O-phthaldialdehyde Derivatives Research Articles

Analysis of Gizzerosine in Foodstuffs by HPLC Involving Pre-column Derivatization with o-Phthaldialdehyde.

Gizzerosine [2-amino-9-(4-imidazolyl)-7-azanonanoic acid] is a toxic amino acid formed from histamine and lysine at high temperatures, and may be present in foodstuffs (e.g., fishmeal and meat-bone meal) for animals including cats and dogs. Here we developed a simple, rapid, sensitive, specific, and automated method for the analysis of gizzerosine in foodstuffs by high-performance liquid chromatography (HPLC) involving pre-column derivatization with o-phthaldialdehyde (OPA) in the presence of N-acetylcysteine (instead of the usual 2-mercaptoethanol or ethanethiol reagent). OPA reacted immediately (within 1 min) with gizzerosine in an autosampler at room temperatures (e.g., 20-25°C), and their derivative was directly injected into the HPLC column. The highly fluorescent gizzerosine-OPA derivative was well separated from the OPA derivatives of all natural amino acids known to be present in physiological fluids (e.g., plasma), proteins and foodstuffs, and was detected at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The total time for chromatographic separation (including column regeneration) was 20 min per sample rather than 40 min and longer in previous HPLC methods. The detection limit for gizzerosine was at least 6 pmol/ml in an assay solution (HPLC vial) or at least0.09pmol per injection into the HPLC column. The analysis of gizzerosine was linear between 1 and 100pmol per injection. When gizzerosine was extracted from foodstuffs, its detection limit was at least 875pmol/g foodstuff or at least0.21 mg/kg foodstuff. Our routine HPLC technique does not require any cleanup of samples or the OPA derivatization products (including the OPA-gizzerosine adduct), and is applicable for the analysis of gizzerosine in bothfoodstuffs and animal tissues.

Determination of enantiomeric excess of some amino acids by second-order calibration of kinetic-fluorescence data

In this investigation a new non-separative kinetic-spectroflourimetric method is proposed for the determination of lysine (lys), leucine (leu) and phenylalanine (phe) enantiomers as their o-phthaldialdehyde (OPA) derivatives in the presence of an optically active chiral thiol compound, 1-mercapto-2-propanol (MP). At ambient temperature and in the borate buffer media of pH 9.6, MP, OPA, as highly selective fluorogenic reagents, and amino acid (AA) enantiomers reacts with each other to yield two fluorescent diasteriomers of D and L-AA with maximum difference in fluorescence intensity at about 450 nm. To achieve information from the small spectral changes, the data are analyzed by Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) method. Linear calibration curves are achieved to distinct D and L-lys, leu and phe in different mole ratios by applying appropriate constraints in MCR-ALS procedures. This is the first application of MCR-ALS in determination of enantiomeric excess (ee) using OPA/MP adduct as chiral reagent, which benefits from direct time dependent-fluorescence spectral analysis and does not require prior separation of chiral analytes. Both the cross-validated correlation coefficient (Q2) and root mean squares error of prediction (RMSEP) indicated satisfactory prediction ability of this method.

Occurrence of Fumonisins B1 and B2 in homemade medicinal plants: Exposure assessment in northern Turkey

This study was conducted to determine the recent level of contamination with Fumonisin B1 (FB1) and Fumonisin B2 (FB2) in major medicinal plants and to assess consumer exposure in northern Turkey. FB1 and FB2 were investigated by using high performance liquid chromatography (HPLC) with fluorescence detection after derivatization with o-phthaldialdehyde (OPA). A total of 78 homemade medicinal plant samples from 14 species were analysed. The recovery in thyme was 67.2±5.2% for FB1 and 80.8±14.3% for FB2 spiked with 1 μg g–1 of each analyte. The minimum detectable amount for the OPA derivatives of FB1 and FB2 were 1 ng per injection and 2.5 ng per injection, respectively. The limit of quantitation (LOQ) S/N=10 was 0.078 and 0.313 μg g–1, and the limit of detection (LOD) S/N=3 was 0.023 and 0.093 μg g–1 for FB1 and FB2, respectively. FB1 was detected in thyme (0.125) and mint (0.125 and 0.256 μg g–1) samples; however. FB2 toxin was below the detection limit in all samples. These results indicate that toxins might be present in homemade medicinal plants; however, the risk of exposure to fumonisins by the consumption of those plants was lower than the estimated TDI limits (<2 μg kg–1 bw).

Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection.

Studies of protein nutrition and biochemistry require reliable methods for analysis of amino acid (AA) composition in polypeptides of animal tissues and foods. Proteins are hydrolyzed by 6M HCl (110°C for 24h), 4.2M NaOH (105°C for 20 h), or proteases. Analytical techniques that require high-performance liquid chromatography (HPLC) include pre-column derivatization with 4-chloro-7-nitrobenzofurazan, 9-fluorenyl methylchloroformate, phenylisothiocyanate, naphthalene-2,3-dicarboxaldehyde, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and o-phthaldialdehyde (OPA). OPA reacts with primary AA (except cysteine or cystine) in the presence of 2-mercaptoethanol or 3-mercaptopropionic acid to form a highly fluorescent adduct. OPA also reacts with 4-amino-1-butanol and 4-aminobutane-1,3-diol produced from oxidation of proline and 4-hydroxyproline, respectively, in the presence of chloramine-T plus sodium borohydride at 60°C, or with S-carboxymethyl-cysteine formed from cysteine and iodoacetic acid at 25°C. Fluorescence of OPA derivatives is monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Detection limits are 50 fmol for AA. This technique offers the following advantages: simple procedures for preparation of samples, reagents, and mobile-phase solutions; rapid pre-column formation of OPA-AA derivatives and their efficient separation at room temperature (e.g., 20-25°C); high sensitivity of detection; easy automation on the HPLC apparatus; few interfering side reactions; a stable chromatography baseline for accurate integration of peak areas; and rapid regeneration of guard and analytical columns. Thus, the OPA method provides a useful tool to determine AA composition in proteins of animal tissues (e.g., skeletal muscle, liver, intestine, placenta, brain, and body homogenates) and foods (e.g., milk, corn grain, meat, and soybean meal).

Differentiation of Allium subgenera by cysteine sulphoxides and further amino acids

The genus Allium (>800 species) has been divided into several subgenera, sections, species and subspecies [1]. Cysteine sulphoxides are most prominent amino acid derivatives of this genus. These substances are susceptible for the enzyme alliinase. Alllinase reaction and further non-enzymatic steps do result in volatile sulphur compounds, which exhibit various bioactivities, e.g., antibiotic activities. The pattern of cysteine sulphoxides is rather characteristic for some subgenera. For instance, the subgenus Melanocrommyum is characterized by different heteroaromatic cysteine sulphoxides, whereas the subgenus Cepa typically shows rather high amounts of isoalliin. In the now presented study, amino acids as well as cysteine sulphoxides have been analyzed as their corresponding o-phthaldialdehyde (OPA) derivatives. Besides the above mentioned differences in the pattern of cysteine sulphoxides, also further amino acids show some relations between their amounts and subgenera. Most prominent are differences in the content of L-arginine in different subgenera of the genus Allium. L-arginine is a valuable nitrogen storage compound in many angiosperms [2]. But this amino acid has been also found in some Allium species in high concentrations. As an example, the amount of L-arginine in Allium cepa L. was increased during the maturation process [3]. Further on, the amount of L-arginine in samples belonging to the subgenera Allium and Cepa was significant higher (p<0.05) as the amount of L-arginine found in the subgenus Melanocrommyum suggesting different mechanisms of nitrogen storage. These differences should allow chemo-taxonomical classification of subgenera.

Allium Species from Central and Southwest Asia Are Rich Sources of Marasmin

Marasmin, which is especially known from the two South African species Tulbaghia alliacea and Tulbaghia violacea , but was also described for the garlic mushroom Marasmius alliaceus , is the precursor of the thiosulfinate marasmicin. Marasmicin has attracted considerable attention because of its antifungal and tuberculostatic activities. However, many Allium species of the subgenus Melanocrommyum, especially Allium suworowii , are also very rich in marasmin. A. suworowii revealed concentrations of marasmin up to 1.6%, related to the fresh weight of bulbs, and up to 3.0%, related to air-dried fruiting bodies, of the corresponding γ-glutamylmarsmin was found in M. alliaceus. Both species show much higher amounts of marasmin as Tulbaghia and could be considered as natural sources for the isolation of this compound. Further promising Allium species with considerable amounts of marasmin besides other cysteine sulfoxides are Allium stipitatum and Allium altissimum . (R(S),R(C))-Marasmin is typical for the investigated species of the subgenus Melanocrommyum, whereas γ-glutamyl-(S(S),R(C))-marasmin is the only cysteine sulfoxide for the genus Marasmius known until now. Both cysteine sulfoxides were isolated and described as o-phthaldialdehyde (OPA) derivatives. Furthermore, the cysteine sulfoxides methiin, propiin, S-(2-pyrrolyl)-cysteine sulfoxide, eventually S-(2-pyridyl)-cysteine sulfoxide and S-(2-pyridyl)-L-cysteine N-oxide were found.

