Assay of acyclovir in human skin layers by high-performance liquid chromatography

This paper describes an assay procedure for acyclovir quantification in human skin after in vitro transdermal transport experiments. The procedure employs warm distilled water for acyclovir (ACV) extraction and high-performance liquid chromatography (HPLC) as analytical method. The procedure has good reproducibility, sensitivity and specificity, resulting in a reliable method for biopharmaceutical studies of ACV distribution in skin tissue. The chromatographic conditions set up, using distilled water as mobile phase, makes the analytical procedure simple and easy to perform.     

Determination of ivermectin in human plasma by high-performance liquid chromatography

A specific reversed-phase HPLC-assay with sensitive fluorometric detection has been developed to measure the potent new antiparasitic agent ivermectin (CAS 70288-86-7) in human plasma (and urine). The lower limit of the method was 1 ng/ml and the intra-/interassay variability averaged 4.5/6.9%, respectively. The assay was applied for measuring plasma (urine) concentrations of ivermectin upto 56 (72 h) following a single oral dose of 6 and 12 mg. No unchanged or conjugated ivermectin could be detected in urine. Plasma concentrations increased linearly with dose but elimination half-life (12.6/13.4 h) was independent of the administered dose. Thus, the method is applicable for monitoring plasma levels during clinical and pharmacokinetic trials with ivermectin to evaluate its most efficacious dosage regimen.     

Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography

An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 microliters of plasma and 240 microliters of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 microM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierke's disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects.     

Determination of total homocysteine in human plasma by isocratic high-performance liquid chromatography

A simple, sensitive and precise isocratic HPLC method for the determination of total homocysteine in human plasma is described. The thiol compounds were liberated from plasma proteins by reduction with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic marker, 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulphonate. The derivatives were separated isocratically within 7 min by reversed-phase HPLC using a Superspher 100 RP-18 column as stationary phase. By using this approach more than 200 samples a day can be assayed for total homocysteine. The method was linear up to 100 mumol/l and proved to be sensitive with a detection limit of 0.1 mumol/l and the lowest limit of reliable quantification of 0.5 mumol/l for homocysteine in buffer. Intra- and inter-assay coefficients of variation were both < 4% at a concentration of 10 mumol/l homocysteine. Similar results were obtained for homocysteine concentrations between 0.5 and 100 mumol/l. The analytical recovery for these concentrations ranged from 94.9 to 117.0%. As compared to other protocols published so far, this modified method is less complicated but equally sensitive and reproducible and allows a rapid determination of total homocysteine and cysteine in human plasma under routine conditions.     

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

A subfamily of small GTP-binding proteins, rab, has been shown to be involved in regulation of vesicular traffic in eukaryotic cells. The goal of this study was to identify the rab proteins associated with atrial secretory granules. A [32P]GTP-overlay assay showed the presence of multiple small GTP-binding proteins on the atrial granules. By biochemical analysis, we have demonstrated that one of the small GTP-binding proteins associated with the atrial granules is a rab12 protein (rab12p), one of the rab proteins that are most closely related to a Sec4 protein of yeast. Association of rab12p with the atrial granules was confirmed by immunogold electron microscopy. Immunoprecipitation followed by immunoblot analysis with anti-rab12 antibody showed that in addition to atria, rab12p was expressed in multiple other organs and cell lines. These results suggest that rab12p may function in vesicular traffic in multiple diverse types of cells.     

Assay of bropirimine in rat plasma by means of robotic solid-phase extraction and high-performance liquid chromatography

Vacuous jaw movements induced by the muscarinic agonist pilocarpine and striatal dopamine depletions were examined using a slow motion videotape system. With this procedure, rats were videotaped in a Plexiglas tube so that the profile of the head region could be seen. Vacuous jaw movements were analyzed by examining the tape at 1/6 normal speed. An observer recorded each jaw movement using a computer, and the computer program re-calculated the temporal characteristics of jaw movement responses back to normal speed. The interresponse time was recorded for each jaw movement, and each jaw movement interresponse time was assigned to a 50 ms wide time bin. Thus, the distribution of interresponse times could be used to analyze the temporal characteristics of jaw movement responses. In the first experiment, rats were administered saline vehicle, 1.0 mg/kg and 2.0 mg/kg pilocarpine. The rats were videotaped 10-15 min after injection, and the data were analyzed as described above. Pilocarpine induced very high levels of vacuous jaw movements, and the vast majority of all movements occurred in "bursts" with interresponse times of 1.0 s or less. Analysis of the interresponse time distributions showed that most of the jaw movements were within the 150-350 ms range. The modal jaw movement interresponse time was in the 150-200 ms range, which corresponds to a local frequency of 5-6.66 Hz. In the second experiment, the neurotoxic agent 6-hydroxydopamine was injected directly into the ventrolateral striatum in order to produce a local dopamine depletion. The dopamine-depleted rats were observed for jaw movements 7 days after surgery. The overall level of jaw movement activity resulting from dopamine-depletion was much lower than that produced by pilocarpine. There was a significant inverse correlation between ventrolateral striatal dopamine levels and total number of vacuous jaw movements. Videotape analysis indicated that the temporal characteristics of jaw movements induced by dopamine depletions were similar to those shown with pilocarpine. These experiments indicate that vacuous jaw movements induced by pilocarpine and striatal dopamine depletion occur in a frequency range similar to that shown in parkinsonian tremor.     

