Stereoselective high-performance liquid chromatographic analysis of ketoprofen and its acyl glucuronides in chronic renal insufficiency

A rapid, sensitive method was developed for the quantification of the R- and S-enantiomers of ketoprofen and their acyl glucuronide conjugates in the plasma and dialysate of hemodialysis-dependent anephric patients. Unconjugated R- and S-ketoprofen plasma concentrations were determined directly by liquid chromatography using a S,S-Whelk-O1 chiral stationary phase. R- and S-Ketoprofen glucuronide for use as standards were resolved using a C₁₈ reversed-phase HPLC column with a mobile phase containing the ion-pair reagent tetrabutylammonium hydrogen sulfate. Plasma glucuronides, however, could not be directly quantified due to matrix interference. Therefore, the glucuronides were isolated using reversed-phase HPLC and quantified after alkaline hydrolysis using the S,S-Whelk-O1 chiral stationary phase column.     

Isotopic separations of the drug N-0437 and its diastereoisomeric glucuronides by high-performance liquid chromatography

During the investigations of the metabolic pathways of the new dopaminergic drug N-0437 we encountered a substantial difference in HPLC-retention times between the metabolites, detected by a uv spectrophotometer, and their tritium-labeled markers, measured off-line by a scintillation counter. These distinct retention times can be ascribed to a phenomenon known as isotopic fractionation. In this article we quantified the isotopic separation by reversed-phase HPLC of the unlabeled N-0437, its deuterated and tritiated analogs, and their corresponding glucuronides, synthesized in vitro by rat liver microsomes. In the separation of the glucuronides we demonstrated that this isotope effect is dependent largely on the eluent pH.     

Ion-pair high-performance liquid chromatographic separation of two thyroxine glucuronides formed by rat liver microsomes

A simple reversed-phase ion-pair high-performance liquid chromatographic separation method has been developed for thyroxine (T4) and its glucuronide metabolites formed by liver microsomes of untreated and 3-methylcholanthrene-treated rats. Besides the phenol-T4-glucuronide, another, probably acyl-T4-glucuronide, formation has been detected. The effect of pH and temperature on the stability of the acyl-T4-glucuronide was also investigated. The lowering of pH to 2 and cooling the samples to 5°C is necessary to prevent the hydrolysis of acylglucuronide, while both pH and temperature do not affect the stability of the phenol-T4-glucuronide. The retention times of T4 and phenol-T4-glucuronide are highly influenced by the pH of the mobile phase, but not that of acyl-T4-glucuronide.     

Identification of quercetin glucuronides in human plasma by high-performance liquid chromatography–tandem mass spectrometry

After intake of food or herbal medicinal products containing quercetin glycosides, the systemic availability of the genuine glycoside, as well as the systemic occurrence of the aglycone or conjugates of this polyphenol has been a matter of dispute. Consequently, we designed this study to develop a reliable method for determination of quercetin and its metabolites. Following consumption of fried onions five different glucuronides of quercetin could be identified in human plasma samples by means of HPLC–UV–MS/MS. Selective determination of the target compounds was achieved by simultaneous UV (254 nm) and MS/MS detection with selected reaction monitoring experiments using positive mode electrospray ionisation. In contrast, neither the free flavonol nor the genuine glycoside could be detected in plasma. Identification of the quercetin glucuronides detected in vivo was confirmed by comparison with authentic reference compounds synthesised enzymatically using glucuronyl transferase from rabbit liver.     

High-performance liquid chromatographic method for the determination of benzodiazepines in plasma or serum using the column-switching technique

A column-switching high-performance liquid chromatographic method for the simultaneous determination of five frequently prescribed benzodiazepines: clonazepam, diazepam, flunitrazepam, midazolam and oxazepam was developed. A 50-μl plasma sample was directly injected into a BioTrap 500 MS (hydrophobic polymer) column. After a washing step with a mixture of phosphate buffer and acetonitrile, the retained benzodiazepines were back-flushed into a reversed-phase (LiChrospher Select B C₈) column with a mobile phase of acetonitrile–phosphate buffer. The method showed excellent linearity from 50 to 1000 ng/ml for clonazepam, flunitrazepam and midazolam and from 50 to 5000 ng/ml for diazepam and oxazepam. The recoveries were around 98% for all the benzodiazepines studied. The relative standard deviation for between- and within-day assay was <20% for low concentrations close to the values of the limit of quantification and <4% for high concentrations. The procedure described is relatively simple and rapid because no off-line manipulation of the sample is required: the total analysis time is approximately 30 min.     

