nitric oxide synthase) system could be employed as indirect markers for recombinant human erythropoietin (rHuEPO) administration in doping control. We assessed a predictive value of four proposed new markers for rHuEPO abuse. Preliminary data showed that concentrations of ADMA, symmetrical dimethylarginine (SDMA), citrulline and arginine in human urine were increased after administration of a single intravenous erythropoietin injection (2000 U day(-1), Epocrine, St-Petersburg, Russia). The study of variations of ADMA, SDMA, arginine and citrulline levels before and after rHuEPO administration was performed with two healthy male volunteers. Urine samples were collected before rHuEPO administration and urinary concentrations of ADMA and SDMA were determined at 10.0-40 microg mL(-1) and of arginine and citrulline at 0.5-10 microg mL(-1). A single dose injection of rHuEPO caused an increase in ADMA, SDMA, arginine and citrulline concentrations up to 40-270 microg mL(-1), 40-240 microg mL(-1), 10-60 microg mL(-1) and 12-140 microg mL(-1), respectively. These preliminary results indicated that an indirect approach could be used as a pre-screening of urine samples in order to decrease the number of samples with a low probability of rHuEPO abuse and, thus, save costs and human workload.     

Determination of arginine and asymmetric dimethylarginine (ADMA) in human plasma by liquid chromatography/mass spectrometry with the isotope dilution technique

Arginine (ARG) is a substrate for endogenous nitric oxide (NO) production whereas its metabolite, asymmetric dimethylarginine (ADMA), acts as an inhibitor. Sufficient NO production is essential for cardiovascular key functions, thus elevated concentration levels of ADMA are related to a range of cardiovascular diseases. Owing to the lack of reliable methods for the measurement of ARG and ADMA in human plasma, concentration values determined with these methods can differ considerably. We present here a simple and very robust liquid chromatographic/mass spectrometric method for the determination of ARG and ADMA utilizing isotope-labeled internal standards. Sample preparation requires only protein precipitation; the analytes were derivatized with o-phthalaldehyde-mercaptoethanol and separated on a reversed-phase C(18) column with gradient elution. The analytes were detected with an electrospray ionization ion trap instrument working in the full-scan single mass spectrometry mode. Concentration values obtained with this method for healthy controls were ARG = 63.9 +/- 23.9 microM and ADMA = 0.355 +/- 0.066 microM, with a normal range for ADMA from 0.225 to 0.485 microM. The corresponding values for end-stage chronic renal failure patients are ARG = 48.1 +/- 18.5 microM, p < 0.01 and ADMA = 0.673 +/- 0.134 M, p < 0.001.     

Pharmacokinetic applications of capillary electrophoresis

This review briefly discusses the use of capillary electrophoretic (CE) methods for the investigations of different aspects of pharmacokinetics. In most investigations, CE was the method of choice because of its unique features, including high resolving power for chiral or metabolite separation, small sample volume for pediatric pharmacokinetics or for cell-based investigations, in situ microdialysis sampling for rapid eliminations, low UV wavelength detection for nonderivatized analytes, fast and simplified sample processing for existing methods that require tedious sample preparation, or as a second method for verifications. Moreover, instrumental aspects of CE-based assays for pharmacokinetic studies, such as different modes of CE methods for analyzing biological samples, sample stacking for increasing detection sensitivity, and coupling techniques with microdialysis and mass spectrometry, are also discussed in this review. Furthermore, the advantages and limitations of CE methods as well as the future outlook for pharmacokinetic studies are summarized.     

