High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4-700 ng/ml of quercetin in plasma and 20-1000 ng/ml of quercetin in urine. The lower limit of quantification was approximately 7 ng/ml of quercetin in plasma and approximately 35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was approximately 0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.     

High-performance liquid chromatographic determination of levodropropizine in human plasma with fluorometric detection

The present describes a new high-performance liquid chromatographic method with fluorescence detection for the analysis of levodropropizine [S-(−)-3-(4-phenylpiperazin-1-yl)-propane-1,2-diol] (Levotuss), an anti-tussive drug, in human serum and plasma. A reversed-phase separation of levodropropizine was coupled with detection of the native fluorescence of the molecule, using excitation and emission wavelengths of 240 nm and 350 nm respectively. The analytical column was packed with spherical 5 μm poly(styrene-divinylbenzene) particles and the mobile phase was 0.1 M NaH₂PO₄ pH 3-methanol (70:30, v/v), containing 0.5% (v/v) tetrahydrofuran. For quantitation, p-methoxylevodropropizine was used as the internal standard. Samples of 200 μl of either serum or plasma were mixed with 200 μl of 0.1 M Na₂HPO₄ pH 8.9 and extracted with 5 ml of chloroform-2-propanol (9:1, v/v). The dried residue from the organic extract was redissolved with distilled water and directly injected into the chromatograph. The limit of detection for levodropropizine, in biological matrix, was about 1–2 ng/ml, at a signal-to-noise ratio of 3. The linearity was satisfactory over a range of concentrations from 3 to 1000 ng/ml (r² = 0.99910); within-day precision tested in the range 5–100 ng/ml as well as day-to-day reproducibility proved acceptable, with relative standard deviations better than 1% in most cases. Interferences from as many as 91 therapeutic or illicit drugs were excluded.     

High-performance liquid chromatographic determination of beta-phenylethylamine in human plasma with fluorescence detection

A simple and highly sensitive method for the determination of beta-phenylethylamine in human plasma is investigated. The method employs high-performance liquid chromatography with fluorescence detection. beta-Phenylethylamine and p-methylbenzylamine (internal standard) in human plasma are isolated by cation-exchange chromatography on a Toyopak SP cartridge and then converted into the corresponding fluorescent derivatives with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, a fluorescence derivatization reagent for amines. The derivatives are separated within 30 min on a reversed-phase column, TSK gel ODS-120T, with isocratic elution, and detected fluorometrically. The detection limit of beta-phenylethylamine is 0.3 pmol/ml in plasma (S/N = 3).     

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 μBondapak C₁₈ column preceded by a 4–5 cm × 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 μg/ml. Recoveries from plasma samples seeded with 1.8, 4 and 8 μg/ml isoxicam were 1.86 ± 0.077, 4.10 ± 0.107 and 8.43 ± 0.154 μg/ml with relative standard deviations of 3.3%, 2.5% and 5.4%, respectively. The linearity in urine ranged from 0.125 to 2 μg/ml. The precision of the method was 3.3–9.0% relative standard deviation over the linear range.     

Column-switching high-performance liquid chromatographic assay for determination of asiaticoside in rat plasma and bile with ultraviolet absorbance detection

A column-switching high-performance liquid chromatography (HPLC) method is described for the determination of asiaticoside in rat plasma and bile using column-switching and ultraviolet (UV) absorbance detection. Plasma was simply deproteinated with acetonitrile prior to injection and bile was directly injected onto 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 asiaticoside was accurate and repeatable, with a limit of quantification of 0.125 μg/ml in plasma and 1 μg/ml in bile. The calibration curves were linear in a concentration range of 0.125–2.5 μg/ml and 1–20 μg/ml for asiaticoside in rat plasma and bile, respectively. This method has been successfully applied to determine the level of asiaticoside in rat plasma and bile samples from pharmacokinetics and biliary excretion studies.     

