Simultaneous determination of tiopronin and its metabolites in rat blood by LC-ESI-MS-MS using methyl acrylate for stabilization of thiol group.

A sensitive and reproducible HPLC-electrospray tandem mass spectrometric method has been developed for the analysis of tiopronin (TP) and its metabolites, 2-mercaptopropionic acid (2-mpa) and S-methylated TP (SA13), in rat blood using methyl acrylate (MA) for the stabilization of a thiol group. The thiol groups of TP and 2-mpa in rat blood were immediately derivatized by the addition of MA-acetonitrile solution in 0.1 M Tris HCl (pH 9.1). The purification of the derivatives was accomplished by a simple liquid-liquid extraction procedure involving protein precipitation step. The analysis was performed on a Zorbax SB-C18 analytical column by a gradient elution with methanol-0.05 M acetic acid (15:85 and 7:3, v/v). Negative ion electrospray ionization with selected reaction monitoring was employed for the detection of analytes. Linearity of calibration was observed over the range of 0.5-1000 ng/ml for TP and 2-mpa, and 2-1000 ng/ml for SA13. The intra- and inter-assay variability for all analytes at the limit of quantitation (LOQ) level ranged from 5.47 to 16.75% and 4.95 to 7.23%, respectively. The LOQs estimated for TP, 2-mpa and SA13 were 0.5, 0.5 and 2 ng/ml, respectively. This assay method was successively applied to a pharmacokinetics study after an oral administration of TP (10 mg/kg) to rats.

Isoform selective inhibition and inactivation of human cytochrome P450s by methylenedioxyphenyl compounds.

1. A series of methylenedioxyphenyl compounds were evaluated for their inhibitory and inactivation effects on nine human cytochrome P450 (CYP) activities using microsomes from human B-lymphoblast cells expressing specific human CYP isoforms. 2. Methylenedioxyphenyl compounds which possess a bulky structure such as 1,4-benzothiazine showed substantial inhibition of S-warfarin 7-hydroxylation catalysed by CYP2C9, S-mephenytoin 4'-hydroxylation by CYP2C19, bufuralol 1'-hydroxylation by CYP2D6, and testosterone 6beta-hydroxylation by CYP3A4. Regarding ethoxyresorufin O-deethylation catalysed by CYP1A1 and benzyloxyresorufin O-dealkylation by CYP2B6, the subtle change of a substitution of the 1,4-benzothiazine structure affected the inhibition selectivity. Ethoxyresorufin O-deethylation by CYP1A2, coumarin 7-hydroxylation by CYP2A6, and chlorzoxazone 6-hydroxylation by CYP2E1 were not inhibited by almost any of the methylenedioxyphenyl compounds. The inhibitory effects of methylenedioxyphenyl compounds that possess a short chain amino group on the human CYP isoforms were not significant. 3. The methylenedioxyphenyl compounds inactivated CYP1A1 (k(inact) = 0.034 min(-1) and K(i) = 0.81 microM), CYP2C9 (k(inact) = 0.041 and 0.042 min(-1) and K(i) = 0.56 and 0.15 microM), CYP2D6 (k(inact) = 0.044-0.339 min(-1) and K(i) = 0.21-19.88 microM), and CYP3A4 (k(inact) = 0.076-0.251 min(-1) and K(i) = 0.25-0.69 microM). These results suggested that the methylenedioxyphenyl compounds investigated in this study would be potent mechanism-based inactivators of these human CYP isoforms. In contrast, CYP2B6 and CYP2C19 were not inactivated. 4. The present study suggested that the selectivity of inhibition or inactivation of human CYP isoforms by methylenedioxyphenyl compounds may vary according to the structure of the side chain.

Enantioselective immunoaffinity extraction for simultaneous determination of optically active bufuralol and its metabolites in human plasma by HPLC.

A combined method of immunoaffinity extraction with high-performance liquid chromatography has been developed for the enantioselective determination of bufuralol and its metabolites in human plasma. The antibodies having high affinity toward the asymmetric center at the C-1 position of bufuralol and its 1'-oxidized metabolites and low affinity to their antipodes were elicited by immunization of rabbits with immunogens, (1R)- and (1S)-1'-oxobufuralol O-carboxymethyloxime-bovine serum albumin conjugates, respectively. 0.5 ml Of the immunoaffinity adsorbent (7.6 mg.ml-1 for anti-(1S)-antibody and 28.8 mg.ml-1 for anti-(1R)-antibody) prepared by immobilization of an antibody was capable of retaining up to 1 microgram of (R)- and (S)-bufuralol and up to 500 ng of other metabolites. The adsorbates were recovered quantitatively by elution with methanol-10 mM ammonium acetate buffer (pH 5) (95:5, v/v) without any interfering peaks on the high-performance liquid chromatogram. The proposed method was evaluated to be useful for the simultaneous determination of optically active bufuralol and its metabolite in plasma with acceptable recovery and precision.

Beta adrenergic antagonist permeation across cultured rabbit corneal epithelial cells grown on permeable supports.

