Simple method for reversed-phase high-performance liquid chromatographic analysis of fungal pigments in fruit-bodies of Boletales (Fungi)

A reversed-phase HPLC method has been developed for the analysis of hydroxylated pulvinic acid derivatives which are responsible for the pigmentation of fruit-bodies belonging to the order Boletales (Fungi). Variegatic, xerocomic and atromentic acid as well as variegatorubin were detected and separated in methanolic extracts of Boletus permagnificus and Xerocomus parasiticus, and the pigment profile of these species was compared. The identity of the pigments was confirmed by means of LC-atmospheric pressure chemical ionization (APCI) mass spectrometry.     

A reversed-phase high-performance liquid chromatographic method to analyze retinal isomers

A high-performance liquid chromatographic (HPLC) procedure was developed to separate all-trans-, 13-cis-, 11-cis- and 9-cis-retinal isomers. Two reversed-phase Vydac C18 columns in series were used with an isocratic solvent system of 0.1 M ammonium acetate-acetonitrile (40:60, v/v) as mobile phase and all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-no natetraene-1-ol (TMMP) as internal standard. Prior to HPLC, the retinal isomers were efficiently extracted in their original isomeric conformation using dichloromethane-n-hexane in the presence of formaldehyde. This technique is suitable for the assay of 11-cis- and all-trans-retinal isomers in retina.     

Simple isocratic high-performance liquid chromatographic method for measurement of iodixanol in human plasma.

A simple isocratic high-performance liquid chromatographic method was developed for the determination of iodixanol in human plasma. Samples containing an internal standard were prepared for analysis using a simple clean-up procedure based on Sep-Pak C18 solid-phase extraction and chromatographed using a size-exclusion column with purified water as a mobile phase. The iodixanol peak was completely separated from the peaks of an internal standard and endogenous substances on this column. Three geometric isomers (exo-exo, endo-exo and endo-endo forms) of iodixanol could be eluted as a single peak. The method was found to be applicable to pharmacokinetic studies of iodixanol in human plasma.     

Simple high-performance liquid chromatographic method for the determination of captopril in biological fluids.

A rapid, simple and sensitive column-switching high-performance liquid chromatographic procedure for the determination of captopril in plasma and urine had been developed. p-Bromophenacyl bromide was used as a derivatizing reagent to react with captopril to form a product that showed ultraviolet-absorbing properties. For plasma samples the protein was removed with 6% perchloric acid before injection. The urine samples were directly injected into the chromatograph. The column-switching system was equipped with a pre-column (5 cm x 0.5 cm I.D.) packed with muBondapak C18 (37-50 microns) and an analytical column (15 cm x 0.5 cm I.D.) packed with YWG-C18, 10 microns. Impurities were washed from the pre-column with 0.2% acetic acid and the retained substances were eluted into the analytical column with acetonitrile-water-acetic acid (35:65:0.4, v/v). Captopril was detected at 260 nm. The calibration curve was linear in the range 20-1000 ng/ml for plasma and 10-200 micrograms/ml for urine. The recoveries averaged 103.2 and 99.5% for plasma and urine, respectively. The coefficients of variation were all less than 10%.     

Simple and rapid high-performance liquid chromatographic assay for esmolol.

A procedure for determining esmolol concentrations in blood is described. Dichloromethane was used to extract esmolol from the blood and to inhibit the activity of blood esterases. Blood esmolol concentrations were determined by high-performance liquid chromatography using 3-methoxy-O-demethylencainide as the internal standard. The limit of detection of this assay was 5 ng/ml. The relationship between the peak-height ratio of esmolol and the internal standard was linear in the concentration ranges 10-30,000 ng/ml. The mean absolute and relative recoveries of esmolol from blood were 84 and 89%, with coefficients of variation less than 3%. This method has been used in our laboratory for pharmacokinetic and pharmacodynamic studies.     

Pyrolysis of reversed-phase high-performance liquid chromatographic packing materials

Pyrolysis coupled with gas chromatography and or mass spectrometry, allows the identification of the bonded chain in reversed-phase high-performance liquid chromatographic stationary phases. It is possible to distinguish whether an octadecylated reversed-phase was prepared with a trifunctional(e.g., trichloro or monofunctional dimethyl, e.g., dimethyl-ethoxy) octadecylsilane from the relative heights of the heptadecene and octadecene peaks. The nature of the pyrolysis products was investigated. No carbon chains are formed with more carbon atoms than in the bonded chain. The peak area ratio of methane to that of the combined C₄ products allows one to deduce whether the reversed-phase was deactivated as not by reaction with a trimethylsilylating reagent (end-capping).     

Simple high-performance liquid chromatographic method for the measurement of amiloride in body fluids

A simplified rapid high-performance liquid chromatographic (HPLC) procedure has been developed for the measurement of amiloride in plasma or urine. Solid-phase extraction columns provide quick, clean and simple sample preparation, allowing ten samples to be processed in less than 5 min. The HPLC system uses a standard reversed-phase (C18) column with detection by ultraviolet absorption at 365 nm. The assay has been used to define plasma amiloride concentration-time profiles and to quantitate urine amiloride recovery in healthy men after repeated administration at two doses.     

