Simple high-performance liquid chromatographic method for determination of ketoconazole in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of ketoconazole in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile. The mobile phase comprised 0.05 M disodium hydrogen orthophosphate and acetonitrile (50:50, v/v) adjusted to pH 6. Analysis was run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The method is specific and sensitive with a quantification limit of approximately 60 ng/ml and a detection limit of 40 ng/ml at a signal-to-noise ratio of 3:1. Mean absolute recovery value was about 105%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 14%. The calibration curve was linear over a concentration range of 62.5–8000 ng/ml.     

Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

Simple high-performance liquid chromatographic method for the determination of metformin in human plasma

A simple high-performance liquid chromatographic method using ultraviolet detection was developed for the determination of metformin in human plasma. The method entailed direct injection of the plasma sample after deproteination using perchloric acid. The mobile phase comprised 0.01 M potassium dihydrogen orthophosphate (pH 3.5) and acetonitrile (60:40, v/v). Analyses were run at a flow-rate of 1.0 ml/min with the detector operating at a detection wavelength of 234 nm. The method is specific and sensitive, with a quantification limit of approximately 60 ng/ml and a detection limit of 15 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery value was about 97%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The calibration curve was linear over a concentration range of 62.5–4000 ng/ml.     

Simple liquid chromatographic method for the determination of cefotaxime in human and rat plasma

A high-performance liquid chromatographic method with ultraviolet (UV) detection was developed for measuring cefotaxime in rat and human plasma. The method used direct injection of the plasma supernatant after deproteinization with 70% perchloric acid. Degradation of cefotaxime in acidic medium was retarded by adding phosphate buffer before centrifuging the sample. The mobile phase was 0.05 M aqueous ammonium acetate-acetonitrile-tetrahydrofuran (87:11:2, v/v) adjusted to pH 5.5. Analysis was run at a flow-rate of 1.0 ml/min, and a detection wavelength of 254 nm was used. The method has a quantification limit of 0.20 microgram/ml. The within- and between-day coefficients of variation and accuracy values were less than 8% and +/-3%, respectively, while the recovery values were greater than 87% over the concentration range tested (0.20-50 microgram/ml). The speed, sensitivity, specificity and reproducibility of this method make it particularly suitable for the routine determination of cefotaxime in human plasma. Moreover, only a relatively small sample plasma volume (100 microliter) is required, allowing this method to be applied to samples taken from neonates.     

Simple high-performance liquid chromatographic method for the determination of tocotrienols in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of vitamin E especially δ-, γ- and α-tocotrienols in human plasma. The method entailed direct injection of plasma sample after deproteinization using a 3:2 mixture of acetonitrile–tetrahydrofuran. The mobile phase comprised 0.5% (v/v) of distilled water in methanol. Analyses were run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 296 nm and emission wavelength of 330 nm. This method is specific and sensitive, with a quantification limit of approximately 40, 34 and 16 ng/ml for α-, γ- and δ-tocotrienol, respectively. The mean absolute recovery values were about 98% while the within-day and between-day relative standard deviation and percent error values of the assay method were all less than 12.0% for α-, γ- and δ-tocotrienol. The calibration curve was linear over a concentration range of 40–2500, 30–4000 and 16–1000 ng/ml for α-, γ- and δ-tocotrienol, respectively. Application of the method in a bioavailability study for determination of the above compounds was also demonstrated.     

Simple and reliable method of doxycycline determination in human plasma and biological tissues

Over recent years there has been a resurgence in the use of doxycycline in clinical practice, which does not depend on its antibacterial properties. This paper describes a method of determination of doxycycline in human plasma and atheromatous tissue using high-performance liquid chromatography (HPLC), and a cheap commercially available extraction system. Doxycycline is extracted in the mobile phase and injected directly into the HPLC system, avoiding time consuming drying up steps. A limit of detection of 0.125 μg/ml of plasma, and a relative standard deviation of 3% was achieved, making the method very reliable and useful for assays within the usual therapeutic range. The method has also been applied to the extraction of a mixture of tetracyclines from plasma and atherma with equal efficacy, making it useful for assays of this class of drugs in veterinary practice and assays of food contaminants.     

Simple and rapid liquid chromatography method for determination of moxifloxacin in plasma

A high performance liquid chromatographic method for determination of moxifloxacin in human plasma was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C₁₈ column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.125 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.     