PURPOSE: To determine whether cultured rabbit corneal epithelial cells (RCEC), grown on permeable supports, provide a suitable in vivo model for characterizing transcellular drug permeation and metabolism. METHODS: Primary rabbit corneal epithelial cells grown in DMEM-F12 were seeded on Transwell-COL inserts coated with fibronectin. The epithelial barrier integrity was evaluated, based on measurements of 14C-mannitol and 3H-PEG900, and their transepithelial electrical resistance (TEER). Ultrastructure evaluation was based on scanning electron microscopy and transmission electron microscopy, which were performed 8 days after seeding. Measurements of beta adrenergic antagonist permeability were performed to assess transcellular permeability. RESULTS: Eight days after seeding, the TEER reached a peak of 144 omega.cm2 and the 14C-mannitol and 3H-PEG900 permeabilities were 6.8 x 10(-6) and 2.9 x 10(-6) cm/sec, respectively. Ultrastructural analysis revealed a multilayered structure with numerous microplicae and typical cytoplasmic organelles along with desmosomes. The relationship between permeation of beta-blockers and lipophilicity resembled the intact isolated cornea. CONCLUSIONS: This is the first description of cultured RCEC grown on permeable support. Many of its properties mimic those described in the intact corneal epithelium. Even though its electrical tightness is less than that of the intact cornea, the transcellular permeability to lipophilic beta-antagonists is comparable to the isolated preparation. Therefore, this model will facilitate characterization of ocular permeation mechanisms of hydrophobic drugs whose route of permeation is transcellular.

Different pharmacokinetics of (4R)-hexahydro-7,7-dimethyl-6-oxo-1,2,5-(3-14C)dithiazocine-4-carboxyli c acid between the fasting and non-fasting rat: role of intestinal microorganisms.

1. We report differences in the pharmacokinetics of (4R)-hexahydro-7,7-dimethyl-6-oxo-1,2,5-(3-14C)dithiazocine-4-carb oxylic acid (14C-SA3443) between the normal fasting and non-fasting rat, especially in the blood concentration-time curves and respiratory excretion. Exhalation of 14CO2 was an important route of elimination and accounted for 21.2% of the dose in the non-fasting rat but only 3.7% in fasting animals. 2. In the intestinal microorganism-compromised rat, we found little differences in the pharmacokinetics of 14C-SA3443 between fasting and non-fasting states. No respiratory excretion was observed in the intestinal microorganism-compromised animal. 3. In the reaction mixture of 14C-SA3443 with the cecal contents of rat, 14C-acetic acid and 14C-butyric acid were detected and 14CO2 barely detected. 4. The amounts of 14C-acetic acid and 14C-butyric acid in the reaction mixture of 14C-SA3443 with non-fasting rat cecal contents were more than those with fasting rat cecal contents. 5. We concluded that the reason for the different pharmacokinetics of 14C-SA3443 between the fasting and non-fasting rat was the differences in participation of the metabolism of 14C-SA3443 by intestinal microorganisms.

Distributional change of alpha-tocopherol in the rat lens with age.

Concentration of alpha-tocopherol (alpha-Toc.) in the rat lens (1, 4 and 12 months old) was determined in single lenticular layers using gas chromatography and mass spectrometry. The highest concentration of alpha-Toc. in lens was found in the nucleus, followed by the deeper anterior and deeper posterior cortices, the shallow anterior and shallow posterior cortices, and the equatorial region. The topographic alpha-Toc. distribution in the lens did not differ between lenses of 1-, 4- and 12-month-old rats. A significant decrease of alpha-Toc. concentration was seen in lenses of 4- and 12-month-old rats compared to those 1 month old. Concentration and distribution in 4- and 12-month-old rat lenses were almost the same. The concentration of alpha-Toc. in the lens changes in relation to age.

Determination of pilocarpine in aqueous humour by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A new method has been developed for rapid analysis and determination of pilocarpine in aqueous humour using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. The chromatography was carried out on a reversed-phase phenyl column with 0.1% acetic acid-acetonitrile (95:5, v/v). Pilocarpine and its analogues, isopilocarpine, pilocarpic acid and isopilocarpic acid, were separated. An aqueous humour sample was deproteinized with methanol. After evaporation, the residue was dissolved in the mobile phase. The method was applied to the analysis of the metabolite in aqueous humour after the topical application of 2% pilocarpine (w/v) eye-drops. The main metabolite, pilocarpic acid, was easily identified. The protonated molecular ion of pilocarpine was used for the determination. The calibration curve had a good linearity within the concentration range investigated (2 ng to 10 micrograms/ml). The limit of determination was estimated to be an aqueous humour concentration of ca. 2 ng/ml. The method was applied to the determination of unchanged pilocarpine after the topical application of 2% pilocarpine (w/v) eye-drops.

Gas chromatographic-mass spectrometric determination of tiopronin in human blood using acrylic acid esters as a derivatization reagent for the thiol group.

A gas chromatographic-mass spectrometric method for determining tiopronin, which has a thiol group, in human blood has been described. To prevent the oxidative degradation of tiopronin in the blood, its thiol group was immediately protected by treatment with isobutyl acrylate, which reacted readily with tiopronin in a 0.1 M Na2HPO4 solution (pH 9.1). The reaction was quantitative within 30 min. The blood sample was deproteinized and purified by a combination of liquid-liquid extraction and solid-phase extraction. Finally, the carboxyl moiety of the ester adduct was derivatized to the pentafluorobenzyl ester. The derivatives of tiopronin and the internal standard were analysed with gas chromatography-mass spectrometry. The precision of the method was satisfactory, and the calibration curve had good linearity in the concentration range investigated. The limit of determination of tiopronin in blood was estimated to be ca. 1 ng/ml.