Determination of atractylenolide II in rat plasma by reversed-phase high-performance liquid chromatography

A method for quantitative determination of atractylenolide II in rat plasma using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with UV spectrometry was established. From a variety of compounds and solvents tested, atractylenolide III was selected as the internal standard (IS) and ethyl acetate was found to be the best solvent for extracting atractylenolide II from plasma samples. RP-HPLC analysis of the extracts was performed on an analytical column (DIKMA ODS, 150 x 4.6 mm; i.d., 5 microm) equipped with a security guard pre-column system. There was good linearity over the range 0.05-5.0 microg/mL (r > 0.99). The recoveries were more than 90.0% in plasma, and the intra- and inter-day coefficients of variation were less than 10.0% in all cases. The limit of detection (LOD) was 0.025 microg/mL and the lower limit of quantification (LLOQ) was 0.05 microg/mL. The RP-HPLC method was applied to quantitate atractylenolide II in rat plasma within 24 h in a pharmacokinetics study where experimental rats received a single dose of atractylenolide II (60 mg/kg).     

Improvement of cyclosporin A determination in whole blood by reversed-phase high-performance liquid chromatography

A chromatographic method was developed for the determination of cyclosporin A in human whole blood using reversed-phase HPLC at room temperature. Most previous reports carried out this liquid chromatographic separation at temperatures above 70 degrees C. The present procedure greatly improves the detection limit by controlling peak broadening effects, as well as the lifetime of the column at room temperature. Under optimal conditions and using ketoconazole as an internal standard, the calibration graph was linear in the range of 16-1000 microg/L with a relative standard deviation of 3.72% at 150 microg/L and 2.45% at 300 microg/L (n = 11) of cyclosporin A. The detection limit was of 5.0 microg/L cyclosporin A. By this procedure, cyclosporin A pharmacokinetic parameters in healthy Chinese subjects were studied. The developed method could be applied to the quantification of cyclosporin A in human blood samples and allows the study of its pharmacokinetics in routine laboratories.     

Characterization of Klason lignins by reversed-phase high-performance liquid chromatography using wide-pore octadecylsilica and stepwise gradients of dimethylformamide in water

RP-HPLC using stepwise gradients of N,N-dimethylformamide (DMF) in a phosphate-buffered (pH 3.00) aqueous mobile phase and a wide-pore (30 nm) octadecylsilica column has been applied to the analysis of a variety of lignins--organocell lignin, dioxane lignin, Bj?rkman lignin, and Klason lignins from both softwoods and hardwoods, respectively, in order to demonstrate the usefulness of the approach for their characterization. Tandem combination of spectrophotometric diode array detection and fluorimetric detection was used to acquire more detailed information about the chromatographic behavior of lignins. The results show that a ten-step gradient can reveal distinct features of lignins and humic substances. Combination of good solvating properties of DMF for lignins together with a wide-pore RP sorbent improves surface interactions of the analytes and suppresses the influence of size exclusion effects. Thus it provides reproducibility of characterization profiles and robustness of the method. The calculated repeatability of the retention time of selected peaks was +/- 0.46% RSD. The reproducibility of the data within one week (set of seven data) was +/- 1.1% RSD. These data are also representative of the other well-shaped peaks of analyzed substances.     

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.     

Faster analysis by reversed-phase high-performance liquid chromatography

Summary Many analysts are not taking full advantage of the high speed possibilities of modern LC. Some analytical procedures reported in the literature, and many in regular use in control laboratories, could be achieved in less time without loss in precision. Some factors which affect retention times are discussed and the advantages and disadvantages of employing shorter column lengths and finer packing materials in reversed-phase HPLC are examined. The effect on efficiency of increased flow rates with 10,5 and 3 m ODS materials is shown. The ability to couple shorter column lengths without loss of efficiency is also demonstrated. This allows a minimum length to be selected that gives adequate resolution. Examples of high speed separations are shown and limitations in state of the art HPLC equipment and chromatographic data systems are discussed briefly.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Resolution of triacylglycerol positional isomers by reversed-phase high-performance liquid chromatography

The ability of reversed-phase high-performance liquid chromatography (RP-HPLC) to separate some positionally isomeric disaturated and monounsaturated triacylglycerols (TAGs) as intact species is demonstrated for the first time. Mobile phases of acetonitrile modified with methanol, ethanol, 2-propanol, 1-propanol, 1-butanol, acetone, or dichloromethane were tested for the separation of POP-PPO, PLP-PPL, PEP-PPE, and PDP-PPD (P-palmitic, O-oleic, L-linoleic, E-eicosapentaenoic, D-docosahexaenoic acid residue) on a single RP-HPLC column. The resolution improved with increasing number of double bonds in the acyl residues. While POP and PPO were only partially resolved, PDP and PPD were fully separated with all tested mobile phases, except those containing methanol. Also separated were the four TAGs having the same equivalent carbon number (ECN = 42), PEP, PPE, PDP, and PPD, on a single RP-HPLC column with mobile phase acetonitrile-2-propanol (70:30, v/v) at 0.8 mL/min. In all cases the isomer with the unsaturated acyl residue in either 1- or 3-position was retained more strongly than the respective 2-isomer.     

