Rapid quantitation of ultraviolet-induced thymine-containing dimers in human cell DNA by reversed-phase high-performance liquid chromatography

Rapid and efficient separation of all three types of cyclobutyl pyrimidine dimer (Pyr mean value of Pyr) species induced in cellular DNA by far-ultraviolet (UV) light (chiefly 254 nm) has been achieved by reversed-phase high-performance liquid chromatography using octadecylsilyl stationary phases. The order of elution is: (Ura mean value of Ura) less than (Ura mean value of Thy) less than (Thy mean value of Thy) less than Thy. The determination of Pyr mean value of Pyr species in DNA from UV-irradiated, [3H]thymidine-labelled human skin fibroblasts in tissue culture is demonstrated for far-UV fluences as low as 10 J/m2. The ability to measure specifically individual dimer types allowed demonstration of comparable kinetics of repair for two labelled dimer species (Ura mean value of Thy and Thy mean value of Thy).     

Separation of pyrimidine derivatives by reversed-phase high-performance liquid chromatography

Chromatographic behavior and separation conditions of pyrimidine derivatives were studied by high-performance liquid chromatography using a reversed-phase column and a multiwave UV detector.     

Measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography

A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in combination with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 10(3) concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed.     

Use of optimization software to determine rugged analysis conditions in high-performance liquid chromatography

The ruggedness evaluation of an analytical method is now generally required for further validation. By considering ruggedness at an early stage of method development, major disappointments and amount of work could be avoided. This work shows that the optimization software OSIRIS can be helpful for the chromatographer during a method development, as it takes into account the method ruggedness. The ruggedness of the analysis conditions is then evaluated all along the selectivity optimization procedure. This optimization software belongs to the interpretive methods that consist of predicting the optimum conditions by modeling first the solute retention over the parameter space using a minimum number of preliminary runs. The choice of a response function is studied. This response function must be able to take into account several individual criteria: analysis time, minimal resolution and ruggedness of each parameter. Some optimum separations, determined using a ruggedness criteria or not, are given and compared in terms of long term repeatability.     

Incandescent lamps can produce pyrimidine dimers in DNA

DNA molecules that have been exposed to light from a 150 W incandescent spot lamp are nicked by the Micrococcus luteus endonuclease specific for cyclobutyl-type pyrimidine dimers. The production of these enzyme-sensitive sites increases with increasing spot lamp exposure. These sites have been confirmed to be pyrimidine dimers by their property of being photoreversed by an E. coli photoreactivating enzyme. The emission spectrum of the lamp shows detectable output at wavelengths less than 320 nm. These results indicate that the sensitivity of the techniques utilized in this work can be used to detect low levels of contaminating UV radiation.     

Reversed-phase high-performance liquid chromatography of pyrimidine bases and nucleosides. Application of solvophobic theory

The chromatographic behaviour of some natural and modified pyrimidine bases and nucleosides on an octadecyl stationary phase was studied. The retention and selectivity parameters of the separation of the compounds studied were derived on the basis of solvophobic theory. The mechanism of base and nucleoside interactions with the surface of the hydrocarbonaceous stationary phase is discussed. The best separation is observed at pH 3.5 for the bases and at pH 4.8-5.2 for the nucleosides. An increase in the solute surface tension results in an increased selectivity of separation. When the surface tension and the ionic strength of the mobile phase are not kept constant, there are considerable deviations in retention from that predicted by solvophobic theory.     

Use of 4-bromomethyl-7-methoxycoumarin for derivatization of pyrimidine compounds in serum analysed by high-performance liquid chromatography with fluorimetric detection

Derivatization of the pyrimidine nucleobases and nucleosides with 4-bromomethyl-7-methoxycoumarin was studied with the aim of developing a sensitive and selective column liquid chromatographic method for these substances in serum. The labeling reactions and the nature of derivatives are discussed, together with the chromatographic properties of these derivatives. The derivatives are stable for at least several weeks. Typical detection limits are 50 pg for inosine, 150 pg for uridine, 50 pg for uracil, 50 pg for thymine and 100 pg for fluorodeoxyuridine, respectively. Within-day coefficients of variation averaged 5.0% for the stored-frozen serum pools; the mean day-to-day value was 5.2%. Thirty samples could be processed per working day.     

Determination of 6-methyladenine in DNA by high-performance liquid chromatography.

