Determination of methylated arginines by column-switching high-performance liquid chromatography-fluorescence detection

A column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method for the determination of three methylated arginines, N(G)-monomethyl-L-arginine (L-NMMA), N(G),N(G)-dimethyl-L-arginine (asymmetric dimethyl-L-arginine, ADMA), and N(G),N(G)'-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 micromol l(-1), respectively (n= 5).     

A fully automated amino acid analyzer using NBD-F as a fluorescent derivatization reagent

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.     

A new derivatization method coupled with LC-MS/MS to enable baseline separation and quantification of dimethylarginines in human plasma from patients to receive on-pump CABG surgery

Asymmetric dimethylarginine (ADMA) is an inhibitor of nitric oxide synthase and a risk factor for cardiovascular events. We have developed a new derivatization method to enable baseline separation of the regio-isomers, ADMA, and symmetric dimethylarginine (SDMA), within 15 min on a C18 reverse phase column. Reacting naphthalene-2,3-dicarboxaldehyde with ADMA and SDMA in the presence of 2-mercaptoethanol produces corresponding 2,3-dihydro-benzo[f]isoindol-1-ones that are more stable than previously reported ortho-phthaldialdehyde and 2-mercaptoethanol derivatives. LC-MS/MS quantitation of these derivatives can be used to determine ADMA and SDMA concentrations in the plasma of patients to receive on-pump coronary artery bypass grafting (CABG) surgery. The LOD, LOQ and lower LOQ (LLOQ) of this method were determined to be 2.6, 8.7, and 25 nM for ADMA, and 2.5, 8.3, and 25 nM for SDMA, respectively, with consumption of only 50 μL of plasma. The relative standard deviations and relative errors of the intraday and interday determinations, as measurements of reproducibility and accuracy, are all within 15%. The ADMA and SDMA concentrations in patient plasma are 298.1 ± 11.2 nM (mean ± S.E.M., n = 123) and 457.7 ± 19.8 nM (mean ± S.E.M., n = 123), respectively. Upon unblinding of our clinical trial, these predetermined values might explain patient clinical outcomes associated with on-pump CABG surgery, as ADMA is known to inhibit nitric oxide production. Furthermore, this derivatization reaction in conjunction with LC-MS/MS analysis may open a venue to explore alternative chemical labeling modes for LC-MS/MS applications, such as analysis of other amino acids, metabolites, and peptides containing primary amine group(s).     

Evaluation of mobile phase, ion pairing, and temperature influence on an HILIC-MS/MS method for L-arginine and its dimethylated derivatives detection

Asymmetric N(G),-N(G)-dimethylarginine (ADMA) increases in diseases such as renal failure, diabetes mellitus, and hypercholesterolemia. The feasibility and utility of a hydrophilic interaction chromatography (HILIC) method for the separation of free L-arginine (Arg), ADMA, and symmetric N(G),-N(G')-dimethylarginine (SDMA) on a typical silica column were explored and the impact of some experimental parameters on the chromatographic behavior of these analytes was investigated. The effect of water and TFA content in mobile phase and of column temperature was investigated during the development of a fast and simple HILIC-MS/MS method that might be suitable for the quantification of free Arg, ADMA, and SDMA in plasma for routine analysis. Our results show that a good compromise between efficiency and peak shape with acceptable retention and total chromatographic run time is achieved using an ACN/water (90:10) mobile phase with TFA% as additive ranging from 0.015 to 0.025% and column temperature ranging from 25 to 30 degrees C.     

An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 micromol l(-1) (0.5 pmol on column) to 5.0 micromol l(-1) (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 microl of plasma sample for assay.     

