Formation of glucoside conjugate of acetaminophen by fungi separated from soil.

The phase II metabolite of acetaminophen in filamentous fungi and actinomycetes separated from soil was investigated. Fifty-four filamentous fungi and twenty-seven actinomycetes were screened to transform acetaminophen. The metabolites of acetaminophen were assayed using liquid chromatography-tandem mass spectrometry. The only metabolite was subject to enzymatic hydrolysis to confirm its structure. Acetaminophen was converted into glucoside conjugate, by filamentous fungi JX1-60, LN17-2, LN20-1 and the yield of the conjugate was 60.01%, 44.27%, 100%, respectively, and no phase I metabolites were detected. Glucoside conjugation of acetaminophen in filamentous fungi differs from the phase II metabolism of glucuronidation in humans. The fungus LN20-1 could be a suitable model to synthesize glucoside conjugate of acetaminophen.

Structural identification of bitespiramycin metabolites in rat: a single oral dose study.

Bitespiramycin is a macrolide antibiotic consisting of a mixture of some nine spiramycin ester derivatives. It has a similar spectrum of antibiotic activity to that of spiramycin but has superior pharmacokinetic properties. In this study, a rapid and facile LC/ESI-MSn method was applied to study the metabolism of bitespiramycin in rat following a single oral dose (80 mg kg-1). Concentrations of parent drug constituents and metabolites were determined in plasma, urine, feces and bile. Concentrations of parent drug constituents and metabolites in plasma were very low. In urine, feces and bile, parent drug constituents and 38 metabolites were identified on the basis of their chromatographic and mass spectrometric properties. The identity of 17 metabolites was confirmed by comparison with reference substances. The principal metabolites were the corresponding spiramycins formed by hydrolysis of the 4''-(3-methylbutanoate) groups. Other important metabolic pathways were: hydrolytic loss of the forosamine and mycarose sugars; aldehyde reduction; cysteine conjugation of the aldehyde group; and hydrolysis of the lactone ring. Products formed by lactone ring opening were found only in urine, and those formed by aldehyde reduction were found only in feces. Aldehyde reduction and hydrolytic loss of forosamine represent novel biotransformation pathways for spiramycin derivatives.

Biotransformation of pantoprazole by the fungus Cunninghamella blakesleeana.

To investigate the biotransformation of pantoprazole, a proton-pump inhibitor, by filamentous fungus and further to compare the similarities between microbial transformation and mammalian metabolism of pantoprazole, four strains of Cunninghamella (C. blakesleeana AS 3.153, C. echinulata AS 3.2004, C. elegans AS 3.156, and AS 3.2028) were screened for the ability to catalyze the biotransformation of pantoprazole. Pantoprazole was partially metabolized by four strains of Cunninghamella, and C. blakesleeana AS 3.153 was selected for further investigation. Three metabolites produced by C. blakesleeana AS 3.153 were isolated using semi-preparative HPLC, and their structures were identified by a combination analysis of LC/MS(n) and NMR spectra. Two further metabolites were confirmed with the aid of synthetic reference compounds. The structure of a glucoside was tentatively assigned by its chromatographic behavior and mass spectroscopic data. These six metabolites were separated and quantitatively assayed by liquid chromatography-ion trap mass spectrometry. After 96h of incubation with C. blakesleeana AS 3.153, approximately 92.5% of pantoprazole was metabolized to six metabolites: pantoprazole sulfone (M1, 1.7%), pantoprazole thioether (M2, 12.4%), 6-hydroxy-pantoprazole thioether (M3, 1.3%), 4'-O-demethyl-pantoprazole thioether (M4, 48.1%), pantoprazole thioether-1-N-beta-glucoside (M5, 20.6%), and a glucoside conjugate of pantoprazole thioether (M6, 8.4%). Among them, M5 and M6 are novel metabolites. Four phase I metabolites of pantoprazole produced by C. blakesleeana were essentially similar to those obtained in mammals. C. blakesleeana could be a useful tool for generating the mammalian phase I metabolites of pantoprazole.

A novel metabolic pathway of morphine: formation of morphine glucosides in cancer patients.

