Stability study of Prulifloxacin and Ulifloxacin in human plasma by HPLC–DAD

A new and specific HPLC–DAD method for the direct determination of Prulifloxacin and its active metabolite, Ulifloxacin, in human plasma has been developed. Plasma samples were analysed after a simple solid phase extraction (SPE) clean-up using a new HILIC stationary phase based high-performance liquid chromatography (HPLC) column and an ammonium acetate buffer (5?mM, pH 5.8)/acetonitrile (both with 1% Et₃N, v/v) mobile phase in isocratic elution mode, with Danofloxacin as the internal standard. Detection was performed using DAD from 200 to 500?nm and quantitative analyses were carried out at 278?nm. The LOQ of the method was 1?μg/mL of the cited analytes and the calibration curve showed a good linearity up to 25?μg/mL. For both analytes the precision (RSD%) and the trueness (bias%) of the method fulfil with International Guidelines. The method was applied for stability studies, at three QC concentration levels, in human plasma samples stored at different temperature of?+?25,?+?4 and ?20?°C in order to evaluate plasma stability profiles.     

Determination of ciclosporin A and its six main metabolites in isolated T-lymphocytes and whole blood using liquid chromatography-tandem mass spectrometry

A specific and sensitive method for determination of intracellular ciclosporin A (CsA) and its six main metabolites AM1, AM9, AM1c, AM1c9, AM19 and AM4N, in isolated T-lymphocytes and whole blood is described. T-lymphocytes were separated from whole blood using Prepacyte. The analytes were extracted and purified from isolated lymphocytes and whole blood by protein precipitation followed by solid-phase extraction (SPE). The analytes and the internal standard, ciclosporin C (CsC), were separated on a reversed phase C8 column (30 mm x 2.1mm, 3 microm) with a 10 mm x 2 mm, 5 microm Drop-In Guard Cartridge, using gradient elution chromatography and tandem ion trap mass spectrometry detection. The method has been validated in accordance with FDA guidelines and showed linear range from 0.25 to 500 ng/mL for CsA, 0.5 to 500 ng/mL for AM1, AM9 and AM19, 1 to 500 ng/mL for AM4N, AM1c and AM1c9 in intracellular matrix, and 2.5 to 3000 ng/mL for all analytes in whole blood. The applicability of the method is shown on patient samples.     

Micellar electrokinetic capillary chromatography determination of +S and -R arotinolol in serum using UV detection and solid phase extraction.

A method for the simultaneous determination of +S and -R arotinolol in serum by micellar electrokinetic capillary chromatography is described. Stereoselective resolution of the arotinolol enantiomers was achieved using 5 mM sodium taurocholate in 10 mM sodium dihydrogen phosphate buffer of pH 2.5. A 72-cm uncoated fused-silica capillary at a constant voltage of 15 kV was used for the analysis. The analytes of interest were extracted from serum using solid phase extraction. An octadecyl cartridge gave good recoveries in excess of 87% for both +S and -R arotinolol without any interference. The calibration curves were linear over the range of 50-500 ng ml(-1) with +S propranolol as the internal standard and the coefficient of determination was greater than 0.999 (n = 3). The limit of quantitation was 50 ng ml(-1) for each enantiomer and the detection limit using 1 ml serum and a UV detection set et 220 nm was 25 ng ml(-1) (S/N = 2). Precision and accuracy of the method were in the range 0.8-2.7% and 1.2-6.4%, respectively, for +S arotinolol and 1.1-3.9% and 2.2-6.5%, respectively, for -R arotinolol.     

Analysis of the second generation antidepressant venlafaxine and its main active metabolite O-desmethylvenlafaxine in human plasma by HPLC with spectrofluorimetric detection

A high-performance liquid chromatographic method has been developed for the determination of the recent serotonin and norepinephrine reuptake inhibitor (SNRI) venlafaxine and its main active metabolite, O-desmethylvenlafaxine, in human plasma. Separation was obtained by using a reversed-phase column (C8, 150 x 4.6 mm I.D., 5 microm) and a mobile phase composed of 75% aqueous phosphate buffer containing triethylamine at pH 6.8 and 25% acetonitrile. Fluorescence detection was used, exciting at lambda=238 nm and monitoring the emission at lambda=300 nm. Citalopram was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with C1 cartridges (100 mg, 1 mL). The limit of quantification (LOQ) was 1.0 ng mL(-1) and the limit of detection (LOD) was 0.3 ng mL(-1) for both analytes. The method was applied with success to plasma samples taken from patients undergoing treatment with venlafaxine. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence, the method is suitable for therapeutic drug monitoring of venlafaxine and its main metabolite in depressed patients' plasma.     