Simple and rapid liquid chromatography method for determination of efavirenz in plasma

A simple and rapid high performance liquid chromatographic method for determination of efavirenz in human plasma was developed. The method involved extraction of sample with ethyl acetate and analysis using a reversed-phase C(18) column (150 mm) with UV detection. The assay was linear from 0.0625 to 10.0 microg/ml. The method was specific for efavirenz estimation and the drug was stable in plasma up to one month at -20 degrees C. The average recovery of efavirenz from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring of efavirenz.     

Simple method for determination of paraquat in plasma and serum of human patients by high-performance liquid chromatography

A simple and fast HPLC system is presented for quantifying paraquat in human plasma and serum using 1,1'-diethyl-4,4'-bipyridyldiylium (diethyl paraquat) as an internal standard. An octadecyl-silica column is used with an eluent of 10% acetonitrile (v/v) containing sodium 1-octanesulphonic acid (3.0 mM) and a diethylamine-orthophosphoric acid buffer (pH 3). Unlike with other techniques, sample treatment requires only the precipitation of protein contents by 6% perchloric acid (v/v) in methanol. The method has a limit of detection of 0.1 microg/ml and is linear up to 10 microg/ml. The serum of four patients and the plasma of one patient with paraquat intoxication's were analysed and positive identification and quantification was readily achieved. One of those patients survived, partially given the rapid disclosure of his levels of paraquat. Therefore, this method is suitable for quantification of paraquat in toxicological samples. It may be used as a prognostic tool in critical case detoxification and to quickly identify potentially salvageable patients for enrollment in new hemofiltration studies.     

Simple method for clinical determination of 13 carotenoids in human plasma using an isocratic high-performance liquid chromatographic method

We report a reversed-phase high-performance liquid chromatography method which resolves 13 identified carotenoids and nine unknown carotenoids from human plasma. A Nucleosil C₁₈ column and a Vydac C₁₈ column in series are used with an isocratic solvent system of acetonitrile–methanol containing 50 mM acetate ammonium–dichloromethane–water (70:15:10:5, v/v/v/v) as mobile phase at a flow-rate of 2 ml/min. The intra-day (4.5–8.3%) and inter-day (1.3–12.7%) coefficients of variation are suitable for routine clinical determinations.     

Simple and sensitive high-performance liquid chromatography method for the determination of docetaxel in human plasma or urine

Several methods for quantification of docetaxel have been described mainly using HPLC. We have developed a new isocratic HPLC method that is as sensitive and simpler than previous methods, and applicable to use in clinical pharmacokinetic analysis. Plasma samples are spiked with paclitaxel as internal standard and extracted manually on activated cyanopropyl end-capped solid-phase extraction columns followed by isocratic reversed-phase HPLC and UV detection at 227 nm. Using this system, the retention times for docetaxel and paclitaxel are 8.5 min and 10.5 min, respectively, with good resolution and without any interference from endogenous plasma constituents or docetaxel metabolites at these retention times. The total run time needed is only 13 min. The lower limit of quantification is 5 ng/ml using 1 ml of plasma. The validated quantitation range of the method is 5–1000 ng/ml with RSDs≤10%, but plasma concentrations up to 5000 ng/ml can be accurately measured using smaller aliquots. This method is also suitable for the determination of docetaxel in urine samples under the same conditions. The method has been used to assess the pharmacokinetics of docetaxel during a phase I/II study of docetaxel in combination with epirubicin and cyclophosphamide in patients with advanced cancer.     

Simple GC-FID method development and validation for determination of alpha-tocopherol (vitamin E) in human plasma

This paper describes the development and validation of a novel GC-FID method for the determination of alpha-tocopherol concentration in human plasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. To determine the concentration of alpha-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. alpha-tocopherol was extracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min(-1). Calibration curves were linear over the concentration range 1-30 microg ml(-1) (for standard solutions and solutions without endogenous alpha-tocopherol in plasma) and 5-34 microg ml(-1) (for solutions with endogenous alpha-tocopherol in plasma). Absolute recovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of alpha-tocopherol from plasma averaged 97.44%. The limit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 microg.ml(-1) and 0.30 microg.ml(-1), respectively. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) was better than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of alpha-tocopherol. The endogenous alpha-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with this method.     

