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Determination of conjugated bile acids in human bile and duodenal fluid by reverse-phase high-performance liquid chromatography.

A simple mehtod using reverse-phase liquid chromatography is presented for resolution and quantitation of the major conjugated bile acids of man, including the glycine and taurine conjugates of the dihydroxy bile acids, chenodeoxycholic and deoxycholic acid. Using modern, high-performance chromatographic equipment, analysis time is less than 30 minutes. The quantitative range of the method, with detection by refractive index, is 0.05 to 0.1 mumol of bile acid and the limit of detection for an injection sample is 0.01 mumol. This provides a sensitivity sufficient for analysis of dilute duodenal and gallbladder bile with minimal sample preparation.

Bile

A rapid micromethod for measuring theophylline in serum by reverse-phase high-performance liquid chromatography.

We describe an assay system for measuring theophylline in 25 microliters of serum. The procedure involves extraction with a 95:5 mixture of chloroform:isopropanol containing beta-hydroxypropyltheophylline as internal standard, and reverse-phase chromatography on a 4 mm x 30 cm column containing "micron Bondapak C18." Theophylline and beta-hydroxypropyltheophylline are eluted with a 90:10 mixture of sodium acetate butter (20 mmoles/litre pH 4.0) and acetonitrile at a flow rate of 1.8 ml/min., are detected by their absorbance at 254 nm, and quantitated by measuring peak areas. Column temperature has not been found to be critical in this analysis. Each analysis requires 9 minutes of chromatography time with a total analysis time of 20 minutes. Analytical recoveries were found to be 71 to 75% for theophylline and 94% for beta-hydroxypropyltheophylline. This difference in recovery is corrected when determining the theophylline concentration in unknown samples. The method has good precision (coefficients of variation between 7.0% and 7.9% for therapeutic and toxic concentrations). The results obtained with this method compare favourably with results obtained by a published cation-exchange high-performance liquid chromatographic method. None of the metabolites of theophylline, common compounds related to theophylline in structure or drugs tested have been found to interfere with the analysis described.

Chromatography, High Pressure Liquid

The determination of corticoids and related steroid analogs by high-performance liquid chromatography.

High-performance reverse-phase liquid chromatographic data on 28 corticoids and related steroid analogs, plus methyl and propyl parabens, are presented. Retention values, relative to acetone, are reported for each steroid on Bondapak Phenyl/Corasil, Bondapak C18/Corasil, and micro(micron) Bondapak C18 columns, and methanol/water or acetonitrile/water mobile phases. Ultraviolet detector response data for four of the steroids and calculated efficiencies for the three different reverse-phase columns are given. Chromatograms showing the separations of four steroids plus methyl and propyl parabens obtained on each of the three columns are also presented.

Adrenal Cortex Hormones

Preparation and characterization, using high-performance liquid chromatography, of an enzyme forming glucosides of cytokinins.

Cytokinins can occur naturally as glycosides with beta-D-glucose as the sugar substituent. From radish (Raphanus sativus) cotyledons, an enzyme has been partly purified which synthesizes the 7-glucopyranoside of zeatin [6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine], a compound known to occur in this species. High-performance reverse-phase liquid chromatography was uniquely useful as the analytical procedure for quantitative study of the minute amounts of enzyme available. The enzyme uses UDPglucose as the source of the sugar residue. A large number of derivatives of purine are glucosylated, but adenine derivatives with an alkyl side chain at least three carbon atoms in length at position N6 are preferentially glucosylated. This corresponds to the structural features required for high cytokinin activity. The 7-glucoside of zeatin is known to be very weakly active in cytokinin bioassays. Hence, this enzyme, and others catalyzing the same reaction, have a role in the regulation of cytokinin activity.

Cytokinins

Amikacin assay in serum by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the quantitative determination of amikacin in serum is described. The antibiotic was separated from serum by adsorption on silica gel. The adsorbed drug was then derivatized with o-phthalaldehyde and eluted with ethanol. The derivatized amikacin was separated by reverse-phase, high-performance liquid chromatography and quantitated by fluorometry. A linear response for serum samples containing amikacin ranging from 1.0 to 15 mug/ml was obtained. Other antibiotics including various aminoglycosides did not interfere with the amikacin assay. Comparison with a standard microbiological assay gave a correlation coefficient of 0.99. This chemical assay is sensitive and specific under the conditions tested and can be performed rapidly.

Amikacin

Acyclovir kinetics after intravenous infusion.

