Indirect resolution of baclofen enantiomers from pharmaceutical dosage form by reversed-phase liquid chromatography after derivatization with Marfey's reagent and its structural variants

A high-performance liquid chromatographic (HPLC) method was developed for chiral assay of baclofen enantiomers in pharmaceutical formulations using an indirect approach. Baclofen enantiomers were derivatized with Marfey's reagent (FDNP-L-Ala-NH2) and its structural variants FDNP-L-Phe-NH2, FDNP-L-Val-NH2, FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2. The resultant diastereomers were separated on RP-TLC [triethylammonium phosphate buffer (pH 4.0, 50 mm)-acetonitrile, 50:50] and on a C18 column using a linear gradient (45 min) of acetonitrile and 0.01% aqueous trifluoroacetic acid (TFA) with UV detection at 340 nm. The differences in the retention times (Delta t R) of diastereomers due to the five chiral reagents were compared. The maximum and minimum difference in retention times between separated diastereomers was for FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2, respectively. The effect of flow rate, acetonitrile content and TFA concentration on resolution was studied. The method was validated for linearity, repeatability, limit of detection and limit of quantification.     

A (-)-sparteine-directed highly enantioselective synthesis of boroproline. Solid- and solution-state structure and properties

A two-step, direct asymmetric synthesis of the trifluoroacetate ammonium salt of boroproline is reported. (-)-Sparteine-mediated lithiation of N-Boc-pyrrolidine afforded N-Boc-aminoboronic acid in good yield and enantioselectivity as determined by HPLC (pinanediol ester). Deprotection using TFA yielded the ammonium salt; full characterization data are presented, and the structure in aqueous solution and the occurrence of a B-N species are discussed.     

Preliminary investigation of the application of on-line membrane extraction of trifluoroacetic acid as an aid to improvement of negative ion electrospray mass spectrometry data

We have recently investigated the biodegradation of a number of acidic aromatic compounds that give excellent chromatography using trifluoroacetic acid (TFA) based HPLC methods. Unfortunately HPLC methods using TFA are not usually compatible with detection by negative ion mass spectrometry as TFA suppresses ionisation of the analyte during the electrospray process. We present a preliminary investigation of the use of an anion-exchange micro-membrane suppressor to remove TFA on-line post column with the aim of improvement of mass spectral data using an aromatic acid as an example, Thus LC-MS using a TFA based HPLC method with negative ion mass spectral detection is shown to be possible with good sensitivity.     

Chromatographic purification and characterization of B-phycoerythrin from Porphyridium cruentum. Semipreparative high-performance liquid chromatographic separation and characterization of its subunits

A fast preparative two-step chromatographic method for purification of B-phycoerythrin from Porphyridium cruentum is described. This biliprotein was homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielding three closely migrating bands corresponding to its three subunits. Baseline separation of its alpha-, beta- and gamma-subunits was achieved by a reversed-phase HPLC gradient semipreparative method with a C4 large-pore column and a solvent system consisting of 0.05% trifluoroacetic acid (TFA) in water and 0.05% TFA in acetonitrile. B-Phycoerythrin in different aggregation states and its subunits have been spectroscopically characterized. Hexameric B-phycoerythrin has similar secondary and tertiary structure than dissociated B-phycoerythrin determined by circular dichroism.     

Signal enhancement for gradient reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis with trifluoroacetic and other strong acid modifiers by postcolumn addition of propionic acid and isopropanol

