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.     

High-performance liquid chromatographic determination of acetone in blood and urine in the clinical diagnostic laboratory.

A method for the determination of acetone in plasma or urine by high-performance liquid chromatography (HPLC) was developed. Plasma specimens are deproteinized with acetonitrile (1:1, v/v) 2,4-dinitrophenylhydrazine (DNPH) is added to the supernatant or to filtered urine samples, similarly treated with acetonitrile (2:1, v/v) to prevent crystallization of the synthesized phenylhydrazone. An aliquot (20 microliters) of the reaction mixture was subjected to HPLC at ambient temperature using a reversed-phase Pecosphere 3 x 3 C18 column with acetonitrile-water (45:55, v/v) as eluent at a flow-rate of 1 ml/min and detection at 365 nm. Hydroxyacetone and acetoacetate phenylhydrazone derivatives do not interfere. The identification of acetone by its retention time was confirmed by comparison with a laboratory-synthesized acetone DNPH derivative. The concentration of acetone, eluted within 3 min, was determined by the peak-height method. The detection limit was 0.034 mmol/l; the relative standard deviations were less than 5% within run (n = 20) and less than 10% between run (n = 20).     

Quantification of midodrine and its active metabolite in plasma using a high performance liquid chromatography column switching technique

An automated column-switching HPLC system is described for the simultaneous determination of midodrine, an alpha-adrenergic stimulating drug, and its active metabolite, ST-1059. Serum or plasma (850 microliters) is directly injected onto a RP18 (30 micrograms particle size) pre-column (9 x 4 mm ID) which acts as an on-line liquid-solid extractor and analyte enrichment system. The injection is followed by washing steps. The fraction containing the analytes is transferred onto an analytical RP18 column via step gradient elution where the final analysis is performed. Fluorescence detection is used (lambda ex 290 nm and lambda em 322 nm), and method detection limits of 0.8 ng/mL plasma were reached. These were sufficiently low to determine the plasma concentration-time profiles for both compounds following oral administration of 2.5 mg and 5 mg midodrine hydrochloride. The assay in serum or plasma was linear in the range of 1 to 15 ng analyte/mL, the recovery was greater than 95%, and the reproducibility was sufficient. The assay was rugged and was maintained by routinely changing the home-made, dry packed pre-column every 20th serum injection.     

Study on a new precolumn derivatization method in the determination of metformin hydrochloride

Metformin hydrochloride is successfully determined by reversed-phase high-performance liquid chromatography with a new precolumn derivatization method using 9,10-anthraquinone-2-sulfonyl chloride as the derivatization agent. Several derivatization systems are tried to optimize the derivatization conditions, and a new post-derivatization treatment method is established. The derivatization product is analyzed on a Lichrosper C18 column (6.0 mm x 150 mm, 5 microm) at 256 nm with methanol-water (70:30, v/v) as the mobile phase. The calibration curves of the derivatives for the UV detector (0.01-4 mg/L) are linear with respect to peak area. The detection limit (peak area) for the metformin hydrochloride is 0.01 mg/L for a signal-to-noise ratio of 3:1. In human plasma, the detection limit is 0.02 mg/L. This assay is rapid, sensitive, and highly reproducible.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in plasma after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat plasma has been developed. Racemic atenolol and practolol (internal standard) were extracted from alkalinized plasma (pH 12) into dichloromethane containing 3% (v/v) heptafluoro-1-butanol, and the organic layer was evaporated. The samples were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate at pH 8.5 for 30 min. After removal of excess reagent, the diastereomers were extracted into dichloromethane. The diastereomers were separated on a Microspher C18 column (3 microns) with a mobile phase of acetonitrile-sodium acetate buffer (0.01 M, pH 7) (50:50, v/v) at a flow-rate of 0.8 ml/min. Fluorescence detection (lambda ex = 227 nm, lambda em = 310 nm) was used. When 100 microliters of plasma were used, the quantitation limit was 10 ng/ml for the atenolol enantiomers. The assay was applied to measure concentrations of atenolol enantiomers in plasma after intravenous administration of racemic atenolol to rats.     