Determination of ticlopidine in human plasma by high-performance liquid chromatography and ultraviolet absorbance detection

A simple HPLC method has been developed for the determination of ticlopidine in human plasma. Plasma samples were buffered at pH 9 and extracted with n-heptane-isoamyl alcohol (98.5: 1.5, v/v). Imipramine was used as internal standard. Chromatography was performed isocratically with acetonitrile-methanol-0.05 M KH₂PO₄ (20:25:55, v/v) at pH 3.0 containing 3% triethylamine at a flow-rate of 1 ml/min. A reversed-phase column, Supelcosil LC-8-DB, 15 cm × 4.6 mm I.D., 5 μm particle size, was used. The effluent was monitored by UV absorbance detection at 235 nm. The method showed good accuracy, precision and linearity in the concentration range 5–1200 ng/ml. The limit of quantitation was 5 ng/ml, with a precision (C.V.) of 8.91%, which is the same as that achieved by other authors with a previously published GC-MS method. The procedure described in this paper is simple and allows the routine assessment of ticlopidine plasma concentration in pharmacokinetic studies following therapeutic doses in human subjects.     

Measurement of piperaquine in plasma by liquid chromatography with ultraviolet absorbance detection

Piperaquine (PQ) is an antimalarial drug enjoying a resurgence of use in combination with an artemisinin derivative because of parasite resistance to standard treatments. Its pharmacokinetic properties have not been characterised. An assay for PQ in plasma was developed using solvent extraction and liquid chromatographic separation on a Waters XTerra RP(18) column, with a mobile phase of 7% acetonitrile in water (containing 0.025% trifluoroacetic acid, 0.1% NaCl and 0.008% triethylamine) and UV detection at 340 nm. The assay was linear up to 1000 microg/l. Intra- and inter-day relative standard deviations were <10% (5-500 microg/l) and <21% (5-500 microg/l), respectively. Inter-day limits of quantitation and detection were 5 microg/l and 3 microg/l, respectively. A preliminary pharmacokinetic study in a patient who received 2.56 g of PQ phosphate orally with dihydroartemisinin as four doses over 32 h found an apparent steady-state volume of distribution of 447 l/kg, an apparent oral clearance 0.93 l/h/kg and a terminal half-life of 17.3 days.     

Column-switching high-performance liquid chromatographic assay for determination of apigenin and acacetin in human urine with ultraviolet absorbance detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of apigenin and the 4′-methylated derivative acacetin in human urine using column-switching and ultraviolet (UV) absorbance detection. Urine samples were enzymatically hydrolysed and solid-phase extracted prior to injection onto the HPLC system. Prior to elution of apigenin and the internal standard, 5,7,8-trihydroxyflavone, from the first column used for sample clean-up, the six-port valve was switched to the second column for analysis with UV detection. Detection of apigenin was precise and reproducible, with a limit of quantification of 10 ng ml⁻¹ urine. Detection and quantification of acacetin was linear down to 70 ng ml⁻¹ urine. The method has been successfully applied to determine the level of apigenin in 100 human urine samples from an intervention study with parsley.     

Determination of loratadine in human plasma by high-performance liquid chromatographic method with ultraviolet detection

A HPLC–UV determination of loratadine in human plasma is presented. After simple liquid–liquid extraction with 2-methylbutane–hexane (2:1) and evaporation of organic phase the compounds were re-dissolved in 0.01 M HCl, evaporated again and finally separated on a Supelcosil LC-18-DB column. The analyses were done at ambient temperature under isocratic conditions using the mobile phase: CH₃CN–water–0.5 M KH₂PO₄–H₃PO₄ (440:480:80:1, v/v). UV detection was performed at 200 nm with a limit of quantification of 0.5 ng/ml. The precision was found to be satisfactory over the whole range tested (0.5–50 ng/ml) with relative standard deviations of 2.3–6.3 and 5.2–14.1% for intra- and inter-assays, respectively.     

