Direct high-performance liquid chromatographic enantioseparation of beta-lactam stereoisomers

High-performance liquid chromatographic methods were developed for the separation of the enantiomers of 12 beta-lactams. Direct separations were performed on chiral stationary phases (CSPs) containing cellulose-tris-3,5-dimethylphenyl carbamate (Chiralcel OD-RH and OD-H columns), the macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T column), or teicoplanin aglycone (Chirobiotic TAG column) as the chiral selector. It was clearly established that, with teicoplanin-based columns, the teicoplanin aglycone was most often responsible for the enantioseparation of the beta-lactams. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was in the range between 0.02 and 0.97 kJ mol(-1) for these beta-lactam stereoisomer separations. The separations were carried out with high selectivity and resolution, and the method was therefore suitable for monitoring of the enantiomeric excess after chiral synthesis. The Chirobiotic and Chiralcel columns appear to be highly complementary to one another. The best separation of this class of beta-lactam compound could be obtained using the Chirobiotic TAG in the polar-organic mode plus the Chiralcel OD-H in the normal-phase mode. The elution sequence was also determined.     

Synthesis,chromatographic resolution and chiroptical properties of carboxyibuprofen stereoisomers: Major metabolites of ibuprofen in man

The chromatographic resolution of the four stereoisomers of carboxyibuprofen, a major metabolite of ibuprofen in man, was achieved using a Chiralpak AD chiral stationary phase (CSP) (J.T. Baker, Milton, Keynes, UK). The elution order of the stereoisomers was determined to be 2′S,2R; 2′R,2R; 2′R,2S; 2′S,2S by a combination of stereoselective synthesis of diastereoisomeric mixtures and analysis of the two diastereoisomers isolated from human urine following the administration of (S)-ibuprofen. The individual stereoisomers were isolated by semipreparative chiral phase chromatography and characterized by circular dichroism spectroscopy. Chirality 9:75–87, 1997. © 1997 Wiley-Liss, Inc.     

Stabilization and gas chromatographic analysis of the four stereoisomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman) in rat blood

A method for the stabilization and gas chromatographic analysis of the four stereoisomers of C(+/-)P(+/-)-1,2,2-trimethylpropyl methylphosphonofluoridate (C(+/-)P(+/-)-soman) in rat blood samples is described. Satisfactory stabilization of all four stereoisomers is obtained by (i) acidification of the blood sample to pH 4.2 at 0 degrees C, to stabilize the C(+/-)P(+) isomers, (ii) addition of aluminum ions (2.5 mM) for complexation of fluoride ions, which prevents regeneration of C(+/-)P(-)-soman by free fluoride ions from soman-inhibited aliesterase, and (iii) addition of 2,2-dimethylpropyl methylphosphonofluoridate in order to occupy covalent binding sites for C(+/-)P(-)-soman. The stereoisomers of soman and internal standard are extracted from the blood-stabilizing buffer mixture with a Sep-Pak C18 cartridge and are subsequently eluted with ethyl acetate with overall extraction recoveries of 52 +/- 8%. The four soman stereoisomers are resolved and analyzed on a wide-bore capillary Chirasil Val column, synthesized, and coated in house, which also resolves the internal standard C(+/-)P(+/-)-1,2,2-[U-2H]trimethylpropyl methylphosphonofluoridate from C(+/-)P(+/-)-soman. Alternatively, the gas chromatographic analysis can be performed on a wide-bore capillary Chirasil Val column, identical with the commercially available Chirasil Val column, when combined in series with a Carbowax 20M column. This system resolves the four stereoisomers of soman and the internal standard C(-)P(+)-1,2,2-trimethylpropyl [U-2H]methylphosphonofluoridate. Using an alkali flame ionization detector, the detection limit of our procedure is ca. 250 pg soman isomer/blood sample.     

