Simultaneous determination of chloroquine and metronidazole in human biological fluid by high pressure liquid chromatography

Chloroquine (CQ) and metronidazole (MZ) were measured in human urine and plasma by HPLC with UV detection. This method was used to analyse plasma levels in 4 African volunteers after an oral dose of 1000 mg CQ and 750 mg MZ, in a European on weekly prophylaxis of 500 mg CQ, and on 50 hospital urine samples. In the Africans peak plasma levels were over 1 microgram/ml and peak time was 1 1/2-2 hr. In the European plasma levels ranged from 0.58 to 0.36 microgram/ml. Over 80% of the urine samples contained CQ, MZ or both. The assay system was found flexible and economical for the therapeutic monitoring of these two important tropical drugs.     

Determination of hydroxyproline by high pressure liquid chromatography.

A rapid, precise, and simple HPLC method provides an assay of hydroxyproline from tissue extracts or solutions of collagen. Samples are hydrolyzed with 6 N HCl, derivatized with phenyl isothiocyanate, and chromatographed on a small, C18 reverse-phase HPLC column. Hydroxyproline (Hyp) is separated from other amino acids and detected by absorption at 254 nm. The method detects 0.40 to 36 micrograms of Hyp with a linear response. Separation requires a total of 6 min, including column cleanup and reequilibration. All components are commercially available, making this a convenient method for routine measurement of collagen concentration.     

Measurement of phosphate esters in extract of fetal rat heart by automated high pressure liquid chromatography

Creatine phosphate, nucleotides and glycolytic phosphate esters were estimated in extract of beating, in situ freeze clamped, $\text{131}\text{2}$ to $\text{191}\text{2}$ day fetal rat hearts by automated phosphate ester chromatography. Creatine phosphate increased more than 4-fold to almost 9 n moles per mg. protein at $\text{191}\text{2}$ days, while ATP remained relatively constant at about 19 to 21 n moles per mg. protein. Most other nucleotides decreased as gestation advanced. ATP rather than creatine phosphate appears to be the major energy source of fetal rat heart. Except for glucose-6-phosphate, which increased, the glycolytic phosphate esters decreased only very slightly with advancing gestational age, suggesting a relatively stable basal glycolytic activity. Methodology includes correction for phosphate esters of whole blood trapped in extracts of in situ freeze clamped tissues.     

Determination of plastoquinone-9 in chloroplasts by high pressure liquid chromatography

A method for the specific determination of nmol quantities of plastoquinone-9 in chloroplasts is described. HPLC of a heptane extract of chloroplasts separated the plastoquinone-9 from other material permitted a final spectrophotometric assay. Levels of plastoquinone-9 plastoquinol-9 were determined in dark adapted and illuminated chloroplasts.     

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.     

Identification and quantitative determination of prostaglandins by high pressure liquid chromatography

High pressure liquid chromatography (HPLC) using 4′ × 1/8″ columns of a pellicular silica support (Corasil-II) allows identification of prostaglandins diastereomers based on their characteristic retention relative to a standard, PGE₂ in this study. Surprisingly this simple method allows separation of PGE₂, PGE₁, and PGE_o (dihydro-PGE₁) or PGF₂α, PGF₁α, and PGF_oα without resort to silver nitrate impregnated stationary phases. Even more subtle distinctions such as that between 13,14-dihydro-PGF₂α, PGF₁α and 5,6- -PGF₂α are possible by HPLC. The differential refractometer detector used throughout can also be used for quantitation. This is illustrated by a study of the relative rates of degradation of natural PGF₂α (an oil at the temperature employed, 41°C) and racemic PGF₂α (mp. 63°) on exposure to air.     

Identification by high pressure liquid chromatography and radioimmunoassay of JH-III in Acheta domesticus

Corpora allata culture medium and haemolymph of adult females of Acheta domesticus were purified by thin layer chromatography and/or high pressure liquid chromatography and submitted to a radioimmunoassay for juvenile hormones. The conditions of purification have been established. Only one immunoreactive compound with the retention time of JH-III diol standard was detected in both samples providing evidence that in adult Acheta domesticus JH-III is the only juvenile hormone.     

The analysis of the betaine homarine by high pressure liquid chromatography

A procedure is presented which is suitable for the qualitative and quantitative analysis of the betaine homarine in aqueous tissue extracts. After preliminary purification of the extract by gel permeation chromatography on Sephadex G-25, quantitative analysis of the homarine content is performed by high pressure liquid chromatography on a 1-m column of Corasil II.     