Determination of laquinimod in plasma by coupled-column liquid chromatography with ultraviolet absorbance detection

Laquinimod is an immunomodulator that is currently in clinical trials. For pharmacokinetic and toxicokinetic studies in animals and humans a sensitive and accurate bioanalytical method was required. In this paper a bioanalytical method for the determination of laquinimod by liquid chromatography is described. After a protein precipitation step the plasma sample was injected onto a coupled-column HPLC system. After further purification from macromolecules on a short restricted access material C(18) column the analyte was transferred to a reversed-phase C(18) analytical column and separated from interfering substances. The analyte was detected by UV detection. The method was validated with respect to linearity, selectivity, precision, accuracy, limit of quantitation, limit of detection, recovery and stability. The limit of quantitation was 0.75 micromol/L, the intermediate precision was 1.8-3.6% (C.V.) and the accuracy was 97.7-114.7%. In conclusion, the method was found to perform well and is suitable for use in pharmacokinetic and toxicokinetic studies.     

High-performance liquid chromatographic determination of ambroxol in human plasma

Ambroxol has been determined in biological fluids using a rapid and sensitive high-performance liquid chromatographic method. The samples prepared from plasma by liquid—liquid extraction were analysed on reversed-phase silica gel by competing-ion chromatography with ultraviolet detection. The method was applied to the determination of ambroxol levels in twelve healthy volunteers after oral administration of 90 mg of ambroxol in tablets of Mucosolvan and Ambrosan.     

High-performance liquid chromatographic determination of tramadol in human plasma

Tramadol has been determined in human plasma samples using a sensitive high-performance liquid chromatographic method. The plasma samples were extracted with tert.-butylmethyl ether in one-step liquid-liquid extraction (recovery 86%) and analyses of the extracts were performed on reversed-phase silica gel using ion-pair chromatography (verapamil as an internal standard) and fluorescence detection. The method was applied to the determination of tramadol levels in twelve healthy volunteers after oral administration of 100 mg of tramadol in capsules of Protradon and Tramal.     

Quantitation of Valdecoxib in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection using liquid-liquid extraction

A simple, sensitive and specific HPLC method with UV detection (210 nm) was developed and validated for quantitation of Valdecoxib in human plasma, the newest addition to the group of non-steroidal anti-inflammatory drugs-a highly selective cyclooxygenase-2 inhibitor. The analyte and an internal standard (Rofecoxib) were extracted with diethyl ether/dichloromethane (70/30 (v/v)). The chromatographic separation was performed on reverse phase ODS-AQ column with an isocratic mobile phase of water/methanol (47/53 (v/v)). The lower limit of quantitation was 10 ng/ml, with a relative standard deviation of <20%. A linear range of 10-500 ng/ml was established. This HPLC method was validated with between-batch and within-batch precision of 1.27-7.45 and 0.79-6.12%, respectively. The between-batch and within-batch bias was 0.74-7.40 and -0.93 to 7.70%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Valdecoxib in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is suitable for bioequivalence studies following single dose in healthy volunteers.     

High-performance liquid chromatographic assays with fluorometric detection for mivacurium isomers and their metabolites in human plasma

Two high-performance liquid chromatographic assays coupled with fluorometric detection have been developed for the determination of mivacurium isomers (trans-trans, cis-trans and cis-cis) and their monoester and alcohol metabolites in human plasma. A novel solid-phase extraction procedure allowed good recovery of the mivacurium isomers (mean 98%) and their monoester metabolites (mean 83%), whereas the alcohol metabolites were analyzed after direct precipitation of plasma proteins. For all analytes, these assays proved to be sensitive (LOQ 3.9–15.6 ng/ml), reproducible (C.V. < 15%) and accurate (>94%) over the therapeutic range of concentrations of mivacurium and its metabolites. These two methods were applied successfully to a pharmacokinetic study of mivacurium after a bolus dose of 0.15 mg/kg in anesthetized patients.     

High-performance liquid chromatographic analysis of nitroxoline in plasma and urine

A high-performance liquid chromatographic method has been developed for the determination of nitroxoline in 50-μl plasma and urine samples.A structural analogue of nitroxoline, 8-hydroxyquinoline, was added to the eluent in order to suppress peak asymmetry. Several parameters of the eluent were studied for the optimisation of the chromatographic system.Plasma concentration—time curves were constructed for three volunteers after they had received an oral dose of 100 mg of nitroxoline. Plasma half-life was about 1 h. Within 12 h, about 1% of the dose was excreted in the urine as free nitroxoline and about 30% as conjugated metabolite of the parent compound.