Development of hydrophilic fluorogenic derivatization reagents for thiols: 4-(N-acetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-(N-trichloroacetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole

Sensitive, reactive, and hydrophilic fluorogenic reagents for thiols with the benzofurazan skeleton, 4-(N-acetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (AcABD-F) and 4-(N-trichloroacetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (TCAcABD-F) have been developed. These reagents reacted with thiols within 10 min at 60 degrees C. AcABD-F and TCAcABD-F themselves do not fluoresce but are strongly fluorescent after the reaction with thiol compounds. The generated derivatives were highly water-soluble, since they dissociated a proton and ionized in the neutral pH region. The derivatives with four biologically important thiol compounds were separated on a reversed-phase HPLC column and detected fluorometrically at 504 nm with excitation at 388 nm. The detection limit attained for homocysteine with AcABD-F was 25 fmol on column (11 nM) (signal-to-noise ratio = 3), and that for glutathione with TCAcABD-F was 45 fmol on column (20 nM).     

Sensitive fluorometric detection of prostaglandins by high performance liquid chromatography after precolumn labelling with 4-(N,N-dimethylaminosulphonyl)-7-(1-piperazinyl)-2,1,3-benzoxadiazole (DBD-PZ).

A highly sensitive and simple method for the determination of prostaglandins (PGs) by HPLC with fluorescence detection is described. PGs are converted to the corresponding fluorescence derivatives by the reaction with 4-(N,N-dimethylaminosulphonyl)-7-(1-piperazinyl)-2,1,3-benzoxadiaz ole (DBD-PZ) in the presence of 2,2'-dipyridyl disulphide and triphenyl phosphine in acetonitrile. The reaction is completed at room temperature after 30 min. The DBD derivatives of nine PGs are separated within a single 45 min chromatographic run on a reversed phase ODS column with a linear gradient elution using water and acetonitrile. The detection limits (signal-to-noise ratio of 3) calculated from the standard mixture of PGs (6-keto-F1 alpha, F1 alpha, F2 alpha, E1, E2, D2, limaprost, A1 and B1) are in the range 1.7-5.0 fmol. The applicability of the proposed procedure is evaluated to the detection of PGs added to rat plasma.     

LC-APCI-MS法同时分离测定人体血浆中的精氨酸及二甲基精氨酸的含量

建立了简单、灵敏和快速分离测定人体血浆中L-精氨酸(ARG)、不对称二甲基精氨酸(ADMA)和对称二甲基精氨酸(SDMA)的等度高效液相色谱-质谱联用方法.采用选择性离子检测(SIM)和大气压化学电离离子化(APCI),L-高精氨酸作内标,整个方法测定时间在5min以内.ARG,ADMA和SDMA的分析限均为0.2μmol/L,日间和日内测定的精密度分别为2.9%～6.7%和2.1%～5.2%,标准加入回收率为94.0%～105.0%.采用上述方法测定人体血浆中的精氨酸及二甲基精氨酸的含量,结果令人满意.     

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.     

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.     

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 4-hydroxy-2-nonenal in primary rat hepatocyte cultures by liquid chromatography with laser induced fluorescence detection

An HPLC (high performance liquid chromatography) method with laser induced fluorescence (LIF) detection is described for the determination of 4-hydroxy-2-nonenal (HNE) formed from lipid peroxidation in rat hepatocytes. Carbonyl compounds were fluorescently labelled by incubating the hepatocyte samples with a tagging reagent, 4-(2-carbazoylpyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole (NBD-ProCZ), at 60 degrees C for 10 min. The hydrazone derivatives were extracted with a C18 solid phase extraction (SPE) cartridge and separated on a reversed-phase HPLC column. The detection limit was 2.5 fmol or 0.5 nM (5 microL injection) of HNE in the cell homogenate. Method precision (C.V.) was 5% at the 5 nM level. The method has been used to determine free HNE in rat hepatocyte samples treated with several pro-oxidant toxins. A significant HNE increase (from 4 to 27.6 pmol/10(6) cells) was observed with the samples treated by allyl alcohol. The results were in accordance with those for malondialdehyde formation as measured by a thiobarbituric acid (TBA) assay.     

