High-performance liquid chromatographic method for stability and pharmacokinetic studies of nicorandil

Nicorandil is a unique vasodilator that combines the actions of a potassium channel activator and a nitrovasodilator. Little literature is available on its chemical stability and pharmacokinetics in animals. We developed a simple, specific, sensitive, and precise high-performance liquid chromatographic method for the determination of nicorandil in aqueous solution and rat plasma, achieving a detection limit of 0.3 microgram/ml with 180-microliters samples. Nicorandil was found to be relatively stable between pH 2 and 10 at 37 degrees C (half-life = 461-84 h) and it is subjected to specific base catalysis above pH 10. Both chemical degradation and in vivo metabolism produced N-(2-hydroxyethyl)nicotinamide, the denitrated product. Preliminary pharmacokinetic investigations showed that the assay is capable of quantitating nicorandil in rat plasma over a range of 0.3-100 micrograms/ml. These studies also suggested that the pharmacokinetics of nicorandil are dose-dependent.     

High-performance liquid chromatographic assay for fenoprofen in human plasma

A high-performance liquid chromatographic method is described for the quantitation of fenoprofen, dl-2-(3-phenoxyphenyl)-propionic acid, in human plasma. The proteins in plasma were precipitated by the addition of hydrochloric acid. Fenoprofen and the internal standard, dl-2-(4-phenoxyphenyl)valeric acid, were extracted into butyl chloride and then back-extracted into sodium hydroxide. The aqueous solution was injected onto a reversed-phase alkylphenyl column, and the compounds were eluted using a mobile phase of acetonitrile-water-acetic acid (50:50:2 v/v/v). At a flow rate of 1 ml/min, the retention times of fenoprofen and the internal standard were 8 and 12 min, respectively. The absorbance was monitored at 272 nm. The method requires 1.0 ml of plasma and is sensitive to 0.5 microgram/ml. This procedure has been used for routine assay of multiple samples from bioavailability and compliance studies.     

High-performance liquid chromatographic assay for human liver microsomal omeprazole metabolism

Assays for the measurement of omeprazole metabolites in plasma and urine have been reported, but when applied to the determination of omeprazole metabolites formed by human liver microsomal incubations there were obvious limitations in sensitivity. The present high-performance liquid chromatographic (HPLC) assay, which comprises extraction, evaporation and reconstitution, is several-fold more sensitive with a limit of detection of approximately 2 pmol (2 nM in incubate) for omeprazole sulphone and 25 pmol (25 nM in incubate) for hydroxyomeprazole. Extraction efficiency is essentially quantitative and is highly reproducible (coefficient of variation = 2.1% for both metabolites). The assay is linear over a wide range of concentrations and the formation of the metabolites is linear with respect to both time (to 15 min) and protein concentration (to 1.5 mg/ml). Two minor metabolites, one of which was identified tentatively as 5-O-desmethylomeprazole, were also formed by human liver microsomes and could be determined by this method. Preliminary studies of the formation of omeprazole sulphone and hydroxyomeprazole showed that the formation kinetics in human liver microsomes were biphasic for both metabolites, suggesting that at least two different cytochrome P450 isoforms are involved in their formation.     

Validated high-performance liquid chromatographic assay for the determination of promazine in human plasma. Application to pharmacokinetic studies

A high-performance liquid chromatographic method for the determination of promazine in human plasma is described. The assay involves a single-step liquid-liquid extraction using pentane-2-propanol (98:2, v/v). The analyte of interest and the internal standard chlorpromazine were separated on a Spherisorb CN column using a mobile phase of acetonitrile-50 mM ammonium acetate (9:1, v/v). Electrochemical detection was achieved using an applied potential of +750 mV. The assay was validated according to international requirements prior to application to a pharmacokinetic study and was found to be specific, accurate and precise with a linear range of 0.25-25 ng ml(-1).     

High-performance liquid chromatographic determination of a new oral thrombin inhibitor in the blood of rats and dogs

Donovanosis is a genital ulcer disease that occasionally has extragenital manifestations. This report describes a case of disseminated donovanosis in a 54-year-old woman from northern Australia who had subsequent thoracic vertebral osteomyelitis and spinal cord compression. Malignancy and vertebral tuberculosis were the major differential diagnoses. The patient had no genital lesions at the time of diagnosis of extragenital donovanosis but had undergone a hysterectomy, thus raising the possibility of prior disease of the uterine cervix (most previous cases have been associated with primary cervical disease). Despite treatment with doxycycline, she had no significant neurological improvement. Donovanosis disseminated to bone has been reported in 18 cases in the last 55 years. Awareness of donovanosis in the differential diagnosis of osteomyelitis and prompt pelvic examinations enabling early diagnosis of occult cervical disease are the most important measures in preventing morbidity and mortality due to disseminated donovanosis.     

