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At least 1,279 records · Page 71Linked to original sources
PubMed1995

Determination of thiopurine nucleotides in human lung tissue by high-performance liquid chromatography.

A method for the analysis of thiopurine nucleotides in human transbronchial lung biopsy was developed. The sample treatment procedure is based on perchloric acid homogenisation and deproteinisation with dithiothreitol and hydrolysis of thiopurine nucleotides into their free bases by heating of the acid extract. Then, the free bases were analyzed in the gradient elution mode on a Hypersil ODS, 3-microns column using dihydrogenphosphate buffer-methanol as eluent. Mean analytical recoveries for 6-thioguanosine monophosphate and 6-thioinosinic acid from lung tissue were 97.0 +/- 2.0 and 98.0 +/- 1.8% at a concentration of 3.0 nmol/ml and the minimum detectable amounts were 3.5 and 2 pmol, respectively. The procedure described is simple and represents a suitable method for the investigation of thiopurine nucleotides in tissues.

Chromatography, High Pressure Liquid↗
PubMed1995

Determination of (+)-catechin in plasma by high-performance liquid chromatography using fluorescence detection.

A high-performance liquid chromatographic method, using fluorescence detection, was developed for the determination of (+)-catechin in rabbit plasma. The procedure involved the precipitation of plasma protein using acetonitrile, followed by solid-phase adsorption onto alumina. After washing with water and methanol, the residue was vortex-mixed with perchloric acid solution to release the adsorbed (+)-catechin. Separation was performed on a reversed-phase column using an eluent consisting of phosphoric acid solution with 12% acetonitrile. The excitation and emission wavelengths were set at 280 and 310 nm, respectively. The retention times for (+)-catechin and the internal standard (deoxyhigenamine) were 6.87 and 8.47 min respectively, without any interference. Validations of accuracy and precision were satisfactory in both within- and between-run assays. All coefficients of variance were less than 6% and mean relative errors were within +/- 3.75%. The average recovery was 73.77%. The limit of detection and quantitation were 1 ng and 0.02 micrograms/ml, respectively. Application of this method was successfully assessed by intravenous administration of a 15 mg/kg dose of (+)-catechin in rabbits. This new method provides a simple, specific and sensitive determination for (+)-catechin in rabbit plasma and is suitable for pharmacokinetic studies.

Acetonitriles↗
PubMed1995

Simple and specific reversed-phase liquid chromatographic method with diode-array detection for simultaneous determination of serum hydroxychloroquine, chloroquine and some corticosteroids.

This paper describes a simple, specific, and sensitive high-performance liquid chromatographic (HPLC) method using ion-pair reversed-phase chromatography with photodiode-array detection for the simultaneous determination of hydroxychloroquine (HCQ) and chloroquine (CQ) in serum samples from rheumatoid arthritis patients receiving either HCQ sulphate or CQ diphosphate. The assay is also applicable to the simultaneous determination of corticosteroids. The method consisted of two diethyl ether extractions of 1.0 ml of serum, to which two internal standards (2,3-diaminonaphthalene and 18-hydroxy-11-deoxycorticosterone) and 1.0 ml of 0.25 M sodium hydroxide had been added. After the organic phase was evaporated to dryness at 30-40 degrees C under a stream of nitrogen, the extract was reconstituted with a 1:1 mixture of 0.1 M perchloric acid and methanol, an aliquot of which was injected on to the system. Peak-height ratios at different wavelengths (A245/343, A245/256, A245/265 and A245/275) were utilized as a method of assessing peak homogeneity. Some anti-inflammatory drugs which may be used for rheumatic disorders were shown not to interfere with the assay. The method provides selectivity by using diode-array detection at several wavelengths. The use of two internal standards not only compensates for losses during the sample manipulation but also prevents erroneous results in case of interference.

Adrenal Cortex Hormones↗
PubMed1995

High-performance liquid chromatographic determination of the novel antitumour drug topotecan and topotecan as the total of the lactone plus carboxylate forms, in human plasma.