Sensitive high-performance liquid chromatographic determination of famotidine in plasma : Application to pharmacokinetic study

A sensitive high-performance liquid chromatographic (HPLC) method for the quantitation of famotidine in human plasma is described. Clopamide was used as the internal standard. Plasma samples were extracted with diethyl ether to eliminate endogenous interferences. Plasma samples were then extracted at alkaline pH with ethyl acetate. Famotidine and the internal standard were readily extracted into the organic solvent. After evaporation of ethyl acetate, the residue was analysed by HPLC. The chromatographic separation was accomplished with an isocratic mobile phase consisting of acetonitrile—water (12:88, v/v) containing 20 mM disodium hydrogenphosphate and 50 mM sodium dodecyl sulphate, adjusted to pH 3. The HPLC microbore column was packed with 5 μm ODS Hypersil. Using ultraviolet detection at 267 nm, the detection limit for plasma famotidine was 5 ng/ml. The calibration curve was linear over the concentration range 5–500 ng/ml. The inter- and intra-assay coefficients of variation were found to be less than 10%. Applicability of the method was demonstrated by a bioavailability/pharmacokinetic study in normal volunteers who received 80 mg famotidine orally.     

Validation of a high-performance liquid chromatographic assay method for pharmacokinetic evaluation of busulfan

The development and validation of a high-performance liquid chromatographic (HPLC) assay for determination of busulfan concentrations in human plasma for pharmacokinetic studies is described. Plasma samples containing busulfan and 1,6-bis(methanesulfonyloxy)hexane, and internal standard, were prepared by derivatization with sodium diethyldithio-carbamate (DDTC) followed by addition of methanol and extraction with ethyl acetate. The extract was dried under nitrogen and the samples reconstituted with 100 μl of methanol prior to HPLC determination. Chromatography was accomplished using a Waters NovaPak octadecylsilyl (ODS) (150×3.9 mm I.D.) analytical column, NovaPak ODS guard column, and mobile phase of methanol-water (80:20, v/v) at a flow-rate of 0.8 ml/min with UV detection at 251 nm. The limit of detection was 0.0200 μg/ml (signal-to-noise ratio of 6) with a limit of quantitation (LOQ) of 0.0600 μg/ml for busulfan in plasma. Calibration curves were linear from 0.0600 to 3.00 μg/ml in plasma (500 μl) using a weighting scheme. Precision of the assay, as represented by C.V. of the observed peak area ration values, ranged from 4.41 to 13.5% (13.5% at LOQ). No day-to-day variability was observed in predicted concentration values and the bias was low for all concentrations evaluated (bias: 0 to 4.76%; LOQ: 2.91%). The mean derivatization and extraction yield observed for busulfan in plasma at 0.200, 1.20 and 2.00 μg/ml was 98.5% (range 93.4 to 107%). Plasma samples containing potential busulfan metabolites and co-administered drugs, which may be present in clinical samples, provided no response indicating this assay procedure is selective for busulfan. This method was used to analyze plasma concentrations following administration of a 1 mg/kg oral busulfan dose.     

Immunoaffinity extraction of morphine, morphine-3-glucuronide and morphine-6-glucuronide from blood of heroin victims for simultaneous high-performance liquid chromatographic determination

The development of an immunoaffinity-based extraction method for the determination of morphine and its glucuronides in human blood is described. For the preparation of an immunoadsorber, specific antisera (polyclonal, host: rabbit) against morphine, morphine-3-glucuronide and morphine-6-glucuronide were coupled to 1,1′-carbonyldiimidazole-activated trisacrylgel and used for immunoaffinity extraction of morphine and its glucuronides from coronary blood. The resulting extracts were analysed by HPLC with native fluorescence detection. The mean recoveries from spiked blood samples were 71%, 76% and 88% for morphine, morphine-3-glucuronide and morphine-6-glucuronide, respectively. The limit of detection was 3 ng/g blood and the limit of quantitation was 10 ng/g blood for all three analytes. The results of the analysis of coronary blood samples from 23 fatalities due to heroin are presented.     

Simple and robust high-performance liquid chromatographic method for the determination of ranitidine in microvolumes of human serum

A simple robust high-performance liquid chromatographic method is described for the determination of ranitidine in microvolumes of human serum. The drug of interest was isolated using liquid–liquid extraction with dichloromethane and back-extraction with 0.1% phosphoric acid and separation was obtained using a reversed-phase column under isocratic conditions, with ultraviolet detection at 313 nm. Intra-day and inter-day coefficients of variation ranged from 1 to 6% and 3 to 10%, respectively. Accuracy of the assay was less than 10% at all concentrations. The limit of detection and the limit of quantitation were 2 and 7 ng/ml, respectively. The linearity was assessed in the range 10–1000 ng/ml. It was shown that a group of common drugs co-administered with ranitidine did not interfere with its determination. The applicability of this method for the pharmacokinetic study of ranitidine following i.v. infusion in patients was demonstrated using only 100 μl of serum. The ruggedness of the assay was demonstrated over a three-year period.     