Simple and sensitive method for the determination of chlorpheniramine maleate in human plasma using liquid chromatography-mass spectrometry

A convenient liquid chromatographic-single quadrupole mass spectrometric (LC-MS) method was developed and validated for the determination of chlorpheniramine maleate (INN name: chlorphenamine) in human plasma. The method had advantages of a single liquid-liquid extraction with diethylether and high sensitivity. The linearity was also excellent over the concentration range of 0.52-20.8 ng/ml of chlorpheniramine maleate. The intra- and inter-day precision and accuracy ranged between 0.0 and 13.9%, showing a good reproducibility. This developed method was successfully applied to analysis of chlorpheniramine maleate in clinical studies.     

Simple chromatography method for simultaneous determination of dextromethorphan and its main metabolites in human plasma with fluorimetric detection

Dextromethorphan, the innocuous non-narcotic antitussive agent, is the most widely used probe drug to assess CYP2D6 function both in vivo and in vitro. For this reason a simple and selective high performance liquid chromatography method with fluorimetric detection for simultaneous quantitation of dextromethorphan, and its main metabolites in human plasma was developed and validated. The method involved a simple and rapid protein precipitation protocol, using a mixture of ZnSO(4) and methanol. The analysis was performed on a 3 microm, C(18) Tracer Excel 15 cm x 0.4 cm i.d. column by gradient elution in which Mobile phase A consisted of potassium dihydrogen phosphate buffer (pH = 3, 0.01 M):methanol:tetrahydrofuran (68.5:31:0.5), and mobile phase B consisted of methanol:tetrahydrofuran (93.25:6.75). Linear calibration curves were obtained in the range of 10-500 ng/ml for dextromethorphan, dextrorphan and hydroxymorphinan. The limit of quantitation (LOQ) was 10 ng/ml for each compound. The maximum within and between days precisions were 7.4 and 7.8%, respectively. The accuracies at four different concentration levels ranged from 88.2 to 111.5%. The recoveries were between 88.0 and 108.6%. The assay method was successfully applied to determine dextromethorphan metabolic ratio after an oral dose of 30 mg of dextromethorphan hydrobromide.     

Simplified method for determination of rosiglitazone in human plasma

Rosiglitazone is a thiazolidinedione antihyperglycemic drug used in the treatment of type 2 diabetes mellitus. Rosiglitazone is extensively metabolized by cytochrome P450 2C8 and so may have some utility as an in vivo probe for this enzyme. A liquid chromatographic method using sensitive fluorescence detection and simplified sample processing involving protein precipitation with acetonitrile was developed. The isocratic mobile phase consisted of 10 mM sodium acetate-acetonitrile (pH 5; 60:40, v/v) and was delivered at a flow rate of 1 ml/min to an Alltima phenyl column (250 mm x 4.6 mm, 5 microm). Detection was by fluorescence at (EX/EM) 247/367 for rosiglitazone and 235/310 for the internal standard betaxolol. Intra- and inter-day precision ranged from 3.1 to 8.5% and 2.3 to 5.7%, respectively. No endogenous interference was observed with either rosiglitazone or the internal standard. The assay is simple, economical, precise, and is directly applicable to human pharmacokinetic studies involving single dose rosiglitazone administration.     

HPLC-atmospheric pressure chemical ionization mass spectrometric method for enantioselective determination of R,S-propranolol and R,S-hyoscyamine in human plasma

A method for the simultaneous determination of R- and S-propranolol and R- and S-hyoscyamine in human plasma was developed, validated and applied to the analysis of samples from a clinical study. Sample preparation was performed by solid-phase extraction of 2 ml of human plasma using Oasis MCX cartridges and the enantioselective separations were achieved using a Chirobiotic V chiral stationary phase. The chromatography was carried out using gradient elution with a mobile phase composed of methanol:acetic acid:triethylamine which was varied from 100:0.05:0.04 to 100:0.05:0.1 (v/v/v) over 30 min and delivered at a flow rate 1 ml/min. The internal standard was R,S-propranolol-d7 and the analytes were quantified using a single quadrupole mass spectrometer employing APCI interface operated in the positive ion mode with single ion monitoring. The enantioselective separation factors, alpha, were 1.15 and 1.07 for S- and R-propranolol and R- and S-hyoscyamine, respectively. The standard curves were linear for all target compounds with coefficients of determination (r2) ranging from 0.9977 to 0.9999. The intra- and inter-day precision and accuracy were 相似文献   

Simple and efficient method for isolation and cultivation of endoscopically obtained human colonocytes