Trifluoroacetic acid (TFA) and other volatile strong acids, used as modifiers in reverse-phase high-performance liquid chromatography, cause signal suppression for basic compounds when analyzed by electrospray ionization mass spectrometry (ESI-MS). Evidence is presented that signal suppression is caused by strong ion pairing between the TFA anion and the protonated sample cation of basic sample molecules. The ion-pairing process “masks” the protonated sample cations from the ESI-MS electric fields by rendering them “neutral”. Weakly basic molecules are not suppressed by this process. The TFA signal suppression effect is independent from the well-known spray problem that electrospray has with highly aqueous solutions that contain TFA. This previously reported spray problem is caused by the high conductivity and surface tension of aqueous TFA solutions. A practical method to enhance the signal for most basic analytes in the presence of signal-suppressing volatile strong acids has been developed. The method employs postcolumn addition of a solution of 75% propionic acid and 25% isopropanol in a ratio 1:2 to the column flow. Signal enhancement is typically 10-50 times for peptides and other small basic molecules. Thus, peptide maps that use ESI-MS for detection can be performed at lower levels, with conventional columns, without the need to use capillary chromatography or reduced mass spectral resolution to achieve satisfactory sensitivity. The method may be used with similar results for heptafluorobutyric acid and hydrochloric acid. A mechanism for TFA signal suppression and signal enhancement by the foregoing method, is proposed.     

Malondialdehyde tagging improves the analysis of arginine oligomers and arginine-containing dendrimers by HPLC-MS

The selective modification of arginine residues by malondialdehyde (MDA) was used to improve the mass spectrometric analysis of arginine oligomers (Arg(x), x = 4, 6, 7, 8, 9) and an arginine-containing dendrimeric peptide. MDA tagging significantly increased the hydrophobicity of the arginine side-chain and resulted in improved retention in RP HPLC of the oligoarginines using formic acid as mobile phase additive. This avoided the use of TFA to generate sufficient retention, as TFA was shown to lead to a dramatically reduced sensitivity (up to ten-fold for Arg(8) and Arg(9)) as a result of the strong signal suppression by ion pairing with multiple basic residues. MDA modification of Arg oligomers not only resulted in improved detection sensitivity for most of the peptides studied (e. g., more than six-fold for Arg(7)), but also greatly enhanced the quality of MS/MS spectra, in line with previous results for other peptides. Furthermore, MDA modification helped to identify major side products in a sample of a dendrimeric peptide, a class of peptides that is typically difficult to analyze by MS.     

Free energy calculations on the binding of colchicine and its derivatives with the alpha/beta-tubulin isoforms

Tubulin is the target for numerous small molecule ligands which alter microtubule dynamics leading to cell cycle arrest and apoptosis. Many of these ligands are currently used clinically for the treatment of several types of cancer, and they bind to one of three distinct binding sites within beta-tubulin (paclitaxel, vinca, and colchicine), all of which have been identified crystallographically. Unfortunately, serious side effects always accompany chemotherapy since these drugs bind to tubulin indiscriminately, leading to the death of both cancerous and healthy cells. However, the existence and distribution of divergent tubulin isoforms provide a platform upon which we may build novel chemotherapeutic drugs that can differentiate between different cell types and therefore reduce undesirable side effects. We report results of computational analysis that aims at predicting differences between the binding energies of a family of colchicine derivatives against 10 human alpha/beta-tubulin isoforms. Free energy perturbation method has been used in our calculations and the results provide a proof of principle by indicating significant differences both among the derivatives and between tubulin isoforms.     

Constituent analysis and quality control of anthocyanin constituents of dried Lycium ruthenicum Murray fruits by HPLC–MS and HPLC–DAD

In recent years, many investigations on the anthocyanins of the fresh Lycium ruthenicum Murray fruits have been reported; while few studies about dried fruits have been published. In this study, chemical profile of dried fruits was illustrated by a high-performance liquid chromatography–tandem mass spectrometry method, which provided evidence for the certain identification of the main anthocyanins. Among these compounds, nine of them were selected as marker compounds for the semiquantitative evaluation, using a simple and reliable method by high-performance liquid chromatography–photodiode array detection (HPLC–DAD), with the combination of chromatographic fingerprint analysis. Separation was achieved on a C18 ODS 80TS QA analytical column with linear gradient elution of acetonitrile and 10% formic acid and 0.1% trifluoroacetic acid (TFA) aqueous solution. Our results showed that the contents of anthocyanins of dried L. ruthenicum Murray from different origins were different. We also inferred the anthocyanin compositions of dried L. ruthenicum Murray through analyzing the UV spectrum, retention time, elution order, and MS data. Finally, eight kinds of anthocyanin compositions were identified and different from the anthocyanins in fresh L. ruthenicum Murray. In a word, this study may provide experimental data in further development and utilization of L. ruthenicum Murray.     