A simple and fast liquid chromatography–tandem mass spectrometry method for measurement of underivatized <Emphasis Type="SmallCaps">l</Emphasis>-arginine,symmetric dimethylarginine,and asymmetric dimethylarginine and establishment of the reference ranges

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and an established biomarker for endothelial function, while symmetric dimethylarginine (SDMA), an emerging biomarker for renal function, has been shown to outperform creatinine-based equations for estimated glomerular filtration rate. In order to study these analytes for clinical research, a fast and simple method for measuring arginine (ARG), SDMA, and ADMA in plasma by liquid chromatography–tandem mass spectrometry (LC-MS/MS) has been developed. Plasma (50 μL) was mixed with 50 μL of internal standard of ¹³C-arginine and d₇-ADMA followed by protein precipitation with methanol containing 1% ammonium acetate (300 μL). After centrifugation, the supernatant (100 μL) was mixed with 300 μL of acetonitrile with 1% formic acid, and the mixture was injected onto a silica column monitored by a mass spectrometer. The analytical cycle time was 5.0 min. The method was linear from 5.7 to 489.7 μM for ARG, 0.06 to 5.15 μM for SDMA, and from 0.34 to 5.65 μM for ADMA, with an accuracy of 99.0–120.0%. Total coefficients of variation for all analytes ranged from 2.7% to 7.7% for three concentration levels. The effects of hemolysis, lipemia, uremia, icterus, specimen tube types, storage at different temperature, and freeze/thaw were thoroughly investigated. Reference ranges were established using 51 well-defined reference subjects (12 men and 39 women, age 19–64 years): 53.1–129.7 μM for ARG, 0.32–0.65 μM for SDMA, and 0.36–0.67 μM for ADMA. In conclusion, the validated LC-MS/MS method described here offers a fast and reliable ARG, SDMA, and ADMA quantitation in plasma with minimum sample preparation.     

High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-carbon metabolism using HPLC-MS/MS

Risk of chronic diseases, like cardiovascular disease and cancer, has been associated with biomarkers related to one-carbon metabolism, which comprises a metabolic network of cross-talking pathways. To address this complexity in epidemiological studies, we have established an isotope dilution HPLC-MS/MS method for quantification of 12 biomarkers and metabolites. All sample handling is performed by a robotic workstation. The assay uses 45 μL of plasma, and sample treatment consists of protein precipitation by trichloroacetic acid. The analytes were separated on a Fortis Phenyl column using an isocratic mobile phase that contained water, methanol and acetic acid. Methionine, methionine sulfoxide, choline, betaine, dimethylglycine, arginine, asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine, creatinine, cystathionine and trimethyllysine all showed limits of detection well below the 5th percentile of plasma distributions in healthy humans, coefficients of variation were in the range 2.2–12.3 %, and recoveries were 80–131 %. Simple sample processing, low-volume consumption, multiplexing and high capacity/short run time of this method make it suitable for large-scale metabolic profiling of precious biobank samples.     

Determination of dimethylarginine levels in rats using HILIC-MS/MS: an in vivo microdialysis study

Nitric oxide (NO) is one of the most important mediators and neurotransmitters and its levels change under pathological conditions. NO production may be regulated by endogenous nitric oxide synthase (NOS) inhibitors, in particular asymmetric dimethylarginine (ADMA). Most of the interest is focused on ADMA, since this compound is present in plasma and urine and accumulation of ADMA has been described in many disease states but little is known about cerebrospinal fluid (CSF) concentrations of this compound and of its structural isomer symmetric dimethylarginine (SDMA). To determine the levels of methylarginines, we here present a new hydrophilic interaction chromatography (HILIC)-MS/MS method for the precise determination of these substances in CSF from microdialysis samples of rat prefrontal cortex (PFC). The method requires only minimal sample preparation and features isotope-labelled internal standards.     