The disposition and safety of the antiviral drug acyclovir were studied in 14 subjects with advanced malignancies. Acyclovir was administered by a 1-hr intravenous infusion at doses of 0.5, 1.0, 2.5, and 5.0 mg/kg. At the end of infusion, mean peak plasma levels (+/- SEM), determined by radioimmunoassay, were 6.4 +/- 0.7, 12.1 +/- 2.3, 14.9 +/- 2.7, and 33.7 +/- 7.1 microM. The plasma concentration-time profiles could be described by a biexponential equation. The half-life of acyclovir in the slow disposition phase ranged from 2.2 to 5 hr and the drug was detected in the plasma for at least 18 hr after infusion. The total body clearance ranged from 117 to 396 ml/min/1.73 m2. A proportionality between area under the curve and dose suggests that acyclovir exhibits dose-independent kinetics in the dose range studied. There was wide variation in cumulative urinary excretion of unchanged drug, ranging from 30 to 69% of the dose. From renal clearances of acyclovir, which were higher than creatinine clearances, it appears that both glomerular filtration and tubular secretion contribute to its renal excretion. Analysis of the urine by reverse-phase high-performance liquid chromatography revealed the presence of the metabolite 9-carboxymethoxymethylguanine. There was no indication of toxicity either clinically or from laboratory findings in any of the study subjects. This study demonstrates that in addition to selectivity and low toxicity, the kinetic profile and metabolic disposition of acyclovir make it an attractive candidate for therapy in a variety of herpes infections.

Aged

Proteomic profiling of the aqueous extract from the antennal gland of the Pacific white shrimp, Litopenaeus vannamei.

The antennal gland (AnG) of decapod crustaceans has been proposed as a potential source of bioactive molecules involved in chemical communication; however, its protein composition remains largely unexplored. Here, we present the first reference proteomic map of the aqueous extract from the antennal gland of the Pacific white shrimp Litopenaeus vannamei. Protein extracts from immature and mature females were analyzed using an integrated workflow combining one-dimensional SDS-PAGE, reverse-phase high-performance liquid chromatography (RP-HPLC), and nanoLC-tandem mass spectrometry. Electrophoretic and chromatographic analyses revealed a high degree of qualitative similarity between reproductive stages. SDS-PAGE resolved six major protein bands (∼227, 166, 77, 42, 35, and 17 kDa), most comprising multiple co-migrating proteins as revealed by LC-MS/MS. Hemocyanin was identified as the predominant protein and was detected across several electrophoretic bands. Additional proteins were associated with innate immunity, including β-1,3-glucan-binding protein and coagulable hemolymph protein; reproductive processes, including vitellogenin, spermatogonial stem-cell renewal factor, farnesoic acid O-methyltransferase, estrogen sulfotransferase, and prostaglandin reductase 1; as well as energy metabolism, protein homeostasis, cytoskeletal organization, and intracellular trafficking. Because several identified proteins are widely distributed or known hemolymph components, their detection cannot be assumed to reflect AnG-specific expression or function. Collectively, these findings establish a molecular reference for the L. vannamei AnG and reveal protein components associated with multiple physiological processes. This dataset provides a proteomic framework for future comparative and functional studies aimed at elucidating antennal gland physiology and experimentally evaluating the potential involvement of proteinaceous or peptide-based molecules in chemical communication in decapod crustaceans.

Animals

5',8-cyclo-dAdo and 8-oxo-dAdo DNA Lesions Are Both Substrates of Adenosine Deaminase: A Preliminary Study.

Genetic information, whether inside or outside the nucleus, is exposed to a variety of harmful physico-chemical factors. Although DNA damage repair systems have been extensively studied, little information about post-repair and non-genomic DNA damage metabolism is available in the literature. Adenosine deaminase (ADA) is an abundant enzyme found on both sides of the cell membrane that regulates the concentration of adenine derivatives. In this article, it has been shown that 7,8-dihydro-8-oxo-2'-deoxyadenosine (OXOdAdo) and (5'R/S) 5',8-cyclo-2'-deoxyadenosine ((5'R/S)cdAdo) are suitable substrates for ADA. For this purpose, theoretical Density Functional Tight Binding and RP-HPLC analyses were applied. The products of ADA activity, i.e., OXOdIno (7,8-dihydro-8-oxo-2'-deoxyinosine) and (5'R/S) cdIno ((5'R/S) 8-cyclo-2'-deoxyinosine), were identified and confirmed by high-resolution mass spectroscopy. Although the (5'R) and (5'S)cdAdo enzymatic deamination processes are much slower (34% and 32% after 168 h, respectively) than the process observed for dAdo, 5',8-cyclo-2'-deoxyinosine should be considered when monitoring cyclopurine levels in physiological fluids. The same should be considered in the case of OXOdAdo, which is completely converted to OXOdIno within one minute and may therefore be less visible than OXOdGuo during mass spectroscopy analysis. Both these observations are important, given the abundance of 2'-deoxyadenosine on both sides of the cell membrane and its potential conversion into OXOdAdo and (5'R/S)cdAdo. They may also explain why the observed level of OXOdAdo is much lower than that of OXOdGuo in cells and physiological fluids, even though their difference in ionisation potential is only 0.25 eV. Future studies are needed to further investigate the metabolism of DNA damage and to identify the enzymes involved in nucleic acid biochemistry.