New simple liquid chromatographic method for the determination of trimethoprim, sulfadiazine and N4-acetylsulfadiazine in plasma of broilers

A new method for simultaneous quantification of trimethoprim, sulfadiazine and N4-acetylsulfadiazine in plasma of broilers at levels down to 13-16 ng/ml has been developed. Samples were deproteinized with acetonitrile, defatted with hexane, and extracted with dichloromethane. Chromatographic analysis was carried out on a C18 column in the presence of tetrabutylammonium hydrogen sulfate, a competing base, while detection was performed at 240 nm for trimethoprim, and 270 nm for both sulfadiazine and N4-acetylsulfadiazine. Accuracy and precision data showed recoveries and relative standard deviation values better than 87.3% and 3.1%, respectively, for all three analytes. The good analytical characteristics of the method could allow limits of detection in the low ng/ml range to be realised. The method was successfully applied to determine drug concentrations in plasma samples from broilers administered a combination of sulfadiazine and trimethoprim.     

Column-switching high-performance liquid chromatographic method for the determination of a thymidylate synthase inhibitor, LY231514, an investigational agent for the treatment of solid tumors, in human plasma

A reversed-phase, column-switching high-performance liquid chromatographic (HPLC) method is described for the determination of a new thymidylate synthase inhibitor in human plasma. The compound and an internal standard are extracted from plasma using a Certify II solid-phase cartridge. Extracts are evaporated to dryness and the residue is reconstituted with mobile phase buffer. The analytes are separated from polar interferences and buffer salts originating from the elution step on a 4-mm YMC Basic pre-column. The fraction containing the analytes is further separated on a 25-cm YMC Basic column. The analytes are detected by their absorbance at 250 nm. The limit of quantitation is 10 ng/ml. The method is linear from 10 ng/ml to 80 μg/ml using three standard curve ranges. Validation studies for all three ranges show the method to be reproducible. The method has been successfully used to support pharmacokinetic studies.     

Determination of dapoxetine, an investigational agent with the potential for treating depression, and its mono- and di-desmethyl metabolites in human plasma using column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic (HPLC) method is described for the determination of dapoxetine, and its mono- and di-desmethyl metabolites in human plasma. The analytes, including an internal standard, were extracted from plasma at basic pH with hexane—ethyl acetate. The organic extract was evaporated to dryness and the residue reconstituted with acetonitrile. The analytes were separated from late-eluting endogenous substances on a Zorbax RX-C₈ pre-column. The front-cut fraction containing the analytes was further separated on a second RX-C₈ column. The analytes were detected by their native fluorescence, using excitation and emission wavelengths of 230 and 330 nm, respectively. The limit of quantitation was determined to be 20 ng/ml, and the response was linear from 20 to 200 ng/ml. The method has been successfully applied to human plasma samples in a Phase I study.     

Determination of triethylenetetramine (TETA) and its metabolites in human plasma and urine by liquid chromatography-mass spectrometry (LC-MS)

A liquid chromatography-mass spectrometry (LC-MS) method has been developed to measure triethylenetetramine (TETA) and its metabolites in human samples. We identified two metabolites of TETA, N1-acetyltriethylenetetramine (MAT) and N1,N10-diacetyltriethylenetetramine (DAT), the latter being novel. We further developed this LC-MS method for the measurement of TETA and these metabolites in human plasma and urine in a single injection. Separation of analytes was achieved on a cyano column using 15% acetonitrile, 85% water (18 M Omega), and 0.1% heptafluorobutyric acid as the mobile phase. Simultaneous MS detection was performed at [M+H]+ values of 147, 189, 231 and 245, corresponding to TETA, MAT, DAT, and N1-acetylspermine as the internal standard, respectively. This method was successfully applied to measure TETA, MAT and DAT in plasma and urine of humans receiving oral drug treatment.     

Quantitation of ibogaine and 12-hydroxyibogamine in human plasma by liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic (HPLC) method with fluorimetric detection was developed for the simultaneous determination of ibogaine and noribogaine in human plasma using fluorescein as internal standard. This method involved a solid phase extraction of the compounds from plasma using N-vinylpyrrolidone-divinybenzene copolymer cartridges. Separation of the three analytes was performed on a reversed-phase Supelcosil C18 analytical column (75 mm x 4.6mm i.d., 3 microm particle size). The excitation wavelength was set at 230 nm for the first 15.8min and then at 440 nm for the following 14.2 min; the emission wavelength was set at 336 nm for the first 15.8 min and then at 514 nm for the following 14.2 min. Obtained from the method validation, inter-assay precision was 6.0-12.5% and accuracy was 95.4-104%. The extraction efficiencies of the assay were higher than 94% and were constant across the calibration range. The lower limits of quantitation were 0.89 ng/ml for ibogaine and 1 ng/ml for noribogaine; at these levels, precision was < or =17% and accuracy was 95-105%. In this paper, extensive stability testing was undertaken using a wide range of storage conditions. Special attention must be paid to sample handling to avoid light degradation of the compounds.     

