Separation of Selected Beta Lactam Antibiotic Epimers on Gamma Cyclodextrin,Ion Exchange Ethylvinylbenzene/Divinylbenzene/Copolymer and Poly(Styrene-Divinylbenzene) Copolymer Stationary Phases

Abstract

High performance liquid chromatography (HPLC) stationary phases of gamma cyclodextrin, ion exchange ethylvinylbenzene/divinylbenzene (EVB/DVB) copolymer and poly (Styrene-divinylbenzene) (PRP-1) copolymer were investigated for the separation of beta lactam antibiotic epimers of cephalexin, moxalactam, ticarcillin, and carbenicillin. A combination of ion pair chromatography and inclusion complex formation improved the selectivity of moxalactam epimers on gamma cyclodextrin but had no effect on cephalexin epimers. A 10% increase in resolution was obtained for the moxalactam epimers on gamma cyclodextrin when 3mM tetrapropylammonium bromide was present in the mobile phase. Ion-exchange and reverse phase properties of the ion-exchange EVB/DVB phase coupled with perchlorate or sodium pentane sulfonate ion pair chromatography were also successful in separating some of the epimers. Retention and separation behavior of the analytes could not be easily predicted using this multiphase system. The PRP-1 phase was capable of easily resolving the epimers studied with minor adjustments in the mobile phase pH and organic modifier concentration. The PRP-1 phase would be highly recommended for the separation of antibiotic epimers based on the model compounds studied.     

Separation of chlamydosporol epimers by reversed-phase HPLC using commercial solvent optimisation software

Summary Two epimers of the mycotoxin chlamydosporol were separated by HPLC on an RP-18 column using a quaternary mobile phase consisting of water (79.1%), methanol (10.0%), acetonitrile (10.4%) and tetrahydrofuran (0.5%), with a flow rate of 1 ml min^–1. This optimal composition of mobile phase, with which the resolution value for the two epimers (1 and2) was 2.73 with retention times of 5.88 and 7.12 min, respectively, was achieved by the application of Philips Solvent Optimisation Software PU 6100. The presence of free silanols on the stationary phase was shown to be an essential requirement for the separation of the chlamydosporol epimers.     

Identification and Quantification by NMR Spectroscopy of the 22R and 22S Epimers in Budesonide Pharmaceutical Forms

The authors developed four variants of the qNMR technique (¹H or ¹³C nucleus, DMSO-d6 or CDCl₃ solvent) for identification and quantification by NMR of 22R and 22S epimers in budesonide active pharmaceutical ingredient and budesonide drugs (sprays, capsules, tablets). The choice of the qNMR technique version depends on the drug excipients. The correlation of ¹H and ¹³C spectra signals to molecules of different budesonide epimers was carried out on the basis of a comprehensive analysis of experimental spectral NMR data (¹H-¹H gCOSY, ¹H-¹³C gHSQC, ¹H-¹³C gHMBC, ¹H-¹H ROESY). This technique makes it possible to identify budesonide epimers and determine their weight ratio directly, without constructing a calibration curve and using any standards. The results of measuring the 22S epimer content by qNMR are comparable with the results of measurements using the reference HPLC method.     

Studies on Steroids CCXXXXIV. Separation and Characterization of C-25 Epimers of Unconjugated and Conjugated Trihydroxycholestanoic Acids in Urine from a Patient with Zell Weger Syndrome by High-Performance Liquid Chromatography

Abstract

The separation and characterization of C-25 epimers of unconjugated and glycine- and taurine-conjugated 3α, 7α, 12α - trihydroxy-5β-cholestanoic acid (THCA) in biological fluids by high-performance liquid chromatography (HPLC) are described. The 5β-cholestanoic acid fraction was obtained from a urine specimen from a patient with Zellweger syndrome by passing it through a Sep-pak C₁₈ cartridge. Bile acids were derivatized quantitatively into the fluorescent compounds through the hydroxyl group at C-3 by treatment with 1-anthroyl nitrile. The derivatives were separated into the unconjugated, glycine- and taurine-conjugated fractions by ion-exchange chromatography on alipophilicgel, piperidinohydroxypropyl Sephadex LH-20. Sub-sequent resolution of each fractionin to (25s)- and (25R)-THCA was attained by HPLC on a Cosmosil 5C column. The C-25 epimers of unconjugated and conjugated Tk%A were unequivocally identified on the b asis of the irbehaviors in HPLC using mobile phases of different pHs. The ratios of the unconjugated, glycine- and taurine-conjugated (25RbTHCA to the corresponding (25S)-epimers were 16:1, 5:4 and 3:2, respectively .     

