ABSTRACT
OBJECTIVE: To develop an RP-HPLC method for chiral separation of propranolol (PL) enantiomers and to apply it in enantioselective transdermal permeation study of PL. METHODS: The separation was performed on an amylose derivative chiral stationary phase Chiralpak AD-RH column (4.6 mm × 150 mm, 5 μm) with the mobile phase of 10 mmol · L-1 borate buffer (0.1% triethylamine, pH 9.0)-acetonitrile-ethanol (50:25:25, V/V/V). The flow rate was 0.5 mL · min-1, the column temperature was 25°C, and the UV detection wavelength was 229 nm. Additionally, the enantioselectivity of racemic PL and enantiomers through excised mouse skin was evaluated by using borate buffer as permeation donor solvent. RESULTS: PL enantiomers were entirely separated within 21 min (Rf=2.2) and were found to be linear in the range of 3-200 μg · mL-1. The average recoveries of R-VL and S-PL were 100.84% and 103.24%, respectively. The results of skin permeation experiments indicated that there were no significant permeation differences among racemic PL, S-PL enantiomer and S-PL enantiomer. CONCLUSION: The developed RP-HPLC method is accurate, sensitive and efficient, and it is successfully applied to enantioselective transdermal permeation study of PL.
ABSTRACT
OBJECTIVE: To develop an enantioselective HPLC-MS-MS method for the pharmacokinetic study of flurbiprofen transdermal patches. METHODS: The analyte and internal standard, ketoprofen, were extracted from plasma by methyl tert-butyl ether, analyzed on a Chiralpak AD-RH column(4.0 mm×150 mm, 5 μm)with mobile phase consisting of acetonitrile-ammonium acetate buffer solution(2 mmol·L-1 pH 5.3, 50:50, V/V), and detected with UV detector at 273 nm. RESULTS: The assay was linear over the concentration range of 2.0-1000 ng·mL-1 and the intra- and inter-day precision over this range were not more than 5.8%. The limit of quantification in plasma was 2.0 ng·mL-1. CONCLUSION: The method established here is accurate, precise, rapid, sensitive, and successfully applied to the enantioselective determination of flurbiprofen in the pharmacokinetic study of flurbiprofen transdermal patches in rabbits.
ABSTRACT
The enantioselective hydrolysis of racemic epichlorohydrin for the production of enantiopure (S)-epichlorohydrin using whole cells of Aspergillus niger ZJB-09173 in organic solvents was investigated. Cyclohexane was used as the reaction medium based on the excellent enantioselectivity of epoxide hydrolase from A. niger ZJB- 09173 in cyclohexane. However, cyclohexane had a negative effect on the stability of epoxide hydrolase from A. niger ZJB-09173. In the cyclohexane medium, substrate inhibition, rather than product inhibition of catalysis, was observed in the hydrolysis of racemic epichlorohydrin using A. niger ZJB-09173. The racemic epichlorohydrin concentration was markedly increased by continuous feeding of substrate without significant decline of the yield. Ultimately, 18.5% of (S)-epichlorohydrin with 98% enantiomeric excess from 153.6 mM of racemic epichlorohydrin was obtained by the dry cells of A. niger ZJB-09173, which was the highest substrate concentration in the production of enantiopure (S)-epichlorohydrin by epoxide hydrolases using an organic solvent medium among the known reports.
ABSTRACT
A strain NK13 was screened for a certain extent asymmetric hydrolyze the rac-ketoprofen chloroethyl ester and identified as Bacillus megaterium. For the preparation of gene libraries, a positive clones was obtained from the tributyrin flat. The sequence of this esterase gene had been analysised, and contained the whole ORF of an esterase gene with the length of 933bp. The esterase gene of NK13 was compared with the esterase genes of GenBank and the result showed that the esterase gene of NK13 was a novel gene(GenBank accession nember DQ196347).The new esterase gene was inserted into the plasmid pET21b+, then the recombinant plasmid transformed E.coli BL21. After being induced by IPTG, it was expressed in the host strain. SDS-PAGE analysis showed that the relative molecular mass of the esterase was about 34kDa. The result of TLC and HPLC showed that the recombinant strain had higher conversion ration than templet strain. 47.4%of the rac-ketoprofen Chloroethl ester was hydrolyzed to ketoprofen by the recombinant strain in 45min. The (S)- ketoprofen enantiomeric excess, in the later,was 55.46%, which indicated that the esterase could hydrolyze (S)-ketoprofen chloroethyl ester firstly.