Simultaneous determination of creatinine and pseudouridine concentrations in bovine plasma by reversed-phase liquid chromatography with photodiode array detection

A simple, rapid method for the simultaneous determination of creatinine and pseudouridine in bovine plasma is described. Plasma was de-proteinised, concentrated, and chromatographed for 15 min on a C(18) column. Analytes were detected at an optimum wavelength (262 nm) and the internal standard (cimetidine) was detected at 220 nm. The pH of analysis was between 6.5 and 7 where both analytes exist in single chemical forms giving maximum accuracy. Recoveries of both analytes were above 96%. Lowest detectable amounts of creatinine and pseudouridine were 0.28 nmol and 9.0 pmol, and the typical levels detected (+/-SD) were 60 (+/-2.8) and 2.3 (+/-0.10) micromol/L, respectively.     

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.     

Spectrophotometric resolution of ternary mixtures of tryptophan, tyrosine, and histidine with the aid of principal component-artificial neural network models

A simple and sensitive spectrophotometric method to resolve ternary mixtures of tryptophan (Trp), tyrosine (Tyr), and histidine (His) in synthetic and water samples is described. It relies on the different kinetic rates of the analytes in their oxidative reaction with potassium ferricyanide (K(3)Fe(CN)(6)) in alkaline medium. The absorbance data were monitored on the analytical wavelength (420 nm) of K(3)Fe(CN)(6) spectrum. Synthetic mixtures of the three amino acids were analyzed, and the data obtained were processed by principal component-artificial neural network (PC-ANN) models. After reducing the number of spectral data using principal component analysis, an artificial neural network consisting of three layers of nodes was trained by applying a back-propagation learning rule. Tangent and sigmoidal transfer function were used in the hidden and output layers, respectively. The analytical performance of this method was characterized by relative standard error. The method allowed the determination of Trp, Tyr, and His at concentrations between 10 and 55, 10 and 60, and 10 and 40 microg ml(-1), respectively. The results show that the PC-ANN is an efficient method for prediction of the three analytes.     

Determination of methotrexate, several pteridines, and creatinine in human urine, previous oxidation with potassium permanganate, using HPLC with photometric and fluorimetric serial detection

A novel HPLC method, using UV and fluorimetric serial detection, for the simultaneous determination of methotrexate (MTX), five disease marker pteridines, and the reference metabolic subproduct creatinine (CREA) in human urine was established. A previous oxidation process using 10(-3) M KMnO4 (pH 5.0) and 35min of oxidation time was necessary to transform the analytes in the highly fluorescent pteridinic rings. CREA was not affected by the oxidative medium. Using Tris-HCl/NaCl buffer solution (pH 6.6) as mobile phase, MTX and the assayed pteridines were monitored by fluorescence at lambda(em) = 444 nm and lambda(ex) = 280 nm and creatinine was monitored by absorption measurements at lambda(abs) = 230 nm. All components were well resolved in approximately 7 min. Detection limits, according the criteria of Clayton and co-workers, were 10 ng ml(-1) for MTX, less than 1 ng ml(-1) for all of the pteridines, and 4 microg ml(-1) for CREA.     

Sensitive method for the assay of sertindole in plasma by high-performance liquid chromatography and fluorimetric detection

A simple and highly sensitive normal-phase HPLC method is described for determining sertindole concentrations in human plasma using fluorimetric detection. A short C₈ column was used to extract sertindole and the internal standard from plasma; the column was rinsed with acetonitrile, and the analytes were recovered by elution with methanol. This uncommon selectivity between the two solvents allowed clean extraction and near- quantitative recovery of the analytes (> 89%). Separation was done on a 5-μm silica-gel column and detection was performed by fluorimetry, with emission at 340 nm and excitation at 260 nm. The detection and lower quantifiable limits were 0.01 and 0.025 ng/ml, respectively, with no interference from plasma or potential metabolites.     

Enantioselective determination of zopiclone and its metabolites in urine by capillary electrophoresis

A method has been developed for the stereoselective determination of zopiclone and its main metabolites in urine. After the addition of the internal standard zolpidem the urine samples were extracted at pH 8 with chloroform-isopropanol (9:1). Analyses were carried out using capillary electrophoresis (CE) with β-cyclodextrin as the chiral selector. The analytes were detected using UV laser-induced fluorescence detection with a He-Cd laser operated at 325 nm. Urine samples of two volunteers after oral administration of 7.5 mg zopiclone were investigated. The S-(+)-enantiomers of zopiclone and its metabolites were always excreted in higher amounts than the R-(−)-enantiomers. With the same method the zopiclone enantiomers were quantified in saliva. Compared to high-performance liquid chromatography, the CE method is very fast and simple.     