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 diethyldithiocarbamate (DDTC) followed by addition of methanol and extraction with ethyl acetate. The extract was dried under nitrogen and the samples reconstituted with 100 microl of methanol prior to HPLC determination. Chromatography was accomplished using a Waters NovaPak octadecylsilyl (ODS) (150 x 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 microg/ml (signal-to-noise ratio of 6) with a limit of quantitation (LOQ) of 0.0600 microg/ml for busulfan in plasma. Calibration curves were linear from 0.0600 to 3.00 microg/ml in plasma (500 microl) using a 1/y weighting scheme. Precision of the assay, as represented by C.V. of the observed peak area ratio 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 microg/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.     

High-performance liquid chromatography-mass spectrometry-mass spectrometry analysis of morphine and morphine metabolites and its application to a pharmacokinetic study in male Sprague-Dawley rats

A high-performance liquid chromatography tandem mass spectrometry-mass spectrometry (LC-MS-MS) assay was developed for the analyses of morphine, morphine glucuronides and normorphine in plasma samples from rats. The analytes were extracted by using C2 solid-phase extraction cartridges. The extraction recoveries were 100% for morphine, 84% for morphine-3-glucuronide, 64% for morphine-6-glucuronide and 88% for normorphine. Both intra- and inter-assay variabilities were below 11%. Using a plasma sample size of 100 microliters, the limits of detection were 13 nmol l-1 (3.8 ng ml-1) for morphine, 12 nmol l-1 (5.5 ng ml-1) for morphine-3-glucuronide, 26 nmol l-1 (12 ng ml-1) for morphine-6-glucuronide and 18 nmol l-1 (5.0 ng ml-1) for normorphine, at a signal-to-noise ratio of 3. The present assay was applied to a pharmacokinetic study in rats after intraperitoneal administration of morphine.     

High-performance liquid chromatographic assay for a new fasciolicide agent, alphaBIOF10, in biological fluids

This paper describes a high-performance liquid chromatographic method with ultraviolet absorbance detection at 304 nm for the determination of 6-chloro-5-(1-naphthyloxy)-2-methylthio benzimidazole (alphaBIOF10) -- a new fasciolicide agent -- and its sulphoxide (SOalphaBIOF10), in plasma and urine. It requires 2 ml of biological fluid, an extraction using Sep-Pak cartridges, and methanol for drug elution. Analysis is performed on a microBondapak C18 (10 microm) column, using methanol-acetonitrile-water (40:30:30, v/v) as the mobile phase. Results showed that the assay is sensitive: 7.2 ng/ml for alphaBIOF10 and SOalphaBIOF10 in plasma and 3.6 ng/ml for both compounds in urine. The response was linear between 0.195 and 12.5 microg/ml. Maximum intra-day coefficient of variation was 5.3%. Recovery obtained was 97.8% for both alphaBIOF10 and SOalphaBIOF10. In urine, recovery was 99.6% and 93.1% for alphaBIOF10 and SOalphaBIOF10 respectively. The method was used to perform a preliminary pharmacokinetic study in two sheep and was found to be satisfactory.     

High-performance liquid chromatographic methods for determination of marine biotoxins

Paralysing and diarrhetic shellfish poisonings (PSP and DSP) are important intoxications caused by the consumption of shellfish, mainly bivalve molluscs, contaminated by certain species of toxic dinoflagellates present in the marine phytoplankton. Their appearance and massive reproduction take place in some periods of the year, causing the phenomenon commonly known as 'red tide'. This causes significant problems to health and to the economy of the Galician region. The AOAC mouse bioassay is the most commonly used method of analysis for these toxic compounds, being the official method in most countries. Owing to the lack of sensitivity and selectivity of the biological assay, HPLC methods were developed as an alternative methodology. In this paper work carried out on the improvement of the chromatographic conditions in order to achieve accurate information about the PSP and DSP compounds present in the studied samples is reported.     