Fragmentation study of iridoid glycosides including epimers by liquid chromatography‐diode array detection/electrospray ionization mass spectrometry and its application in metabolic fingerprint analysis of Gardenia jasminoides Ellis

A high‐performance liquid chromatography‐diode array detection/electrospray ionization mass spectrometry (HPLC‐DAD/ESI‐MS) method was applied to the characterization of ten iridoid glycosides in Gardenia jasminoides Ellis, a traditional Chinese medicine. During the process of structural elucidation, two groups of isomers including two epimers were structurally characterized and differentiated according to their distinctive fragmentation patterns which were closely related to their isomeric differentiations. Subsequently, the major compounds were purified by multi‐dimensional chromatography and semi‐preparative HPLC and the structure identification was confirmed with NMR techniques. The major fragmentation pathways of iridoid glycosides in Gardenia jasminoides Ellis obtained through the MS data were schemed systematically, which provided the best sensitivity and specificity for characterization of the iridoid glycosides especially the isomers so far. Based on the fragmentation patterns of iridoid glycosides concluded, seven major iridoid glycosides were characterized in rat plasma after intravenous administration of Gardenia jasminoides Ellis. Copyright © 2010 John Wiley & Sons, Ltd.     

trans-chloro(N,N-dimethyl-d-phenylglycine)-(3-methyl-1-phenylpent-1-ene)platinum(II) complexes. HPLC separation and identification of all the diastereomers

The diastereomeric trans-chloro(N,N-dimethyl-d-phenylglycine)(3-methyl-1-phenylpent-1-ene)platinum(II) complexes, derived by coordination of the enantiomeric and geometric isomers of 3-methyl-1-phenylpent-1-ene (2), were separated by HPLC. Four trans- and two cis-olefin complexes were recognized in the chromatogram. The configuration of all chiral centers of the olefin in the six complexes were assigned. Under the conditions of preparation, the pairs of diastereomers 1R,2R,3S/1S,2S,3S and 1S,2S,3R/1R,2R,3R were formed in a ratio > 1 for the trans-isomer, whereas the cis-isomer gave the 1R,2S,3S and 1S,2R,3R epimers only. The complexes do not epimerize on standing at room temperature in solution; similar behaviour of the corresponding complexes of trans-stilbene (4C) indicates that the conjugated aromatic double bond is coordinated more strongly than those aliphatic and cycloaliphatic olefins.The efficient HPLC separation of the diasteromeric complexes 2C, permits the enantiomeric analysis of 2, as well as the preparative resolution of the olefin.     

Effective separation and C-24 stereochemical assignment of epimeric 24-isopropenylcholesterols

Separation of epimeric 24-isopropenylcholesterols ($\text{1a}$) was effectively achieved by reversed-phase HPLC. The C-24 stereochemistry of these epimers was determined on the basis of chemical and spectroscopic evidence.     

Confluenines A–F, N-oxidized l-isoleucine derivatives from the edible mushroom Albatrellus confluens

The phytochemical study of the fruiting bodies of Albatrellus confluens afforded three pairs of new N-Oxidized l-Isoleucine derivatives epimers, confluenines A–F (1–6). The structures of these compounds were deduced using spectroscopic techniques, and the absolute configurations of the compounds were assigned by comparison between experimental and theoretical NMR and ECD data, respectively. Putative biosynthetic pathways toward the oxime, hydroxamate moieties and epimers were discussed. Compounds 5 and 6 exhibited weak antimicrobial activities against Staphylococcus aureus with MIC₉₀ values at 29.3?μg/mL and 56.7?μg/mL, respectively.     