Simultaneous determination of hydrophilic amino acid enantiomers in mammalian tissues and physiological fluids applying a fully automated micro-two-dimensional high-performance liquid chromatographic concept

A validated two-dimensional HPLC system combining a microbore-monolithic ODS column and a narrowbore-enantioselective column has been established for a sensitive and simultaneous analysis of hydrophilic amino acid enantiomers (His, Asn, Ser, Gln, Arg, Asp, allo-Thr, Glu and Thr) and the non-chiral amino acid, Gly, in biological samples. To accomplish this goal, the amino acids were first tagged with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to the respective fluorescent NBD derivatives which were separated in the first dimension by a micro-reversed-phase column. The automatically collected fractions of the target peaks were then transferred to the second dimension consisting of a Pirkle type enantioselective column generating separation factors higher than 1.13 for all the enantiomeric target analytes. The system was validated using standard amino acids and a rat plasma sample, and analytically satisfactory calibration and precision results were obtained. The present 2D-HPLC system enables the fully automated determination of hydrophilic amino acid enantiomers in mammalian samples. The d-isomers of all the investigated 9 amino acids were found in rat urine but at various enantiomeric ratios.     

Selective determination of cysteines through precolumn double-labeling and liquid chromatography followed by detection of intramolecular FRET

In this paper we introduce a novel approach for highly selective and sensitive analysis of cysteines (glutathione, cysteine, and homocysteine). This method is based on the detection of intramolecular fluorescence resonance energy transfer (FRET) in a liquid chromatography (LC) system after double-labeling of the amino and sulfhydryl groups of the cysteines. In this detection process, we monitored the FRET between the amine-derivatized and thiol-derivatized fluorophores. We screened 16 combinations of fluorescent reagents. As a result, FRET occurred most effectively when the sulfhydryl and amino groups of the cysteines were derivatized with 7-diethylamino-3-[{4'-(iodoacetyl)amino}phenyl]-4-methylcoumarin (DCIA, Ex/Em 390/480 nm) and 4-fluoro-7-nitrobenz-2-oxo-1,3-diazole (NBD-F, Ex/Em 480/540 nm), respectively, in this order. The double-labeled cysteines emitted NBD-F fluorescence (540 nm) through an intramolecular FRET process when they were excited at the wavelength of maximum excitation of DCIA (390 nm). The generation of FRET was confirmed by comparison with analysis of n-amylamine or tryptophan (amines without a sulfhydryl group) and 6-mercaptohexanol (thiol without an amino group) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the double-labeled cysteines (DCIA and NBD-F) when performing LC on an ODS column with isocratic elution. The limits of quantification (signal-to-noise ratio = 10) and detection (signal-to-noise ratio = 3) for the cysteines, for a 20-μL injection volume, were in the range 150-670 fmol and 46-200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of a system which takes advantage of conventional detection of the derivatives. Furthermore, this method provides sufficient selectivity and sensitivity to determine the total cysteines present in the plasma of healthy humans.     

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.     

High-performance liquid chromatography/chemiluminescence determination of biological thiols with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide

The peroxyoxalate chemiluminescence detection of biological thiols combined with high-performance liquid chromatography (HPLC) is described. SH groups of the thiol compounds including glutathione (GSH), cysteine, N-acetylcysteine, cysteamine, and D-penicillamine were labelled with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM), a specific fluorogenic reagent for SH group. The labelling reaction was carried out at 60 degrees C for 30 min and at pH 8.5 and a sample of the resulting reaction mixture was subjected to HPLC. Five kinds of labelled thiols were separated within 12 min on ODS-80 column (150 x 4.6 mm ID; 5 microns) and detected in the ranges from 500 fmol to 2 pmol/100 microL (cysteamine and N-acetylcysteine), to 3 pmol/100 microL (cysteine) and to 5 pmol/100 microL (GSH and D-penicillamine). The lower detection limits were from 7 fmol (cysteamine) to 113 fmol (GSH) per 100 microL (S/N = 2). The method was applied to the determination of thiols in a rat liver. The amounts of glutathione and cysteine were 1.23 +/- 0.15 mumol/g (n = 5) and 0.15 +/- 0.04 mumol/g (n = 5), respectively.     