A sensitive high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantitation of the novel anticancer agent topotecan and topotecan as the total of its lactone and carboxylate forms in human plasma. Linear response in analyte standard peak area were observed over the concentration range 0.05-10 ng/ml using 100-microliters plasma samples. The instability of the drug in the biological matrix necessitated that the plasma fraction was obtained within 5 min after blood sampling by centrifugation, immediately followed by protein precipitation with cold methanol (-30 degrees C). Stability studies have indicated that topotecan is stable in these methanolic extracts for at least 4.5 months at -30 degrees C and 2 months at -70 degrees C. For the total determination of the lactone plus lactone ring-opened forms of the drug as topotecan, plasma samples were deproteinated with methanol and, subsequently, acidified with 7% (v/v) perchloric acid. Plasma samples for the measurement of total levels of the lactone and the ring-opened forms of topotecan were stable for at least 4.5 months when stored at -30 degrees C. After centrifugation, the supernatants were analysed by HPLC using a Zorbax SB-C18 Stable Bond column and methanol-0.1 M hexane-1-sulfonic acid in methanol-0.01 M N,N,N',N'-tetramethylethylenediamine (TEMED) in distilled water pH 6.0 (25:10:65, v/v) as the mobile phase. Detection was performed fluorimetrically. Within-run and between-run precision was always less than 12.1% in the concentration range of interest (0.05-10.0 ng/ml). The limit of quantitation is 0.05 ng/ml. Accuracy measurements ranged between 87.6 and 113.5%.

Antineoplastic Agents↗
PubMed1995

Determination of plasma activities of purine nucleoside phosphorylase by high-performance liquid chromatography: estimates of nonparenchymal cell injury after porcine liver transplantation.

An assay is described for measurement of purine nucleoside phosphorylase (PNP) in plasma by high-performance liquid chromatography (HPLC). A plasma sample was incubated with hypoxanthine and ribose-1-phosphate in phosphate-free medium at pH 7.4 to catalyse the production of inosine by plasmatic PNP. The reaction was stopped by addition of perchloric acid to inactivate the enzyme and to precipitate plasma proteins. After centrifugation and neutralization of the supernatant with NaOH the increase in the substrate inosine was determined by HPLC. Plasma activities of PNP averaged 5.0 mU/ml before and 12.3 mU/ml (p < 0.001), 5 min after porcine liver transplantation. At the same time points, the plasma activities of the frequently used liver enzymes lactate dehydrogenase or alanine aminotransferase remained virtually unchanged. Thus, plasmatic activities of PNP may be a suitable and early indicator of ischemic alterations to the graft in vivo.

Alanine Transaminase↗
PubMed1995

Simultaneous quantitation of oxidised and reduced glutathione in equine biological fluids by reversed-phase high-performance liquid chromatography using electrochemical detection.

A change in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) can be used to indicate oxidative stress in vivo. A rapid and highly sensitive isocratic reversed-phase high-performance liquid chromatographic method using coulometric electrochemical detection (LCEC) has been developed to simultaneously detect GSH and GSSG in equine biological fluids. Perchloric acid was used to extract GSH and GSSG from equine plasma and haemolysates, and methanol was used to deproteinise bronchoalveolar lavage fluid samples. Injection of extracts onto a Hypersil ODS HPLC column produced well resolved peaks corresponding to GSH and GSSG. The concentrations of GSH and GSSG found in equine haemolysates were similar to those previously found in humans and laboratory animals, although, to the authors' knowledge, previous attempts to measure GSH and GSSG in bronchoalveolar lavage fluid using LCEC have been unsuccessful. This method can be used to measure the GSH redox ratio in biological fluids during physiological conditions that may induce oxidative stress, such as exercise and disease.

Animals↗
PubMed1995

Determination of midazolam and its unconjugated 1-hydroxy metabolite in human plasma by high-performance liquid chromatography.

A selective and sensitive high-performance liquid chromatographic method for the analysis of midazolam and its unconjugated 1-hydroxy metabolite in plasma samples was developed. The compounds were extracted from plasma by a liquid-liquid extraction procedure with diethyl ether. Mean analytical recoveries were 87% and 86% at a concentration of 300 ng/ml for midazolam and 1-hydroxymidazolam, respectively, and the quantification limit was 2 ng/ml for a plasma volume of 1 ml. The separation of midazolam, 1-hydroxymidazolam and flurazepam (internal standard) was achieved on a Spherisorb 5 CN column using methanol-2(r)propanol (75:25, v/v) containing 0.015% perchloric acid at a flow-rate of 1.5 ml/min. The method is sensitive enough for monitoring midazolam and also the unconjugated form of the active metabolite in plasma during pharmacokinetic studies.

