High-performance liquid chromatographic determination of taurine in biological fluids by post-column fluorescence reaction with thiamine.

A high-performance liquid chromatographic method is described for the selective determination of taurine in biological fluids by post-column fluorescence reaction. Taurine was separated on an adsorption-distribution type Shodex Ionpac KC-811 column. Then it was converted with hypochlorite into the corresponding N-chloramine, which was allowed to react with thiamine to give fluorescent thiochrome. As little as 6 ng per injection of taurine could be determined. The average recoveries of spiked taurine in serum and urine were 99.5 +/- 2.7 and 101.8 +/- 2.9%, respectively. The method could be applied to the assay of taurine in human serum and urine with simple pretreatment.     

Analysis of Taurine in Blood Plasma of Epileptic Patients Using an Improved Isocratic HPLC Method for Amino Acids

Abstract

An efficient, reversed-phase HPLC method is described in which 21 derivatized amino acids were isocratically separated in less than 90 minutes. An internal standard was used to improve precision and accuracy. The method, which separated taurine from α-, β-, and γ-aminobutyric acids, was used to quantify taurine levels in 83 blood plasma samples. A statistical comparison was made between taurine levels found in the plasma of epileptic and non-epileptic children.     

Determination of taurine in plasma by capillary zone electrophoresis following derivatisation with fluorescamine

A novel capillary zone electrophoresis method is described for the determination of taurine in plasma. The method is rapidly executed and is highly selective for taurine as separation is based on the difference in ionisation of this amino acid from that of other amino acids. Following addition of homotaurine as internal standard, plasma proteins were precipitated with acetonitrile and the supernatant was derivatised with fluorescamine in the presence of a borate buffer. Capillary electrophoresis (CE) separations were carried out in reverse polarity mode at 27.5 kV on a Beckman P/ACE MDQ CE instrument, equipped with a diode array detector (DAD) set at 266 nm. The sample tray was cooled to 5 degrees C and separations were carried out at 20 degrees C. The fused-silica capillary was 50.2 cm in length (40.2 cm to detector) with an internal diameter of 75 microm. A capillary conditioning solution was applied daily in order to suppress the residual electroosmotic flow (EOF). The method, which was validated using feline plasma as the blank matrix, was shown to be linear and reproducible over the concentration range 2.5-100 microg/mL. The coefficients of variation (CVs) of replicate analyses were less than 4.5% at 1 microg/mL taurine in feline plasma and less than 3% for 2.5 microg/mL in human plasma. Recovery was estimated at 99.2% with a CV of 4.85%. It has been demonstrated that quantitation in aqueous solution yields similar results to those obtained by interpolation on a plasma calibration curve provided that subtraction for the taurine peak in unspiked plasma is carried out and that a suitable internal standard is employed.     

Two high‐performance liquid chromatography–tandem mass spectrometry methods for determination of edaravone and taurine in human plasma: Application to drug–drug interaction and pharmacokinetic studies

Two high‐performance liquid chromatography–tandem mass spectrometry methods were developed and validated for the quantification of edaravone (method A) or taurine (method B) in human plasma. After protein precipitation, separations were achieved on an Ultimate XB‐C8 (2.1 × 50 mm, 3.0 µm) column for edaravone and a ZORBAX SB‐Aq column (2.1 × 100 mm, 3.5 µm) for taurine, respectively. The detection used electrospray ionization source via multiple reaction monitoring in positive‐ion mode for edaravone and negative‐ion mode for taurine, respectively. The lower limits of quantification were 10.0 ng/mL for edaravone and 3.00 μg/mL for taurine. The selectivity, accuracy, and precision of the methods were all within acceptable limits. Two methods were successfully applied to a drug–drug interaction study and a pharmacokinetic study of edaravone and taurine in healthy Chinese volunteers after intravenous infusion of single or compound injection. The results showed that co‐administration of edaravone with taurine increased the C_max and AUC_0‐24 of taurine in human plasma while taurine did not affect the systemic exposure of edaravone. Edaravone and taurine have the dose‐dependent pharmacokinetic profiles in human.     

HPLC determination of plasma taurine

Summary The taurine content in plasma was determined by high performance liquid chromatographic analysis of its dansyl derivative with fluorometric detection. After the reaction with dansyl chloride, the derivative was extracted from an aqueous mixture by using tetrabutylammonium as a counter-ion. The influences of different tetrabutylammonium salts and of the eluent mixture composition were studied. Omotaurine, added as the internal standard to the plasma samples, assured good reproducibility. The procedure was applied to the determination of taurine in specimens from different mammalian species.     