A method for the determination of 6-methyladenine (6MA) by high-performance liquid chromatography (HPLC) has been developed. DNA bases were separated by using the strong cation-exchange resin Zipax SCX. Purine bases were obtained by hydrolysis and dialysis of DNA and analysed by HPLC. 6MA in DNA from Escherichia coli was determined by the proposed method. It is suggested that the method could be applicable to analyses of 6MA from other biological sources.     

Separation of polyamines, conjugated to DNA, by reversed-phase high-performance liquid chromatography

Genomic DNA was isolated from the lichen Evernia prunastri in order to analyze by high-performance liquid chromatography the occurrence of polyamines conjugated to the macromolecule. The acid-insoluble (PH) fraction of this DNA contained mainly conjugated spermidine, although small amounts of free putrescine and spermidine were also present. The PH fraction of DNA also contained conjugated evernic acid, the main phenol produced by this lichen species. Conjugation of polyamines to calf thymus DNA was carried out under in vitro conditions. Conjugation was to spermidine and mainly to spermine and produced DNA compactation. Evernic acid enhanced the action of polyamines in order to produce DNA aggregation.     

Chemical fingerprint and quantitative analysis of fructus psoraleae by high-performance liquid chromatography

Fructus Psoraleae, a widely used traditional Chinese medicine, is well known as a health supplement ingredient. In our study, an improved and comprehensive HPLC fingerprint of Fructus Psoraleae was established. Two important new benzofuran glycosides, psoralenoside and isopsoralenoside, were identified as characteristic constituents for the first time. HPLC separation was performed on an RP-C8 column. The mobile phase was acetonitrile and 0.1% acetic acid solution with linear gradient change of acetonitrile from 10 to 82% in 40 min. The flow rate was 1.0 mL/min, and the detection wavelength was set at 310 nm. The HPLC chromatograms of twenty-six samples from different regions of China showed a similar pattern. Twelve peaks were selected as characteristic peaks and further identified as psoralenoside, isopsoralenoside, psoralen, isopsoralen, bavachromene, corylifolin, corylin, psoralidin, isobavachalcone, bavachinin, corylifol A, and bakuchiol, respectively. Nine of them were simultaneously quantitatively analyzed for the first time. A more comprehensive analytical method was established for the fingerprint of Fructus Psoraleae. It is very useful for authentication and quality assessment of the crude drug.     

Use of narrow-bore high-performance liquid chromatography for microanalysis of protein structure

We report here systematic approaches to microanalysis of protein structures using narrow-bone high-performance liquid chromatography of protein, peptide and derivatized [phenylthiocarbamyl (PTC-) and phenylthiohydantoinyl (PTH-)] amino acids. The utilization of columns with small diameters (2 mm or less) has improved resolution and sensitivity and enabled protein structure analysis at the low pmol level. Preparative isolation of proteins and peptides of pmol quantity is achieved and separation and identification of PTC- and PTH-amino acids can be routinized at the low pmol to subpmol level. The use of diode-array detection enables simultaneous multiple-wavelength monitoring and spectral retreat, which greatly enhances flexibility and usefulness of the present methods. In combination with protein microsequencing techniques, these narrow-bore high-performance liquid chromatographic procedures can facilitate structural analysis of minutely available proteins of biochemical and physiological significance.     

Assay of human erythrocyte pyrimidine and deoxypyrimidine 5'-nucleotidase by isocratic reversed-phase high-performance liquid chromatography

We report a rapid and reproducible assay for activity of human erythrocyte pyrimidine 5'-nucleotidase and deoxypyrimidine 5'-nucleotidase. The nucleotides CMP, UMP, dUMP, dCMP or dTMP are individually incubated 30 min at 37 degrees C with erythrocyte hemolysate and 4 mM magnesium chloride in Tris, pH 7.5. Data are provided for standardization of the reaction with each substrate. Individual nucleoside products are assayed in less than 10 min by reversed-phase high-performance liquid chromatography at 280 nm with 0-14% methanol in 0.01 M potassium dihydrogen phosphate. This is the first report of a high-performance liquid chromatographic assay system which allows quantitation of the activity of pyrimidine 5'-nucleotidase isozymes using five individual pyrimidine and deoxypyrimidine nucleotides as the substrates.     