Analysis of enzymatic steroid conversions by high pressure liquid chromatography.

Enzyme catalyzed introduction of the 1–2 double bond into a steroid can be monitored through spectrophotometric changes accompanying electron acceptor reduction or through paper or thin-layer chromatographic analysis of the reaction product. The spectrophotometric method is not applicable to cases in which the oxidized form of the electron acceptor is continually regenerated. In studying such cases, we have found high pressure liquid chromatography (HPLC) to be a method of direct analysis more convenient than paper chromatography or tlc. Use of a water based eluant and a reverse phase column for the HPLC analysis allows direct injection of a sample of the aqueous reaction solution after acidification, and no extraction with an organic solvent is necessary.     

Quantitation of anthracyclines in human hematopoietic cell subpopulations by flow cytometry correlated with high pressure liquid chromatography

The major white cell subpopulations present in bone marrow and peripheral blood can be discriminated by forward and perpendicular light scatter two-parameter flow cytometry (FCM). Fluorescent properties of anthracycline antibiotics allow measurement of the concentration of these cytotoxic drugs in hematopoietic cells by FCM as a third parameter. Analysis of scatter-gated fluorescence histograms provides quantitative information about the cellular concentration of at least four cell categories in human blood and bone marrow cells. A good correlation was found between the mean cellular fluorescence measured by FCM and the overall cellular concentration of adriamycin, daunomycin, and their main metabolites determined with high-pressure liquid chromatography (HPLC). In incubation experiments with human hematopoietic tissues, the final concentration of various anthracyclines in subpopulations of white cells appeared to be dependent on cell density, incubation time, temperature, and type of compound and its concentration. FCM analysis is a rapid, sensitive, and quantitative method for measurement of cellular anthracycline concentrations in subpopulations and therefore provides an useful new tool in monitoring chemotherapy.     

Determination of puerarin in human plasma by high performance liquid chromatography

Puerarin, an isoflavone C-glycoside, has been identified as the major active component isolated from Pueraria lobata (Kudzu) responsible for suppression of alcohol drinking. In order to conduct clinical studies of Kudzu's efficacy, a method for measuring its bioavailability and pharmacokinetic profile is needed. We have developed a gradient reversed-phase HPLC system for pharmacokinetic study of puerarin in human plasma. Solid-phase extraction was performed on an abselut Nexus cartridge (60 mg/3 ml) possessing adsorbent function with a recovery of >97% and 4-hydroxybenzoic acid was used as an internal standard. The HPLC assay was performed on a YMC ODS-A column (150 mm x 4.6mm i.d., 5 microm particle size). The HPLC mobile phase consisted of methanol/0.5% acetic acid with 20-35% methanol gradient at a flow-rate of 0.8 ml/min. The UV wavelength was set at 254 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a puerarin concentration range of 5-500 ng/ml in human plasma. The lower limit of quantification was ca. at 8 ng/ml of puerarin in plasma. The detection limit (defined as signal-to-noise ratio of about 3) was approximately 3 ng/ml. The preliminary pharmacokinetic study after oral administration of the Kudzu capsules containing 400mg of puerarin to a healthy volunteer confirmed that the present method was suitable for determining puerarin in human plasma.     

Analysis of sofalcone in human plasma by high performance liquid chromatography

A simple, rapid, sensitive and reliable high performance liquid chromatography (HPLC) method for the determination of the anti-ulcer drug sofalcone in human plasma was developed. Plasma was extracted with ethyl acetate under acidic conditions and sofalcone was determined by HPLC using a C18 column and (methanol-0.1% formic acid aqueous 80:20) mobile phase. The linear calibration curves of sofalcone in human plasma were obtained over the concentration range of 0.01-5.0 microg/ml. The lower limit of quantitation (LLOQ) was 10 ng/ml in human plasma. The precision measured for plasma was within 15%. Extraction recovery was over 85% in blood. The method was successfully applied to the identification and quantification of sofalcone in pharmacokinetic studies.     

Plasma catecholamines in resting rainbow trout,Salmo gairdneri Richardson,by high pressure liquid chromatography*

Plasma norepinephrine and epinephrine from cannulated trout were measured by high pressure liquid chromatography with electrochemical detection. The catecholamines were extracted with acid-washed alumina using a microfilter assembly which permitted analysis of small volumes of plasma. Mean (± S.D.) values for plasma norepinephrine and epinephrine were 1.83 (0.97) pmol ml^?1 and 8.95 (4.94) pmol ml^?1, respectively. These values are compared with catecholamine levels from other vertebrate species.     