Liquid chromatographic analysis of propafenone enantiomers in human plasma

A convenient and sensitive high-performance liquid chromatographic method for analysis of the enantiomers of propafenone (PPF) in human plasma was developed. Racemic propafenone and (-)-ephedrine (internal standard) were first extracted from plasma samples into a mixture of hexane-2-propanol-heptafluorobutanol (95:5:1.25, v/v). After evaporation of the organic layer, the samples were derivatized with R(-)-naphthylethyl isocyanate. The derivatization reached its maximum within 30 s at room temperature with an efficiency of 93.9 +/- 2.8% (mean +/- S.D.). The formed diastereomers were subsequently separated on a silica column with a mobile phase of hexane-2-propanol-isobutanol (96:2:2, v/v) at a flow-rate of 1.5 ml/min. The ultraviolet detection wavelength was set at 220 nm. Using 1 ml plasma, the detection limit was 6.25 ng/ml for the propafenone enantiomers. The assay was successfully employed to measure propafenone enantiomers in plasma samples of a healthy subject after oral administration of a single 150-mg dose of the racemate.     

Immunomagnetic separation and detection of Salmonella cells using newly designed carriers

A new method for simultaneous determination of alkoxyethanols (2-methoxyethanol, 2-ethoxyethanol, 2-isopropoxyethanol, and 2-butoxyethanol) by high-performance liquid chromatography (HPLC) with fluorescence detection has been developed. The alkoxyethanols and an internal standard (2-phenoxyethanol) were derivatized by treatment with 1-anthroylnitrile to give the anthroyl esters. The esterification was completed in 30 min in the presence of quinuclidine as base catalyst at room temperature. After stopping the reaction, an aliquot of the final solution was injected into the HPLC. The resulting anthroyl esters of the alkoxyethanols and the internal standard were separated on a C18 reversed-phase column with acetonitrile-water-acetic acid (65:35:0.1, v/v) as the mobile phase and detected fluorimetrically at excitation and emission wavelengths of 360 nm and 460 nm, respectively. The detection limits of the derivatives as alkoxyethanols at a signal-to-noise ratio of 3 were in the range of 1-3 pg per injection. The minimal amounts of alkoxyethanols derivatized in the reaction mixture for derivatization to determine the limits of detection were approximately 0.5 ng. This HPLC method was applied to the determination of some of alkoxyethanols in the air of the workplace where the thinner containing alkoxyethanols was used for painting.     

Determination of gaseous nitrogen in gas mixtures using low pressure microwave induced plasma emission spectrometry

A microwave induced plasma emission spectrometer operating at low energy (30 W) and low plasma gas pressure (2.5.10(-2) Pa) has been used for the quantitative measurement of molecular nitrogen in natural gases. The samples have been introduced into the plasma using a counterflow principle to produce emission spectra of diatomic molecular fragments in low excitation states (advantage: minimized interferences). The N(2) concentration has been determined by measuring the intensity of the N(2)-line at 337.13 nm (C(3)Pi(u)-B(3)Pi(g)-system; (0,0)vibrational transition) and of the NH-line at 336.03 nm (A(3)Pi(i)-X(2)Sigma(-)-system; (0,0)-vibrational transition). A linear correlation between concentration and signal intensities has been obtained in the range of 0.00% to 14.24% (v/v). The method possesses a detection limit of 0.01 ppm (v/v) for the determination of N(2), and a reproducibility of 1.33% (RSD).     

Simultaneous liquid chromatographic measurement of melatonin and related indoles through post-column electrochemical demethylation and fluorescence derivatization.

In this paper we describe a highly sensitive and selective liquid chromatographic method for the determination of 5-methoxyindoles (5-methoxyindole-3-acetic acid, 5-methoxytryptamine, 5-methoxytryptophol, and melatonin) using a post-column technique involving electrolytic demethylation followed by fluorescence derivatization with benzylamine. We separated these compounds within 30 min by reversed-phase liquid chromatography using acetate buffer (pH 6.5)-acetonitrile-methanol [8:1:1 (v/v); isocratic elution] and then demethylated them, using a commercial coulometric system, to give the corresponding 5-hydroxyindoles. Next, we converted the 5-hydroxyindole products into fluorescent derivatives by their reactions with benzylamine in the presence of potassium hexacyanoferrate(III). We detected the derivatives spectrofluorometrically at 480 nm upon excitation at 345 nm. The detection limits (signal-to-noise ratio = 3) of the 5-methoxyindoles were in the range from 12 to 93 fmol per 20-microL injection.     