Simple liquid chromatographic method for the determination of physostigmine and its metabolite eseroline in rat plasma: application to a pharmacokinetic study

Physostigmine, an anticholinergic drug, and its metabolite eseroline can be quantitated by high-performance liquid chromatography (HPLC) with photodiode-array detection. After addition of the structurally related internal standard (-)-N-methylphysostigmine, rat plasma samples were extracted and cleaned using a Varian Bond Elut C(18) column. The methanol-ammonia (98:2) eluate was evaporated to dryness and reconstituted with 0.01 M sodium dihydrogenphosphate (pH 3). Physostigmine and eseroline were separated on an Alltech Ultrasphere Silica column (250x4.6 mm I.D.; particle size 5 micrometer) at a flow-rate of 1 ml/min, with a mobile phase of 0.01 M sodium dihydrogenphosphate (pH 3)-acetonitrile (85:15). The limits of detection were 10 and 25 ng/ml for physostigmine and eseroline, respectively; the signal-to-noise ratio for this concentration was approximately 3:1. Spiked rat plasma containing 0.1-2.5 microgram/ml of physostigmine and eseroline gives good linearity. The average percentage recovery from five spiked plasma samples was 88.0+/-2.9 and 61.1+/-5.6% for physostigmine and eseroline, respectively. Within the concentration range 0.1-2.5 microgram/ml, the within-day precision was 1.9-8.3 and 3.0-7.7% for physostigmine and eseroline, respectively, and the between-day precision was 4.1-9.3 and 3.7-11% for physostigmine and eseroline, respectively. The method is rapid, simple and reliable, thus it is suitable for pharmacokinetic studies in the rat.     

Simultaneous separation of atovaquone, proguanil and its metabolites on a mixed mode high-performance liquid chromatographic column

An isocratic high-performance liquid chromatographic (HPLC) method for simultaneous separation of the components in the antimalarial combination drug Malarone^® with UV detection is described. An HPLC system using a mixed mode column composed of 50% C₁₈ phase and 50% strong cation-exchanger has been optimised for the simultaneous separation of atovaquone, proguanil and its two main metabolites. The mobile phase was optimised for factors such as pH, counter ion concentration and acetonitrile. Elimination of interferences from other antimalarial drugs was achieved by adding sodium perchlorate to the mobile phase. With a mobile phase of acetonitrile-phosphate buffer (60:40, v/v) pH 6.8, 50.7 mmol l⁻¹ K⁺ and 10 mmol l⁻¹ Na·ClO₄, separation was achieved within a run time shorter than 17 min.     

Development of a plasma high-performance liquid chromatographic assay for LY303366, a lipopeptide antifungal agent, and its application in a dog pharmacokinetic study

An HPLC assay for plasma analysis of LY303366 (I), a semi-synthetic lipopeptide antifungal related to echinocandin B (ECB), was developed to support the selection and subsequent preclinical development of I. The method involved extraction of I from plasma with the aid of solid-phase extraction (SPE) cartidges followed by reversed-phase HPLC with UV detection at 300 nm. The method is simple, selective and is applicable to dog, rat, mouse and rabbit plasma. Validation studies using dog plasma showed that the values obtained for parameters of linearity, precision and accuracy were within acceptable limits. Based on analysis of 0.3 ml of plasma, the lower limit of quantitation was 20 ng/ml. The method has been successfully applied to determine the pharmacokinetic parameters of I in the dog following intravenous (i.v.) and oral administration. Compared to first generation ECB antifungal agents, the results of the i.v. dog study indicated a 50% reduction in clearance of the drug from plasma (0.1 l/h/kg) and an 18-fold increase in the volume of distribution at steady state (1.8 l/kg). When administered orally, compound I had an absolute bioavailability of 9%; however, plasma levels remained above the MIC for C. albicans (0.005 μg/ml) through 48 h. Given the excellent potency of I and its broad spectrum of activity relative to first generation ECB antifungal agents, the assay results for I indicate the potential for its use as a broad spectrum i.v. and oral antifungal agent.     