Adenosine Deaminase

Reverse-phase chromatography of polar biological substances: separation of catechol compounds by high-performance liquid chromatography.

Catecholamines and their metabolites have been separated isocratically by reverse-phase chromatography with aqueous (no organic solvent admixed) eluents. Unlike ion-exchange or ion-pair chromatography, mixtures of both acidic and basic substances can be separated in a single chromatographic run, because the retention is governed by hydrophobic interactions between the nonpolar moiety of the solute molecules and the octadecyl-silica stationary phase. The relative retention values strongly depend on the pH of the eluent, which governs the degree of dissociation of ionogenic solutes. The reproducibility of the results and the stability and efficiency of the chromatographic systems make this approach particularly attractive for use in clinical analysis.

3,4-Dihydroxyphenylacetic Acid

High-performance liquid chromatographic determination of benzodiazepines in human plasma.

A simple and sensitive method for determination of benzodiazepines in plasma has been developed using high-performance liquid chromatography in a reverse-phase mode. The method is illustrated by application to plasma samples containing diazepam and N-desmethyldiazepam at concentrations which would be encountered during therapy, with limits of detection of 10 ng/ml and 2 ng/ml for diazepam and N-desmethyldiazepam, respectively.

Chromatography, High Pressure Liquid

Analysis for indole compounds in urine by high-performance liquid chromatography with fluorometric detection.

We describe a chromatographic system involving a high-performance chemically-bonded reverse-phase column and fluorescence detection for measurement of indoles in urine. We controlled retention and selectivity by optimizing the methanol content and pH of the mobile phase. Six reference indoles were separated in less than 20 min; three 5-hydroxyindoles were eluted in less than 7 min. About 5-15 ng of aqueous solutions of these compounds can be detected. The combination of selectivity (from use of the chromatographic column) and fluorescence detection permitted analysis for five of the six indoles after a single urine-deproteinization step.

5-Hydroxytryptophan

The extraction and analysis of benzodiazepines in tissues by enzymic digestion and high-performance liquid chromatography.

A simple, rapid and sensitive method is described for the analysis of unchanged benzodiazepine drugs and their metabolites, in human and animal tissues, using the proteolytic enzyme subtilisin Carlsberg followed by reverse-phase liquid chromatography. The enzymic digestion yields far higher recoveries than conventional extraction methods, while the liquid chromatogrpahic analysis allows the rapid separation and detection of nanogram quantities of these drugs without the need for elaborate "clean-up" procedures.

Animals

Analysis for procainamide and N-acetyl procainamide in plasma or serum by high-performance liquid chromatography.

A high-performance liquid chromatography method is presented for simultaneous analysis for procainamide and N-acetyl procainamide in plasma or serum. The procedure involves internal-standard addition, organic extraction, and separation on a reverse-phase column. The detection limit for procainamide is 0.1 mg/liter and the calibration plot is linear to at least 30 mg/liter. Comparison with a colorimetric assay for procainamide gave a correlation coefficient of 0.989. We checked for interference by a large series of appropriate drugs, and found none, nor did icteric or lipemic sera present problems.

Chromatography, High Pressure Liquid

Purification and analysis of the purity of NADH.

We developed an analytical reverse-phase high-performance liquid chromatographic procedure for rapid assessment of the purity of NADH. The method completely separates adenosine monophosphate and adenosine diphosphoribose from NADH. By use of this analytical technique we found that preparative chromatography on DEAE-cellulose gives NADH that is free from adenine nucleotides as well as other impurities that commonly are present in NADH. The absorbance ratio at 260 and 340 nm of the purified NADH in 1.8 mmol/liter ammonium carbonate is 2.261 +/- 0.002 (+/- 1 SD).

Bicarbonates

Assay of sulfonylureas in human plasma by high-performance liquid chromatography.

A sensitive and specific high-performance liquid chromatographic procedure for the determination of chlorpropamide or tolbutamide in plasma in the presence of their metabolites is described. The ether extract of acidified plasma is redissolved in the mobile phase, 17% acetonitrile in 0.05 M aqueous ammonium formate, and chromatographed on a reverse-phase column on a high-performance liquid chromatograph fitted with a UV absorbance detector. Quantitation of plasma samples containing less than 0.5 mug/ml of chlorpropamide and 5 mug/ml of tolbutamide is reported, using these drugs as mutual internal standards. The retention times of the metabolites are such that they do not interfere in the procedure. The assay method was tested in a human volunteer with both drugs and found suitable for single-dose pharmacokinetic studies.

Chlorpropamide