Validated HPLC method for determination of carboxylic acid metabolite of clopidogrel in human plasma and its application to a pharmacokinetic study

A new, simple, and reproducible method for determination of carboxylic acid metabolite of clopidogrel in human plasma has been developed. After liquid-liquid extraction in acidic medium with chloroform, samples were quantified on a Nova-pak C(8), 5 microm column using a mixture of 30 mM K(2)HPO(4)-THF-acetonitrile (pH = 3, 79:2:19, v/v/v) as mobile phase with UV detection at 220 nm. The flow rate was set at 0.9 mL/min. Ticlopidine was used as internal standard and the total run time of analysis was about 12 min. The method was linear over the range of 0.2-10 microg/mL of clopidogrel metabolite in plasma (r(2) > 0.999). The within-day and between-day precision values were in the range 1.0-4.8%. The limit of quantification of the method was 0.2 microg/mL. The method was successfully used to study the pharmacokinetics of clopidogrel in healthy volunteers.     

Determination of memantine in plasma and vitreous humour by HPLC with precolumn derivatization and fluorescence detection

A new HPLC procedure with precolumn derivatization and rimantadine as the internal standard for determining memantine, a candidate agent for the treatment of glaucoma in plasma and vitreous humour, has been developed and validated. Precolumn derivatization was performed with 9-fluorenylmethyl-chloroformate-chloride (FMOC-Cl) as the derivatization reagent and followed by a liquid-liquid extraction with n-hexane. Optimal conditions for derivatization were an FMOC-Cl concentration of 1.5 mM, a reaction time of 20 min, the temperature at 30°C, the borate buffer pH 8.5, and a borate buffer-acetonitrile ratio of 1:1. The derivatives were analyzed by isocratic HPLC with the fluorescence detector λex 260 nm λem 315 nm on a Novapack C(18) reversed-phase column with a mobile phase of acetonitrile-water (73:27, v/v), 40°C, and a flow rate of 1.2 mL/min. The linear range was 10-1000 ng/mL with a quantification limit of ～ 10 ng/mL for both types of samples. This analytical method may be suitable for using in ocular availability studies.     

Stability indicating methods for the determination of some anti-fungal agents using densitometric and RP-HPLC methods

Two chromatographic methods were developed for the determination of some anti-fungal drugs in the presence of either their degradation products or cortisone derivatives. The densitometric method determined mixtures of each of ketoconazole (KT), clotrimazole (CL), miconazole nitrate (MN) and econazole nitrate (EN) with the degradation products of each one. Mixtures of MN with hydrocortisone (HC) and of EN with triamcinolone acetonide (TA) were also successfully separated and determined by this technique. For KT and CL, a mixture of methanol:water:triethylamine (70:28:2 v/v) was used as a developing system and the spots were scanned at 243 nm and 220 nm for KT and CL, respectively. For MN and EN, a mixture of hexane:isopropyl alcohol:triethylamine (80:17:3 v/v) was used as a developing system and the spots were scanned at 225 nm for both drugs. The HPLC method determined mixtures of CL or EN with their degradation products which were separated and quantified on a Zorbax C8 column. Elution was carried out using methanol:phosphate buffer pH 2.5 (65:35 v/v) as a mobile phase at a flow rate of 1.5 ml/min and UV detection at 220 nm for CL. For EN, a mixture of methanol:water containing 0.06 ml triethylamine pH 10 (75:25 v/v) was used as a mobile phase at a flow rate of 1.5 ml/min and UV detection at 225 nm. The methods were also used to separate mixtures of CL with betamethasone dipropionate (BD) and EN with TA in a laboratory prepared mixture and in pharmaceutical preparations. The methods were sensitive, precise and applicable for determination of the drugs in pharmaceutical dosage forms.     

Quantification of glutathione in plasma samples by HPLC using 4-fluoro-7-nitrobenzofurazan as a fluorescent labeling reagent

A rapid and highly sensitive high-performance liquid chromatograpy method with fluorescence detection has been developed for determination of glutathione (GSH) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. The separation of the derivatized glutathione was performed using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)-acetonitrile (77:23, v/v) at a flow rate of 1.0 mL/min with the column temperature 2°C. The eluted derivatives were fluorometrically detected at an excitation wavelength 470 nm and an emission wavelength 530 nm. Under the optimum chromatographic conditions, the calibration curve was linear over the range of 0.1 μmol/L to 10.0 μmol/L with the correlation coefficient of 0.9988. The precision of the method was satisfactory with the intra- and inter-day coefficient of variation being 6.3%, 6.9%, respectively. This method has been used to determine glutathione concentrations in plasma samples from healthy individuals.     