Calibration↗
PubMed1996

Simple reversed-phase high-performance liquid chromatography quantitation of ganciclovir in human serum and urine.

A fast, simple, and cost-effective HPLC method for the quantitation of the antiviral drug ganciclovir is described. The serum samples are extracted with perchloric acid and neutralized with potassium phosphate buffer, and urine samples are diluted with distilled water. A reversed-phase column with isocratic elution by 15 mM potassium phosphate buffer (pH 2.5) containing 0.25% acetonitrile is used to separate ganciclovir; quantitation is by UV absorbance at 254 nm. Total turnaround time is 22 min; more than 3000 samples can be run on a single column without loss of peak quality. The limit of quantitation is 0.05 micrograms/ml. Recoveries varied from 91 to 107% with coefficients of variation ranging from 0.387 to 7.95%.

Antiviral Agents↗
PubMed1996

Determination of glibenclamide in human plasma by solid-phase extraction and high-performance liquid chromatography.

A sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma has been developed. Sample clean-up prior to chromatographic analysis was accomplished by extraction of the drug using a solid-phase RP-8 or RP-18 cartridge instead of the conventional liquid-liquid extraction methods described. For the separation of the drug from the endogenous components a reversed-phase column (LiChrosorb RP-8) of 5 microns particle size and 250 x 4 mm I.D., together with a mobile phase consisting of aceronitrile-12 mM perchloric acid (47:53) was selected. The method employs progesterone as an internal standard, and a reversed-phase column combined with UV detection of the drug at 230 nm. The detector response was linear up to the concentration of 400 ng/ml and the average recovery was 100.36%. The sensitivity of the method was 5 ng/ml.

Chromatography, High Pressure Liquid↗
PubMed1996

Reversed-phase high-performance liquid chromatography method for the analysis of nitro-arginine in rat plasma and urine.

Nitro-L-arginine (L-NNA) is an inhibitor of the enzyme nitric oxide synthase (NOS). We developed a simple, sensitive and reproducible reversed-phase high-performance liquid chromatographic method for detection of nitro-arginine (L- and D-enantiomer) in rat plasma and urine. Samples were treated with perchloric acid, neutralized and eluted through a C8 reversed-phase column with a mobile phase of 18.5 mM heptanesulfonic acid-10% methanol in water, using theophylline as an internal standard. Plasma recovery for both isomers was complete, and the sensitivity limit was 0.5 micrograms/ml. This method may be used for disposition studies of L-NNA in small animals.

Animals↗
PubMed1996

Fluorimetric determination of nicorandil in human plasma by a high-performance liquid chromatographic-postcolumn ultraviolet irradiation [corrected] system equipped with on-line back-pressure tubing.

For the determination of nicorandil in plasma, a fluorometric technique using HPLC-postcolumn UV radiation [corrected] has been developed. The chromatographic system consisted of a single pump, photoreactor and on-line back-pressure tubing. The system was suitable for the separation of nicorandil under the present reaction conditions. The calibration graph was linear over the range 6.5-1170 ng ml-1 using an injected volume of 100 microliters. The pretreatment of the plasma samples consisted only of deproteinizing steps by adding perchloric acid. The mean recovery from plasma was 90.2%.

Administration, Oral↗
PubMed1996

Determination of 2-chloro-2'-deoxyadenosine nucleotides in leukemic cells by ion-pair high-performance liquid chromatography.

A specific isocratic ion-pair HPLC method for the quantitation of mono-, di- and triphosphates of 2-chloro-2'-deoxyadenosine (CdA) in leukemic cells from patients is described. The method is based on an extraction of nucleotides from cells with a solution of perchloric acid containing triethylammonium phosphate followed by an isocratic separation on an Ultrasphere ODS column (250 x 4.6 mm, 5 microns) with a mixture of 89% triethylammonium phosphate buffer (0.08 M, pH 6.1) and 11% methanol as the eluent. UV absorbance at 265 nm was used. The limit of detection was 65 nM. Standard curves for the CdA triphosphate (CdATP) were linear within the concentration range of 200 nM to 12 microM. The mean overall recovery of CdATP was 90% within a concentration range of standard curves. The within-day and day-to-day coefficients of variation at concentrations of 1.44 microM and 6.25 microM CdATP were < 10%. The applicability of the method was demonstrated by in vitro studies of the accumulation of CdA mono-, di- and triphosphates in CCRF-CEM cells and by determination of the cellular pharmacokinetics of CdA nucleotides in leukemic cells from a patient treated with CdA.