Direct chromatographic resolution of racemic cyclohexylaminoglutethimide and its acetylated metabolite using cellulose-based chiral stationary phase

Racemic cyclohexylaminoglutethimide (±ChAG) and its acetylated metabolite (±ChAG) were resolved by a direct chromatographic method using a Chiracel OD column without derivatization. Maximum resolutions (R) of 4.89 and 0.74 were obtained for the enantiomers of cyclohexylaminoglutethimide and its acetylated metabolite, respectively.     

Direct high-performance liquid chromatographic resolution of the enantiomers of tiaprofenic acid using immobilized human serum albumin

Resolution of racemic tiaprofenic acid (TA) has been performed using immobilized human serum albumin as the stationary phase. The eluent was phosphate buffer—acetonitrile—n-octanoic acid (90:10:0.015, v/v). Detection was achieved at 305 nm. The pharmacokinetics of the enantiomers were studied following oral administration into humans and after subcutaneous injection in rats. Plasma concentrations of (+)-TA were much greater than those of (−)-TA. For the rat, the pharmacokinetic parameters between (−)-TA and (+)-TA were all statistically different (p < 0.005).     

Direct isolation of neuronal microtubule skeletons.

The microtubule skeletons of cultured neuronal cells have been isolated in their original form utilizing taxol and rigorous extraction conditions. Continuous microtubules are visible throughout the cell body and into the processes. These preparations include the complex set of microtubule-associated proteins of 69,000 and 80,000 daltons previously identified in these cells.     

Single-step isolation and resolution of pancreatic carboxypeptidases A and B.

Carboxypeptidases A and B have been isolated individually from aqueous extracts of mammalian pancreatic acetone powders by affinity chromatography on [N-(epsilon-aminocaproyl)-p-aminobenzyl]succinyl-Sepharose 4B (CABS-Sepharose). The affinity ligand was synthesized from DL-benzylsuccinic acid, purified, and characterized by UV absorption and NMR spectroscopy. Both enzymes from the various species were homogeneous by NaDodSO4-polyacrylamide gel electrophoresis and displayed high specific activities. No cross contamination of one enzyme species with the other was found. The ease of synthesis of the ligand from its commercially available precursor, its stability, and the mild elution conditions render CABS-Sepharose an excellent affinity support for the single-column isolation of both carboxypeptidases A and B. The procedures extend the utility of this resin previously demonstrated for carboxypeptidase A from human pancreatic juice [Peterson, L. M., Sokolovsky, M., & Vallee, B. L. (1976) Biochemistry, 15, 2501]. The use of CABS-Sepharose as a general affinity matrix for the isolation of metallocarboxypeptidases is suggested.     

A rapid column chromatographic method for the isolation of catechol-type siderophores.

The ester groups of glycerophospholipids in tissue extracts can be cleaved in less than 10 min at room temperature if the lipids are extracted with hexane-isopropanol and the filtrate is treated with methanolic NaOH. The resulting mixture can be treated with aqueous Na-sulfate containing sulfuric acid and partitioned to remove the inorganic reagents and hydrophilic ester degradation products. When the procedure is applied to brain lipid extracts, the addition of alkali produces a second, lower phase that contains much of the hydroxycerebroside, virtually all of the sulfatide in the extract, and small amounts of other lipids. The sulfatide can be isolated from the lower phase by neutralizing it with HCl in aqueous methanol, and partitioning with chloroform to remove nonlipid components. The lower phase is evaporated to dryness and treated with periodic acid to convert the cerebroside to a less polar product. The lipids recovered from the reaction mixture are then fractionated with a Florisil column, which yields highly purified sulfatide. Starting with 300 g of pig brain one can obtain about 1.1 g of sulfatide in 4 working days, using conventional, compact equipment. Since the precipitation step is almost complete, and the procedure can be scaled down to very low levels, the method has promise for quantitation methods and isotopic studies of sulfatide metabolism.     