Simple High-Performance Liquid Chromatographic Method for the Analysis of Quinine in Human Plasma without Extraction

Abstract

A simple, rapid and sensitive assay, capable of quantitating quinine (Q) in human plasma samples is reported. The assay uses a reversed-phase C18 HPLC column packed with 5 μ ODS Hypersil. The chromatographic separation was accomplished with an isocratic mobile phase comprising acetonitrile-aqueous phosphate buffer pH 2 (50:50, v/v) containing 25 mM sodium dodecyl sulfate and 3 mM tetrabutylammonium bromide at a flow rate of 0.5 ml/min. The eluant was monitored by a fluorescence detector (excitation wavelength at 350 nm and emission wavelength at 450 nm). The assay was based on a simple plasma protein precipitation technique. To 200 μ of plasma sample, 400 μ of internal standard (cinchocaine 30 μ/ml in methanol) was added. After brief vortexing and centrifugation, the clear supernatant was injected onto the HPLC column. The inter- and intra-assay coefficients of variation were found to be less than 10%. The lowest limit of detection for Q in plasma was 18 ng/ml.     

A sensitive liquid chromatographic method for the analysis of isovaleric and valeric acids in urine as fluorescent derivatives

A simple and sensitive isocratic liquid chromatographic method was developed for the analysis of isovaleric and valeric acids in human urine as biomarkers in metabolic acidosis. The method is based on the derivatization of isovaleric and valeric acids with a fluorescent reagent 2-(2-naphthoxy)ethyl-2-(piperidino)ethanesulfonate for labeling the analytes with the naphthoxy fluorophore. The resulting fluorescent derivatives of isovaleric and valeric acids were separated on a phenyl-hexyl column, using a mixed solvent of methanol-water-tetrahydrofuran (55:31:14, v/v) as the mobile phase. The separated derivatives were monitored with a fluorimetric detector (excitation at 225 nm and emission at 360 nm). The linear range of the method for the determination of isovaleric acid or valeric acid derivative was over 0.2 approximately 8.0 microM. The detection limit (signal to noise ratio=3 with 10 microl injected) of isovaleric acid or valeric acid was about 0.04 microM. Application of the method to the analysis of isovaleric acid in the urine of a patient with isovaleric acidemia proved feasible.     

Solid-phase extraction with liquid chromatography and ultraviolet detection for the assay of antidepressant drugs in human plasma

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase high-performance liquid chromatography (HPLC) procedure for the assay of five antidepressant drugs (trazodone, doxepin, desipramine, maprotiline and imipramine) is reported. The drugs were recovered from plasma buffered at a suitable pH using C18 Bond-Elut cartridges and mixtures of methanol-aqueous buffer as washing and elution solvents. The recoveries of the drugs using other sorbent materials (C8, C2, cyclohexyl, cyanopropyl and phenyl Bond Elut and copolymer HLB waters cartridges) were also examined. The selectivity of SPE was examined by using spiked plasma samples and the CH cartridge gave rise to the cleanest extracts. Cyclohexyl cartridges were conditioned successively with 2 ml of methanol and 1 ml of acetic acid-sodium acetate buffer (0.1 M, pH 4.0). Plasma sample was buffered at pH 4.0 and then applied to the sorbent. The washing step was performed subsequently with 1.5 ml of acetate buffer (0.1 M, pH 4.0), 100 microl of acetonitrile and 1 ml of methanol-acetate buffer (30:70, v/v). Finally, the analytes were eluted with 0.5 ml of methanol-acetate buffer (70:30, v/v). The extract was evaporated to dryness, reconstituted in mobile phase, and chromatographed on a reversed-phase C18 column with ultraviolet detection at 215 nm. The recoveries of trazodone, doxepin, desipramine, maprotiline and imipramine from spiked plasma samples using the CH cartridge were 58 2, 84 3, 83 3, 83 3 and 82 2%, respectively. The within-day and between-day repeatabilities were lower than 6% and 9%, respectively. The linearity of calibrations for the five antidepressants was between 0.005 and 2 microg/ml. The limits of detection were 1 ng/ml for trazodone, doxepin and desipramine and 2 ng/ml for maprotiline and imipramine.     

Determination of anti-virus drug, ganciclovir, in human serum by HPLC with precolumn fluorescence derivatization using phenylglyoxal.