High-performance liquid chromatographic method for the determination of norfloxacin glutamate and glucuronate in solid and liquid dosage forms and its application to stability testing

A simple, precise, stability-indicating reversed-phase high-performance liquid chromatographic method for norfloxacin glutamate and norfloxacin glucuronate in liquid and solid dosage forms is described. Chloronitrodiazepine was used as the internal standard. The eluent used with a C18 bonded phase column was methanol-water-diethylamine (50:50:0.4, v/v/v) (pH* 5.5). The effects of the eluent pH*, the ratio of methanol to water, and the quantity of diethylamine on the retention times of the sample and internal standard were investigated. The method showed good linearity in the range 1-45 micrograms ml-1 for norfloxacin. Solid samples were ground, dissolved in the eluent, filtered, and then determined by this method. Liquid samples were dissolved in the same solvent. The average recoveries of norfloxacin glutamate and norfloxacin glucuronate in their simulated preparations were 99.5% for solid products and 99.8% for liquid products. The method was applied to the study of the thermal stability of the drugs by following the degradation of norfloxacin glutamate and glucuronate in the four products in accelerated tests at 37-80 degrees C for up to 3 months. Shelf-lives at 25 degrees C of the four products were predicted from the results assuming zero- and first-order kinetics of decomposition, and were at least 1.5 years for liquid products and 2 years for solid products.     

High-performance liquid chromatographic determination of furosemide in plasma and urine and its use in bioavailability studies

Steroid hydroxylase cytochrome P450c17 has been previously purified from microsomal fractions of immature rat livers. In this study, we investigated the expression of P450c17 in rat livers to understand a role of steroidogenesis in the extrasteroidogenic tissue. Upon immunoblot analysis utilizing liver microsomes from rats, P450c17 was detected in 1 and 3 week old rats but not in adult rats. Data from immunohistochemical studies also showed a similar age-dependent expression of P450c17 and indicated that P450c17 detected in immature rat livers is localized in cells surrounding interlobular veins. This age-dependent expression of P450c17 in rat livers was observed in both sexes. Upon enzymatic analysis utilizing microsomal fractions from livers, levels of 17alpha-hydroxylase and 17,20-lyase activity for pregnenolone and progesterone increased by 3 weeks and dramatically reduced at 7 weeks, which is consistent with the expression level of P450c17. These data clearly indicate that P450c17 is expressed in immature rat liver to produce 17alpha-hydroxysteroids and C19-steroids. Based upon immunoblot analysis, the expression level of P450c17 in immature rat livers was approximately one third of that in testis. Compared expression level of P450c17 and total volume of organs between liver and testis, the total amount of steroid metabolites produced by liver P450c17 could be greater than that produced by gonadal P450c17. Because of the absence of P450c17 in rat adrenal glands, rat liver could be the major site for producing 17alpha-hydroxysteroids and C19-steroids in this particular period of life. Although physiological products formed by P450c17 in liver and their roles remain to be elucidated, this study suggests a large capacity of prepubertal rat liver for participating the production of steroid hormones and a putative importance of 17alpha-hydroxysteroids and C19-steroids, such as cortisol and androstendione, which are generally believed to be minor components of steroid hormones in rodents.     

High-performance liquid chromatographic method for measuring total plasma homocysteine levels

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri-n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.65+/-2.3 and 8.9+/-2.4 micromol/l, while post-methionine load gave tHcy levels of 19.9+/-5.5 and 26.8+/-5.5 micromol/l, for women and men, respectively (n=40).     

High-performance liquid chromatographic analysis of porphyrins in clinical materials

Methods for the isolation of porphyrins as their methyl esters from porphyric urine and faeces as well as other biological materials are described. Quantitative analyses can be carried out by high-performance liquid chromatography (HPLC), using appropriate internal standards; hence excretion patterns in the various types of porphyria can be obtained which may facilitate clinical diagnosis more effectively than the earlier qualitative thin-layer chromatographic methods. Use of the newer microparticulate column packing materials has improved the efficiency of the HPLC analyses, and enables the more convenient isochratic elution techniques to be used (rather than gradient elution). Separations of some porphyrin isomers on these columns are also described.     

High-performance liquid chromatographic separation and electrochemical detection of cephalosporins

It was found that both normal human myometrium and uterine leiomyoma contain several glycosaminoglycans. In contrast to many normal and tumour tissues the amount of hyaluronic acid is very low and the proportional amount of sulphated glycosaminoglycans is distinctly higher. It is of interest that heparan sulphate is the major glycosaminoglycan component both in normal myometrium, and in leiomyoma. The amount of hyaluronic acid in myometrium and in the leiomyoma is very low. No significant change in hyaluronate content was observed during the tumour growth. In contrast to that the amount of some sulphated glycosaminoglycans (heparan sulphate, keratan sulphate, chondroitin sulphates and heparin) distinctly increased. It is suggested that some of the GAGs participate in the creation of a storage depot for biologically active molecules (growth factors, enzymes) which are thereby stabilized and protected. Hydrolytic degradation of some GAGs may result in the release of some cytokines which may promote the tumour growth and stimulate collagen biosynthesis by tumour cells.     

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.     

