Automated determination of hypoxanthine and xanthine in urine by high-performance liquid chromatography with column switching

We report a high-performance liquid chromatographic method with column switching for urinary hypoxanthine and xanthine. Analyses were carried out with both a reversed-phase column and an anion-exchange column connected by a column switch and controlled automatically by a computerized system controller. The relationships between standard concentrations and peak heights were linear in a concentration range of 1 to 1000 nmol/ml. The recovery of hypoxanthine added to urine was 101.1%, and that of xanthine was 98.1%. With our method urinary hypoxanthine and xanthine can be measured accurately without any sample preparation other than filtration.     

Determination of meropenem in serum by high-performance liquid chromatography with column switching

A rapid, accurate and sensitive liquid chromatographic assay with on-line solid-phase extraction for determination of meropenem in serum is described. Sample was directly injected onto the extraction column for sample clean-up and extraction. Thereafter, using an on-line column-switching system the drug was quantitatively transferred and separated on a C18 analytical column. Ultraviolet absorption at 298 nm was used for detection. The assay was linear from 1 to 100 micrograms/ml. Recovery was 98.5%. Based on a 20-microliters sample volume (serum- water, 1:1, v/v), detection limit was 0.1 microgram/ml. An application of the method to study the pharmacokinetics of meropenem is given.     

Quantitative determination of pamoic acid in dog and rat serum by automated ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography

Pamoic acid is used as a counter ion to obtain long-acting pharmaceutical formulations of certain basic drugs. In order to investigate the pharmacokinetics of pamoic acid, a simple, sensitive and reliable method has been established for the quantitative determination of pamoic acid in serum from dog and rat. The method uses ion-pair solid-phase extraction followed by ion-pair reversed-phase high-performance liquid chromatograpy. The influence on recovery of the addition of different agents (tetrabutylammonium acetate, methanol, sodium hydroxide) to the serum samples prior to solid-phase extraction was studied and the analytical method was validated. The method was found to be valid for accurate, precise and selective determination of pamoic acid in the tested concentration range of 5-200 ng/ml serum. The overall performance of the HPLC method was found to be satisfactory for the purpose of determining concentrations of pamoic acid in serum samples from pharmacokinetic studies with pamoic acid in dogs and rats.     

Quantitative determination of domperidone in rat plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and selective analytical method for the determination of domperidone in rat plasma is described. The procedure involves liquid-liquid extraction followed by reversed-phase high-performance chromatographic analysis with fluorometric detection at 282 nm for excitation and 328 nm for emission. The detection limit was 1 ng ml(-1) using 1 ml of plasma. This assay procedure should be useful for the pharmacokinetic study of domperidone in small animals such as rats.     

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.     

Determination of sumatriptan succinate in human plasma by high-performance liquid chromatography with coulometric detection and utilization of solid-phase extraction

Sumatriptan succinate (the analyte) and naloxone (the internal standard) were extracted from plasma with a solid-phase extraction technique. Chromatography and detection were performed by isocratic reversed-phase high-performance liquid chromatography with coulometric end-point detection. The standard curve was linear over the range 0-100 ng/ml of sumatriptan succinate in plasma. The reproducibility (as defined by the coefficient of variation, C.V.) over the range of the standard curve was 4.9-7.3%. The recovery averaged 83%. The sensitivity was 0.25 ng of sumatriptan on column (allowing a concentration of 0.5 ng/ml to be determined from a 1-ml plasma sample volume). Plasma profiles of the analyte following subcutaneous (s.c.) administration in eight normal male volunteers, are presented.     

Simultaneous determination of GTS-21 and its metabolite in rat plasma by high-performance liquid chromatography using solid-phase extraction

It has been suggested that GTS-21 can improve the learning deficits and inhibit the neuro-degeneration in patients with Alzheimer's disease. This paper describes a reversed-phase high-performance liquid chromatographic assay with visible detection at 405 nm for determination of GTS-21 and its metabolite, 4-hydroxy-GTS-21 in rat plasma. The method uses solid-phase extraction with a Bond Elut C18 column. A quantitation limit of 1.0 ng/ml was achieved using 0.5 ml of rat plasma. In the validation study, the coefficients of variation and the relative errors of each compound were less than 10%. Also freeze-thaw and storage stability were confirmed. This method has proved to be applicable to the pharmacokinetic study of GTS-21 in rats.     

Stability and determination of aflatoxins by high-performance liquid chromatography with amperometric detection

A method based on reversed-phase high-performance liquid chromatography (RP-HPLC) with amperometric detection with a glassy carbon electrode at a constant potential of 1.4 V is reported for the separation and identification of aflatoxins B1, B2, G1 and G2 in a model mixture. The chromatography was performed on a PAH-Baker column with a ternary mobile phase containing methanol, acetonitrile and aqueous LiClO4 electrolyte. Aflatoxin G1 showed the highest electroactivity in the compound series studied. Calibration curves of aflatoxins G1 and B2 were linear up to 0.2 and 0.3 mmol/l, respectively. Sensitivity varied between 7 and 10 ng for the different aflatoxins. The combination of different HPLC detectors in the analysis of these compounds was applied to investigate the stability of aflatoxins G1 and B2.     

