Gradient chromatofocusing high-performance liquid chromatography. I. Practical aspects

In this work, a versatile method for generating linear pH gradients using weak anion-exchange HPLC has been developed, which is termed gradient chromatofocusing high-performance liquid chromatography. This method utilizes a linear external pH gradient generated in the mobile phase entering the column (inlet pH gradient), superimposed on an internally-generated pH gradient within the column (column pH gradient), which results from the buffering action of the ion exchanger on the mobile phase and vice versa. The method shows significant advantages over conventional chromatofocusing, including: decreased expense due to the use of common buffer components, ease of adjusting the slope of the pH gradient produced at the outlet of the column (outlet pH gradient) through the manipulation of the inlet pH gradient and the ability of using high concentration buffers in the mobile phase. Chromatography of fibrinogen degradation products was done using gradient chromatofocusing. Bandwidths comparable to conventional chromatofocusing were obtained in the separation of fibrinogen degradation products.     

Simultaneous variation of temperature and gradient steepness for reversed-phase high-performance liquid chromatography method development. I. Application to 14 different samples using computer simulation

The optimized reversed-phase HPLC separation of 14 different samples is reported, based on simultaneous changes in temperature and gradient steepness. Four experimental runs are required for each sample, following which preferred conditions can be predicted using computer simulation software (DryLab). The overall accuracy and effectiveness of this method development approach is discussed, with particular attention to the use of resolution maps provided by the software. These maps are useful for maximizing resolution for the total sample, for optimizing the separation of a smaller number of selected sample compounds, and as an initial step in the separation of more demanding samples.     

Analysis of the Morgan-Elson chromogens by high-performance liquid chromatography

The Morgan-Elson method for quantitative N-acetylhexosamine analysis is a two-step procedure comprising alkali treatment of the sugar and subsequent condensation of the resulting chromogens with p-dimethylaminobenzaldehyde (Ehrlich's reagent) to yield a colored product. In the present investigation, the products formed in the first step of the procedure were analyzed by high-performance liquid chromatography (HPLC) on a reversed-phase (C18) column, which was eluted with a water-methanol gradient; the absorbance of the effluent was monitored at 229 nm. The profile generated from alkali-treated N-acetylglucosamine exhibited two major peaks, in a ratio of approximately 2.5:1, which accounted for 94% of the total peak area. A third peak, accounting for 3% of the peak area, was eluted in an intermediate position, and several smaller peaks were also observed. The three predominant components, isolated by preparative HPLC, all gave a purple color on addition of Ehrlich's reagent, indicating that they were Morgan-Elson chromogens. The HPLC profile of alkali-treated N-acetylmannosamine was identical to that of the products generated from N-actylglucosamine, as was expected because of the elimination of the asymmetry at C-2 during formation of the chromogens. N-Acetylgalactosamine yielded two major peaks, which were eluted in the same positions as the two major products formed from N-acetylglucosamine, but the intermediate peak seen in the N-acetylglucosamine pattern was absent. The HPLC procedure allowed detection of as little as approximately 25 ng of N-acetylglucosamine and may therefore be of value as an alternative to the complete Morgan-Elson procedure when only small amounts of sample are available for quantitative analysis.     

Analysis of the imidazoacridinone C1311 by high-performance liquid chromatography

Natural polyphenols found in rosemary have not only potent antioxidant activities but also anticarcinogenic properties. We have studied some of the molecular mechanisms involved in their chemopreventive action using in vitro human liver and bronchial cell models. Rosemary extract, or its active components, carnosol or carnosic acid are potent inhibitors of DNA adduct formation induced by benzo(a)pyrene or aflatoxin B1. At least two mechanisms are involved in the anticarcinogenic action of rosemary extract: (i) inhibition of the metabolic activation of procarcinogens catalysed by the phase I cytochrome P450 enzymes; (ii) induction of the detoxification pathway catalysed by the phase II enzymes such as glutathione S-transferase.     