Combination of solid-phase extraction and large-volume stacking with polarity switching in micellar electrokinetic capillary chromatography for the determination of traces of nonsteroidal anti-inflammatory drugs in saliva

A reliable MEKC method for the identification and quantitation of traces of the nonsteroidal anti-inflammatory drugs (NSAIDs) ketoprofen, fenbufen and indomethacin in saliva is proposed. Using CE to analyze biological samples often requires suppressing the interferences and peak broadening typically resulting from high-conductivity sample matrices. We addressed this problem by using Microcon, a centrifugal filter device, to reduce the viscosity of saliva and exclude most higher-molecular-mass substances. This initial pretreatment was followed by the combined used of off-line SPE to isolate and concentrate the analytes, and large-volume stacking with polarity switching (LVSS) in the capillary. These two preconcentration steps allow the determination of NSAIDs at concentrations above 0.1 microg/L; therefore, the proposed SPE/LVSS/MEKC method affords a 500-fold sensitivity enhancement relative to conventional CE injection. The LODs obtained afford the determination of NSAIDs in saliva, where analytes can be present at the microgram-per-liter level.     

Determination of mercaptopurine and its four metabolites by large-volume sample stacking with polarity switching in capillary electrophoresis

This study describes approaches for stacking a large volume of sample solutions containing a mixture of mercaptopurine monohydrate, 6-methylmercaptopurine, thioguanine, thioguanosine, and thioxanthine in capillary electrophoresis (CE). After filling the run buffer (60 mM borate buffer, pH 8.5), a large sample volume was loaded by hydrodynamic injection (2.5 psi, 99.9 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-15 kV). Monitoring the current and reversing the polarity when 95% of current recovered, the separation of anionic analytes was performed in a run buffer < 20 kV. Around 44- to 90-fold improvement of sensitivity for five analytes was achieved by large-volume stacking with polarity switching when compared with CE without stacking. This method was feasible for determination of the analytes spiked in plasma. Removing most of electrolytes from plasma is a key step for performing large-volume sample stacking. Solid-phase extraction was used for pretreatment of biological samples. To our knowledge, this study is one of few applications showing the possibilities of this stacking procedure to analyze biological samples by large-volume sample stacking with polarity switching (LVSSPS) in CE.     

Thirty years of capillary electrophoresis in food analysis laboratories: potential applications

CE has generated considerable interest in the research community since instruments were introduced by different trading companies in the 1990s. Nowadays, CE is popular due to its simplicity, speed, highly efficient separations and minimal solvent and reagent consumption; it can also be included as a useful technique in the nanotechnology field and it covers a wide range of specific applications in different fields (chemical, pharmaceutical, genetic, clinical, food and environmental). CE has been very well evaluated in research laboratories for several years, and different new approaches to improve sensitivity (one of the main drawbacks of CE) and robustness have been proposed. However, this technique is still not well accepted in routine laboratories for food analysis. Researching in data bases, it is easy to find several electrophoretic methods to determine different groups of analytes and sometimes they are compared in terms of sensitivity, selectivity, precision and applicability with other separation techniques. Although these papers frequently prove the potential of this methodology in spiked samples, it is not common to find a discussion of the well-known complexity of the matrices to extract analytes from the sample and/or to study the interferences in the target analytes. Summarizing, the majority of CE scientific papers focus primarily on the effects upon the separation of the analytes while ignoring their behavior if these analytes are presented in real samples.     

Probing AGXT2 enzyme activity in mouse tissue by applying stable isotope‐labeled asymmetric dimethyl arginine as substrate

Asymmetric dimethylarginine (ADMA) is a metabolite of the amino acid l ‐arginine. It competitively inhibits the enzymatic production of the cell‐signaling substance nitric oxide. Therefore, increased levels of ADMA are associated with a range of cardiovascular and other diseases. ADMA is biologically eliminated by direct renal excretion and hydrolysis by the enzyme DDAH. Recently, a further elimination pathway via the transamination by the enzyme AGXT2 to α‐keto‐δ‐(N^G,N^G‐dimethylguanidino)valeric acid (DMGV) has come into the focus of biological research. In this work, we describe an assay for the AGXT2 activity in mouse liver and kidney tissue. It is based on the transformation of isotope‐labeled ADMA‐d₆ to DMGV‐d₆. The quantification of the DMGV‐d₆ produced by this reaction in tissue homogenate samples was accomplished by chromatographic separation on a porous graphitic carbon column and tandem mass spectrometric detection. DMGV‐d₆ with the deuterium labels in different molecular positions was used as internal standard. The overall production rates of DMGV‐d₆ in mice were 195.37 pmol/min/mg total protein in liver and 85.21 pmol/min/mg total protein in kidney tissue, with coefficients of variation of 6.31% and 11.25%, respectively. This method can be applied as a tool for the characterization of the ADMA elimination by the AGXT2 pathway. Copyright © 2012 John Wiley & Sons, Ltd.     