反相高效液相色谱法分离蛋白质的研究

采用反相高效液相色谱法考察了几种大孔硅胶烷基键合固定相在等度淋洗条件下进行蛋白质分离的色谱性能。研究了冲洗剂中有机溶剂异丙醇的浓度、离子对酸（ＴＦＡ）浓度对蛋白质保留时间的影响。探讨了蛋白质在ＲＰ－ＨＰＬＣ中的保留机理。结果表明,大孔硅胶（２０～３０ｎｍ）短链（Ｃ４和Ｃ８）烷基键合固定相适合蛋白质的分离。     

The Use of Perfluorinated Carboxylic Acids in the Reversed-Phase HPLC of Peptides

Abstract

The relative effectiveness of trifluoroacetic acid (TFA), pentafluoropropanoic acid (PFPA), heptafluorobutyric acid (HFBA) and undecafluorocaproic acid (UFCA) as hydrophobic counter-ions in the reversedphase high performance liquid chromatography (RP-HPLC) of peptides was assessed. Four solvent systems were compared each containing 0.01M of a perfluorocarboxylic acid throughout. Twelve standard peptides and proteins were loaded onto the RP-HPLC column which was eluted with a linear gradient of 20-58.4% aqueous acetonitrile over 90 minutes. The retention times of the peptide standards were different in each solvent system. In progressing from TFA to PFPA, HFBA and UFCA all the peptides showed greater retention times. However, the effect was most marked with peptides having the greatest number of basic groups. By exploiting this behaviour a different type of chromatography can be introduced into the RP-HPLC purification of peptides. For instance, column fractions obtained from the use of the TFA solvent system can be re-chromatographed in a solvent system containing HFBA. It is possible by this procedure to purify naturally occurring peptides on the basis of their overall positive charges. At 0.01M each acid solution is sufficiently U.V. transparent to permit monitoring of column effluents at 210 nm. TFA, PFPA, HFBA and UFCA solvent systems are also completely volatile and this property facilitates the bioassay, radioimmunoassay and amino acid analysis of column fractions.     

Separation of Periodate, Iodate and Iodide on a C-18 Stationary Phase. Dependence of the Retention on the Temperature and Solvent Composition. Monitoring of an Oxidative Cleavage Reaction

The separation of periodate, iodate and iodide can be easily achieved by HPLC on a C-18 column with a mobile phase consisting of acetonitrile and aqueous phosphoric acid, without the need of any mobile phase additives that are often employed. All three iodine species are well separated. Separation factors as high as 16 are observed, and retention factors k between ～0.5 and 8. The retention time of all three ions increases with increasing acetonitrile concentration. The retention time of periodate and iodide decreases with increasing temperature, however, it increases slightly for iodate. An application of the HPLC method that was developed is the monitoring of an oxidative cleavage reaction, the cleavage of the enone-group of the steroid 4-androstene-3,17-dione. Periodate is used as oxidative reagent for this reaction. During the reaction periodate is reduced to iodate, and eventually both iodine species will be present in the reaction mixture, besides the starting material and product. In the final product the remaining iodate ions will be reduced to iodide which can be quantified accurately using the developed HPLC method.     