Solid-phase extraction with end-capped C2 columns for the routine measurement of racemic citalopram and metabolites in plasma by high-performance liquid chromatography

An assay based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for the measurement of citalopram and its main metabolites desmethylcitalopram and didesmethylcitalopram. The best extraction procedure was performed with end-capped C2 column utilising secondary silanol interactions to obtain clean extract. The HPLC analysis was done on a phenyl column with a mobile phase without any amine additives. Fluorescence detection gave a limit of detection of 0.8 nmol/l plasma for the compounds analysed.     

The influence of temperature on the multiple separation of estrogenic steroids using mobile phases modified with beta-cyclodextrin in high-performance liquid chromatography

The effect of temperature on the retention and multiple separation of six estrogenic steroids in reversed-phase liquid chromatography has been studied. Capacity factors (k') of estriol, 17 beta-estradiol, 17 alpha-estradiol, d-equilenin, equilin and estrone were measured using mobile phase modified with different concentrations of beta-cyclodextrin (from 0-16 mM), a fixed solvent composition (acetonitrile-water) and a wide range of column temperatures (from 5 to 80 degrees C). The plots of capacity factors vs. reciprocal of absolute temperature are nonlinear in each case when mobile phase modified with beta-cyclodextrin was used. Particularly strong nonlinearity was observed at lower temperature and at higher beta-cyclodextrin concentration. The complex chromatograms were evaluated using optimization parameters such as capacity factor of the last-eluted peak (k'max), the smallest resolution between adjacent peaks (Rs,min) and relative resolution product (r). The results presented describe precisely the role of temperature in high-performance liquid chromatography systems in which mobile phases modified with cyclodextrin were used. Moreover, the elution order of estrogenic steroids on modified and unmodified mobile phases has been discussed.     

Multiple peaks in high-performance liquid chromatography of proteins. beta-Lactoglobulins eluted in a hydrophobic interaction chromatography system

The hypothesis of Geisler (Brain Res. 212 (1981) 198-201), in which the different spontaneous-rate classes of primary auditory neurons were accounted for by the different sizes of uniquantal EPSPs relative to the gap between resting membrane and threshold potentials, was represented with an expanded model which included relative refractory effects. The spike rates generated by the expanded model, when plotted vs. estimated sound level, are qualitatively similar to those of experimentally obtained rate-level curves. The hypothesis is also consistent with recent ultrastructural data which suggest that average quantal-release rates for any particular primary auditory neuron are inversely related to its spontaneous rate. The model's recovery processes following spike generation (hazard functions) are also similar to those observed experimentally.     

Effect of geometrical conditions on the mass-transfer parameters near a polarized liquid metallic electrode: I. Electrode size

The behavior of liquid metallic electrode-chloride melt systems is studied as a function of the electrode size. The precipitation of lead on a solid substrate and the effect of the liquid electrode diameter on the mass-transfer intensity are investigated in the Pb-NaCl-KCl (1: 1)-PbCl₂ system in the circulation cell and laminar flow regimes.     

Simultaneous determination of the stereoisomers of guggulsterone in serum by high-performance liquid chromatography

Simultaneous separation of E- and Z-guggulsterone, which is the main ingredient of 'Guggulip', an ayurvedic drug, was accomplished by HPLC on a C18 column using methanol, acetonitrile, buffer and tetrahydrofuran as a mobile phase. The compounds were monitored at 248 nm on a photodiode array detector. The assay method was used for the simultaneous determination of stereoisomers (E and Z) of guggulsterone in spiked serum and dosed (50 mg/kg, p.o.) rats. The recoveries of E- and Z-isomers from serum samples were always greater than 90%. The calibration graph was linear over the range of 25-2500 ng/ml for Z- and E-isomers. Lowest quantitation limit of Z- and E-guggulsterones was 25 ng/ml.     

Effect of temperature on the structure of liquid iron

Review of published data on the properties of liquid iron at different temperatures. Conclusion supporting a proposal that a structural change takes place in the melt.     