Analysis of selected ionic liquid cations by ion exchange chromatography and reversed-phase high performance liquid chromatography

The chromatographic behavior of 8 ionic liquids - 7 homologues of 1-alkyl-3-methylimidazolium and 4-methyl-N-butylpyridinium - has been investigated with a strong cation exchange adsorbent. In particular, the dependence of the retention properties of these solutes on mobile phase composition, pH, and buffer concentration was evaluated with the aim of optimizing and improving the selectivity and retention of solute separation. While using the SCX stationary phase, several interactions occurred with varying strengths, depending on the mobile phase composition. Cation exchange, nonspecific hydrophobic interactions, and adsorption chromatography behavior were observed. Reversed phase chromatography occurred at low concentrations of acetonitrile, electrostatic and adsorption interactions at higher organic modifier concentrations. Elevated buffer concentrations lowered the retention factors without affecting the selectivity of ionic liquids. Obtained results were further compared to the chromatographic behaviour of ionic liquids in the reversed phase system. All analyzed ionic liquids follow reversed-phase behavior while being separated. Much lower selectivity in the range of highly hydrophilic compounds is obtained. This suggests preferred use of ion chromatography for separation and analysis of compounds below 4 carbon atoms in the alkyl side chain.     

Determination of human plasma protein binding of baicalin by ultrafiltration and high-performance liquid chromatography

A simple and selective HPLC assay was developed and utilized for determination of human plasma protein binding of baicalin. The method involved solid-phase extraction and reversed-phase chromatographic separation with a mobile phase of acetonitrile-0.02 mol/L phosphate buffer (pH 2.5; 25:75, v/v) and UV detection at 276 nm. The standard curve for baicalin was linear over the concentration range 0.1-20 microg/mL and the limit of detection was 0.02 microg/mL. The absolute recovery was greater than 76%. The intra-day and inter-day variations were less than 10%. Ultrafiltration technique was applied to determining the plasma protein binding of baicalin in human plasma. Results show the plasma protein binding of baicalin was in the range 86-92% over all the concentrations studied and the protein binding association constant was determined to be 1.21 x 10(5) L/mol at 4 degrees C.     

Simultaneous quantification of berberine and lysergol by HPLC-UV: evidence that lysergol enhances the oral bioavailability of berberine in rats

A sensitive and simple HPLC method was developed for the simultaneous quantification of berberine and lysergol in rat plasma. The chromatographic separation was achieved on a C₁₈ column using isocratic elution with methanol–acetonitrile–0.1% ortho‐phosphoric acid (25:20:55, v/v/v), pH adjusted to 6.5 with triethylamine and detected at a UV wavelength of 230 nm. The extraction of the berberine and lysergol from the rat plasma with methylene chloride resulted in their high recoveries (82.62 and 90.17%). HPLC calibration curves for both berberine and lysergol based on the extracts from the rat plasma were linear over a broad concentration range of 50–1000 ng/mL. The limit of quantification was 50 ng/mL. Intra‐ and inter‐day precisions were <15% and accuracy was 87.12–92.55% for berberine and 87.01–92.26% for lysergol. Stability studies showed that berberine and lysergol were stable in rat plasma for short‐ and long‐term period for sample preparation and analysis. The described method was successfully applied to study the pharmacokinetics of berberine as well as lysergol following oral administration in Sprague–Dawley rats. The results of the study inferred that lysergol improved the oral bioavailability of berberine. Copyright © 2011 John Wiley & Sons, Ltd.     