Acid catalyzed monodehydro-2,5-diketopiperazine formation from N-α-ketoacyl amino acid amides

We have developed a new synthetic method for monodehydro-2,5-diketopiperazines (monodehydroDKPs), which is based on an acid catalyzed cyclization of N-α-ketoacyl amino acid amides. Using this cyclization reaction, monodehydroDKP was formed with no or slight racemization in case that N-α-ketoacyl amino acid amides with β-aliphatic-α-ketoacyl groups and sterically unhindered N-substituting groups at the C-terminal amide nitrogen were used in the presence of catalytic amount of p-TsOH (3-5 mol %) or 10% TFA. In the case of β-aryl-α-ketoacyl amino acid derivatives, in which an enol form predominantly exists by conjugation with the aromatic ring, racemization could be minimized by optimizing the reaction conditions (5 mol % p-TsOH, reflux for 6 h), although the chemical yield could not be dramatically improved. However, this reaction condition was successfully applied to the synthesis of a tubulin depolymerization agent, (−)-tert-butyl-oxa-phenylahistin, with no racemization.     

Separation of lidocaine and its metabolites by capillary electrophoresis using volatile aqueous and nonaqueous electrolyte systems

The separation of the basic drug lidocaine and six of its metabolites has been investigated both by using volatile aqueous electrolyte system, at low pH and by employing non-aqueous electrolyte systems. In aqueous systems, the best separation of the compounds under the investigated conditions was achieved by using the electrolyte 60 mM trifluoroacetic acid (TFA)/triethylamine (TEA) at pH 2.5 containing 15% methanol. With this electrolyte, all seven compounds were well separated with high efficiency and migration time repeatability. The separations with bare fused-silica capillaries and polyacrylamide-coated capillaries were compared with higher separation efficiency with the latter. On the other hand, near baseline separation of all the seven compounds was also obtained by employing the non-aqueous electrolyte, 40 mM ammonium acetate in methanol and TFA (99:1, v/v), with comparable migration time repeatability but lower separation efficiency relative to the aqueous system.     

Interference of chitosan in glucose analysis by high-performance liquid chromatography with evaporative light scattering detection

The purpose of this work was to quantify glucose in aqueous solutions containing chitosan by high-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). Chitosan is a natural compound that is used alone or as an additive in several formulations. Microencapsulation of bioactive compounds such as glucose, by means of chitosan, is being explored, but difficulties arise when glucose needs to be determined in the presence of chitosan. HPLC is the technique most commonly used for glucose analysis, and ELSD may offer advantages (e.g. sensitivity and the possibility of operating in gradient mode) compared with other detectors. The influence of chitosan in the analysis of glucose by HPLC with ELSD was investigated at different pH values of the aqueous solutions. Isocratic elution with an acetonitrile/water mixture (80:20, v/v) and water washing between runs were the best options to avoid the mucoadhesive properties of chitosan, which are responsible for column degradation and variability of the retention time of glucose. The developed methodology was considered completely adequate for rapid glucose analysis in aqueous solutions with low pH (< 3), in the presence of chitosan.     

Polymers containing anthraquinone units: Polyimidazoles and polypyrrolones from 1,2,5,6-tetraaminoanthraquinone

1,2,5,6-Tetraaminoanthraquinone has been condensed with isophthalaldehyde and terephthalaldehyde and their bisulfite addition compounds to yield new heat-stable polymers. It has also been condensed with pyromellitic anhydride to give the pyrrolone. The highest viscosities were obtained in polymers prepared with acid catalysts. The polymers were nearly all soluble in concentrated sulfuric acid but not in organic solvents. Those soluble in sulfuric acid could also be solubilized by reduction with sodium dithionite and potassium hydroxide in aqueous organic solutions. A few polymers were apparently crosslinked, since they would not dissolve in either sulfuric acid or in base on reduction. Weak fibers were obtained by spinning the reduced alkaline solutions of the polymers into aqueous acid.     

Determination by hplc of trifluoroacetate levels in plasma and urine of patients anaesthetized with halothane

A new method is described for determination of trifluoroacetic acid (TFA) in biological fluids. Optimum extraction is achieved by addition of 18-crown-6 ether and acidification of the sample. The 4-bromomethyl-7-methoxycoumarin derivatives of the carboxylic acid are prepared and a sample is subjected to HPLC. A linear analytical curve of peak area against TFA concentrations ranging between 0.2 and 20 mug ml is obtained, and the minimum detectable concentration is estimated to be 0.1 mug ml .     