Determination of nanogram levels of the anticoagulant acenocoumarin in serum by high-performance liquid chromatography

A simple, rapid, and sensitive method for the determination fo the short-acting coumarin anticoagulant acenocoumarin in human serum is described. This drug is estracted from the acidified biological matrix together with the internal standard 5-methoxypsoralen. Separation and quantitation are performed on a high-performance liquid chromatograph with a reversed-phase column and an ultraviolet detector operating at 308 nm. Accuracy and precision are good. The lowest limit of detection is 15 microgram/L, which means that acenocoumarin concentrations can be measured in serum from the subtherapeutic to the toxic range. This method can also be used for rapid measurement of warfarin serum concentrations.     

Determination of the major urinary metabolite of flurazepam in man by high-performance liquid chromatography

A high performance liquid chromatographic method for the determination of N-1-hydroxyethylflurazepam, the major urinary metabolite of flurazepam, in human urine is described. Urine specimens were incubated enzymatically to deconjugate N-1-hydroxyethylflurazepam glucuronide (metabolite) and were then extracted at pH 9.0 to extract the metabolite. The extracts were chromatographed on a microparticulate silica gel column using automatic sample injection, isocratic elution at ambient temperature and UV monitoring at 254 nm. The internal standard was 7 chloro-5(2'-chlorophenyl) 1,3-dihydro-1-2-dimethylaminoethyl-2H-1,4-benzodiazepine-2-one. The recovery from urine, in the 0.5-25.0 microgram/ml range, was 96.5 +/- 11.5% (S.D.), and the sensitivity limit was 0.5 microgram/ml. The method was found to be specific for N-1-hydroxyethylflurazepam in the presence of intact flurazepam and other possible urinary metabolites of flurazepam. The method was successfully applied to urine specimens collected from human subjects following the administration of 30-mg single oral doses of flurazepam dihydrochloride.     

Method for the extraction of riboflavin for high-performance liquid chromatography and application to casein

Three different approaches, including the commonly used acid digestion, were compared for the efficient extraction of very low concentrations of riboflavin (vitamin B2) from casein. An extraction method that used pepsin to release riboflavin was the most efficient. The pepsin extraction method was then further optimized using a factorial design to yield the maximum amount of riboflavin. The enzyme takadiastase was used for the conversion of all forms of riboflavin to the free form, and this conversion was found to be time dependent. A reversed-phase HPLC system with fluorescence detection and a methanol-water mobile phase containing acetate buffer were used for the separation and quantification of riboflavin in the extracts. A limit of detection of less than 0.1 mg kg-1 casein and a repeatability RSD of 3% were achieved. The recovery of the method was satisfactory.     

Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping

A two-dimensional liquid chromatographic system is described here which uses size-exclusion liquid chromatography (SEC) followed by reversed-phase liquid chromatography (RPLC) to separate the mixture of proteins resulting from the lysis of Escherichia coli cells and to isolate the proteins that they produce. The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions. Peaks eluting from the first dimension are automatically subjected to reversed-phase chromatography to separate similarly sized proteins on the basis of their various hydrophobicities. The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet region at 215 nm regardless of the SEC mobile phase used. The two-dimensional (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y axis and intensity represents quantity of analyte. Following chromatographic separation, the analytes are deposited into six 96-well (576 total) polypropylene microtiter plates via a fraction collector. Interesting fractions are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mass spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact proteins without any additional sample preparation, electroblotting, destaining, etc. The remaining 97% of a fraction can then be used for other analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry. This 2D HPLC protein purification and identification system was used to isolate the src homology (SH2) domain of the nonreceptor tyrosine kinase pp60c-src and beta-lactamase, both inserted into E. coli, as well as a number of native proteins comprising a small portion of the E. coli proteome.     

Effect of a temperature gradient on bubble growth in tungsten

Bubble distributions in doped tungsten wires have been examined following high temperature anneals (2500° to 3000°C) both in the presence and absence of a thermal gradient. Electron and optical microscopy show that much more bubble growth occurs during gradient anneals, and the results suggest that the gradient is a driving force for bubble growth. This process takes place predominantly on the grain boundaries. Semiquantitative estimates of the bubble flux were made by measuring the change in aggregate bubble surface areas found at various positions in the gradient. Stress induced bubble growth was shown not to be a factor.     