Determination of (15R)- and (15S)-15-methylprostaglandin E2 in human plasma with picogram per milliliter sensitivity by column-switching high-performance liquid chromatography

(15R)-15-Methylprostaglandin E2 (PGE2) is a pro-drug under evaluation for the treatment of acute upper gastrointestinal hemorrhage and gastrointestinal cytoprotection. It is converted in acid (e.g., gastric fluid) to its active 15S epimer. Both epimers are found in human plasma at low pg/ml levels following oral dosing. A high-performance liquid chromatographic (HPLC) method was developed for the simultaneous analysis of (15R)- and (15S)-15-methyl-PGE2 in human plasma. The method combined off-line solid-phase extraction and reversed-phase HPLC clean-up with panacyl bromide derivatization and subsequent analysis using a heteromodal column-switching technique. Assay linearity was demonstrated over a range of 10-200 pg/ml for both 15-methyl-PGE2 epimers (r greater than or equal to 0.995). There were no significant inter-day differences in assay results for either epimer at 50 and 25 pg/ml (p greater than 0.05), with pooled estimates of precision at these levels producing relative standard deviations of less than or equal to 8% and less than or equal to 12%, respectively. The method quantitation limit (signal-to-noise ratio 5:1) for both epimers was 10 pg/ml when processing 3 ml of plasma. The analysis procedure was shown to be useful for quantifying at or below 10% of the (15R)-15-methyl-PGE2 maximum plasma concentration following a 50-micrograms oral dose in three human volunteers. For the same three subjects, however, the plasma concentration of (15S)-15-methyl-PGE2 did not exceed the quantitation limit of 10 pg/ml.     

A short way to invert configuration of the 2,3-hydroxy groups in ecdysteroids

3-epi-2-Dehydro-20-hydroxyecdysone and its 20,22-acetonide were reduced with lithium tris(secbutyl) hydridoborate selectively at the 2-oxo group with formation of 2β,3β-dihydroxy derivatives and the corresponding 2α,3α epimers which were separated by chromatography.     

A high performance liquid chromatographic system for the analysis of tetracycline drug standards, analogs, degradation products and other impurities

This paper describes the high performance liquid chromatographic (HPLC) analysis of eight parent tetracycline standards: tetracycline, chlortetracycline, rolitetracycline, oxitetracycline, minocycline, doxycycline, democlocycline, methacycline; and three tetracycline epimers: epitetracycline, epianhydrotetracycline, and anhydrotetracycline. The HPLC system employs an octadecylsilane reverse phase column and an isopropanol-diethanolamine-phosphate-ammonium EDTA-water mobile phase. This system produced at least partial resolution of all eight parent compounds and many of their degradation products.     

Selective extraction of flavonoids from Ginkgo biloba leaves using human serum albumin functionalized magnetic nanoparticles

In a novel procedure, human serum albumin timctionalized magnetic nanoparticles （ HSA-MNPS ） were used to selectively extract nine flavonoids in the extract of Ginkgo biloba leaves. The chemical structures of those flavonoids were characterized with high performance liquid chromatography coupled with mass spectrometry （HPLC-MS）. The selective extraction with HSA-MNPs coupled with structural elucidation with HPLC-MS shows powerful potential for analysis of bioactive components in traditional Chinese medicines.     

Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins,three purine alkaloids,and gallic acid in tea by high-performance liquid chromatography with diode array detection