Development of 7-(N,N-dimethylaminosulfonyl)-5-N-(4-N-aminoethyl)piperazino-2,1,3-benzoxadiazole as a water-soluble fluorogenic reagent for the sensitive liquid chromatographic determination of saturated carboxylic acids

A reversed-phase high-performance liquid chromatographic (HPLC) method for the femtomole determination of nine saturated carboxylic acids, n-butyric (C4), n-hexanoic (C6), n-caprylic (C8), n-decanoic (C10), lauric (C12), n-tetradecanoic (C14), palmitic (C16), stearic (C18) and arachidic (C20), based on the condensation reaction of these acids with a newly synthesized water-soluble benzofurazan fluorescent reagent, 7-(N,N-dimethylaminosulfonyl)-4-N-(4-N-aminoethyl)piperazino-2,1,3-benzoxadiazole (DBD-PZ-NH2), was developed. The derivatization reaction proceeds with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) in the presence of the catalyst 4-(dimethylamino)pyridine (DMAP). A model derivative of the reagent with n-caprylic acid (C8) was synthesized for fluorescence excitation/emission characterization. Depending on the solvents, including water, methanol, acetonitrile, 1,4-dioxane or N.N-dimethyformamide (DMF), the C8 derivative has a fluorescence emission with a fluorescence quantum yield (phi) ranging from 0.01 to 0.20 in the region from 545 to 580 nm. An exponential increase in phi was observed with increasing acetonitrile content. The calculated detection limits (signal-to-noise ratio = 3:1) of the proposed method for the above nine carboxylic acids were 9.1, 4.0, 2.5, 2.2, 2.0, 1.8, 1.2, 1.0 and 1.3 fmol, respectively. Biological samples including Intralipos 20% and rat plasma were analysed satisfactorily.     

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.     

A sensitive determination method for mexiletine derivatized with dansyl chloride in rat plasma utilizing a HPLC peroxyoxalate chemiluminescence detection system.

A sensitive determination method for a non-fluorescent anti-arrhythmic drug, mexiletine, in rat plasma is presented utilizing a HPLC peroxyoxalate chemiluminescence (PO-CL) detection system. After an internal standard (4-methylmexiletine, 4.35 pmol) and 0.1 N sodium hydroxide solution were added to 5 microL rat plasma, the solution was poured onto an Extrelut 1 column. Both mexiletine and the internal standard were eluted with diethy ether and then the eluate was evaporated to dryness. The residue was dissolved in 0.2 M borate buffer (pH 8.5) and mixed with dansyl chloride (75 nmol) in acetronitrile. After standing of 90 min at room temperature, 0.5 N HCl was added to the reaction mixture to stop the reaction and a 2/45 aliquot of the mixture was subjected to a HPLC PO-CL detection system using bis(4-nitro-2(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) and hydrogen peroxide. The calibration curve for mexiletine in rat plasma was linear over the range 20-100 ng/mL plasma (20.6-103 fmol/injection). The detection limit (S/N = 2) was 1.0 fmol over the whole procedure. The method was applied to the measurement of the time courses of plasma mexiletine concentration after oral administration of the drug [25 mg (115.9 mumol)/kg] to rats.     

Determination of bisphenol-A in water by semi-micro column high-performance liquid chromatography using 2-methoxy-4-(2-phthalimidinyl)phenylsulfonyl chloride as a fluorescent labeling reagent.

A highly sensitive method for the determination of bisphenol-A in water with semi-micro column high-performance liquid chromatography using 2-methoxy-4-(2-phthalimidinyl)phenylsulfonyl chloride as a fluorescent labeling reagent has been developed. The labeling reaction was carried out at 70 degrees C for 20 min in borate buffer (pH 9.5). The derivative eluted at 11.6 min on a reversed-phase column with methanol-water (78:22, v/v) at a flow-rate of 0.2 ml/min. The fluorescence was monitored at 308 nm for excitation and 410 nm for emission. The detection limit (S/N = 3) was 10 fmol per injection. The labeling yield was about 95%.