Simultaneous determination of ketamine and xylazine in canine plasma by liquid chromatography with ultraviolet absorbance detection

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of ketamine and xylazine in canine plasma is described. Plasma samples (500 microl) are cleaned up via liquid-liquid extraction. The analytes and the internal standard clonidine are separated on a cyano (CN) column using a mobile phase containing acetonitrile-0.005 M phosphate buffer adjusted to pH 5.5 (3:2) at a detection wavelength of 215 nm. The method was validated according to specificity, sensitivity, accuracy and reproducibility and was used to determine the plasma concentrations of both compounds in dogs after intramuscular injection.     

Double column-switching high-performance liquid chromatographic method for the determination of TAK-603 and its metabolites in human serum

A double column-switching high-performance liquid chromatographic (HPLC) method for the determination of concentrations for TAK-603 (T) and its metabolites, T-72258 (M-I) and T-72294 (M-III), in human serum was developed. The analytes were extracted with ethyl acetate from human serum samples treated with triethylamine and injected into the HPLC system. Separation of the analytes was performed on the HPLC system with double column-switching technique. The mobile phases A and B for the first column and the mobile phase C for the second column used were a mixture of methanol–10 mM aqueous ammonium acetate solution (1:1, v/v), methanol and a mixture of methanol–10 mM aqueous ammonium acetate solution (11:9, v/v), respectively. The eluate was monitored with a UV detector at a wavelength of 253 nm. The work-up procedure was reproducible and more than 90% of the analytes could be recovered from human serum. The lower limits of quantitation were all 1 ng/ml for the analytes when 0.5 ml of human serum was used. Standard curves were linear with a correlation coefficient (R) of more than 0.999 in the range of 1–500 ng/ml for T, M-I and M-III in human serum. The intra- and inter-day precision of the method for the various analytes were below 4.8%. The accuracy was good with the deviations between spiked and calculated concentrations of the analytes being within 11.0%. The method was successfully applied to analyze serum samples after an oral administration of T to healthy male volunteers.     

High-performance liquid chromatographic determination of tiapride and its phase I metabolite in blood plasma using tandem UV photodiode-array and fluorescence detection

New bioanalytical SPE-HPLC-PDA-FL method for the determination of the neuroleptic drug tiapride and its N-desethyl metabolite was developed, validated and applied to xenobiochemical and pharmacokinetic studies in humans and animals. The sample preparation process involved solid-phase extraction of diluted plasma spiked with sulpiride (an internal standard) using SPE cartridges DSC-PH Supelco, USA. Chromatographic separation of the extracts was performed on a Discovery HS F5 250 mm × 4 mm (Supelco) column containing pentafluorophenylpropylsilyl silica gel. Mobile phase (acetonitrile-0.01 M phosphate buffer pH=3, flow rate 1 ml min(-1)) in the gradient mode was employed in the HPLC analysis. Tandem UV photodiode-array→fluorescence detection was used for the determination of the analytes. Low concentrations of tiapride and N-desethyl tiapride were determined using a more selective fluorescence detector (λ(exc.)/λ(emiss.)=232 nm/334 nm), high concentrations (500-6000 pmol ml(-1)) using a UV PDA detector at 212 nm with a linear response. Each HPLC run lasted 15 min. Lower limits of quantification (LLOQ) for tiapride (N-desethyl tiapride) were found to be 8.24 pmol ml(-1) (10.11 pmol ml(-1)). The recoveries of tiapride ranged from 89.3 to 94.3%, 81.7 to 86.8% for internal standard sulpiride and 90.9 to 91.8% for N-desethyl tiapride.     

Determination of p-phenylenediamine and its metabolites MAPPD and DAPPD in biological samples using HPLC-DAD and amperometric detection

A sensitive and selective HPLC method using a diode array detector (DAD) and an electrochemical detector (ECD) in series has been developed and validated for the quantitative measurement of p-phenylenediamine and its acetylated metabolites N-acetyl-p-phenylenediamine (MAPPD) and N,N′-diacetyl-p-phenylenediamine (DAPPD) in biological samples. The separation was carried out on a hydrophilic modified AQUA C₁₈ column and the mobile phase was composed of acetonitrile: ammonium acetate solution (5:95, 25 mM, v/v). Spectrophotometric detection was performed at 240 or 255 nm and amperometric detection was carried out using a positive oxidation potential of 400 mV. The quantification of the three analytes was validated in the range of 0.05–50 μM and the established limits of determination were 0.5 μM for PPD and MAPPD and 1 μM for DAPPD. The standard deviations (N = 9) were lower than 7.5% at a concentration of 1 μM. The samples were stabilised with ascorbic acid to prevent PPD from oxidizing. Pretreatment of samples or analyte enrichment before sample injection is not required. The method proved to be accurate, sensitive and sufficiently specific. It was applied to the ecotoxicological study of the kinetics of the PPD N-acetylation in cell lysates in two different media.