Antineoplastic Agents↗
PubMed1996

Pharmacokinetic studies of amiloride and its analogs using reversed-phase high-performance liquid chromatography.

We have studied the pharmacokinetics of amiloride and its analogs. A high-performance liquid chromatographic method has been adapted for the measurement of amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and 5-(N, N-hexamethylene)amiloride (HMA) in mouse plasma, kidney, liver and tumor tissues. The method uses a C8 preparative solid-phase column, followed by separation using a reversed-phase C18 column (250 x 4 mm I.D., 5 microns particle size) with detection by ultraviolet absorption at 365 nm. Reversed-phase separations were performed at ambient temperature using a non-linear gradient method with two different mobile phases: mobile phase A was 100% acetonitrile while mobile phase B was 0.15 M perchloric acid at pH 2.20 (flow-rate was 1.2 ml/min). The retention times for amiloride, benzamil (used as an internal standard), EIPA and HMA are 13.4, 19.5, 21.8 and 23.5 min, respectively. The calibration curves are linear over the range of 0.1-50 microM in plasma and in tissues. The half-lives of amiloride, EIPA and HMA (and their confidence intervals) in plasma after intraperitoneal injection of drugs into mice were 68.8 +/- 0.2, 31.2 +/- 2.5 and 39.3 +/- 7.9 min, respectively. Amiloride was detected as a metabolite of EIPA but not of HMA. When EIPA was injected at a dose of 10 micrograms/g body weight, it was cleared rapidly from liver, but concentrations > 1 microM were sustained for at least 2 h in murine kidney and in a transplantable tumor.

Amiloride↗
PubMed1991

Quantitative analysis of chlorpromazine by electron spin resonance (ESR) spectroscopy.

A simple and sensitive method is described for quantitative analysis of chlorpromazine in blood, serum, urine and tissue homogenate. The chlorpromazine cation radical produced by adding perchloric acid and 2,3-dichloro-5,6-dicyano-p-benzoquinone to the sample can be detected by the ESR method at room temperature. The sensitivity limit is 10 ng, that is, 20 microliters of the solution containing 0.5 microgram chlorpromazine/ml. The time needed for the measurement is within 10 min. The chlorpromazine radical thus produced is very stable; for example, 95% of the radical was observed after 24 h. The advantage of this method is discussed by comparing with the ordinary spectrophotometry which requires the purification of the sample.

Chlorpromazine↗
PubMed1992

Proton magnetic resonance imaging and phosphorus-31 magnetic resonance spectroscopy studies of bromobenzene-induced liver damage in the rat.

Respiratory-gated proton magnetic resonance imaging was used to study the response of the rat liver in situ to bromobenzene, a classic hepatotoxicant. A localized region of high proton signal intensity in the perihilar region of the liver was seen 24-48 hr after an intraperitoneal injection of bromobenzene. Localized proton magnetic resonance spectra from within this region indicated that the increased proton signal intensity was not due to accumulation of fat in the liver, but primarily due to a longer T2 for the proton resonance of water. This is consistent with acute edema in this localized region. In vivo 31P magnetic resonance spectroscopy studies of the same rat livers in situ were performed. Spectroscopic conditions were determined whereby localized, quantitative 31P spectra could be obtained. Using these methods, 10 mmol/kg bromobenzene was found after 24 hr to cause a number of statistically significant (p less than 0.05) effects: a decrease in adenosine 5'-triphosphate levels from 4.1 +/- 0.5 to 3.0 +/- 0.5 mM, a decrease in phosphodiester levels from 11.3 +/- 0.9 to 9.3 +/- 0.7 mM and an increase in the phosphomonoesters from 3.0 +/- 0.4 to 5.5 +/- 1.2 mM (mean +/- standard deviation). High resolution in vitro 31P spectra of perchloric acid extracts of these rat livers showed that the increased phosphomonoester resonance was due to a selective 4.3-fold increase in phosphocholine. Thus, our in vivo and in vitro 31P magnetic resonance spectra are consistent with the hypothesis that a phosphatidylcholine-specific phospholipase C (generating phosphocholine and diacylglycerol) is activated during tissue damage. Both the imaging and spectroscopy results obtained with bromobenzene closely resemble CCl4-induced liver changes previously reported, and may reflect a generalized response of the liver to any acutely acting toxic chemical.

