DNA base composition of Rickettsia tsutsugamushi determined by reversed-phase high-performance liquid chromatography

The DNA base composition of Rickettsia tsutsugamushi was determined by reversed-phase high-performance liquid chromatography and compared with that of Rickettsia rickettsii. The G+C contents were 28.1 to 30.5 mol% for R. tsutsugamushi and 32.1 mol% for R. rickettsii.     

Determination of serum retinol by reversed-phase high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic method was developed for the determination of serum levels of retinol in humans. A direct serum injection technique after deproteinisation was used to avoid lengthy pretreatment steps which can result in degradation of retinol during analysis. The column used was CLC-ODS, the mobile phase was acetonitrile-water and detection wavelength was 328 nm. Deterioration in column performance was not observed even after injection of 300 samples. The lower detection limit was 10 μg/l. On analyzing a serum pool six times, a C.V of 0.7% was obtained. The method is quantitative, reproducible, rapid and highly accurate for routine analysis.     

核酸酶P1催化水解热变性DNA的研究

用桔青霉菌株IM0 2 (PenicilliumcitrinumIM0 2 )发酵制得的核酸酶P1催化水解热变性DNA ,在研究温度、底物浓度、pH、酶加入量等因素对水解结果的影响的实验基础上,通过正交实验获得最佳的工艺条件:温度5 8℃,pH6 5 ,DNA质量浓度4 0g/L ,酶加入量4 % ,脱氧核苷酸的得率92 %。在此基础上推导出其催化米氏常数Km=5 818×10 -2 g/L。     

Measurement of genome wide DNA methylation by reversed-phase high-performance liquid chromatography

Intrinsic affinity tags are useful tools for the study of macromolecular targets. Although polypeptide affinity tags are routinely used in purification and detection of protein complexes, there has been a relative lack of powerful RNA affinity tags that can be embedded within RNA sequences. Here, the preparation and use of two RNA affinity tags against Sephadex or streptavidin are described. The two tags have different strengths that make them appropriate for slightly different uses. One is a high-affinity ligand for streptavidin that can be specifically eluted by competition with biotin under otherwise native binding conditions. The other tag binds selectively to Sephadex beads, and can be eluted by competition with the soluble dextran that composes Sephadex. When properly placed within another RNA molecule, the tags can be used to effect dramatic purification of RNA or ribonucleoprotein complexes from complex mixtures of cellular RNA.     

Determination of O-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic assay for O⁶-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were constituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O⁶-Benzylguanine peak heights were compared to peak heights of O⁶-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O⁶-benzylguanine.     

Determination of temazepam and temazepam glucuronide by reversed-phase high-performance liquid chromatography

A rapid and sensitive method for extracting temazepam from human serum and urine is presented. Free temazepam is extracted from plasma and urine samples using n-butyl chloride with nitrazepam as the internal standard. Temazepam glucuronide is analyzed as free temazepam after incubating extracts with β-glucuronidase. Separation is achieved using a C₈ reversed-phase column with a methanol—water—phosphate buffer mobile phase. An ultraviolet detector operated at 230 nm is used and a linear response is observed from 20 ng/ml to 10 μg/ml. The limit of detection is 15.5 ng/ml and the limit of quantitation is 46.5 ng/ml. Coefficients of variation are less than 10% for concentrations greater than 50 ng/ml. Application of the methodology is demonstrated in a pharmacokinetic study using eight healthy male subjects.     

Analysis and purification of plasmid DNA by reversed-phase high-performance liquid chromatography

Large nucleic acids can be separated by reversed-phase high-performance liquid chromatography. Under our experimental conditions, the retention time depends not on the chain length but rather on the base composition and the secondary structure of the molecule. Because of the torsional strain caused by the supercoiling of the plasmid, more of its bases are accessible for interaction with the hydrophobic stationary phase. This increases the retention time of the supercoiled DNA compared to the relaxed or linear DNA. We have exploited these properties to analyze the quality of plasmid preparations. The method is more sensitive to contaminants than common electrophoretic techniques. Furthermore, we describe a convenient and rapid procedure for purifying plasmid DNA. The highly pure plasmid is biologically more active for most of the enzymatic reactions commonly used in genetic engineering.     