Targeted analysis of sphingoid precursors in human biofluids by solid-phase extraction with in situ derivatization prior to μ-LC-LIF determination

A method for determination of two relevant sphingoid precursors such as sphingosine and sphinganine and the corresponding conjugates sphingosine 1-phosphate and sphinganine 1-phosphate in human urine and serum is here presented. The method is characterized by a solid- phase extraction step with in situ derivatization of the sphingolipids in the eluate (o-phthaldialdehyde derivatives) to obtain fluorescent compounds. In this way, sample preparation was completely performed in a single automated step by means of a lab-on-valve system. Derivatized analytes were injected into a liquid chromatography system operating at micro regime and detected by laser-induced fluorescence. For determination of sphingoid phosphates, they were enzymatically converted to free sphingoids to obtain stable fluorescent derivatives. The detection limits were in the range 4.2-10.2ng mL(-1) for serum and 0.56-1.36ng mL(-1) for urine, with repeatability ranging from 3.9% to 6.2% expressed as relative standard deviation. The method was validated by direct infusion tandem mass spectrometry in multiple reaction monitoring to compare results provided by analysis of biofluids and to confirm the identity of the target compounds. Sensitivity and precision were better than or similar to those provided by the confirmatory method. The automation of sample preparation enables to scale-down this step and improves precision by minimization of human intervention, being thus suitable for clinical analysis.

ChemInform Abstract: NHC‐Catalyzed Spiro Bis‐Indane Formation via Domino Stetter—Aldol—Michael and Stetter—Aldol—Aldol Reactions.

Two novel domino NHC-catalyzed spirocyclizations are described herein, enabling the rapid construction of three new carbon−carbon bonds and a quaternary center with high diastereoselectivity. A variety of spiro bis-indane structures are assembled in a single step from simple o-phthaldialdehyde derivatives.

NHC-Catalyzed Spiro Bis-Indane Formation via Domino Stetter−Aldol−Michael and Stetter−Aldol−Aldol Reactions

Two novel domino NHC-catalyzed spirocyclizations are described herein, enabling the rapid construction of three new carbon-carbon bonds and a quaternary center with high diastereoselectivity. A variety of spiro bis-indane structures are assembled in a single step from simple o-phthaldialdehyde derivatives.

Cysteine Sulfoxides and Volatile Sulfur Compounds fromAllium tripedale

Allium tripedale Trautv. belonging to the subgenus Nectaroscordum grows naturally in the mountainous areas of northwest Iran. Leaves have a very strong and somewhat unpleasant taste and are widely used by the local population as a spicy vegetable. Three new cysteine based compounds were identified in A. tripedale as the first examples of sulfur containing 1-butenyl derivatives in nature. The compounds have been described as o-phthaldialdehyde (OPA) derivatives and identified as (+)-S-(1-butenyl)-L-cysteine sulfoxide (homoisoalliin) and its gamma-glutamyl derivative as well as the gamma-glutamyl derivative of S-(1-butenyl)-L-cysteine (desoxyhomoisoalliin). These cysteine sulfoxides have been also found in Allium siculum Ucria. As volatile compounds, di-(1-butenyl)-disulfide and the cepaene-like compounds di-(1-S-sulfoxymethyl-butyl)-disulfide, 1-S-sulfoxymethyl-butyl-1'-S-sulfoxy-1-butenyl-butyl-disulfide and 1-S-sulfoxymethyl-butyl-1'-S-sulfoxybutyl-butyl-disulfide could be tentatively identified by various MS experiments. Primary products resulting from the alliinase reaction of homoisoalliin seem to be highly unstable and were rapidly converted to the volatile compounds listed above.

