Simultaneous determination of tranilast and metabolites in plasma and urine using high-performance liquid chromatography

A method has been developed for the simultaneous determination of Tranilast, N-(3',4'-dimethoxycinnamoyl)anthranilic acid (N-5'), and metabolites in plasma and urine from humans, dogs and rodents administered N-5'. Total N-5' and metabolite N-3 conjugates were determined in human urine. Detection limits in plasma were 0.2 micrograms/ml for metabolite N-3-S and N-5' and 0.1 micrograms/ml for metabolites N-3 and N-4. In urine, detection limits were 2 micrograms/ml for metabolite N-3-S and N-5' and 1 micrograms/ml for metabolites N-3 and N-4. Metabolite N-4 was not identified in any sample assayed.     

Simultaneous determination of aceclofenac and three of its metabolites in human plasma by high-performance liquid chromatography

Aceclofenac [[2-(2',6'-dichlorophenyl)amino]phenylacetoxyacetic acid] is a phenylacetic acid derivative with potent analgesic and anti-inflammatory properties and an improved gastro-intestinal tolerance. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of aceclofenac and three of its metabolites (4'-hydroxy-aceclofenac, diclofenac, 4'-hydroxy-diclofenac) in human plasma. The analytes were separated using an acetonitrile-phosphate buffer gradient at a flow rate of 1 mL/min, and UV detection at 282 nm. The retention times for aceclofenac, diclofenac, 4'-hydroxy-aceclofenac, 4'-hydroxy-diclofenac and ketoprofen (internal standard) were 69.1, 60.9, 46.9, 28.4 and 21.2 min, respectively. The validated quantitation range of the method was 10-10000 ng/mL for aceclofenac, 4'-hydroxy-aceclofenac and diclofenac, and 25-10000 ng/mL for 4'-hydroxy-diclofenac. The developed procedure was applied to assess the pharmacokinetics of aceclofenac and its metabolites following administration of a single 100 mg oral dose of aceclofenac to three healthy male volunteers.     

Simultaneous measurement of serotonin, catecholamines and their metabolites in cat and human plasma by in vitro microdialysis-microbore high-performance liquid chromatography with amperometric detection.

A method for the simultaneous measurement of norepinephrine, epinephrine, dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin and 5-hydroxyindole-3-acetic acid in cat and human plasma by in vitro microdialysis-microbore high-performance liquid chromatography with electrochemical detection is described. The detection limit (signal-to-noise ratio = 3) is about 0.05-0.1 pg per injection. The volume of plasma samples required is very small (< 200 microliters), hence there is minimal blood loss in repeated blood sampling, especially in experiments using small animals. Within 15 min, a fast isocratic separation of these analytes by using a microbore reversed-phase ODS column is achieved, hence over 90 analyses can be performed in a single working day. As microdialysis per se is not destructive to plasma samples, the remaining plasma sample and perfusate can be repeatedly analysed for other substances. This simple, efficient and sensitive method can therefore be used as a routine clinical and basic research technique in the investigation of blood biogenic amines and their metabolites.     

Simultaneous determination of caffeine and its primary demethylated metabolites in human plasma by high-performance liquid chromatography.

An improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its three primary metabolites (theophylline, theobromine and paraxanthine) in human plasma is described. The four substances were separated on a reversed-phase column (5 microns TSK gel ODS-80TM, 150 mm x 4.6 mm I.D.) by use of the mobile phase methanol-0.1 M NaH2PO4 (30:70, v/v) with a flow-rate of 0.8 ml/min. Absorbance was monitored at 274 nm. The detection limit was 5 ng/ml for theobromine and caffeine and 10 ng/ml for paraxanthine and theophylline. The linearity and reproducibility were sufficient for drug monitoring of caffeine and its primary methylxanthines.     

Simultaneous determination of catecholamines and dobutamine in human plasma and urine by high-performance liquid chromatography with fluorimetric detection.

We report a reliable fluorimetric assay for the simultaneous determination of norepinephrine, epinephrine, dopamine and dobutamine in human plasma and urine, based on liquid-liquid extraction and derivatization with the fluorogenic agent 1,2-diphenylethylenediamine prior to chromatography. The method is sensitive (detection limit 0.3-0.8 pg injected) and reproducible (coefficients of variation 1-10%), and shows good accuracy (93-98%). The method should also be used when one only wants to measure the concentrations of the natural catecholamines, in order to avoid interference by metabolites of dobutamine and by the late-eluting dobutamine itself.     

Determination of L-3,4-dihydroxyphenylalanine in blood by high-performance liquid chromatography after solvent extraction

The use of high-performance liquid chromatography combined with solvent extraction for sample purification is described for the determination of L-3,4-dihydroxyphenylalanine in blood plasma. It is extracted into n-hexanol via complexation of its catechol moiety with diphenyl borate and ion-pair formation of its carboxylic group with tetrapentylammonium ion in an alkaline buffer. Under optimal extraction conditions, L-3,4-dihydroxyphenylalanine and 3,4-dihydroxybenzylamine used as an internal standard are extracted from blood plasma by a simple procedure and in a short time and then separated by reversed-phase ion-pair chromatography. The analytical recovery (100.8%) and reproducibility (coefficient of variation = 2.3% for n = 6) from plasma samples are good enough for routine analysis. L-3,4-Dihydroxyphenylalanine levels in blood can be monitored by this method after oral intake of the substance.     