A selective and highly sensitive liquid chromatographic method for the determination of ganciclovir (anti-virus drug) in human serum was described. After ganciclovir and acyclovir (internal standard; IS) were extracted with solid-phase extraction cartridge from serum, they were converted into fluorescent derivatives by reaction with phenylglyoxal in a phosphate buffer (pH 5.8) at 20 degrees C for 30 min. The derivatives were separated by reversed-phase column with a mixture of acetonitrile-1 mM phosphate buffer (pH 6.2) (18:82, v/v), and were then detected spectrofluorometrically at 512 nm with excitation at 365 nm. Extraction recoveries were 87.0-91.6% for ganciclovir and 86.8-92.3% for IS. The detection limit for ganciclovir spiked to serum was 5 ng ml-1 serum (306 fmol on column) at a signal-to-noise ratio of three. The accuracy and precision of this method were 7.6% and 5.0% even at low concentration (20 ng ml-1). The within- and between-day variations are lower than 7.6% and 8.1%, respectively.     

Determination of the beta-blocker atenolol in plasma by capillary zone electrophoresis

A capillary zone electrophoretic method was optimised for the determination of the beta-blocker atenolol in plasma. Separation was performed in an uncoated silica capillary of 58.5 cm (effective length 50 cm) x 75 microm I.D., and detection was at 194 nm. The effects of the buffer (concentration and pH), the injection time, the voltage applied and the plasma clean-up procedure were studied. The determination of atenolol was achieved in less than 3 min, using an electrolyte of 50 mM H3BO3-50 mM Na2B4O7 (50:50, v/v) pH 9, injected hydrodynamically for 4 s at 50 mbar and applying a voltage of +25 kV. This method was applied to the determination of atenolol in plasma of nine hypertensive patients (male and female, aged from 39 to 73 years). Atenolol concentrations found vary from 30 to 585 ng/ml.     

Determination of amino acids in overlapped capillary electrophoresis peaks by means of partial least-squares regression

Amino acid derivatives of 1,2-naphthoquinone-4-sulfonate (NQS) can be separated by capillary electrophoresis at 30 kV in a fused-silica capillary by using a 40 mM sodium tetraborate-isopropanol (3:1, v/v) solution as background electrolyte. This procedure was suitable for the most common amino acids. However, the peaks of three amino acids (phenylalanine, isoleucine and tyrosine) were only partially resolved and peaks of histidine and leucine derivatives overlapped completely. Partial least-squares regression (PLS) may overcome the lack of selectivity for these amino acids. Spectroelectropherograms of the corresponding amino acid derivative peaks were monitored with a diode-array spectrophotometer in the range 225 to 540 nm. Both spectra and electropherograms can be used as multivariate data for further analysis. In general, the best predictions were obtained using the time domain.     

Stability-indicating methods for determination of tiapride in pure form, pharmaceutical preparation, and human plasma

Four different stability-indicating procedures are described for determination of tiapride in pure form, dosage form, and human plasma. Second derivative (D2), first derivative of ratio spectra (1DD), spectrofluorimetric, and high-performance column liquid chromatographic (LC) methods are proposed for determination of tiapride in presence of its acid-induced degradation products, namely 2-methoxy-5-(methylsulfonyl) benzoic acid and 2-diethylaminoethylamine. These approaches were successfully applied to quantify tiapride using the information included in the absorption, excitation, and emission spectra of the appropriate solutions. In the D2 method, Beer's law was obeyed in the concentration range of 1.5-9 microg/mL with a mean recovery of 99.94 +/- 1.38% at 253.4 nm using absolute ethanol as a solvent. In 1DD, which is based on the simultaneous use of the first derivative of ratio spectra and measurement at 245 nm in absolute ethanolic solution, Beer's law was obeyed over a concentration range of 1.5-9 microg/mL with mean recovery 99.64 +/- 1.08%. The spectrofluorimetric method is based on the determination of tiapride native fluorescence at 339 nm emission wavelength and 230 nm excitation wavelength using water-methanol (8 + 2, v/v). The calibration curve was linear over the range of 0.2-3 microg/mL with mean recovery of 99.66 +/- 1.46%. This method was also applied for determination of tiapride in human plasma. A reversed-phase LC method performed at ambient temperature was validated for determination of tiapride using methanol-deionized water-triethylamine (107 + 93 + 0.16, v/v/v) as the mobile phase. Sulpiride was used as an internal standard at a flow rate of 1 mL/min with ultraviolet detection at 214 nm. A linear relation was obtained over a concentration range of 2-30 microg/mL with mean recovery of 99.66 +/- 0.9%. Results were statistically analyzed and compared with those obtained by applying the reference method. They proved both accuracy and precision.