On-line system for peptide mapping by capillary electrophoresis at sub-micromolar concentrations

Peptide mapping has been widely used for the identification of modified proteins involved in certain diseases. Despite the fact that capillary electrophoresis (CE) has been shown to be a powerful tool for the separation and detection of tryptic peptide fragments after protein digestion, this technique lacks sensitivity for mapping proteins isolated in very small quantities from biological samples. Consequently, it has been necessary to preconcentrate the protein before adding the proteolytic enzyme for digestion in solution. These experimental steps are quite long, labor intensive and require a lot of sample handling. In this paper, we describe an on-line system allowing digestion of the protein, followed by preconcentration, separation and detection of the tryptic fragments in 4 h. Up to an 800-fold preconcentration factor was achieved for cytochrome c, despite a loss of separation efficiency induced by the multiple-valve design of the system and dispersion of the 60-nl desorption plug. Moreover, our system showed good migration time reproducibility between peptide maps and could be reused for several samples.     

Fast and easy coating for capillary electrophoresis based on a physically adsorbed cationic copolymer

In this work, a new copolymer synthesized in our laboratory is used as physically adsorbed coating for capillary electrophoresis (CE). The copolymer is composed of ethylpyrrolidine methacrylate (EPyM) and methylmethacrylate (MMA). The capillary coating is easily obtained by simply flushing into the tubing an EPyM/MMA solution. It is demonstrated that the composition of the EPyM/MMA copolymer together with the selection of the background electrolyte (BGE) and pH allow tailoring the direction and magnitude of the electroosmotic flow (EOF) in CE. It is also shown that the EOF obtained for the EPyM/MMA-coated capillaries was reproducible in all cases independently on pH or polymer composition. Thus, RSD values lower than 1.9% (n=5) for the same capillary and day were obtained for the migration time, while the repeatability interdays (n=5) was observed to provide RSD values lower than 0.5%. The stability of the coating procedure was also tested between capillaries (n=3) obtaining RSD values lower than 0.6%. It is demonstrated with several examples that the use of EPyM/MMA coatings in CE can drastically reduce the analysis time and/or to improve the resolution of the separations. It is shown that EPyM/MMA-coated capillaries allow the separation of basic proteins by reducing their adsorption onto the capillary wall. Also, EPyM/MMA-coated capillaries provide a faster separation of samples containing simultaneously positive and negative analytes. Moreover, it is demonstrated that the use of EPyM/MMA-coated capillaries can incorporate an additional chromatographic-like interaction with nucleosides that highly improves the separation of this group of solutes.     

Arginylation and methylation double up to regulate nuclear proteins and nuclear architecture in vivo

Protein arginylation and arginine methylation are two posttranslational modifications of emerging importance that involve Arg residues and their modifications. To test a hypothesis that posttranslationally added arginines can be methylated, we used high-precision mass spectrometry and metabolic labeling to find whether posttranslationally added arginines can serve as methylation sites. We identified?a number of proteins in?vivo, on which posttranslationally added Arg have undergone mono- and dimethylation. This double modification predominantly affects the chromatin-containing nuclear fraction and likely plays an important regulatory role in chromatin-associated proteins. Moreover, inhibition of arginylation and Arg methylation results in?a significant reduction of the nucleus size in cultured cells, suggesting changes in chromatin compaction and nuclear architecture. Our findings suggest?a functional link between protein regulation by arginylation and methylation that affects nuclear structure in?vivo.