Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C₁₈ column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5–4000 ng/ml. The recovery of internal standard “8212” at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.     

Routine determination of plasma catecholamines using reversed-phase,ion-pair high-performance liquid chromatography with electrochemical detection

A procedure is described for the determination of plasma catecholamines using reversed-phase, ion-pair high-performance liquid chromatography coupled with electrochemical detection. Optimisation of chromatographic conditions with respect to detector performance and adherence to procedures and precautions described, render the method applicable to both neurochemical research and routine clinical analysis. The limit of quantitative detection of the method was found to be approximately 30 pg per injection for individual catecholamines. A single chromatographic run, providing adequate resolution of each component, could be completed in approximately 12 min.     

Determination of ketorolac in human plasma by reversed-phase high-performance liquid chromatography using solid-phase extraction and ultraviolet detection

An improved high-performance liquid chromatographic method has been developed to measure human plasma concentrations of the analgesic nonsteroidal anti-inflammatory drug ketorolac for use in pharmacokinetic studies. Samples were prepared for analysis by solid-phase extraction using Bond-Elut PH columns, with nearly complete recovery of both ketorolac and the internal standard tolmetin. The two compounds were separated on a Radial-Pak C₁₈ column using a mobile phase consisting of water–acetonitrile–1.0 mol/l dibutylamine phosphate (pH 2.5) (30:20:1) and detected at a UV wavelength of 313 nm. Using only 250 μl of plasma, the standard curve was linear from 0.05 to 10.0 μg/ml.     

Determination of stavudine, a new antiretroviral agent, in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive high-performance liquid chromatographic assay has been developed to determine the levels of a new antiretroviral agent, stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T), in human plasma. Didanosine (2′,3′-dideoxyinosine, ddI) was used as the internal standard. The very selective sample pretreatment involved solid-phase extraction using silica gel columns. Chromatography was carried out on a μBondapak phenyl column, using a mobile phase of 0.005 M phosphate buffer (pH 6.8)—methanol (90:10, v/v) and ultraviolet detection at 265 nm. The method has been validated, and stability tests under various conditions have been performed. The detection limit is 10 ng/ml (using 500-μl human plasma samples). The bioanalytical assay has been used in a single pharmacokinetic experiment in a rat to investigate the applicability of the method in vivo.     

Sensitive determination of nitrotyrosine in human plasma by isocratic high-performance liquid chromatography

A highly sensitive and simple isocratic high-performance liquid chromatography method was developed for determination of 3-nitrotyrosine in human plasma with precolumn derivatization with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole. The precision of the method was satisfactory (coefficient of variation 4.8%), and the detection limit was established at 0.1 pmol of 3-nitrotyrosine allowing the determination at the level of 6 pmol/ml in human plasma. The recoveries of 3-nitrotyrosine and α-methyltyrosine, an internal standard, were 89.3 +-7.1 and 85.7±7.6%, respectively. The 3-nitrotyrosine level was 31±6 pmol/ml (mean±S.D., n=9) in plasma from healthy volunteers. Since 3-nitrotyrosine is a stable product of peroxynitrite, an oxidant formed by a reaction of nitric oxide and superoxide radicals, the measurement of its plasma concentration may be useful as a marker of nitric oxide-dependent oxidative damage.     

Analysis of perillic acid in plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A simple and sensitive isocratic high-performance liquid chromatographic (HPLC) method with UV detection for the quantitation of perillic acid, a major circulating metabolite of perillyl alcohol and d-limonene, in plasma is described. Sample preparation involved protein precipitation and subsequent transfer and dilution with 10 mM NaHCO₃. The mobile phase consisted of acetonitrile (36%) and 0.05 M ammonium acetate buffer pH 5.0 (64%). Separations were achieved on a C₁₈ column and the effluent monitored for UV absorption at the analytes' respective UV_max. Separation was excellent with no interference from endogenous plasma constituents. This method was found suitable for quantifying drug concentrations in the range of 0.25 to 200.0 μg/ml using a 0.05-ml plasma sample, and was used to study the plasma pharmacokinetics of perillic acid in mice.     

Determination of fluvastatin and its five metabolites in human plasma using simple gradient reversed-phase high-performance liquid chromatography with ultraviolet detection

A simple gradient reversed-phase high-performance chromatographic method with ultraviolet detection for the determination of fluvastatin (FV) and its five metabolites, (M-2, M-3, M-4, M-5 and M-7) in human plasma was developed and validated. The limit of quantification of FV and its five metabolites in human plasma was 10 ng ml⁻¹. The assay had satisfactory selectivity, recovery, linearity and precision accuracy. Stability studies showed that FV and its five metabolites were stable in plasma up to at least 1 month of storage at −30°C.     

Simultaneous determination of celecoxib,hydroxycelecoxib, and carboxycelecoxib in human plasma using gradient reversed-phase liquid chromatography with ultraviolet absorbance detection

A new HPLC method for the simultaneous determination of celecoxib, carboxycelecoxib and hydroxycelecoxib in human plasma samples has been developed. Following a solid-phase extraction procedure, the samples were separated by gradient reversed-phase HLPC (C(18)) and quantified using UV detection at 254 nm. The method was linear over the concentration range 10-500 ng/ml. The intra-assay variability for the three analytes ranged from 4.0 to 12.6% and the inter-assay variability from 4.9 to 14.2%. The achieved limits of quantitation (LOQ) of 10 ng/ml for each analyte allowed the determination of the pharmacokinetic parameters of the analytes after administration of 100 mg celecoxib.     