Analysis of polar peptides using a silica hydride column and high aqueous content mobile phases

The retention behavior of a set of polar peptides separated on a silica hydride stationary phase was examined with a capillary HPLC system coupled to ESI‐MS detection. The mobile phases consisted of formic acid or acetic acid/acetonitrile/water mixtures with the acetonitrile content ranging from 5 to 80% v/v. The effects on peptide retention of these two acidic buffer additives and their concentrations in the mobile phase were systematically investigated. Strong retention of the peptides on the silica hydride phase was observed with relatively high‐organic low‐aqueous mobile phases (i.e. under aqueous normal‐phase conditions). However, when low concentrations of acetic acid were employed as the buffer additive, strong retention of the peptides was also observed even when high aqueous content mobile phases were employed. This unique feature of the stationary phase therefore provides an opportunity for chromatographic analysis of polar peptides with water‐rich eluents, a feature usually not feasible with traditional RP sorbents, and thus under conditions more compatible with analytical green chemistry criteria. In addition, both isocratic and gradient elution procedures can be employed to optimize peptide separations with excellent reproducibility and resolution under these high aqueous mobile phase conditions with this silica hydride stationary phase.     

Large-scale separation of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza by pH-zone-refining countercurrent chromatography

pH-Zone-refining countercurrent chromatography was successfully applied to the separation of salvianolic acid B from the Chinese medicinal plant, Salvia miltiorrhiza Bunge, using a multilayer coil planet centrifuge. A 2.0 g quantity of sample was separated using the following two-phase solvent system: methyl tert-butyl ether (MtBE)-water, 10 mM TFA in organic stationary phase and 10 mM ammonia in aqueous mobile phase. The obtained fractions were analyzed by HPLC and ESI-MS. The separation yielded 572 mg of the main component of salvianolic acid B with a purity of 94.1%.     

Influence of the mobile phase on salmon calcitonin analysis by reversed-phase liquid chromatography

The retention properties of calcitonins on a reversed-phase column are examined using salmon calcitonin as the model compound. The effect of the concentration of organic modifier, buffer strength, pH of the mobile phase, and ion-pair reagent are studied. In the absence of an ionic modifier in the eluent the calcitonin peak shapes are not symmetrical. The addition of 0.1% trifluoroacetic acid (TFA), however, results in good peak characteristics without the need to add nonvolatile salts. The retention of the calcitonins was found to be very sensitive to the concentration of the organic modifier (acetonitrile) present in the mobile phase. A change of pH between 2 and 5 has only a slight effect of the k' of salmon calcitonin, but the k' increases significantly at higher pH values. The addition of a phosphate buffer to the mobile phase and an increase in the buffer concentration (0-0.2 M) causes a decrease in the retention of salmon calcitonin. Evidence shows that reproducible, quantitatively measurable data can be obtained using reversed-phase chromatography if the ion-pairing reagent and organic modifier concentrations are carefully controlled. The system also shows a good selectivity for the calcitonin series. Therefore, both highly selective methods (qualitative) as well as quantitative methods for analytical, pharmaceutical, and manufacturing use can be developed by adjusting the high-performance liquid chromatography (HPLC) conditions as discussed.     

Liquid structure of acetic acid-water and trifluoroacetic acid-water mixtures studied by large-angle X-ray scattering and NMR

The structures of acetic acid (AA), trifluoroacetic acid (TFA), and their aqueous mixtures over the entire range of acid mole fraction xA have been investigated by using large-angle X-ray scattering (LAXS) and NMR techniques. The results from the LAXS experiments have shown that acetic acid molecules mainly form a chain structure via hydrogen bonding in the pure liquid. In acetic acid-water mixtures hydrogen bonds of acetic acid-water and water-water gradually increase with decreasing xA, while the chain structure of acetic acid molecules is moderately ruptured. Hydrogen bonds among water molecules are remarkably formed in acetic acid-water mixtures at xA相似文献