Contribution to the quantitative and enantioselective determination of kavapyrones by high-performance liquid chromatography on ChiraSpher NT material

A simultaneous HPLC separation of the enantiomers of kavain, dihydrokavain, methysticin and dihydromethysticin, as well as the achiral dienolides yangonin and desmethoxyyangonin was carried out on a ChiraSpher NT column. For quantitative determinations, calibration curves with correlation coefficients between 0.9982 and 0.9996 were established for the genuine kavapyrones. Detection limits between 0.25 microg and 0.5 microg per injection were measured at 240 nm. The defined scopes of work corresponded with the different kavapyrone amounts, depending on growth factors of distinct plant locations. The precision of the method was verified by analysing a phytopharmacon with a nominal value of 40 mg kavapyrones per tablet. The evaluation revealed 39.62 mg per tablet by the sum of single calculated kavapyrones. Relative standard deviations between 1.06% and 2.39% were found for the compounds under investigation. The accuracy of the method was proved by a recovery of 99.7%. To simplify the determination of the total kavapyrone amount, response factors and correlation factors for (+)-dihydrokavain, (+)-methysticin, (+)-dihydromethysticin, yangonin and desmethoxyyangonin were calculated relative to (+)-kavain.     

Studies of the oxidation of ethanol to acetaldehyde by oxyhemoglobin using fluorigenic high-performance liquid chromatography

We noted a rise in acetaldehyde levels in clinical samples of venous whole blood containing ethanol that did not occur in samples from teetotalers. Experiments were performed to define the mechanism involved in acetaldehyde production. The addition of 0.10% ethanol to whole blood produced an immediate increase in acetaldehyde due to acetaldehyde in the stock solution followed by a subsequent increase that became statistically significant by 48 hr. Separation of blood into components documented that the increase in acetaldehyde was associated with the red cell but not plasma fraction. Incubation of isolated hemoglobin with ethanol produced a rise in acetaldehyde levels. Incubation of oxygenated whole blood with ethanol produced a linear increase in acetaldehyde, whereas nitrogen-exposed blood produced no increase. The rise of acetaldehyde in the presence of ethanol was dependent on the concentration of oxygenated hemoglobin A0. Addition of inhibitors of catalase, alcohol dehydrogenase, and glycolytic enzymes (aminotriazole, azide, pyrazole, sodium fluoride, sodium citrate, and iodoacetate) did not inhibit the rise of acetaldehyde, but addition of the hemoglobin ligand cyanide abolished the rise in acetaldehyde. Kinetic analysis with oxygenated whole blood plus inhibitors revealed a Km of 2.5 mM and Vmax of 1.42 microM/min. We conclude that oxyhemoglobin contributes to the metabolism of ethanol to acetaldehyde. These findings may explain in part the high levels of acetaldehyde found in red cells compared with plasma. The results also have implications for the optimum storage of blood samples for acetaldehyde analysis.     

Enzymatic method for the simultaneous determination of hyaluronan and chondroitin sulfates using high-performance liquid chromatography

Biological samples have a high dielectric constant that can shorten RF wavelengths by a factor of 8 relative to the vacuum. At high field strengths, finite wavelength effects within larger samples are the dominant cause of RF field nonuniformity. A coil design is presented that can reduce and even eliminate this inhomogeneity; 4-T images in phantoms and in the head of a normal volunteer are presented, which demonstrate improved homogeneity relative to a standard coil. This coil design should aid in realizing the potential advantages of imaging large samples at high field strengths.     

Determination of synephrine in Oriental pharmaceutical decoctions containing aurantii nobilis pericarpium by ion-pair high-performance liquid chromatography. II

A simple method using ion-pair high-performance liquid chromatography was established for the rapid and precise determination of synephrine in thirty three species of oriental pharmaceutical decoctions containing Aurantii Nobilis Pericarpium. An ODS column and a mixed solvent system of water, acetonitrile, sodium dodecyl sulfate and phosphoric acid as a mobile phase were used for the separation. Synephrine was eluted without interference of other coexisting components within 15 min.