Analysis of prednisolone in plasma by high-performance liquid chromatography

A sensitive, specific high-performance liquid chromatographic procedure for the determination of prednisolone in plasma is described. The organic solvent extract from plasma is chromatographed on a silica gel column using a mobile phase of 0.2% glacial acetic acid, 6% ethanol, 30% methylene chloride in n-hexane on a high-performance liquid chromatograph fitted with an ultraviolet dector (254 nm). Quantitation of plasma samples containing 25 ng/ml prednisolone is reported. Metabolites and endogenous hydrocortisone do not interfere with prednisolone. The determination of prednisolone concentration in plasma following administration of a 10-mg single oral dose to a human subject is described.     

Rapid quantitation of free fatty acids in human plasma by high-performance liquid chromatography

Large quantities of a number of man-made chemicals with the potential to disrupt the developing endocrine and nervous systems in wildlife and humans have been released into the environment. These chemicals are particularly damaging during the embryonic, fetal, and early postnatal periods because they resemble or interfere with the hormones, neurotransmitters, growth factors, and other signaling substances that normally control development. The effects are in many cases irreversible and often are expressed as changes in function rather than as obvious birth defects or clinical diseases. Functional changes pose challenges in documenting the extent of the lesion, especially in the case of neuroendocrinological damage. In the past decade, researchers have added new dimensions to their research strategies in order to compensate for these difficulties. The new approaches reveal more about the extent of the distribution of and exposure to chemicals that interfere with the endocrine and nervous systems and strengthen the links between exposure and damage in developing wildlife and humans. Based on this new knowledge, opportunities abound for extensive multi-disciplinary research involving developmental neurotoxicity.     

Quantification of flumequine enantiomers in plasma by high-performance liquid chromatography

A 62-year-old woman with pulmonary hypertension underwent mitral valve re-replacement through right thoracotomy. Severe adhesion occurred to the right lung. During pleural dissection the lung collapsed under single-lung ventilation, rapidly elevated pulmonary vascular resistance caused hemodynamic instability. When pulmonary hypertension is preoperatively present, this approach under single-lung ventilation is not recommended.     

Determination of sinefungin in rat plasma by high-performance liquid chromatography

This is the 31st article in a continuing series of objectives to direct emergency medicine resident experiences on off-service rotations. Neck and torso trauma accounts for a large portion of injuries, and its management is an essential part of training in emergency medicine. Due to the often life-threatening presentations of trauma victims, resident instruction may be conducted at the bedside in difficult and demanding situations. Therefore, it is essential for residents to have specific goals and objectives to guide their acquisition of knowledge required to make critical decisions for patients with major trauma.     

Determination of pyridonecarboxylic acids in plasma by reverse-phase high-performance liquid chromatography

A simple qualitative and quantitative determination for pyridonecarboxylic acids including nalidixic acid (NA), oxolinic acid (OA) and pipemidic acid (PPA) in chicken plasma was carried out by microbiological, spectrophotometric, thin-layer chromatographic (TLC) and reverse-phase high-performance liquid chromatographic (HPLC) methods. As a test organism for bacteriological bioassay, Bacillus subtilis ATCC-6633 was the most sensitive of seven organisms investigated. Using the cup and the disc methods, a standard curve was obtained by determining the relationship between various drug concentrations and the diameter of the inhibition zone. The three drugs had two strong UV absorbance wavelengths (257 and 330 nm) on spectrophotometry. TLC analysis using a silica gel 60 F254 plate was investigated, and a solution of methanol:chloroform:acetic acid (3:1:1, v/v/v) was found to be the most suitable solvent for separation. The minimum concentration of drug detectable by this method was 0.5 microgram/ml for NA, 0.075 microgram/ml for OA and 0.39 microgram/ml for PPA. For HPLC analysis, a solution of acetonitrile:0.2 M phosphoric acid (1:1, v/v) was superior, and simultaneous determination of all three drugs was possible under the HPLC conditions used. The lowest measurable amount of drug in chicken plasma was 0.01 microgram/g. Recovery from extracts spiked with each drug at a known concentration was close to 100% for NA and OA, but only about 50% for PPA.     