Determination of mycophenolic acid and its phenol glucuronide metabolite in human plasma and urine by high-performance liquid chromatography

Simultaneous determination of mycophenolic acid (MPA) and mycophenolate phenol glucuronide (MPAG) in plasma and urine was accomplished by isocratic HPLC with UV detection. Plasma was simply deproteinated with acetonitrile and concentrated, whereas urine was diluted prior to analysis. Linearity was observed from 0.2 to 50 μg/ml for both MPA and MPAG in plasma and from 1 to 50 μg/ml of MPA and 5 to 2000 μg/ml MPAG in urine with extraction recovery from plasma greater than 70%. Detection limits using 0.25 ml plasma were 0.080 and 0.20 μg/ml for MPA and MPAG, respectively. The method is more rapid and simple than previous assays for MPA and MPAG in biological fluids from patients.     

Simple and rapid high-performance liquid chromatographic method for determination of celecoxib in plasma using UV detection: application in pharmacokinetic studies

A rapid, sensitive and reproducible HPLC method was developed and validated for the analysis of celecoxib in human plasma. The analysis was carried out on a monolithic silica column using UV detection at 254 nm. The assay enables the measurement of celecoxib for therapeutic drug monitoring with a minimum quantification limit of 10 ng ml(-1). The method involves simple, one-step extraction procedure, and analytical recovery was 100.5 +/- 1.3%. The calibration curve was linear over the concentration range of 10-800 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 8%. We also demonstrate the applicability of this method for pharmacokinetic studies in humans.     

Stereospecific high-performance liquid chromatographic analysis of ibuprofen in rat serum

A simple, rapid and sensitive high-performance liquid chromatographic method was developed for determination of ibuprofen, (+/-)-(R, S)-2-(4-isobutylphenyl)-propionic acid, enantiomers in rat serum. Serum (0.1 ml) was extracted with 2,2,4-trimethylpentane/isopropanol (95:5, v/v) after addition of the internal standard, (S)-naproxen, and acidification with H(2)SO(4). Enantiomeric resolution of ibuprofen was achieved on ChiralPak AD-RH column with ultraviolet (UV) detection at 220 nm without interference from endogenous co-extracted solutes. The calibration curve demonstrated excellent linearity between 0.1 and 50 microg/ml for each enantiomer. The mean extraction efficiency was >92%. Precision of the assay was within 11% (relative standard deviation (R.S.D.)) and bias of the assay was lower than 15% at the limit of quantitation (0.1 microg/ml). The assay was applied successfully to an oral pharmacokinetic study of ibuprofen in rats.     

Ion-pair high-performance liquid chromatographic assay of 4-aminopyridine in serum

An assay for the quantitative estimation of 4-aminopyridine in biological fluids has been developed using 2-aminopyridine as internal standard and ion-pair reversed-phase (C_1$$$) high-performance liquid chromatography with detection at 263 nm. A 7.5% solution of acetonitrile in water containing tetrabutylammonium iodide and sodium heptanesulfonate buffered at pH 3.0 provided excellent separation of the analytes from each other and from an interfering peak that was occasionally observed in the outdated human sera used in these studies. Sensitivity, specificity, precision, accuracy and reproducibility all were judged sufficient for the routine use of this assay for pharmacokinetic and pharmacodynamic studies.     

Simple high-performance liquid chromatographic analysis of phenol and p-cresol in urine and feces

Previous reports which present methods of analysis of phenol and p-cresol by HPLC are usually designed for the detection of these compounds in urine, can be complicated by the use of uncommon equipment or additional techniques such as steam distillation or derivatisation, or concentrate on the detection of phenol rather than p-cresol. In this paper we report a simple method suitable for the analysis of phenol and p-cresol in both urine and feces, based upon extraction into ether following acid hydrolysis and UV detection.     

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.     

Simultaneous determination of coumarin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in human serum and plasma by high-performance liquid chromatography

A HPLC method was developed for the determination of the metabolites of coumarin and 7-hydroxycoumarin in plasma and serum. Separation was based on gradient elution of 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin, coumarin and finally 4-hydroxycoumarin (which is used as an internal standard). Standards, prepared in plasma or serum, and samples were treated with trichloroacetic acid, mixed and centrifuged. The supernatant was removed and analyzed by reversed-phase high-performance liquid chromatography on a C₁₈ column. The limit of detection was 50 ng/ml for 7-hydroxycoumarin and 200 ng/ml for coumarin and 7-hydroxycoumarin glucuronide. The linear range was 0.5–100 μg/ml for each of the analytes. The percentage relative standard deviation about the mean measured concentrations were all below 10%. There was no statistical difference between the standard curves prepared in plasma or serum. The method developed was applied to the determination of each of the three compounds in serum, after the administration of 7-hydroxycoumarin, and in plasma after the administration of coumarin. The concentrations of total 7-hydroxycoumarin in the serum samples were also determined by another HPLC method and the results were compared. There was no statistical difference between the results determined.