Analysis of taurine in feline plasma and whole blood by liquid chromatography with fluorimetric detection and confirmation by thermospray mass spectrometry

A liquid chromatographic method with fluorimetric detection was developed to measure taurine (2-aminoethanesulfonic acid) in feline plasma and whole blood. Plasma or lysed whole blood was diluted with a mixture of acetonitrile-methanol-triethylamine-water (25:22:3:50, v/v), filtered through a 10,000 dalton exclusion filter and derivatized with dansyl chloride for 30 min at room temperature. Dansyl taurine was separated from other compounds by reversed-phase liquid chromatography using an octadecyl column and a methanol-acetic acid-triethylamine (30:0.5:0.025, v/v) aqueous mobile phase. The effluent was monitored fluorimetrically at an excitation wavelength of 329 nm and an emission wavelength of 530 nm. The presence of mono-dansylated taurine in feline plasma was confirmed by thermospray mass spectrometry. The limit of detection was 16 nmol/ml and the detector response was linear from 40 to 4000 nmol/ml taurine.     

Determination of catecholamines and their 3-O-methyl metabolites in mouse plasma

The determination of catecholamines and their 3-O-methyl metabolites in a single mouse plasma is necessary to understand the role of the sympathetic nervous activity, while the inactivation of catecholamines by catechol-O-methyltransferase indicates the activity of blood pressure regulation in animals. Here we report the basal catecholamines and their 3-O-methyl metabolite concentrations obtained from 15 microL of mouse plasma utilizing semi-microcolumn high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence detection system. The concentrations were 6.63 +/- 1.37 pmol/mL plasma, 0.49 +/- 0.10 pmol/mL plasma, 5.25 +/- 2.30 pmol/mL plasma, 3.23 +/- 0.84 pmol/mL plasma, 0.44 +/- 0.11 pmol/mL plasma, and 3.39 +/- 1.67 pmol/mL plasma for norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine, respectively (n = 5-7). Further, when blood pressure was reduced by minoxidil, plasma catecholamines were found to be significantly increased by the baroreflex-mediated response in mouse.     

Study on the interaction of plasma protein binding rate between edaravone and taurine in human plasma based on HPLC analysis coupled with ultrafiltration technique

In this work, two high‐performance liquid chromatography (HPLC) assays were developed and validated for the independent determination of edaravone and taurine using 3‐methyl‐1‐p‐tolyl‐5‐pyrazolone and L ‐glutamine as internal standards. In in vitro experiments, human plasma was separately spiked with a mixture of edaravone and taurine, edaravone or taurine alone. Plasma was precipitated with acetonitrile containing 0.1% formic acid. Ultrafiltration was employed to obtain the unbound ingredients of the two drugs. The factors that might influence the ultrafiltration effiency were elaborately optimized. Plasma supernatant and ultrafiltrate containing taurine were derivated with o‐phthalaldehyde and ethanethiol in the presence of 40 mmol/L sodium borate buffer (pH 10.2) at room temperature within 1 min. Chromatographic separations were achieved on an InertSustain C₁₈ column (250 × 4.6 mm, 5 µm). Isocratic 50 mmol/L ammonium acetate–acetonitrile and gradient 50 mmol/L sodium acetate (pH 5.3)–methanol were respectively selected as the mobile phase for the determination of edaravone and taurine. All of the validation data including linearity, extraction recovery, precision, accuracy and stability conformed to the requirements. Results showed that there were no significant alterations in the plasma protein binding rate of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of aromatic and branched-chain amino acids in plasma by HPLC with electrogenerated Ru(bpy)3(3+) chemiluminescence detection

An HPLC method is described for the electrochemiluminescence (ECL) detection of amino acids, following cycloaddition reaction of their amino groups with divinyl sulfone (DVS), using electrogenerated tris(bipyridine)ruthenium(III). The derivatization reaction conditions were examined, with the optimum conditions found to be 40 mM DVS (pH 8.0) at 50 degrees C for 15 min. Detection limits for the 15 amino acids examined varied greatly (0.04-8.0 pmol) using a standard solution by flow injection analysis (FIA). These optimized conditions were used for HPLC determination of the amino acids in human plasma. A linear relationship was obtained up to 100 pmol on a column for aromatic and branched-chain amino acids. Recoveries of Tyr, Met, Val, Leu, Ile, Phe and Trp when added to human plasma (1 micromol/10 ml plasma, n=5) were 101.5+/-1.1, 99.0+/-1.2, 98.0+/-1.4, 101.1+/-1.6, 95.1+/-1.6, 99.2+/-1.5 and 97.7+/-1.3 % (mean+/-S.D.) respectively. The concentrations of the amino acids in the plasma are in good agreement with other published data.     