Determination of a novel sigma receptor antagonist, DuP 734, in rat plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A selective high-performance liquid chromatographic (HPLC) assay for a sigma receptor antagonist, DuP 734 (I), in rat plasma has been developed. Compound I and internal standard, XC031 (I.S.), were first extracted from plasma into an ethyl acetate—toluene mixture (3:7, v/v) and then back-extracted into freshly prepared phosphoric acid (0.03 M). Separation of I and I.S. with no interference from endogenous substances was achieved on a reversed-phase octyl column and detection was by UV at 229 nm. The mobile phase consisted of acetonitrile—glacial acetic acid—triethylamine—0.05 M ammonium acetate (670:4:2:2000, v/v). Using 0.5 ml of rat plasma for extraction, the limit of quantitation was 43 ng/ml and the assay was linear from 43 to 8536 ng/ml. The intra- and inter-day coefficients of variation ranged from 0.7 to 3.0%, and from 1.4 to 14.5%, respectively, over the entire concentration range. The accuracy was within 16.1% of the spiked concentrations. I was stable in frozen plasma at −20°C for at least 68 days.     

Quantitative determination of the cholinesterase inhibitor physostigmine in brain tissue samples using reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method, based on a dynamic cation-exchange system was used for the determination of physostigmine in brain tissue extracts. The precision and detection limit of the method as well as the extraction efficiency were established. The distribution of physostigmine over several parts of the brain after intravertebral application is reported.     

Improved determination of bovine glutaminyl cyclase activity using precolumn derivatization and reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive, rapid and reproducible assay for the determination of glutaminyl cyclase activity is reported. This method is based on the monitoring of the absorption of l-pyroglutamic acid beta-naphthylamide at 235 nm, enzymatically formed from the substrate l-glutaminyl-beta-naphthylamide, after separation by high-performance liquid chromatography using a C-18 reversed-phase column by isocratic elution. The detection limit of this method is at a level as low as 0.08 nmol/ml and, the time consumed for analysis is <6.5 min per sample for separation and quantification. The optimum pH for glutaminyl cyclase activity was 8.0-8.5. The K(m) and V(max) values were 100.2+/-2.9 microM and 332 +/-21.7 pmol/(h microg protein), respectively, with the use of enzyme extract obtained from bovine pituitary. Glutaminyl cyclase activity was strongly inhibited by zinc(II) ion and 1,10-phenanthroline. By using this assay, the stimulatory effect of bacterial lipopolysaccharide on this enzyme activity was observed in macrophage cell line RAW 264.7. Our newly developed assay would be useful for clarification of the physiological role of this enzyme.     

Simultaneous determination of clofibrate and its active metabolite clofibric acid in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet absorbance detection

A reversed-phase high-performance liquid chromatographic (HPLC) using ultraviolet (UV) absorbance detection method for simultaneous determination of clofibrate (I) and its major metabolite clofibric acid (II) in human plasma has been developed to support a clinical study. I, II and internal standard (I.S., III) are isolated from human plasma by 96-well solid-phase extraction (SPE) C(18)z.ccirf;AR plate and quantified by direct injection of the SPE eluent onto the HPLC with UV detection wavelength at 230 nm. Two chromatographic methods, isocratic and step gradient, have been validated from 1.0 to 100.0 microg/ml and successfully applied to plasma sample analysis for a clinical study. The lower limit of quantitation (LLOQ) is 1.0 microg/ml for both I and II when 500 microl plasma sample is processed. Sample collection and preparation is conducted at 5 degrees C to minimize the hydrolysis of I to II in human plasma.     

Quantitative determination of thalidomide in human serum with high-performance liquid chromatography using protein precipitation with trichloroacetic acid and ultraviolet detection

A validated and precise reversed-phase high-performance liquid chromatographic method for the determination of thalidomide in serum, with phenacetin as an internal standard, is described. Protein precipitation, using trichloroacetic acid, was used for clean-up. The aliquot was chromatographed on a octadecyl column, using an eluent composed of 250 ml 0.01 M potassium dihydrogenphosphate, adjusted to a pH of 3.0 with a 43% phosphoric acid solution, mixed with 750 ml methanol. Ultraviolet detection was used at an operation wavelength of 220 nm. Hydrolytic degradation was prevented during analysis by acidification of samples with the precipitation reagent. Thalidomide and phenacetin were found to have retention times of 7.9 and 15.0 min, respectively. Recoveries ranging from 79 to 84% were found for both components, with reproducibility relative standard deviations of 0.8–3% and repeatability coefficients of 1.2–3%. A mean correlation coefficient of 0.9995 was found for the linear calibration curve (n=2) of thalidomide with limits of quantitation of 0.222–21 mg/l. The method appeared to be feasible for pharmacokinetic studies with thalidomide.     

Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of donepezil, a centrally and selectively acting acetyleholinesterase inhibitor, in human plasma. After sample alkalinization with 0.5 ml of NaOH (0.1 M), the test compound was extracted from I ml of plasma using isopropanol-hexane (3:97, v/v). The organic phase was back-extracted with 75 microl of HCl (0.1 M) and 50 microl of the acid solution was injected into a C18 STR ODS-II analytical column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (6 M) and acetonitrile (59.5:0.5:40, v/v) and was delivered at a flow-rate of 1.0 ml/min at 40 degrees C. The peak was detected using a UV detector set at 315 nm, and the total time for a chromatographic separation was approximately 8 min. The method was validated for the concentration range 3-90 ng/ml. Mean recoveries were 89-98%. Intra- and inter-day relative standard deviations were less than 7.3 and 7.6%, respectively, at the concentrations ranging from 3 to 90 ng/ml. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.     

Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C₁₈ Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and μBondapak C₁₈ column (150 × 3.9 mm I.D., 10 μm particle size). The detection limit (A_{375 nm}) for quercetin in plasma was 0.1 μg/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.