Tritium isotope effect in reversed-phase high-performance liquid chromatographic analysis of dopamine and dihydroxyphenylacetic acid

A tritium isotope effect has been demonstrated in the high-performance liquid chromatographic analysis of dopamine and its acidic metabolite dihydroxyphenylacetic acid. The chromatographic system consisted of tributyl-n-phosphate, bound to a ChromSpher C8 column, as stationary phase, and a citrate buffer, containing the ion-pairing agent perchlorate, as the mobile phase. For detection we used continuous electrochemical monitoring (for the total amount of solutes) and discontinuous liquid scintillation counting (for radiolabelled molecules) of the column effluent. [3H]Dopamine and [3H]dihydroxyphenylacetic acid were biosynthesized by incubation of homogenates of striatal tissue from rat brains with 3H-labelled L-tyrosine. The tritium-labelled compounds were eluted before the corresponding unlabelled analogues. The capacity factor reduction increased with the number of tritium atoms incorporated in the molecules: for single, double and triple tritium-labelled dopamine the separation factors amounted to 1.015, 1.028 and 1.033, respectively. No isotope separation was observed for 7-14C-labelled dopamine and dihydroxyphenylacetic acid. The isotope effect observed is ascribed to a decrease in lipophilicity following tritium substitution for hydrogen.     

Ultrafast reversed-phase high-performance liquid chromatographic separations: an overview

Ultrafast reversed-phase high-performance liquid chromatographic (HPLC) separations are often needed for analyses related to combinatorial chemistry, studies in liquid chromatography-mass spectrometry, and other applications in which very rapid sample turnaround is paramount. Unfortunately, no consensus exists regarding the best column technology for optimally performing the desired rapid separations. This overview compares the advantages and limitations for columns of ultramicroporous, ultramicrononporous, and superficially porous particles and monolith structures for the very fast separation of solutes by reversed-phase HPLC. Data from literature and the author's laboratory are used to illustrate the strengths and limitations of the various approaches that can be used for ultrafast separations.     

Rapid high-performance liquid chromatographic method for the determination of peroxyacetic acid

The selective oxidation of methyl p-tolyl sulfide (MTS) to the corresponding sulfoxide (MTSO) by peroxyacetic acid and the subsequent rapid separation of the sulfide and sulfoxide are the basis for a fast and reliable HPLC method for the determination of this oxidizing agent in the presence of hydrogen peroxide. The time required for chromatographic separation was reduced to less than 1 min. To improve the long-term stability of the sulfoxide solution, hydrogen peroxide was decomposed catalytically by manganese dioxide. Even in the presence of a tenfold molar excess of hydrogen peroxide, a storability of at least 20 h without a significant increase in MTSO concentration was achieved. External calibration can be performed using the stable and commercially available MTSO. Real samples from a brewery cleaning-in-place disinfection process were analysed and the results were compared with those of the classical two-step titration.     

Simple and rapid high-performance liquid chromatographic method for endogenous alpha-tocopherol determination in human plasma

A simple and rapid reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of endogenous alpha-tocopherol in human plasma. Following addition of alpha-tocopheryl acetate as the internal standard, the plasma was deproteinized using acetonitrile and isopropanol mixture prior to HPLC analysis. Methanol was used as the mobile phase and the effluent was quantitated at 292 nm. By this developed method, the concentrations of alpha-tocopherol were linearly related to their responses in the range of 0.8-30 microg/mL. The relative standard deviations intra-day and inter-day for alpha-tocopherol in plasma were less than 10%. The percentage of bias was within +/-4%, which confirmed the accuracy of the method. The method has been successfully applied for determining endogenous alpha-tocopherol in healthy Thai male volunteers.     

Simple high-performance liquid chromatographic method for the determination of medroxyprogesterone acetate in human plasma

The high-performance liquid chromatographic (HPLC) method was developed as a simple, reliable alternative to available methods for measuring plasma concentrations of medroxyprogesterone acetate (MPA). The HPLC method has been successfully automated and is suitable for the rapid, inexpensive analysis of large batches of plasma samples. The best approach involves a solvent extraction followed by HPLC separation and analysis. MPA can be efficiently extracted, at all pH values, by nonpolar solvents. The Spherisorb 5-ODS2 HPLC column provides excellent separation of MPA from endogenous steroids of similar structure and from extraneous plasma blank peaks. A batch of 30-40 samples can be prepared by HPLC analysis in 2-3 hours, with a chromatographic run time of 10 minutes/sample. Calibration curves between 5-250 ng/ml show a good correlation between peak height ratio and MPA concentration, even at low levels. Plasma concentrations of MPA in patients receiving 1 g/day were between 12.6-270 ng/ml in this study, suggesting that the sensitivity of this method, 10 ng/ml, is sufficient for monitoring therapeutic concentrations of MPA. The results show a wide individual variation in plasma concentrations following similar dosing schedules--a finding reported by other workers.