AIMS: To characterize directly the conjugated metabolites of morphine in urine samples of cancer patients. METHODS: Urine samples from the patients were treated by solid-phase extraction method and chromatographed using three high-performance liquid chromatography systems. Conjugated metabolites were directly detected with liquid chromatographic/ion trap mass spectrometric (LC/MSn) technique by selected ion monitoring, full scan MS/MS and MS3 modes. RESULTS: Six conjugated metabolites including two new metabolites M5 and M6 were found. Morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) were identified by comparing their l.c. retention times and multistage mass spectra with those of the reference substances. Two novel metabolites, morphine-3-glucoside and morphine-6-glucoside, as well as normorphine glucuronides were identified by comparing their mass fragment patterns and l.c. retention times with those of M-3-G and M-6-G. Hydrolysis of urine samples with beta-glucosidase and beta-glucuronidase provided further evidence of the metabolites M5 and M6 as morphine glucosides. The excretion amounts of morphine conjugates in urines were in the order of morphine glucuronides, morphine glucosides and normorphine glucuronides. CONCLUSIONS: In the present study, the applications of l.c. separation and multistage mass spectra have permitted the direct identification of conjugated metabolites of morphine. To our knowledge, this is the first report about O-linked glucosides of morphine at 3-aromatic and 6-aliphatic hydroxyl groups.

LC-MS-MS analysis of 2-pyridylacetic acid, a major metabolite of betahistine: application to a pharmacokinetic study in healthy volunteers.

1. A sensitive liquid chromatographic-tandem mas spectrometric assay was developed and validated to determine the major metabolite of betahistine, 2-pyridylacetic acid, in human plasma. 2. The analyte was extracted from plasma samples by liquid-liquid extraction and analysed using liquid chromatography-tandem mass spectrometry with an electrospray ionization interface. The method has a lower limit of quantitation of 1 ng ml(-1) fir a 0.5-ml plasma aliquot. The intra- and interday precision (relative standard deviation), calculated from quality control (QC) samples, was less than 10%. Accuracy as determined from QC samples was within +/-7%. 3. The validated method was successfully applied to a pharmacokinetic study of betahistine in healthy volunteers. After oral administration of a single dose of 24 mg betahistine mesylate to 20 healthy Chinese male volunteers, Cmax was 339.4 ng ml(-1) (range 77.3-776.4 ng ml(-1)). The t(1/2) was 5.2 h (range 2.0(-1)-11.4h). The AUC(0-t) obtained was 1153.5 ng ml(-1) h (range 278.5-3150.8 ng ml(-1)). The disposition of the metabolite exhibited a marked interindividual variation. 4. The plasma concentrations of the parent drug were less than 0.5 ng ml(-1), suggesting that it undergoes almost complete first-pass metabolism. The reported two active metabolites were not detected in the plasma of any volunteer. Although there is no evidence that the major metabolite has pharmacological activity, the clinical importance of 2-pyridylacetic acid in humans should be reinvestigated.

Demethylation metabolism of roxithromycin in humans and rats.

AIM: To investigate the demethylated metabolites of roxithromycin (RXM) in humans and rats, and to study the antibiotic activity of these metabolites in vitro. METHODS: The demethylated metabolites of RXM in humans and in rats were identified by liquid chromatography-mass spectrometry (LC-MS), and the in vitro antibiotic activities of them against three standard strains were also studied compared with those of the parent drug and some other metabolites of RXM. RESULTS: O-Demethylation of RXM was one of the main metabolic routes of RXM in humans, whereas N-demethylation metabolism was more predominant in rats. O-Demethyl-RXM appeared to be equally effective with RXM. CONCLUSION: The O-demethyl-RXM was an active metabolite in humans, and there were some species differences in RXM demethylation metabolism between humans and rats.

[Rapid analysis of terbutaline by combined solid phase extraction/liquid chromatography tandem mass spectrometry].

AIM: To develop a liquid chromatography-electrospray ionization tandem mass spectrometry method for rapid analysis of terbutaline at level of 50 pg.mL-1 in human plasma. METHODS: Samples containing terbutaline and salbutamol (internal standard, IS) were extracted using C18 solid-phase extraction cartridges, followed by liquid chromatographic separation and mass spectrometric detection. The mobile phase consisted of acetonitrile-water-formic acid (20:80:1), at a flow-rate of 0.4 mL.min-1. Selected reaction monitoring with mass transitions m/z 226-->151 and m/z 240-->148 were used for terbutaline and IS, respectively. RESULTS: The chromatographic analysis time for each sample was approximately 3.8 min. The assay was linear from 0.05 to 8.0 ng.mL-1. The between-run precision and accuracy of the quality controls (QCs, 0.1, 0.4 and 4.0 ng.mL-1) were characterized by relative standard deviation (RSD) of 2.5% to 7.1% and relative errors of -3.1% to 5.7%, respectively. The within-run precision of QCs was characterized by RSD of 4.2% to 6.6%. This method was applied to the analysis of samples taken up to 60 h after oral administration of 10 mg bambuterol in healthy volunteers. CONCLUSION: The method is shown to be suitable for clinical investigation of terbutaline pharmacokinetics, which offers advantages of specificity, speed, and greater sensitivity over previously reported methods.