Determination of fumonisins in maize by HPLC with ultraviolet detection of o-phthaldialdehyde derivatives

Fumonisins are mycotoxins that are produced by various Fusarium species and occur naturally in maize and maize-based foods. Fumonisins are carcinogenic, causing liver cancer in rats, and are associated with oesophageal cancer and neural tube defects in humans. Analytical methods for individual fumonisin analogues in maize rely on reversed-phase high-performance liquid chromatographic (HPLC) separation after suitable extraction and clean-up. As fumonisins lack a useful chromophore or fluorophore, HPLC detection is achieved by suitable derivatization and sensitive, specific fluorescence detection. A widely used and validated method involves extract clean-up on strong anion exchange solid phase extraction cartridges and pre-column derivatization with o-phthaldialdehyde (OPA). However, many laboratories requiring infrequent fumonisin analysis are only equipped with HPLC with ultraviolet (UV) detection. A HPLC system equipped with both UV and fluorescence detectors connected in series was used to determine the extent to which UV offers an alternative to fluorescence detection in the above analytical method. Comparison of the detection systems using fumonisin standards indicated that fluorescence is about 20-times more sensitive than UV. Analysis of maize samples with differing fumonisin contamination levels indicated that, at fumonisin B1 levels above 1,000µg/kg, the two detection systems were comparable and gave repeatabilities equal or less than 10% on six replicate analyses. Although a sensitive fumonisin analysis requires fluorescence detection, UV may offer an alternative in certain circumstances.

Cysteine sulphoxides, amino acids and alliinase activity of Allium nigrum

Allium nigrum L. belongs to the subgenus Melanocrommyum of the genus Allium. This subgenus is widely distributed in Southwest and Central Asia. However, A. nigrum is a typical plant of east Mediterranean islands. Many species of this subgenus were traditionally used. A. nigrum is reported to be active against helminthiasis. Typical for the genus Allium is a rather high content of cysteine sulphoxides. Most abundant is the cysteine sulphoxide methiin (1). Recently, an unusual cysteine sulphoxide containing a pyrrole ring system (2) could be reported [1]. Both compounds were also present in A. nigrum. Amino acid derivatives were analysed as corresponding o-phthaldialdehyde derivatives by means of HPLC-MS/MS. For methiin (1) and the pyrrole derivative (2), average concentrations of 0.04% and 0.01%, respectively, were found (concentrations related to the fresh weight of bulbs). If these cysteine sulphoxides would be incubated with the enzyme alliinase, a number of thiosulphinates can be expected. Enzymatic incubations were also performed for the low molecular weight extract of A. nigrum and resulting compounds were analyzed by HPLC-MS/MS. Interestingly, only the pyrrole derivative (3) could be detected. It is completely unclear in which manner methiin (1) reacts with the alliinase of A. nigrum. It can be supposed that this alliinase acts different from the well described alliinase of A. sativum (garlic).

A new pyridine cysteine-sulphoxide identified in Allium stipitatum

Allium stipitatum Regel is known as ornamental plant in Europe. In Central Asia it is used as spice and in folk medicine and known as „Musir“ or „Anzur“. Allium stipitatum belongs to subgenus Melanocrommyum section Megaloprason. A newly developed HPLC-MS/MS method was used for screening on amino acids, amines and sulphoxides. Amino acid derivatives were analysed as corresponding o-phthaldialdehyde derivatives. Besides the already described cysteine sulphoxide methiin, a new compound was identified, which is a 2 pyridyl-S-L-cysteine sulphoxide (Figure). Structure elucidation was performed by HRESI, NMR, IR and polarometric measurements. This compound could also be found in further members of the section Megaloprason and is probably useful as chemical marker for this group. Interestingly, the recently described 3 pyrrol-S-(+)-L-cysteine sulphoxide [1], which is also characteristic for the subgenus Melanocrommyum, could not be found in these species carrying the corresponding pyridine derivative. It can be assumed that both biogenetic pathways are excluding each other. Additionally to the above described structure elucidation, the alliinase of A. stipitatum could be partially characterized.

Photodecomposition of o-phthaldialdehyde-derivatized amino acids by the photodiode array detector during their high-performance liquid chromatographic analysis

During the high-performance liquid chromatographic (HPLC) analysis of o-phthaldialdehyde (OPA)-derivatized amino acids such as arginine and homoarginine, we observed that the response of the fluorescence detection (FLD) system is decreased when the photodiode array detection (DAD) system (located before the FLD system) was simultaneously switched on. The decrease in the FLD response, i.e., the difference in the FLD peak area (Δ A) obtained with DAD modes off ( A off) and on ( A on) was dependent upon the flow rate, but the relative FLD response decrease (Δ A/ A off) was practically independent of the amount of analyte injected. For example, decreasing the flow rate from 1 to 0.5 mL/min resulted in the relative decrease of FLD response from ∼5% to 11%. It was concluded that DAD caused a photoinduced partial decomposition of the OPA-derivatized amino acids flowing through the cell. The photoinduced dissociation of OPA derivatives was independently supported by using off-line photodiode array spectrometric measurements with long and short irradiation pulses. Based on the experimental results, for description of the variation of FLD responses with the flow rate upon the irradiation by DAD a simple mathematical model is proposed and reported.