Simultaneous determination of diclofenac potassium and drotaverine hydrochloride in human plasma using reversed-phase high-performance liquid chromatography

A simple high-performance liquid chromatographic method with ultraviolet detection is proposed for the estimation of diclofenac potassium and drotaverine hydrochloride in human plasma. Liquid-liquid extraction was carried out with a mixture of dichloromethane-isopropyl alcohol (80:20, v/v). Chromatographic separation of the analytes and internal standard was achieved on an analytical 250 × 4.6 mm i.d. reversed-phase Thermo BDS Hypersil C8 (5 μm particle size) column using a mobile phase of acetonitrile-0.02M ammonium acetate buffer (53:47, v/v) at pH 3.5. The run time was less than 15 min. Column eluate was monitored at 230 nm. The linearity over the concentration ranges of 25-1500 ng/mL and 32-960 ng/mL was obtained for diclofenac potassium and drotaverine hydrochloride, respectively. The limit of quantification was 25 and 32 ng/mL for diclofenac potassium and drotaverine hydrochloride, respectively. Recoveries of diclofenac potassium and drotaverine hydrochloride from plasma were 97.45% and 98.27%, respectively.     

Simultaneous determination of flucytosine and fluorouracil in human plasma by high-performance liquid chromatography

A validated, sensitive and precise reversed-phase high-performance liquid chromatographic method for the simultaneous determination of 5-flucytosine (5-FC) and 5-fluorouracil (5-FU) in human plasma is described. Two compounds, 5-methylcytosine (5-MC) and 5-chlorouracil (5-CU), were used as internal standards for the determination of 5-FC and 5-FU, respectively. Plasma samples were deproteinized with trichloroacetic acid and chromatographed on an octylsilica column, maintained at 30 degrees C during elution, using a 0.04 M phosphate buffer, pH 7.0, as eleunt. Spectrophotometric diode array detection was used at 266 nm. 5-FC, 5-FU, 5-MC and 5-CU were found to have retention times of 4.8, 5.8, 7.7 and 11.0 min respectively. Recoveries of 91-120% with reproducibility and repeatability coefficients of variation of 0.8-6% were obtained. Mean correlation coefficients of 0.99989 and 0.9995 were found for the linear calibration curves (n = 2) of 5-FC (4.816-192.6 mg/l) and 5-FU (0.05368-5.368 mg/l), respectively. The limits of quantitation were 0.3 mg/l for 5-FC and 0.05 mg/l for 5-FU.     

Simultaneous quantitation of loganin and sweroside in plasma using column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of loganin and sweroside, which are the active ingredients of purified herbal extract from Lonicera japonica (SKL JI), in rat plasma using column-switching and ultraviolet (UV) absorbance detection. Plasma was simply filtrated prior to injection to the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with two six-port switching valves. Detection of loganin and sweroside was accurate and repeatable, with a limit of quantitation of 0.05 μg ml⁻¹ in plasma. The calibration curves for both loganin and sweroside were linear over the concentration ranges of 0.05-40.0 and 0.02-40.0 μg ml⁻¹ in rat plasma, respectively. The intra- and inter-day precision over the concentration range for loganin and sweroside were lower than 8.1 and 10.9% (relative standard deviation, R.S.D.), and accuracy was between 94.7 and 113.5 and 95.0 and 113.1%, respectively. This method has been successfully applied to determine the levels of loganin and sweroside in rat plasma samples from pharmacokinetics studies.     

Simultaneous determination of itraconazole and hydroxyitraconazole in human plasma by high-performance liquid chromatography

The development and validation of a high-performance liquid chromatography (HPLC) method for the simultaneous determination of itraconazole and its metabolite, hydroxyitraconazole, in human plasma is described. The method involved liquid-phase extraction of itraconazole and hydroxyitraconazole using a hexane-dichloromethane (70:30) mixture, after addition of loratidine as an internal standard (IS). Separation was achieved with a reversed-phase C18 column (250 mm x 4.6 mm) employing fluorescence detection (excitation: 264 nm, emission: 380 nm). The mobile phase consisted of [0.01% triethylamine solution adjusted to pH 2.8 with orthophosphoric acid-acetonitrile (46:54)]-isopropanol (90:10, v/v) at a flow rate of 1.0 ml/min. For both the drug and metabolite, the standard curve was linear from 5.0 to 500 ng/ml with goodness of fit (r2) greater than 0.98 observed with four precision and accuracy batches during validation. An observed recovery was more than 70% for drug, metabolite and internal standard. The applicability of this method to pharmacokinetic studies was established after successful application during 35 subjects bioavailibity study. The method was found to be precise, accurate and specific during the study.     

Simultaneous determination of rofecoxib and celecoxib in human plasma by high-performance liquid chromatography

An innovative reversed-phase high-performance liquid chromatographic method is validated for the simultaneous determination of rofecoxib and celecoxib in human plasma. The internal standard is 4-n-pentyl-phenyl-acetic acid. Good chromatographic separation is achieved using a Zorbax SB-CN (5 microm) analytical column operated at room temperature and mobile phase consisting of acetonitrile and water containing 0.1M potassium dihydrogen orthophosphate buffer adjusted to pH 2.4 with 85% orthophosphoric acid (42:58, v/v). UV detection is performed at 254 nm, and the flow rate is maintained at 1.0 mL/min. Plasma samples are extracted into an organic solvent (1-chlorobutane) and evaporated under an air flow. The calibration curve for rofecoxib is linear over the range of 10 to 500 microg/L, and the celecoxib calibration curve is linear over the range of 20 to 2000 microg/L. The lower limit of quantitation for rofecoxib and celecoxib is 10 and 20 microg/L, respectively, using 1.0 mL of human plasma. The validation data show that the assay is sensitive, accurate, specific, and reproducible for the determination of rofecoxib and celecoxib. This method is therefore appropriate for pharmacokinetic studies to quantitate these therapeutic agents in patients with arthritis conditions.