Comparison of chaotropic salt and ionic liquid as mobile phase additives in reversed-phase high-performance liquid chromatography of biogenic amines

Highly hydrophilic compounds belonging to biogenic amines were analysed in the reversed-phase system, modified with the addition of ionic liquids: 1-ethyl-3-methyl-imidazolium hexafluorophosphate (EMIM PF(6)) and chaotropic salt NaPF(6) on Discovery HS C18 column at acidic conditions. The effect of the additives concentration and the presence of organic solvent on the analytes' chromatographic parameters such as retention factor, tailing factor and theoretical plates number were all determined. On the basis of k versus ionic liquid concentration in aqueous-organic mobile phase with constant amount of phosphate buffer, retention mechanism was studied. It was established that the presence of organic solvent with low dielectric constant and ionic liquid with both chaotropic ions allows achieving typical Langmuir shape of this relationship. Investigated mobile phase additives are comparable according to efficiency and selectivity towards biogenic amines analysis. However, the sensitivity was found to be better for the eluent system modified with chaotropic salt.     

Quantification of myrislignan in rat plasma by solid-phase extraction and reversed-phase high-performance liquid chromatography

A rapid and simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for determination of myrislignan in rat plasma after intravenous administration. The analytes extracted from plasma samples by solid-phase extraction were successfully carried out on a Diamonsiltrade mark ODS C(18) column (250 x 4.6 mm i.d., 5 microm) with an RP(18) guard column (8 x 4.6 mm i.d., 5 microm) and a mobile phase of MeOH-H(2)O (4:1, v/v). The UV detector was set at a single wavelength of 270 nm. The linear ranges of the standard curves were 0.5-30.0 microg/mL with the correlation coefficients greater than 0.9992. The lower limits of detection and quantification were 0.1 and 0.3 microg/mL for myrislignan. Intra- and inter-day precisions were 2.4-7.5 and 1.3-5.7%, respectively. The extraction recovery from plasma was more than 90%. This assay method has been successfully used to study the pharmacokinetics of myrislignan in rats.     

Analyses of tetracyline antibiotics by reversed-phase high-performance liquid chromatography

Summary A high-performance liquid chromatographic (HPLC) separation was developed that is generally applicable to nine commercially important tetracyline antibiotics. The general method uses an isocratic system and mobile phase consisting of 0.001MEDTA, pH 6.6, and methanol and a Vydac C₁₈ reversed-phase column. Quantitation of the particular tetracyline in some commercial preparations is accomplished by adjusting the mobile phase composition. Quantitative assays were developed for small amounts of 4-epitetracycline in tetracycline (TC) preparations, demeclocycline in minocycline preparations and TC in chlortetracycline (chlor-TC) preparations. A fast HPLC assay for potency was also developed for chlor-TC and minocycline in these commercial preparations.     

Rapid determination of beta-aminoisobutyric acid by reversed-phase high-performance liquid chromatography

For the determination of beta-aminoisobutyric acid (BAIBA) in urine samples in which the beta-alanine concentrations are higher than those of BAIBA, the resolution between these two amino acids, separated by reversed-phase liquid chromatography on an octadecylsilane column, was optimized. The chromatographic analysis included precolumn derivatization of amino acids with o-phthalaldehyde, followed by a 15-min isocratic elution and detection at 340 nm. Because of its simplicity, this method should be useful for monitoring urinary excretion of BAIBA.     

Quantitative determination of celanide by high-performance liquid chromatography

Methods have been developed for the quantitative determination of celanide [lanatoside C] in substances, in solutions for injection, and in tablets by high-performance liquid chromatography, with the aid of which it is possible to determine small amounts of celanide (0.025 mg/ml) with adequate accuracy. The relative error of the determination does not exceed ±4.0%.All-Union Scientific-Research Institute of Physical Culture, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 476–479, July–August, 1983.     

Stability study and quantitative determination of mebeverine hydrochloride in tablets by means of reversed-phase high-performance liquid chromatography

Summary A rapid and specific reversed-phase high-performance liquid chromatographic method (RPHPLC) is described for the determination of mebeverine hydrochloride in tablets. Elution was performed on an octyl silane column with a methanol-water mixture (75-25), containing 0.05% hexylamine as silanol-blocking agent, adjusted to pH 5.0 with phosphoric acid. The method gave accurate, precise and reproducible results. The mean recovery of the drug from six synthetic tablet mixtures was 100.0% with a relative standard deviation (RSD) of 0.94%. In order to test the specificity of the method, the interference of the degradation compounds of mebeverine hydrochloride and of the intermediates from the synthesis was investigated. None of them did interfere. By means of mass spectrometry and UV-spectrophotometry, the degradation compounds of mebeverine were identified as veratric acid and as 4-|ethyl-[2-(4-methoxyphenyl)-1-methylethyl]amino| 1-butanol. The stability study proved that mebeverine hydrochloride is very stable in tablets; the tablets still contain more than 95% of the declared drug potency after storage for more than one year at 50°C.Colofac; Duspatal; Duspatalin