Postcolumn reactor using a laser‐drilled capillary for light‐emitting diode‐induced fluorescence detection in CE

This study investigated a novel postcolumn reactor for fluorescence detection in CE. A laser-drilled capillary, with an aperture made by laser ablation, was used for mixing derivatization reagents with the analytes separated by CZE. The derivatization reagents, o-phthaldialdehyde (OPA), and 2-mercaptoethanol, were introduced into the capillary through the aperture and reacted with the analytes after CZE separation. High voltages were applied to both the inlet reservoir and the reservoir filled with the derivatization reagents. Thus, the flow rate of the derivatization reagents was controlled by the electric potential that was applied to the reservoir of the derivatization reagents. A UV light-emitting diode was used as an excitation light source for the fluorescence detection of OPA derivatives. A commercially available tee connector was compared with the laser-drilled capillary. The results implied that the dead volume of the laser-drilled capillary was less than that of the tee connector, since the laser-drilled capillary suppressed band broadening more efficiently. The LODs for amino acids were determined to be approximately 5 microM. The method was applied to the determination of amino acids in a Japanese beverage.

Structure and Natural Occurrence of Stereoisomers of the Fumonisin B Series Mycotoxins

1H and 13C NMR spectroscopy of both fumonisin B3 and B4, as well as high-performance liquid chromatography (HPLC) analysis of samples of fumonisin B3 used as standards, showed in each case the presence of two stereoisomers, which could not be separated by preparative chromatography. The 2,3-anti relative configuration for the two minor stereoisomers of fumonisin B3 and B4 was deduced from the NMR data, and their 2S,3R absolute configurations were established by application of Mosher's method using the fumonisin B3 sample. Samples of fumonisin B3 and B4 can contain between 10 and 40% of fumonisin B compounds of the 3-epi series. The 3-epi-FB3, determined by HPLC with fluorescence detection of the o-phthaldialdehyde derivative and confirmed by liquid chromatography-tandem mass spectrometry, was found to occur naturally in a range of maize samples at levels much lower than FB3 (< 20%). The identification of members of the 3-epi-fumonisin B series provides insight into the order and selectivity of steps in fumonisin biosynthesis.

A comparative study of amino acid measurement in leaf extracts by gas chromatography-time of flight-mass spectrometry and high performance liquid chromatography with fluorescence detection

Gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) has become a promising technique for simultaneous and rapid analysis of small metabolites in complex mixtures. The aim of this work was to establish the quantitative nature of the information generated by amino acid analysis of crude leaf extracts using GC-TOF-MS. Dried aliquots of methanol/water extracts of Arabidopsis leaves were analysed in parallel by GC-TOF-MS following trimethylsilylation or high performance liquid chromatography and fluorescence detection of o-phthaldialdehyde derivatives (OPA-HPLC). Twenty amino acids could be routinely detected in leaf extracts by both methods. Because of instability of some trimethylsilylated derivatives, all GC-TOF-MS analyses were performed within a window of 2 h 30 min following derivatization. Repeatability studies showed that relative standard deviations for multiple injections of a single extract were below 20% for both techniques, though significantly smaller for OPA-HPLC. Similar between-extract variability and condition-independent biological variation were detected by OPA-HPLC and GC-TOF-MS, and both techniques detected similar environmentally induced changes in four major amino acids. Recovery of standard compounds through the extraction procedure was between 80% and 120% for OPA-HPLC but more variable when analysed by GC-TOF-MS. When quantified on the basis of tissue fresh weight according to response factors of mixed standards, the two techniques gave consistent values for a number of amino acids but divergent values for others. Taken together, the results suggest GC-TOF-MS analysis of Arabidopsis leaves with the present protocol can be used for absolute quantification of 4–7 amino acids, accurate relative quantification of 8–11 amino acids, and more limited quantification for five compounds of this class.