Measurements by microdialysis of free tissue concentrations of propranolol.

To determine the free concentration of a drug (propranolol) in the interstitial space in humans in vivo, seven male students were investigated by microdialysis of the periumbilical subcutaneous tissue. The microdialysis catheters were calibrated in vivo and the propranolol concentration was determined by high-performance liquid chromatography. Ten hours after intake of 80 mg of propranolol, the total plasma and free interstitial propranolol concentrations were 80 +/- 43 and 7 +/- 2 nM, respectively. After a second dose, maximum concentration was reached after 80 +/- 10 min and 98 +/- 12 min, in plasma, and the concentrations in the interstitial water were 594 +/- 138 and 27 +/- 7 nM, respectively. In a second study, microdialysis was performed on the left ventricular wall in six pigs receiving an intravenous injection of 5 mg of propranolol followed by a constant propranolol infusion for 40 min (5 mg propranolol per h). The maximum concentrations of propranolol were 97 +/- 29 and 6 +/- 2 nM in plasma and in interstitial water, respectively. The data suggest that microdialysis is a useful tool for recording the free concentrations of a drug in the interstitial space.     

Determination of free intracellular amino acids in single mouse peritoneal macrophages after naphthalene-2,3-dicarboxaldehyde derivatization by capillary zone electrophoresis with electrochemical detection.

A method is described for the direct identification and quantification of amino acids in individual mouse peritoneal macrophages by capillary zone electrophoresis with electrochemical detection after on-column derivatization with naphthalene-2,3-dicarboxaldehyde (NDA) and CN-. In this method, individual macrophages and then the lysing/ derivatizing buffer are injected into the front end of the separation capillary by electromigration with the aid of an inverted microscope. The front end of the separation capillary is used as a chamber to lyse the macrophage and derivatize its contents, which minimizes dilution of amino acids of a single macrophage during derivatization. Six amino acids (serine, alanine, taurine, glycine, glutamic acid, and aspartic acid) in single mouse peritoneal macrophages have been identified. Quantitation has been accomplished through the use of calibration curves, where the concentration ratios of these standard amino acids are similar to the concentration ratios of amino acids in macrophages. Cellular levels of the amino acids in these cells range from 0.27 +/- 0.20 fmol/ cell for alanine to 6.4 +/- 4.6 fmol/cell for taurine.     

Spectrophotometric determination of taurine in food samples with phenol and sodium hypochlorite as reagents and an ion-exchange clean-up

A simple and accurate spectrophotometric method is proposed for the determination of taurine in food samples using phenol and sodium hypochlorite as reagents, which form a blue colour with taurine at room temperature and pH 10.35. Ion exchange was used to improve the selectivity of the method. Absorbance measurements were made at 630 nm and the calibration graph was linear from 0 to 180 micrograms ml-1 of taurine with a slope of 0.00242 A (p.p.m.)-1. The precision for the determination of taurine (156 micrograms ml-1) was 0.8% (n = 10). The method was applied successfully to the determination of taurine in milk products and energy drinks.     

Simple and sensitive determination of plasma N tau-methylhistidine by high-performance liquid chromatography using pre-column derivative formation with o-phthalaldehyde-2-mercaptoethanol

A simple, rapid and sensitive assay method for plasma N tau-methylhistidine by isocratic high-performance liquid chromatography has been developed. The deproteinized plasma was treated with o-phthalaldehyde-2-mercaptoethanol. The derivatives were separated on a LiChrospher 100 RP-18 column within 10 min. The detection limit for N tau-methylhistidine was 0.5 pmol. The plasma N tau-methylhistidine content of beef cattle and dairy cows was 0.038 +/- 0.004 and 0.017 +/- 0.002 nmol/ml/kg, respectively.     