Determination of rat liver triglycerides by gas–liquid chromatography and reversed-phase high-performance liquid chromatography

Rats fed with a fat-free or an olive oil-rich diet were employed to compare the response of two chromatographic techniques in the determination of rat liver triglyceride (TG) molecular species composition. Gas–liquid chromatography (GLC) on polarizable liquid phase and reversed-phase high-performance liquid chromatography (RP-HPLC) have been commonly employed for TG analysis, obtaining a similar number of chromatographic peaks when used for animal tissue TG determination. In the present study similar results were achieved with regard to most relevant chromatographic peaks, however, important differences were found in the content of minor TGs. Indeed, RP-HPLC permitted separation of long chain polyunsaturated fatty acids, which were not detected by GLC, while the latter technique reported a higher number of myristoyl-containing TG species. RP-HPLC analysis reported a greater number of TGs, with more similarity to a random composition, made up from the liver fatty acid composition. Therefore, it was concluded that utilization of both techniques would be helpful for liver TG analysis as the use of only one of them does not provide a complete profile of liver TGs. Nevertheless RP-HPLC seems to be more useful for this purpose since revealed a more extensive profile.     

Determination of p-chloronitrobenzene in plasma by reversed-phase high-performance liquid chromatography

A simple, accurate and precise isocratic reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of p-chloronitrobenzene (p-CNB) in rat plasma. A plasma sample was deproteinized with methanol containing the internal standard (p-bromonitrobenzene). The resulting methanol eluate obtained after centrifugation was filtered and injected into a high-performance liquid chromatograph (50 μl each). A column packed with 5 μm octadecylsilane (ODS) spherical particles was used with isocratic elution of methanol—water (45:55, v/v) at a flow-rate of 1.0 ml/min. The compounds were detected by ultraviolet absorbance at 280 nm. The retention times of p-CNB and the internal standard were 12.5 and 15.5 min, respectively, at a column oven temperature of 30°C. The results were linear from 0.05 to 100 μg/ml (r = 0.999), and the detection limit was 0.01 μg/ml. The relative error and the coefficient of variation on replicate assays were less than 7 and 10%, respectively, for all concentrations studied. The overall recoveries of p-CNB were between 97 and 105%. Plasma samples could be stored for up to one month at −20°C.     

Ion-pair reversed-phase high-performance liquid chromatography of adenine nucleotides and nucleoside using triethylamine as a counterion

An isocratic HPLC method for the simple and selective determination of adenine nucleoside and nucleotides has been developed. The separation is achieved at room temperature by reversed-phase chromatography (Shiseido, Capcell Pak C18). A mixture of 0.1 M triethylamine (TEA) phosphate buffer and methanol (95:5, v/v) is used as a standard eluent. Influence of pH and concentrations of organic modifiers and TEA ion on capacity factors of adenine compounds has been investigated. It has been also found that the TEA ion in the eluent is adsorbed onto the reversed-phase surface. The results clearly demonstrate that ion-pair formation with TEA ion occurs probably both in the mobile phase and on the stationary phase and governs the retention of adenine and nucleotides in the present system. The HPLC system is applied to the analysis of adenine nucleotides formed as intermediates in the synthesis of 3′-phosphoadenosine 5′-phosphosulphate (PAPS) and to the assays of ATPases and 5′-nucleotidase activities in rat liver plasma membrane. This method is a new type of ion-pair reversed-phase HPLC system and is suitable for the separation of highly polar organic anions, especially for adenine nucleotides.     

Determination of inulin in plasma and urine by reversed-phase high-performance liquid chromatography

We report a new HPLC procedure for measuring inulin in plasma and urine. Samples after dilution are boiled in mild acidic conditions and then analyzed on a C₁₈ column. Solvent system A is 3.2 mM HCl, pH 2.5, and B is acetonitrile-3.2 mM HCl (60:40, v/v), pH 2.5. The separation is carried out in 8 min with a flow-rate of 1.0 ml/min and the absorbance monitored at 280 nm. The relationship between inulin and the recorded peak area is linear from 0.2 to 3.2 mg/ml with a correlation coefficient of 0.999 for plasma and 0.999 for urine. Within-run precision, measured at three inulin concentrations, ranged from 0.9 to 1.7% in plasma and from 0.8 to 1.2% in urine. Between-run precision varied in plasma from 2.7 to 3.2% and in urine from 3.0 to 3.3%. Analytical recovery ranged from 102 to 107% in plasma and from 101 to 105% in urine, respectively. The method is sensitive, selective and only 30-μl samples are required. Therefore, it could be used to evaluate the glomerular filtration rate even in small babies and to perform studies in animals.     

Determination of quinolone antibiotics in growth media by reversed-phase high-performance liquid chromatography

A simple, accurate, precise, and versatile high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of three quinolone antibiotics in Mueller–Hinton broth. The fluoroquinolone agents studied were ciprofloxacin, ofloxacin, and sparfloxacin; other quinolone agents have been identified using this method but not validated in this matrix (levofloxacin, clinafloxacin, temafloxacin, and trovafloxacin). In addition, several other biological growth mediums have been investigated (human serum, human urine, Todd–Hewitt growth media, Ensure enteral feeding solution, and Haemophilus growth media). This method uses UV detection (280 nm), a simple, one-step protein precipitation extraction, and separation using a C₁₈ column with an isocratic, ion-pairing mobile phase. An appropriate internal standard was obtained by using another quinolone antibiotic of differing retention time. The calibration curves were linear (r²≥0.999) over a concentration range of 0.0625–20.0 μg/ml with a lower limit of quantification of 0.1 μg/ml. The intra-day and inter-day coefficients of variation were less than 15%.     