Separation and Identification of Major Plant Sphingolipid Classes from Leaves

Sphingolipids are major components of the plasma membrane, tonoplast, and other endomembranes of plant cells. Previous compositional analyses have focused only on individual sphingolipid classes because of the widely differing polarities of plant sphingolipids. Consequently, the total content of sphingolipid classes in plants has yet to be quantified. In addition, the major polar sphingolipid class in the model plant Arabidopsis thaliana has not been previously determined. In this report, we describe the separation and quantification of sphingolipid classes from A. thaliana leaves using hydrolysis of sphingolipids and high performance liquid chromatography (HPLC) analysis of o-phthaldialdehyde derivatives of the released long-chain bases to monitor the separation steps. An extraction solvent that contained substantial proportions of water was used to solubilized >95% of the sphingolipids from leaves. Neutral and charged sphingolipids were then partitioned by anion exchange solid phase extraction. HPLC analysis of the charged lipid fraction from A. thaliana revealed only one major anionic sphingolipid class, which was identified by mass spectrometry as hexose-hexuronic-inositolphosphoceramide. The neutral sphingolipids were predominantly composed of monohexosylceramide with lesser amounts of ceramides. Extraction and separation of sphingolipids from soybean and tomato showed that, like A. thaliana, the neutral sphingolipids consisted of ceramide and monohexosylceramides; however, the major polar sphingolipid was found to be N-acetyl-hexosamine-hexuronic-inositolphosphoceramide. In extracts from A. thaliana leaves, hexosehexuronic-inositolphosphoceramides, monohexosylceramides, and ceramides accounted for approximately 64, 34, and 2% of the total sphingolipids, respectively, suggesting an important role for the anionic sphingolipids in plant membranes.

Quantification of ADMA: analytical approaches

Methylated L-arginine analogs are involved in nitric oxide synthase activity regulation. Methods based on high-performance liquid chromatography with fluorescence, capillary electrophoresis, or ion exchange chromatography with absorbance detection were first applied for the quantitative determination of N-monomethyl-L-arginine (NMMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in human blood and urine. These assays revealed elevated circulating levels of ADMA in various diseases and gave accumulating evidence of the usefulness of ADMA as a cardiovascular risk factor. However, the methods used are hampered by the fact that NMMA, ADMA and SDMA can be distinguished from L-arginine only by means of chromatographic separation. This has promoted the development of alternatives that involve mass spectrometry (MS) technology. Today, various MS-based approaches such as liquid chromatography (LC)-MS, LC-MS/MS, gas chromatography (GC)-MS, and GC-MS/MS are available. L-arginine and its analogs have been subjected to LC-MS analysis with and without further derivatization to their o-phthaldialdehyde derivatives. For these methods, labelled L-arginine was used as the internal standard. The first MS-based method that distinguishes NMMA, ADMA, SDMA and L-arginine by mass-to-charge (m/z)-ratio has been reported by Tsikas et al. This GC-MS approach has been further improved by Albsmeier et al by introducing labelled ADMA as an internal standard. As an alternative to existing methods, a commercially available ELISA kit has recently been developed and validated.

Quantification of ADMA: analytical approaches

Methylated L-arginine analogs are involved in nitric oxide synthase activity regulation. Methods based on high-performance liquid chromatography with fluorescence, capillary electrophoresis, or ion exchange chromatography with absorbance detection were first applied for the quantitative determination of N-monomethyl-L-arginine (NMMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in human blood and urine. These assays revealed elevated circulating levels of ADMA in various diseases and gave accumulating evidence of the usefulness of ADMA as a cardiovascular risk factor. However, the methods used are hampered by the fact that NMMA, ADMA and SDMA can be distinguished from L-arginine only by means of chromatographic separation. This has promoted the development of alternatives that involve mass spectrometry (MS) technology. Today, various MS-based approaches such as liquid chromatography (LC)-MS, LC-MS/MS, gas chromatography (GC)-MS, and GC-MS/MS are available. L-arginine and its analogs have been subjected to LC-MS analysis with and without further derivatization to their o-phthaldialdehyde derivatives. For these methods, labelled L-arginine was used as the internal standard. The first MS-based method that distinguishes NMMA, ADMA, SDMA and L-arginine by mass-to-charge (m/z)- ratio has been reported by Tsikas et al. This GC-MS approach has been further improved by Albsmeier et al by introducing labelled ADMA as an internal standard. As an alternative to existing methods, a commercially available ELISA kit has recently been developed and validated.