High-performance liquid chromatographic analysis of isoxicam in human plasma and urine

A sensitive, selective, and rapid high-performance liquid chromatographic procedure was developed for the determination of isoxicam in human plasma and urine. Acidified plasma or urine were extracted with toluene. Portions of the organic extract were evaporated to dryness, the residue dissolved in tetrahydrofuran (plasma) or acetonitrile (urine) and chromatographed on a mu Bondapak C18 column preceded by a 4-5 cm X 2 mm I.D. column packed with Corasil C18. Quantitation was obtained by UV spectrometry at 320 nm. Linearity in plasma ranged from 0.2 to 10 micrograms/ml. Recoveries from plasma samples seeded with 1.8, 4 and 8 micrograms/ml isoxicam were 1.86 +/- 0.077, 4.10 +/- 0.107 and 8.43 +/- 0.154 micrograms/ml with relative standard deviations of 3.3%, 2.5% and 5.4%, respectively. The linearity in urine ranged from 0.125 to 2 micrograms/ml. The precision of the method was 3.3-9.0% relative standard deviation over the linear range.     

A Rapid Determination of Taurine in Human Plasma by LC

Taurine is an amino acid which is not incorporated into proteins but found in the cytosol of many mammalian cells, in high concentrations (2–30 mM). Increase in plasma taurine concentration has already been reported after surgical trauma, X-radiation, muscle necrosis, carbon tetrachloride-induced liver damage, and paracetamol overdose. Plasma taurine concentration was measured using LC with fluorescence detection following derivatization by o-phtalaldehyde plus 3-mercapto-propionic acid and α-aminobutyric acid as internal standard. Under these conditions the retention time of taurine was 10 min. This method was sensitive enough, to quantify 150 pg mL^?1 and detect 50 pg mL^?1 of taurine ranging normally between 65 and 179 mmol L^?1 (8–22 μg mL^?1). The validated method allowed simple determination of human plasma taurine in pharmacokinetic and biomarker studies.     

High-performance liquid chromatographic separation and dual competitive binding assay of corrinoids in biological material

Corrinoids were extracted with hot ethanol from human plasma and faeces and separated by high-performance liquid chromatography. The corrinoids (cobalamin and cobalamin analogues) were quantified in the eluted fractions by a dual radioisotope assay using as binders intrinsic factor and haptocorrin to detect cobalamin and total corrinoids, respectively. Recoveries ranged from 37.7 +/- 5.1% for hydroxycobalamin to 75.0 +/- 9.1% for cyanocobalamin. In plasma, the main forms of cobalamin were the coenzymes methylcobalamin and 5'-deoxyadenosylcobalamin (32.1 +/- 13.4 and 28.4 +/- 12.3%, respectively, of total corrinoids). The cobalamin analogue fraction of plasma was eluted with a retention time close to that of cobinamide and of deoxyadenosylcobalamin. In the faeces, most of the corrinoids separated were detected better by the haptocorrin assay than by the intrinsic factor assay. One corrinoid peak was eluted with the same retention time as cobinamide. This peak was detected by haptocorrin assay but not by intrinsic factor assay. It could therefore correspond to cobinamide.     

Rapid and sensitive determination of donepezil in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of donepezil in human plasma samples. Diphenhydramine was used as the internal standard. The collision-induced transition m/z 380 --> 91 was used to analyze donepezil in selected reaction monitoring mode. The signal intensity of the m/z 380 --> 91 transition was found to relate linearly with donepezil concentrations in plasma from 0.1-20.0 ng/mL. The lower limit of quantification of the LC/MS/MS method was 0.1 ng/mL. The intra- and inter-day precisions were below 10.2% and the accuracy was between -2.3% and +2.8%. The validated LC/MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 5 mg donepezil hydrochloride. The non-compartmental pharmacokinetic model was used to fit the donepezil plasma concentration-time curve. Maximum plasma concentration was 12.3 +/- 2.73 ng/mL which occurred at 3.50 +/- 1.61 h post-dosing. The apparent elimination half-life and the area under the curve were, respectively, 60.86 +/- 12.05 h and 609.3 +/- 122.2 ng . h/mL. LC/MS/MS is a rapid, sensitive and specific method for determining donepezil in human plasma samples.     

High-performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct in biological materials (plasma and human cells) using a commercially available reagent.