Few comparative and validated reports exist on the isolation and growth of colonoscopically obtained colonic epithelium. The aim of this study was to develop and validate a simple method for the cultivation of colonoscopically obtained colonocytes. Forty patients, who underwent routine colonoscopy and where the diagnosis of irritable bowel syndrome was later reached, were included. Seven colon biopsies were taken and incubated at varying time periods of 10-120 min and temperatures of 4-37 degrees C in a chelating buffer. The epithelium was then harvested and cultivated under three different conditions: 1) on a collagen coating, 2) embedded in a collagen gel, or 3) embedded in a gel put on a porous well insert. The effect of conditioned medium (CM), insulin, transferrin, selenium, and the oxygen content was assessed. Viability was tested by the metabolic dimethylthiazol-diphenyl-tetrazolium bromide assay, by flowcytometry, by phase contrast microscopy, and by transmission electron microscopy. Incubation at 21 degrees C for 75 min gave an optimal yield of 3 x 10(6) (2.0-3.8 x 10(6)) viable epithelial cells in intact crypts per seven biopsies. Embedding of crypts in a collagen gel put on a porous membrane was superior to the other methods applied [P < 0.003; median viability 71% (62-100%) compared with preculture values] after 24 h, which was a 160% increase in viability compared with coat-cultivated cells. CM had similar viability supporting effects to FCS. Other supplements had no effects. A simple method is presented, which makes cultivation of colonocytes obtained at endoscopy possible for up to 72 h.     

Simple and sensitive high-performance liquid chromatographic method for the determination of diltiazem and six of its metabolites in human plasma

A sensitive, specific and reproducible high-performance liquid chromatographic technique is described for the simultaneous determination in human plasma of diltiazem (DZ) and six of its primary and secondary metabolites which are products of N- and O-demethylation, deacetylation and N-oxidation. The method involves addition of excess KHCO₃ to 1 ml of plasma, followed by extraction with 4 ml of ethyl acetate. The organic layer was extracted with 0.01 M HCl and the aqueous layer was dried under nitrogen and then reconstituted with 0.002 M HCl. DZ and its metabolites were free from interference and were baseline-separated. Calibration curves were linear in the concentration range studied (5–500 ng/ml for all the species). The lower limit of quantification of the assay was 5 ng/ml for DZ and the metabolites. Inter-day and intra-day coefficients of variation were less than 10%. The applicability of this procedure is shown by evaluating the kinetics of DZ and its metabolites in three patients receiving chronic DZ therapy. N-Demethyldiltiazem, deacetyldiltiazem and N-demethyldeacetyldiltiazem were found to be the major metabolites, as previously described. Deacetyldiltiazem N-oxide was found in two of the patients. The other two known but unreported metabolites in human, O-demethyldeacetyldiltiazem and N,O-didemethyldeacetyldiltiazem, were found in the plasma of all three patients.     

Simple method for determination of terbutaline plasma concentration by high-performance liquid chromatography

A method is described in which low nanomolar concentrations of terbutaline in plasma can be quantitated by use of a standard isocratic high-performance liquid chromatography system with electrochemical detection. Samples were prepared for injection by solid-phase extraction and preserved from degradation by addition of glutathione. Terbutaline and internal standard metaproterenol were resolved from plasma constituents on a single C₁₈ column by ion-pairing chromatography. The method is precise and accurate for measurement of freebase concentrations as low as 4.4 nmol/l (1 ng/ml).     

Quantitative determination of ondansetron in human plasma by enantioselective liquid chromatography-tandem mass spectrometry

A sensitive and enantioselective method was developed and validated for the determination of ondansetron enantiomers in human plasma using enantioselective liquid chromatography-tandem mass spectrometry. The enantiomers of ondansetron were extracted from plasma using ethyl acetate under alkaline conditions. HPLC separation was performed on an ovomucoid column using an isocratic mobile phase of methanol-5 mM ammonium acetate-acetic acid (20:80:0.02, v/v/v) at a flow rate of 0.40 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/z 294-->170 for ondansetron enantiomers, and m/z 285-->124 for tropisetron (internal standard). The method was linear in the concentration range of 0.10-40 ng/mL for each enantiomer using 200 microL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.10 ng/mL. The intra- and inter-assay precision was 3.7-11.6% and 5.6-12.3% for R-(-)-ondansetron and S-(+)-ondansetron, respectively. The accuracy was 100.4-107.1% for R-(-)-ondansetron and 103.3-104.9% for S-(+)-ondansetron. No chiral inversion was observed during the plasma storage, preparation and analysis. The method was successfully applied to characterize the pharmacokinetic profiles of ondansetron enantiomers in healthy volunteers after an intravenous infusion of 8 mg racemic ondansetron.