Determination of polyamines in human prostate by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of polyamines in human prostate has been developed. This method is based on pre-column derivatization with dansyl chloride (Dns-Cl). The derivatives were separated on a μBondapak C₁₈ column (250×4.6 mm I.D.; 10 μm), and eluted with methanol and distilled water using a one-step linear gradient. The column eluate was monitored by fluorescence detection (excitation, 370 nm; emission, 506 nm). The within-assay precision of the study (C.V.) was as follows: putrescine (PUT) 2.88%, spermidine (SPD) 2.94% and spermine (SP) 1.17%. The between-assay precision (C.V.) was: PUT 2.66%, SPD 3.06%, SP 2.79%. The recovery was greater than 97%. The detection limit for PUT, SPD and SP were 0.05, 0.08 and 0.06 nmol/ml, respectively. In contrast to other studies, sample or polyamine derivatives did not require extraction with an organic solvent such as ethanol, evaporation under vacuum or other condensation procedures. This is a simple, rapid and sensitive method that can be applied to the determination of polyamines in nearly all biological tissues and body fluids, such as urine and serum.     

Degradation of cholecystokinin-like peptides by a crude rat brain synaptosomal fraction: a study by high pressure liquid chromatography

Degradation of CCK-8, CCK-4, and related peptides by a crude synaptosomal fraction of rat brain was investigated by monitoring the tryptophan fluorescence of reaction products after HPLC fractionation. At 20 degrees C, the half disappearance time was 52 min for CCK-8, 35 min for unsulphated CCK-8, 20 min for unsulphated CCK-7, 6 min for Tyr(SO3H)-Trp-Met-Asp-Phe-NH2, and 3 min only for CCK-4. Caerulein was much more resistant than CCK-8, and Boc-CCK-4 and Aoc-CCK-4 remained stable for at least 3 h. The apparent Km for CCK-8 and CCK-4 was 40 microM and maximal activity on CCK-8 was observed at pH 7.0. Zn2+ was strongly inhibitory. The protease inhibitors puromycin and bacitracin, the metal chelator 1,10-phenanthroline, and the sulphydryl blocking agents N-ethylmaleimide and p-chloromercuribenzoate greatly reduced the release of tryptophan from CCK-8. Puromycin inhibition of CCK-8 degradation provoked the accumulation of a CCK-7-like peptide, and that of CCK-4 degradation was of a competitive type (Ki = 2 microM). The CCK-8 degrading activity of brain synaptosomes was present in the cytosol as well as in synaptic membranes.     

Changes in monogalactosyl diacylglycerols, alkylgalactolipids and cerebroside fatty acid esters in maturing rat brain measured by high-performance liquid chromatography

A newly developed high-performance liquid chromatography method was used to measure four nonpolar glycolipids in developing rat brain. The accumulation patterns of nonhydroxy- and hydroxycerebroside fatty acid esters were similar to those of myelin cerebrosides. In contrast, both monogalactosyl diacylglycerol and alkylgalactolipids reached maximal levels at the early stages of myelination.     

Determination of meso-alanopine and D-strombine by high pressure liquid chromatography in extracts from marine invertebrates

meso-Alanopine and D-strombine are separated by high pressure liquid chromatography using a cation exchange resin and 2.5 X 10(-5) M sulfuric acid as eluant, at a flow rate of 1.0 ml/min, 20 degrees C column temperature and a pressure of 4 500 kPa. Both opines were detected by conductivity. Separation and quantitation was possible in the range of 0.05 to 25 nmol of meso-alanopine and D-strombine. Chemically or enzymatically synthesized opines were quantitated using alanopine/strombine dehydrogenase from Crassostrea angulata. The enzyme was purified by ammonium sulfate precipitation, Sephadex G-100 filtration and fast-protein-liquid chromatography. Specific activity of the final preparation was 500 U/mg protein with glycine as substrate. The formation of meso-alanopine and D-strombine was demonstrated in neutralized perchloric acid extracts from muscle tissue of Arenicola marina L. following enhanced muscular activity and in Mytilus edulis L., Nucula nitida and Crassostrea angulata after 24 h of anoxia.