The assay of malondialdehyde (MDA) is widely used in clinical chemistry laboratories to investigate lipid peroxidation in oxidative pathologies. In the present work, the thiobarbituric acid (TBA) reaction was carried out on plasma, human erythrocytes and fibroblasts. The reagents used were those of the fluorimetry MDA kit manufactured by Sobioda. We have defined the application of this kit to high-performance liquid chromatography. This adaptation satisfied the criteria of good analytical practice. The detection limit was 2.5 pmol per injection. The retention time of the MDA-TBA2 peak (4.96 +/- 0.07 min) led to excellent resolution of the complex. The within-assay (6-12%) and between-assay (11-12%) precisions were satisfactory. The analytical recovery of MDA after spiking samples of human plasma with tetraethoxypropane standards varied from 70 to 100%. The mean lipoperoxide concentration determined in 32 healthy adults (20-40 years) was 1.04 +/- 0.23 mumol l-1 in plasma. Applied to the erythrocytes of fifteen laboratory workers, the method furnished physiological values of 0.59 +/- 0.21 mumol l-1. Concentrations were significantly higher in chronic renal dialysis patients (4.15 +/- 2.35 mumol l-1. The MDA content of fibroblasts cultured in standard medium was 0.38 +/- 0.04 mumol per g of protein and increased (5.78 +/- 1.38 mumol per g of protein) if the cells were grown in an iron-enriched medium. This accurate high-performance liquid chromatographic method for detection of MDA is the first one which can be applied to plasma, red blood cells and cultured cells. This technique will prevent false positives and should make inter-laboratory comparisons possible.     

Octapeptide-specific and sensitive assay for angiotensin II in plasma

Angiotensin-(1-8)octapeptide (angiotensin II) is the active principle of the renin-angiotensin system. Crossreaction of angiotensin II-antisera with inactive precursors and metabolic fragments prevented the specific quantitation of this hormone in biological fluids. Peptide-extraction on bonded-phase silica followed by peptide-separation using isocratic reverse-phase high performance liquid chromatography and subsequent radioimmunoassay rendered possible the octapeptide-specific measurement of angiotensin II in 2 ml plasma with a detection limit of 0.4 fmol/ml. The coefficient of variation for intra-assay precision was 0.06 and for inter-assay precision 0.13. 125I-angiotensin II was recovered from plasma by solid-phase extraction to 99 +/- 2% (mean +/- S.D.). The overall recovery of 5, 10 and 20 fmol unlabeled angiotensin II added to plasma was 80 +/- 10%. Plasma concentrations in supine normal humans averaged 4.1 +/- 1.6 fmol/ml and were suppressed below the detection limit by angiotensin I converting enzyme inhibition.     

Ion-pair reversed-phase HPLC method for determination of sodium tanshinone IIA sulfonate in biological samples and its pharmacokinetics and biodistribution in mice

The ion-pair reversed-phase HPLC method for determination of sodium tanshinone IIA sulfonate (STS) in various biological samples was for the first time developed and validated, and was applied for pharmacokinetics and tissue distribution studies of intravenously administrated STS in mice. A linear relation was found between peak area and STS concentrations within the ranges of 0.1-5 micraog/ml for plasma, 0.1-5 microg/g of tissue for kidney homogenate, 0.1-20 microg/g of tissue for liver homogenate, 0.1-1 microg/g of tissue for heart, spleen and lung homogenates, respectively. In plasma and tissues, the limit of quantification (LOQ) and the limit of detection (LOD) for STS were 100 ng/ml and 20 ng/ml. In all biological specimens, the average inter- and intra-day precision of STS were within 4.9%. The recoveries were more than 92% at all concentration levels in each type of biological specimens. STS plasma concentration-time data were best fitted with a two-compartment model, characterized by an initial rapid phase of drug concentration decrease, and a slower terminal elimination phase. The pharmacokinetics of STS was characterized with a distribution half-life (t(1/2alpha)) of 1.2+/-0.18 min, a terminal half-life (t(1/2beta)) of 21.6+/-2.4 min, a distribution volume (V) of 0.057+/-0.011 l/kg, a plasma clearance (CL) of 0.86+/-0.12 l/h/kg and an AUC(0-infinity) of 58.41+/-6.21 microg x h/ml. STS was widely distributed into most tissues and was obviously accumulated in liver. This results indicated that STS may be promising to treat liver disease.