Determination of growth hormone-releasing hexapeptide by reversed-phase high-performance liquid chromatography with electrochemical detection

A novel HPLC method with electrochemical detection is described for the determination of a growth-hormone-releasing hexapeptide (GHRP-6). HPLC conditions, such as the column, mobile phase, and oxidation potential, were optimized for sensitivity and selectivity of analysis. GHRP-6 was separated on a reversed-phase CN column with 37% acetonitrile in 100 mM phosphate buffer (pH 7.0) as the mobile phase. The optimum electrochemical oxidation signal was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to two electroactive tryptophans and a histidine residue. Solid-phase extraction using octadecyl cartridges was optimized for sample cleanup of GHRP-6 from serum samples and the method was successfully applied over the concentration range of 5 to 100 ng/ml of analyte.     

Determination of transdermal sildenafil in nude mouse skin by reversed-phase high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method was developed for the determination of sildenafil transdermal permeation of nude mouse skin. A reversed-phase column with UV detection at 224 nm was used for chromatographic separation. The mobile phase consisted of 32% acetonitrile with 0.2% phosphoric acid in water at pH 5.3 adjusted with 10 M NaOH with the flow-rate set at 1.0 ml/min. The limit of quantitation achieved was 5 ng/ml, and the calibration curve showed good linearity over the concentration range of 5–500 ng/ml. The relative standard deviations of within- and between-day analyses were all within 15%. Sildenafil was found to be stable between pH 3 and 12 during 24-h incubation with skin. After transdermal administration of 15.8 μg/ml of sildenafil to nude mouse skin, it was detected as early as 15 min. The transport amount of sildenafil could be quantitated and, at pH 8–11, had the highest permeation rate in nude mouse skin.     

Determination of intracellular glutathione in human skeletal muscle by reversed-phase high-performance liquid chromatography

A chromatographic method for the specific determination of cellular low molecular mass thiols has been applied to human muscle tissue. The method is based on the derivatisation of thiols using monobromobimane, which is a specific reagent for the sulphydryl group. The glutathione and cysteine bimane adducts were separated by reversed-phase HPLC, whilst quantitation of the cysteine and glutathione adducts was achieved by fluorescence spectroscopy. The method was found to yield a quantitative recovery of glutathione (ca. 96%), to be sensitive (down to 20 pmol glutathione/per injection) and reveal a low intra-individual coefficient of variation (C.V. < 5%) of the glutathione concentrations in human skeletal muscle. The concentrations of reduced and total glutathione were 1320 ± 37 μmol/kg wet weight (mean ± S.E.M.) and 1525 ± 66 μmol/kg wet weight, respectively. The method was also applied to tissues from nine healthy volunteers to determine if fluctuations in glutathione level occurred over a 24-h period. No diurnal variation of glutathione level in human skeletal muscle was observed.     

Determination of cholesteryl 14-methylhexadecanoate in blood serum by reversed-phase high-performance liquid chromatography

A simple reversed-phase HPLC method has been developed for the determination of cholesteryl 14-methylhexadecanoate (CMH) in the blood serum. Lipids are extracted from 0.1 ml of blood serum and after centrifugation, the extract is chromatographed and individual cholesteryl esters, including CMH are separated and eluted with an acetonitrile—2-propanol mixture. The quantification of cholesteryl 14-methylhexadecanoate is precise and highly reproducible and the analysis may be completed within 35 min. The level of CMH in the blood of cancer patients appears to be a useful marker of malignant tumors.     

Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C₁₈ Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and μBondapak C₁₈ column (150 × 3.9 mm I.D., 10 μm particle size). The detection limit (A_{375 nm}) for quercetin in plasma was 0.1 μg/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.     

Isolation of teratogenic alkaloids by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography was used for both analytical and preparative separations of several steroidal alkaloids which occur in extracts of Veratrum californicum. The inclusion of 0.1% trifluoroacetic acid in the mobile phase improved the efficiency of the chromatography and the solubility of the compounds in aqueous acetonitrile. Nuclear magnetic resonance was used to assist the identification of the isolated steroidal alkaloids. The effect of the interaction of trifluoroacetic acid with the alkaloids could be clearly seen by changes in the chemical shifts in the nuclear magnetic resonance spectra.