Simultaneous resolution of underivatized regioisomers and stereoisomers of arachidonate epoxides by capillary electrophoresis

cis-Epoxyeicosatrienoic acids (EETs) and their hydrolysis products (threo-DHETs) have been proposed to be endothelial-dependent hyperpolarizing factors (EDHFs) which upregulate blood flow when tissue perfusion is impaired. Various EET regioisomers and enantiomers are formed from arachidonate by inducible cytochrome P450 epoxygenase isoforms, and tissue EET profiles may vary with diet, time, and disease. Because EET actions and metabolism may be regio- and stereospecific, convenient methods to measure profiles of EET isomers in tissues are needed. In the current studies, we describe two simple capillary electrophoretic methods for resolving EETs. The first method involves capillary electrophoresis with a mixture of neutral and anionic beta-cyclodextrins, which in one step baseline-resolves underivatized EET regioisomers and their enantiomers. Low picogram amounts of EET enantiomers were identified based on migration times and UV spectra. The method was also used to assess the antipode purity of EET standards, and to determine murine hepatic levels of EET enantiomers. The second method involves capillary electrochromatography, which also baseline-resolves underivatized EET and DHET regioisomers in one step. We conclude that in EET assays the major advantages of capillary electrophoresis over reversed-phase HPLC are improved peak efficiency, sensitivity, and resolution, plus precise coelution of deuterated and nondeuterated EETs.     

An improved isolation procedure for the gas chromatographic analysis of urinary polyamines.

The isolation of polyamines from urinary hydrolysates in a sufficiently pure state for subsequent analysis by gas chromatography has proved to be difficult. However, by using columns of Porapak-Q and ion-exchange resins, urinary hydrolysates are readily purified and formation of trifluoroacetyl derivatives of polyamines proceeds in high yield without carryover of artifacts in the gas chromatographic elution profile. Good yields from the trifluoroacetylation reaction are not achieved if large quantities of salts or urinary pigments are present. By obtaining the polyamine carbonates in the final stages of the method described, the trifluoroacetylation reaction yields excellent derivatives of nanogram or microgram amounts, particularly after standing over-night at room temperature. The procedure described in detail should permit routine urinary polyamine analysis where rapidity, ease of handling many samples, freedom from complications and artifacts are a consideration.     

Direct isolation of Francisella spp. from environmental samples

Aims:  To develop a selective medium for isolation of F. tularensis, F. novicida and F. philomiragia from environmental samples.
Methods and Results:  A selective media, cysteine heart agar with 9% chocolatized sheep blood, containing polymyxin B, amphotericin B, cyclohexamide, cefepime and vancomycin (CHAB-PACCV) was developed and evaluated for growth of Francisella spp. No differences were observed in recovered colony forming units (CFUs) for F. tularensis , F. novicida and F. philomiragia on CHAB-PACCV vs nonselective CHAB. Growth of non- Francisella species was inhibited on CHAB-PACCV. When environmental samples were cultured on CHAB and CHAB-PACCV, only CHAB-PACCV allowed isolation of Francisella spp. Three new Francisella strains were isolated directly from seawater and seaweed samples by culture on CHAB-PACCV.
Conclusions:  CHAB-PACCV can be used for direct isolation of Francisella spp from environmental samples.
Significance and Impact of the Study:  Francisella spp. show a close association with environmental sources. Future utilization of CHAB-PACCV for isolation of Francisella spp. directly from environmental samples should prove valuable for investigating outbreaks and human infections attributed to environmental exposure.     

Neighboring group participation. Part 16. Stereoselective synthesis and receptor-binding examination of the four stereoisomers of 16-bromomethyl-3,17-estradiols