Quantitative reversed-phase high-performance liquid chromatography of major and modified nucleosides in DNA

Improved, highly accurate high-performance liquid chromatographic methods for the measurement of the major and modified nucleosides in enzymatic digests of DNA using a single column are described. Four high resolution separation protocols (isocratic, binary, ternary and high speed) with specifically improved selectivity for 5-methyldeoxycytidine (m5dCyd) from Ade, dIno and Guo are presented. From a detailed study of the various factors contributing to the precision and accuracy of the measurement, optimized conditions and quantitative protocols were established. The ternary buffer allows for the first time the determination of N6-methyldeoxyadenosine (m6dAdo) in the same chromatographic analysis with the other deoxyribonucleosides. The binary system allows quantitation of the absolute amounts of each ribo- and deoxyribonucleoside as well as the mole % of each as the second buffer elutes 5'dA and the internal standard 8-bromoguanosine. The isocratic system allows precise quantitation of the mole % of each ribo- and deoxyribonucleoside while eliminating the need for buffer change valves, buffer cycling and column re-equilibration. Also, a high-speed isocratic system is described which permits separation of the deoxyribonucleosides in 6 min. The quantitative, enzymatic hydrolysis of DNA was evaluated by comparing a 40-h, three-enzyme system with a 4-h, two-enzyme procedure. The latter protocol proved to be an excellent hydrolysis method. These high resolution liquid chromatography techniques provide the most precise, sensitive and accurate measurement of m5dCyd available, in a straightforward method using as little as 1 microgram of DNA, and have allowed us to demonstrate: the existence of tissue-specific differences in levels of m5dCyd in DNA of humans, monkeys, rats and mice; that m5dCyd levels in DNA change during fetal development; that genomic undermethylation of DNA is correlated with cancer and the presence of m6dAdo in DNA of thermophilic organisms.     

Use of high-performance liquid chromatography to assess airborne mycotoxins. Aflatoxins and ochratoxin A

An HPLC analytical method combining methanol-deionised water (80:20, v/v) extraction, methanol-acetonitrile (50:50, v/v) extraction and fluorescence detection was implanted to analyse ochratoxin A and aflatoxins B1, B2, G1 and G2 of air samples collected during the usual production process in a number of workplaces of a coffee factory to assess the occupational exposure of the engaged workers. The average levels of airborne ochratoxin A and aflatoxins were less than 1.2 and 0.4 ng/m3, respectively, using 50 L air samples. When 150 L air samples were used, levels lower than 0.04 ng/m3 ochratoxin A and 0.013 ng/m3 for aflatoxins B1, B2, G1 and G2, could be detected.     

Use of high-performance liquid chromatography coupled with high-resolution mass spectrometry for the identification and quantitative determination of tetrodotoxin in pharmaceuticals

A procedure is developed for the identification and determination of tetrodotoxin in pharmaceuticals by liquid chromatography coupled with high resolution mass spectrometry (HPLC?HRMS). The components of a pharmaceutical sample were separated in the gradient elution mode on a HILIC hydrophilic sorbent and detected by HRMS with electrospray ionization in the mode of registration of selected ion transitions. The calibration curve is linear in the range 0.75?1.25 μg/mL with the correlation coefficient > 0.9995.     

Simultaneous quantitative determination of amiloride hydrochloride and hydrochlorothiazide in tablets by high-performance liquid chromatography

Summary A procedure for the simultaneous quantitative determination of amiloride hydrochloride and hydrochlorothiazide by high-performance liquid chromatography is proposed. A reversed-phase LiChrosorb C8 stationary phase is used. The eluent consisted of an acetonitrile/0.1M phosphate buffer pH3 (15∶85) mixture, containing 50mM propylamine hydrochloride. In this system amiloride hydrochloride, a basic drug, eluted with a acceptable asymmetry factor (Asf=2.1). A simple extraction procedure with methanol is used. Relative standard deviations of 0.87% and 1.6% were obtained for amiloride hydrochloride and hydrochlorothiazide respectively. Chlorothiazide, a thiazide diuretic, is a suitable internal standard. Furthermore the method is also specific for other thiazide diuretics, potassium-sparing diuretics and loop diuretics and for the respective hydrolysis productes of both drugs. Analysis time is reduced to a minimum; the chromatographic separation is complete within 6 minutes.     

Indirect detection in high-performance liquid chromatography

Indirect detection is a technically simple method to follow and quantify compounds without inherent detector response in high-performance liquid chromatography. The underlying principle can be expressed in simple equations. These equations indicate clearly how the detection sensitivity can be optimized and how disturbance effects can be avoided when the mobile phase gives detector response.