Animals↗
PubMed1995

Symbiosis between in vivo and in vitro NMR spectroscopy: the creatine, N-acetylaspartate, glutamate, and GABA content of the epileptic human brain.

High resolution 1H NMR spectroscopy was used to analyze temporal lobe biopsies obtained from patients with epilepsy. Heat-stabilized cerebrum, dialyzed cytosolic macromolecules, and perchloric acid extracts were studied using one- and two-dimensional spectroscopy. Anterior temporal lobe neocortex was enriched in GABA, glutamate, alanine, N-acetylaspartate, and creatine. Subjacent white matter was enriched in aspartate, glutamine, and inositol. The N-acetylaspartate/creatine mole ratio was lower in anterior temporal neocortex with mesial (0.66) than neocortical (0.80) temporal lobe epilepsy. Human brain biopsy samples were separated into crude and refined synaptosomes, neuronal cell bodies, and glia using density gradient centrifugation. Neuronal fractions were enriched in glutamate and N-acetylaspartate. Glial cell fractions were enriched in lactate, glutamine, and inositol. The creatine content was the same in biopsied epileptic cortex (8.8-8.9 mmol/kg) and normal in vivo occipital lobe (8.9 mmol/kg). Glutamate content was higher in epileptic cortex at biopsy (10.1-10.5 mmol/kg) than normal in vivo occipital lobe (8.8 mmol/kg). GABA content was higher in biopsies of epileptic cortex (2.3-2.2 mmol/kg) than in normal in vivo occipital lobe (1.2 mmol/kg). N-acetylaspartate content was lower in biopsied epileptic temporal cortex (5.8-6.8 mmol/kg) than normal in vivo occipital lobe (8.9 mmol/kg). Paired in vivo and ex vivo measurements are critical for a firm understanding of the changes seen in the 1H-spectra from patients with epilepsy.

Aspartic Acid↗
PubMed1989

Quantitative analysis of chlortetracycline and related substances by high-performance liquid chromatography.

Isocratic high-performance liquid chromatography on Zorbax C8 7 microns allows quantitative determination of chlortetracycline, 4-epichlortetracycline, tetracycline, demethylchlortetracycline and isochlortetracycline using a mobile phase containing dimethylsulphoxide, 1 M perchloric acid and water (35:5:60). The minor impurities anhydrochlortetracycline and 4-epianhydrochlortetracycline, which are more strongly retained can be determined using a second isocratic system with a mobile phase containing more organic modifier. The method has been used for the comparison of official standards and for the analysis of a number of commercial samples.

Chlortetracycline↗
PubMed1994

Simultaneous determination of chloroquine, proguanil and their metabolites in human biological fluids by high-performance liquid chromatography.

A reversed-phase ion-pair high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous measurement of chloroquine, proguanil and their major metabolites in human plasma, erythrocytes and urine. After a liquid-solid extraction on a Bond Elut C8 cartridge, the compounds are separated on a C8 Lichrospher 60 RP select B column by isocratic elution; the mobile phase is water-acetonitrile-methanol (78:28:4, v/v/v) with 0.5 M ammonium formate and 0.075 M perchloric acid. The eluent is monitored with an ultraviolet detector at 254 nm. The lower limits of quantification in plasma are near 6.0 ng ml-1 for chloroquine and near 9.0 ng ml-1 for proguanil. No chromatographic interference can be detected from endogenous compounds or from other antimalarial drugs. The method is accurate and precision is good with inter- and intra-assay relative standard deviations lower than 6.8% for plasma samples. N-(2-6 dichlorobenzylidene amino)guanidine is used as an internal standard. The chromatographic procedure takes 35 min and can be used for therapeutic drug monitoring and clinical studies.

Antimalarials↗