The four possible isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a-4a) with proven configurations were converted into the corresponding 3-benzyloxy-16-bromomethylestra-1,3,5(10)-triene-3,17-diols (5e-8e). Depending on the reaction conditions the cis isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a and 2a) were transformed into 3-benzyloxy-16-bromomethylestra-1,3,5(10)-trien-17-yl acetate (5b and 6b) or 16-bromomethyl-3-hydroxyestra-1,3,5(10)-trien-17-yl acetate (5c and 6c) on treatment with HBr and acetic acid. The mechanism of the process can be interpreted as involving front-side neighboring group participation. Under similar experimental conditions, the trans isomers (3a and 4a) yielded only 3-benzyloxy-16-acetoxymethylestra-1,3,5(10)-trien-17-yl acetates (3b and 4b) or 16-acetoxymethylestra-1,3,5(10)-triene-3,17-diyl diacetates (3d and 4d). Both the cis (1a and 2a) and the trans (3a, and 4a) isomers were transformed into 16-bromomethylestra-1,3,5(10)-trien-17-ol (5a-8a) by the Appel reaction on treatment with CBr4/Ph3P. Debenzylation of 5a-8a was carried out with HBr and acetic acid to yield 5e-8e. The debenzylation process in the presence of acetic anhydride produces the diacetates 5d-8d. The structures of the compounds were determined by means of MS, 1H NMR and 13C NMR spectroscopic methods. Compounds 5c-8c and 5e-8e were tested in a radioligand-binding assay. Except for the affinity of 7e for the estrogen receptor (Ki=2.55 nM), the affinities of the eight compounds (5c-8c and 5e-8e) for the estrogen, androgen and progesterone receptors are low (Ki > 0.55, 0.52 and 0.21 microM, respectively).     

Synthesis of all four stereoisomers of 3-amino-2-hydroxybutanoic acids

All four stereoisomers of 3-amino-2-hydroxybutanoic acids were been obtained as single enantiomers via stereospecific reactions from D-gulonic acid gamma-lactone and D-glucono-delta-lactone.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat.

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Direct high-performance liquid chromatographic separation of the enantiomers of an aromatic amine and four aminoalcohols using polysaccharide chiral stationary phases and acidic additive

The HPLC enantiomeric separation of N-benzyl-alpha-methyl-benzylamine, phenylalaninol, tryptophanol, 2 (diphenylhydroxymethyl)pyrrolidine, and isoproterenol was accomplished in the normal-phase mode using two polysaccharide-derived chiral stationary phases (CSPs) and various n-hexane/2-propanol mobile phases with acidic (TFA) or basic (DEA) additive. The compounds were separated without any derivatization and separation factor range between 2.09 and 1.09 with resolution factor 3.4 and 0.4, respectively. The best separation of the enantiomers of the amine was achieved on amylose tris (3, 5-dimethylphenylcarbamate) CSP with TFA additive in the mobile phase; in acidic conditions, instead, the best enantioseparation of the aminoalcohols was achieved on cellulose tris (3, 5-dimethylphenilcarbamate). A long equilibration time of the CSP when switching from an undoped mobile phase to a doped one is required to obtain reproducible results.     

High-pressure liquid chromatographic resolution of vitamin A compounds

An electrochemical system has been devised to measure phenol concentrations in aqueous solutions. The unit employs the immobilized enzyme, tyrosinase, to oxidize phenol in the presence of saturating levels of oxygen. The oxidation product, ortho-benzoquinone, is then chemically reduced in the presence of an excess of ferrocyanide ions. The coupled oxidation of ferrocyanide ions to ferricyanide ions results in a measurable potential difference in the electrochemical system. The resulting zero current potentials in these steady-state potentiometric measurements are shown to be directly proportional to the logarithm of phenol concentration over the range of 3.8 × 10^?7 to 1 × 10^?4m. The results of studies carried out with alternate substrates for the enzyme and interfering compounds are also presented.     

Direct isolation of mycoplasmas and acholeplasmas from sera and kidneys of calves.

Kidneys and sera of calves from various farms and primary kidney tissue cultures were tested for mycoplasma and acholeplasma contamination. Altogether 24 strains belonging to Mycoplasma arginini and Acholeplasma axanthum were isolated from tissue cultures, kidneys and sera. The same species were detected from lungs and peribronchial lymph nodes of calves, together with A. laidlawii, A. modicum and M. bovirhinis species. There was a close correlation between the mycoplasma content of tissue cultures, sera and kidneys and the clinical picture observed in the farm, as well as between the quality of tissue cultures and the mycoplasma content of organs, sera and tissue cultures.