Simultaneous determination of primidone and its three major metabolites in rat urine by high-performance liquid chromatography using solid-phase extraction

Sialosyl-fucosyl poly-LacNAc without sialosyl-Lex epitope in myeloid cell line HL60 was shown to be the ligand for E-selectin-dependent adhesion, particularly under dynamic flow conditions, in our previous study (Handa K, Stroud MR, Hakomori S, Biochemistry 36, 12412-12420, 1997). HL60 cells express only fucosyl-transferase (FT) IV and VII. X3NeuAcVII3FucnLc10, a representative component showing E-selectin-dependent binding under dynamic flow conditions, is not alpha 1-->3 fucosylated at the penultimate GlcNAc catalyzed by FT-VII, but is alpha 1-->3 fucosylated at the internal GlcNAc catalyzed by FT-IV. VI3NeuAcnLc6 is converted to VI3NeuAcIII3FucnLc6 by FT-IV, but is also converted to VI3NeuAcV3FucnLc6 by FT-VII. Thus, penultimate fucosylation catalyzed by FT-VII is not restricted for nLc6 backbone, but is highly restricted for nLc10 backbone. The cooperative effect of FT-IV and FT-VII for synthesis of poly-LacNAc having sialosyl-Lex with internal fucosylation may be blocked or highly restricted in poly-LacNAc having more than two LacNAc units, because preferential alpha 1-->3 fucosylation by FT-IV takes place at internal GlcNAc, inhibiting penultimate fucosylation by FT-VII.     

Improved one-step solid-phase extraction method for morphine, morphine-3-glucuronide, and morphine-6-glucuronide from plasma and quantitation using high-performance liquid chromatography with electrochemical detection

OBJECTIVE: The purpose of this survey was to study the sensation of oral dryness and its underlying factors in the 55-year-old population of Oulu (a medium-sized Finnish town), 780 of whom (77%) participated. METHOD: In addition to the examination of oral health status and salivary flow rate measurements, depressive symptoms were determined on the basis of the Zung Self-Rating Depression Scale (ZSDS). The participants were also interviewed about their health, medication, physical health, physical activity, smoking habits, alcohol consumption, and factors related to their work. RESULTS: The prevalence of subjective sensations of dry mouth was 25.8% among men and 33.3% among women (p = 0.025). A statistically significant association was found between a subjective sensation of dry mouth and a low unstimulated flow rate, regular smoking, xerogenic medication, and the presence of at least one illness connected with dry mouth. Those who had a sensation of dry mouth also thought their physical condition and their health to be poorer and more often had a high rate of depressive symptoms. After the confounding factors had been added stepwise into the logistic regression model, depressive symptoms were still significantly associated with the sensation of oral dryness. CONCLUSIONS: When evaluating the causes of the sensation of dry mouth, the possibility of depression as an underlying factor should be considered.     

Determination of p-hydroxymandelic acid enantiomers in urine by high-performance liquid chromatography with electrochemical detection

High-performance liquid chromatography (HPLC) with electrochemical detection using a chiral ligand-exchange column was developed for the enantioselective determination of p-hydroxymandelic acid (HMA), a metabolite of synephrine, with high sensitivity. A good linear relationship between current ratio and amount was noted for 0.5-500 pmol HMA, with a correlation coefficient of 0.999 for each HMA enantiomer. The relative standard deviation (R.S.D.) was 1.6% at 100 pmol d-HMA and 2.2% at 100 pmol l-HMA. The detection limit of each HMA enantiomer was 0.5 pmol (signal to noise ratio, S/N = 3). By this method, HMA in Citrus unshiu and in urine following the ingestion of C. unshiu was determined. Although no HMA was found in c. unshiu, d- and l-HMA were present in urine after the ingestion of C. unshiu. The time courses of HMA and conjugated synephrine enantiomers excreted in urine following the ingestion of C. unshiu for 24 h could be monitored. This method should prove applicable to the study of synephrine metabolism.     

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection

A method is described which permits the trace analysis of 10 chlorobenzenes in aqueous samples. Chlorobenzenes were extracted from water samples by solid-phase extraction with a C18 cartridge and analysis was carried out by gas chromatography-mass spectrometry in the selected-ion monitoring mode. The recovery and precision of the method were evaluated by extraction of spiked reagent-grade water at concentration levels of 0.1, 1.0 and 10.0 micrograms/l. This method was applied to the determination of chlorobenzenes in tap, ground and river water. By preparing 200 ml of environmental water samples, the detection limits of the compounds studied were in the range of 0.010-0.042 microgram/l.     

Determination of dopexamine hydrochloride in human blood by high-performance liquid chromatography with electrochemical detection

A method is described for the determination of dopexamine hydrochloride at concentrations of 5 to 100 ng/ml in human blood using electrochemical detection. The method uses a Hypersil ODS column and a mobile phase containing heptane sulphonate, orthophosphoric acid, diisopropylamine and disodium EDTA. Blood samples are stabilised immediately after collection by the use of dipotassium EDTA and a high concentration of sodium metabisulphite. The sample preparation procedure consists of a simple de-proteinisation with perchloric acid. The method is accurate, with inter-assay accuracies ranging from 100 to 104%, and is free of interference by blood from different individuals. Known and potential metabolites of dopexamine hydrochloride and a wide range of drugs do not interfere with the method. The method is precise with inter-assay coefficients of variation of 10.6% at 5 ng/ml and of less than 4.2% at higher concentrations. Stabilised blood samples may be stored for over six months at -25 degrees C prior to analysis.     