Monomers of (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCG), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) and (−)-3-O-methyl epicatechin gallate (ECG3′Me) (purity, >97%) were successfully prepared from extract of green tea by two-time separation with Toyopearl HW-40S column chromatography eluted by 80% ethanol. In addition, monomers of (−)-catechin (C), (−)-gallocatechin (GC), (−)-gallocatechin gallate (GCG), and (−)-catechin gallate (CG) (purity, >98%) were prepared from EC, EGC, EGCG, and ECG by heat-epimerization and semi-preparative HPLC chromatography. With the prepared catechin standards, an effective and simultaneous HPLC method for the analysis of gallic acid, tea catechins, and purine alkaloids in tea was developed in the present study. Using an ODS-100Z C₁₈ reversed-phase column, fourteen compounds were rapidly separated within 15 min by a linear gradient elution of formic acid solution (pH 2.5) and methanol. A 2.5–7-fold reduction in HPLC analysis time was obtained from existing analytical methods (40–105 min) for gallic acid, tea catechins including O-methylated catechins and epimers of epicatechins, as well as purine alkaloids. Detection limits were generally on the order of 0.1–1.0 ng for most components at the applied wavelength of 280 nm. Method replication generally resulted in intraday and interday peak area variation of <6% for most tested components in green, Oolong, black, and pu-erh teas. Recovery rates were generally within the range of 92–106% with RSDs less than 4.39%. Therefore, advancement has been readily achievable with commonly used chromatography equipments in the present study, which will facilitate the analytical, clinical, and other studies of tea catechins.     

Perylene bisimide atropisomers: synthesis, resolution, and stereochemical assignment

The macrocyclization of the tetra-hydroxyphenoxy-substituted perylene bisimide 4 bearing two (R)-configured 2-octyl substituents in the imide positions by etherification with diethylene glycol ditosylate afforded both the diagonally bridged (1,7- and 6,12-linkage) and laterally bridged (1,12- and 6,7-linkage) regioisomers 6 and 7. The atropo-diastereomers of the diagonally bridged macrocycle 6 were separated by semipreparative HPLC on a chiral column, and their absolute configurations were determined by circular dichroism (CD) spectroscopy in combination with quantum chemical CD calculations. The isolated epimers (P,R,R)-6 and (M,R,R)-6 represent the first examples of diasteriomerically pure perylene bisimide atropisomers. The optical and chiroptical properties of these epimers were investigated by UV/vis, fluorescence, and CD spectroscopy, and their conformational properties have been explored by temperature-dependent 1H NMR studies.     

Synthetic studies of quinolizidine 195C and derivatives

Sulfur-substituted dihydropyridones prepared by aza-Diels–Alder reactions were converted to the cis-2,6-disubstituted derivatives, which could then proceed through ring-closing metathesis (RCM) and cross metathesis (CM) reactions to give many quinolizidine derivatives, including two epimers of (±)-195C.     

A pair of tirucallane C27-triterpenoid cyclopentenone epimers from the stem barks of Aphanamixis grandifolia

Two novel tirucallane C₂₇-triterpenoid epimers, aphagranins A (1) and B (2), featuring an unprecedented enolized cyclopentenone presented in the side-chain at C-17, were isolated from the stem barks of Aphanamixis grandifolia. Extensive spectroscopic analyses helped the establishment of the structures of the two isolates, whose absolute configurations were determined using density functional theory (DFT) calculations of optical rotation, and electronic circular dichroism (ECD). Remarkable discrepancies in the inhibitory activities against the growth of six lines of human cancer cells (MCF-7, A549, HepG2, Bel-7402, SGC-7901, and BGC-823) were found for the two epimers: with IC₅₀ less than 10 μM, aphagranin A exhibited much stronger antiproliferative activity than aphagranin B, showing no such activities with IC₅₀ over 20 μM.     

A concise synthesis of (2S,4R)- and (2S,4S)-4-methylglutamic acid

A concise, multi-gram scale method for producing the bioactive and enantiomerically pure epimers, (2S,4R)- and (2S,4S)-glutamic acids, in a single synthetic scheme is described.     

Preparative production and separation of 2-acetamido-2-deoxymannopyranoside-containing saccharides using borate-saturated polyolic exclusion gels

A new separation method based on the combination of exclusion and ion exchange chromatography in borate buffer was developed. It allows semi-preparatory and preparatory separation of isobaric N-acylhexosamines (C-2 epimers) and corresponding methyl glycosides (anomers and tautomers). Three types of polyolic gels were tested for these separations. Ion-exchange HPLC was used as a rapid and reliable method for the quantification of the respective analytes. NMR studies of the interactions of N-acetylhexosamines with borate confirmed the importance of a proper stereochemical arrangement of acetamido sugars for their interactions with borate anions.