Determination of norfloxacin in human plasma and urine by high-performance liquid chromatography and fluorescence detection

A method for the determination of norfloxacin in human plasma and urine is described. Plasma samples were deproteinized using acetonitrile. The supernatant was analysed by C18 HPLC. Fluorescence detection at an excitation wavelength of 300 nm and an emission wavelength of 450 nm was utilized. The assay was validated in the concentration range of 31 to 2507 ng/ml when 0.5-ml aliquots of plasma were handled. The intra-day precision of the spiked quality control samples ranged from +/- 0.37 to +/- 4.14% in plasma (concentration range: 70.3-2109.2 ng/ml) and from +/- 0.51 to +/- 1.56% in urine (concentration range: 7.5-299.4 micrograms/ml). The intra-day accuracy obtained for norfloxacin in the quality control samples ranged from -5.18% to -9.47% in plasma and from -10.56% to - 5.91% in urine. The assay has been used to support human pharmacokinetic studies.     

Determination of dimethindene in human tears by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of dimethindene in human tears. The tear samples were diluted in a 0.01 M hydrochloric acid-n-propanol mixture to prevent the irreversible adsorption of dimethindene. The diluted samples were directly injected into the chromatographic system to avoid sample pretreatment. The validation data demonstrate that the method is specific, precise and accurate within the calibration range of 12 to 1000 ng/ml dimethindene free base.     

Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma

We report a very rapid and simple isocratic reversed-phase HPLC separation of malondialdehyde (MDA) in normal human plasma without previous purification of the MDA-2-thiobarbituric acid (TBA) complex. The separation of MDA-TBA complex was performed using a 250x4.6 mm Nucleosil-5C18 column with a mobile phase composed of 35% methanol and 65% 50 mM sodium phosphate buffer, pH 7.0. Samples of 50 microl (composed of 100 microl plasma mixed with 1.0 ml of 0.2% 2-thiobarbituric acid in 2 M sodium acetate buffer containing 1 mM diethylenetriaminepentaacetic acid, pH 3.5, and 10 microl of 5% 2,6-di-tert.-butyl-4-methylphenol in 96% ethanol, incubated at 95 degrees C for 45 min [K. Fukunaga, K. Takama and T. Suzuki, Anal. Biochem., 230 (1995) 20] were injected into the column. The MDA-TBA complex was eluted at a flow-rate of 1 ml/min and monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Analysis of groups of normal male and female volunteers gave plasma levels of MDA of 1.076 nmol/ml with a coefficient of variation of about 58%. No significant statistical differences were found between male and female groups, and no correlation was discovered on the age.     

Determination of leucovorin and 5-fluorouracil in plasma by high-performance liquid chromatography

A method was developed for the determination of (6R)- and (6S)-leucovorin and 5-fluorouracil in plasma. As leucovorin diastereoisomers cannot be separated on a classical reversed-phase column, it was necessary to use a chiral stationary phase. The method presented is based on the same principle as the method described by Wainer and Stiffin [J. Chromatogr., 424 (1988) 158], i.e., coupling of a bovine serum albumin phase to an achiral stationary phase. Before the chromatography, the drug was isolated from the plasma matrix by solid-phase extraction. For 5-fluorouracil, chromatography was performed on a classical RP-18 column after extraction from the plasma by liquid-liquid extraction. Both methods were validated and applied to the analysis of patients' samples.     

Determination of heptylphysostigmine in plasma by high-performance liquid chromatography with electrochemical detection

P0, the major structural protein of peripheral myelin, is a homophilic adhesion molecule with a single immunoglobulin (Ig) domain, which contains a single N-linked glycosylation site and two cysteines. We have previously reported four different mutations of the myelin P0 gene in four families of Charcot-Marie-Tooth neuropathy type 1 (CMT1). In this study we found a new mutation of the myelin P0 gene in a small family of CMT1. The affected persons had an A - to - G substitution of nucleotide 245 of the myelin P0 gene in one allele, leading to a cysteine substitution for tyrosine82 in the extracellular Ig-domain. An additional cysteine in the extracellular domain may form a disulfide bond and cause an inappropriate change in the tertiary structure of the functional Ig-domain of P0.