Rapid alcohol determination in plasma and urine by column liquid chromatography with biosensor detection

An enzyme based amperometric biosensor used as a selective and sensitive detection unit in column liquid chromatography for the determination of ethanol and methanol in biological fluids such as plasma and urine is described. The reagentless enzyme electrode is based on the co-immobilisation of alcohol oxidase and horseradish peroxidase in carbon paste. The selectivity of the biosensor was found to vary when four various alcohol oxidase enzyme preparations from Candida boidinii, Pichia pastoris, and Hansenula polymorpha were used in the biosensors described. High sensitivity could be obtained for a number of alcohols, organic acids, and aldehydes. Optimisation regarding the sensitivity and selectivity of the four alcohol oxidase co-immobilised biosensors are outlined. A fast and reliable liquid chromatographic separation system with a PLRP-S polymer based separation column used with a phosphate buffer as the mobile phase was optimised using the best biosensor which was based on alcohol oxidase from P. pastoris and which showed the highest turnover rate for alcohols, as the detector for the determination of ethanol and methanol in human urine and plasma samples. The selectivity and stability of the biosensor were retained by working at an applied potential of -50 mV versus Ag/AgCl, the optimal operational potential, and by the casting of a protective membrane on the electrode surface. High selectivity of the enzyme electrode was also found towards other easily oxidisable interfering species normally present in biological fluids. It was found that stable and reliable determinations of ethanol and methanol in plasma and urine could be performed with only a simple dilution and centrifugation step prior to injection into the liquid chromatographic system. An analysis time of 4 min was required for the assay, with a sample throughput of 13 samples h(-1).     

Automated and sensitive method for the determination of formoterol in human plasma by high-performance liquid chromatography and electrochemical detection

An automated high-performance liquid chromatography (HPLC) method for the determination of formoterol in human plasma with improved sensitivity has been developed and validated. Formoterol and CGP 47086, the internal standard, were extracted from plasma (1 ml) using a cation-exchange solid-phase extraction (SPE) cartridge. The compounds were eluted with pH 6 buffer solution-methanol (70:30, v/v) and the eluate was further diluted with water. An aliquot of the extract solution was injected and analyzed by HPLC. The extraction, dilution, injection and chromatographic analysis were combined and automated using the automate (ASPEC) system. The chromatographic separations were achieved on a 5 microm, Hypersil ODS analytical column (200 mm x 3 mm I.D.), using (pH 6 phosphate buffer, 0.035 M + 20 mg/l EDTA)-MeOH-CH3CN (70:25:5, v/v/v) as the mobile phase at a flow-rate of 0.4 ml/min. The analytes were detected with electrochemical detection at an operating potential of +0.63 V. Intra-day accuracy and precision were assessed from the relative recoveries of calibration/quality control plasma samples in the concentration range of 7.14 to 238 pmol/l of formoterol base. The accuracy over the entire concentration range varied from 81 to 105%, and the precision (C.V.) ranged from 3 to 14%. Inter-day accuracy and precision were assessed in the concentration range of 11.9 to 238 pmol/l of formoterol base in plasma. The accuracy over the entire concentration range varied from 98 to 109%, and precision ranged from 8 to 19%. At the limit of quantitation (LOQ) of 11.9 pmol/l for inter-day measurements, the recovery value was 109% and C.V. was 19%. As shown from intra-day accuracy and precision results, favorable conditions (a newly used column, a newly washed detector cell and moderate residual cell current level) allowed us to reach a LOQ of 7.14 pmol/l of formoterol base (3 pg/ml of formoterol fumarate dihydrate). Improvement of the limit of detection by a factor of about 10 was reached as compared to the previously described methods. The method has been applied for quantifying formoterol in plasma after 120 microg drug inhalation to volunteers. Formoterol was still measurable at 24 h post-dosing in most subjects and a slow elimination of formoterol from plasma beyond 6-8 h after inhalation was demonstrated for the first time thanks to the sensitivity of the method.     

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.     

Sensitive and selective determination of methylenedioxylated amphetamines by high-performance liquid chromatography with fluorimetric detection

A rapid, sensitive and selective liquid chromatographic method with fluorimetric detection was developed for the separation and quantification of four methylenedioxylated amphetamines without interference of other drugs of abuse and common substances found in illicit tablets. The method was validated by examining linearity, precision and accuracy as well as detection and quantification limits. Methylenedioxylated amphetamines were quantified in eight tablets from illicit drug seizures and results were quantitatively compared to HPLC-UV analyses. To demonstrate the better sensitivity of the fluorimetric detection, methylenedioxylated amphetamines were analyzed in serum after a liquid-liquid extraction procedure and results were also compared to HPLC-UV analyses.