High-performance liquid chromatographic determination of midazolam in rat brain

The authors tested whether perfectionism dimensions interact with specific stress to predict depression over time. A sample of 103 current and former patients completed measures of perfectionism and depression at Time 1 and measures of stress and depression 4 months later. After controlling Time 1 depression, self-oriented perfectionism interacted only with achievement stress to predict Time 2 depression. Socially prescribed perfectionism did not interact with achievement or social stress to predict Time 2 depression, but it did predict Time 2 depression as a main effect. The results provide support for the contention that perfectionism dimensions are involved in vulnerability to depression over time.     

High-performance liquid chromatographic analysis of isoniazid and its dosage forms

A high-performance liquid chromatographic analysis is described for isoniazid as a drug entity and in its tablet and injectable dosage forms. After incorporation of the drug or dosage form in a solvent mixture and addition of an internal standard, tribenzylamine, an aliquot is chromatographed using a pellicular silica gel medium followed by UV spectrophotometric detection at 254 nm. The response of the chromatographic system was linear over a concentration range corresponding to 20-200% of the labelled amount of isoniazid. Comparison of the results with those obtained by the official USP XIX method indicates similar accuracy and precision. The advantages of the proposed method are its simplicity and rapidity, its potential for automation, and its specificity. The specificity was demonstrated in the presence of potential degradation products of isoniazid, other drugs used with isoniazid in combination dosage forms, and an adduct formed by the reaction of isoniazid with lactose in the tablet.     

High-performance liquid chromatographic analysis of chlorhexidine in saliva after mouthrinsing

BACKGROUND: Neutrophils contribute to the host defense mechanism, but they can cause remote organ injury in peritonitis. The purpose of this study was to examine neutrophil adhesion to the peritoneum and remote organs simultaneously in peritonitis using a fluorescence microscopic method. STUDY DESIGN: Experiment 1: Sprague-Dawley rats (n = 16) were injected intraperitoneally (ip) with saline solution or 10(5), 10(7), or 10(9) Escherichia coli. Five hours after challenge, 1 x 10(6) fluorescein-labeled neutrophils were infused. Two minutes after neutrophil injection, five peritoneal samples (the greater omentum, mesentery, parietal peritoneum, colon, and ileum), both lungs, the liver, and the right kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy. Lung myeloperoxidase (MPO) activity was also determined. Experiment 2: Rats (n = 23) were given 10(9) E. coli ip. Before challenge (0 h) or at 1, 5, or 10 h after challenge, labeled neutrophils were infused. Then, the labeled neutrophil numbers in organs and lung MPO activities were assessed as described for Experiment 1. Hemodynamic and arterial blood gas data were also obtained in another set of rats before and at 1, 5, 8 and 10 h after 10(9) E. coli ip challenge. RESULTS: Experiment 1: The labeled neutrophil numbers in the peritoneum, lungs, and kidney showed significant positive correlations with the injected bacterial numbers. Lung MPO also positively correlated with E. coli number and labeled neutrophil number in the lungs. Experiment 2: Labeled neutrophil numbers in the peritoneum and kidney peaked at 5 h. The pulmonary labeled neutrophil number rose, reaching a plateau at 5 h. No remarkable change was observed in the hepatic labeled neutrophil number. There was a positive correlation between lung MPO activity and pulmonary labeled neutrophil number. Hemodynamic and blood gas data reflected a hyperdynamic state. CONCLUSIONS: Concomitant dose-dependent increases in neutrophil adhesion in the peritoneum, lungs, and kidney were observed in this peritonitis model. Increased neutrophil adhesion was transient in the peritoneum and kidney but persistent in the lungs. Strategies modulating neutrophil adhesion in organs are anticipated to be useful for the treatment of peritonitis.     

High-performance liquid chromatographic method for the determination of fenofibric acid and reduced fenofibric acid in human blood, plasma and urine

In this study, a very reliable HPLC method was developed for the determination of fenofibric acid and reduced fenofibric acid in the biological samples described as follows. After addition of the internal standard solution and 0.5 M HCl to the biological sample, fenofibric acid, reduced fenofibric acid and the internal standard were extracted with a mixed solvent of n-hexane and ethyl acetate (90:10) from the mixture. The acids were back-extracted from the organic phase with 0.1 M Na2HPO4 and then re-extracted from the aqueous phase with a mixed solution of n-hexane and ethyl acetate (95:5) after addition of 0.5 M HCl. The organic phase was evaporated to dryness under the vacuum. The residue was dissolved in MeOH and diluted with distilled water. An aliquot of the resulting solution was injected on the HPLC. High reproducibility was observed in this HPLC method (C.V.% less than 4%). Moreover it was confirmed that the conjugates in the urine could be hydrolyzed by incubation at 37 degrees C for 18 h after addition of 400 IU of beta-glucuronidase.