[Determination of amlodipine in human plasma by liquid chromatography-tandem mass spectrometry].

AIM: To develop a sensitive and specific LC/MS/MS method for determination of amlodipine in human plasma. METHODS: Amlodipine and internal standard 4'-hydroxypropafenone were extracted from plasma using liquid-liquid extraction, then separated on a Zorbax C8 column. The mobile phase consisted of acetonitrile-water-formic acid (75:35:1), at a flow-rate of 0.4 mL.min-1. A Finnigan TSQ tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring (SRM) using the precursor-->product ion combinations of m/z 409-->238 and m/z 358-->116 was used to quantify amlodipine and internal standard, respectively. RESULTS: The linear calibration curves were obtained in the concentration range of 0.4-16.0 ng.mL-1. The limit of quantification was 0.4 ng.mL-1. Each plasma sample was chromatographed within 3.7 min. The method was successfully used in several pharmacokinetic studies for amlodipine. More than 1,500 plasma samples were assayed within two weeks. CONCLUSION: The method is proved to be suitable for clinical investigation of amlodipine pharmacokinetics, which offers advantages of specificity, speed, and greater sensitivity over the previously reported methods.

[Pharmacokinetics of meloxicam in healthy Chinese volunteers].

AIM: To assess the pharmacokinetic profile of single doses of meloxicam in healthy Chinese volunteers. METHODS: The plasma concentrations of meloxicam after an oral dose of 15 mg to twenty healthy male volunteers were analyzed by means of a validated HPLC method. The pharmacokinetic parameters were subjected to Shapiro-Wilk test to determine whether these data were fitted to a normal distribution. RESULTS: The twenty volunteers can be classified into extensive metabolizers and poor metabolizers according to pharmacokinetic parameters. The main parameters in the two groups obtained were as follows: T 1/2 were 21 +/- 4 and 38 +/- 9 h, AUC0-infinity were 49 +/- 10 and 110 +/- 8 micrograms.h.mL-1, respectively. Even the AUC data in extensive metabolizers were 1.7 times as that reported in White volunteers following the same doses of meloxicam. CONCLUSION: There were significant individual differences in the pharmacokinetics of meloxicam in Chinese volunteers, which may be due to the genetic polymorphism of CYP2C9.

[Determination of bambuterol in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to pharmacokinetic study].

AIM: To develop a sensitive, specific and accurate method for quantifying bambuterol in human plasma and to study pharmacokinetics of bambuterol in male healthy Chinese. METHODS: Plasma samples were prepared based on a simple liquid-liquid extraction. The extracted samples were analyzed on liquid chromatography using a Zorbax SB C18 column interfaced with a triple quadrupole tandem mass spectrometer and detected by use of selected reaction monitoring mode. RESULTS: The linear calibration curves were obtained in the concentration range of 0.05-4.0 ng.mL-1. The limit of quantification was 0.05 ng.mL-1. The intra- and inter-run precision was measured to be below 7%. The inter-run accuracy was less than 8% for the analyte. After an oral administration of 10 mg bambuterol hydrochloride to 18 healthy Chinese volunteers the main pharmacokinetic parameters of bambuterol were as follows: Tmax was (2.3 +/- 1.3) h; Cmax was (3.95 +/- 2.20) ng.mL-1; T1/2 was (11.4 +/- 6.1) h and AUC0-t was (26.85 +/- 11.77) ng.h.mL-1. CONCLUSION: The method is shown to be accurate, robust and convenient, and suitable for pharmacokinetic studies of bambuterol. It was found that there was marked inter-individual difference in the pharmacokinetics of bambuterol in Chinese volunteers after a single oral dose, which may be attributed to the difference of activity of cholinesterase, an enzyme catalyzing bambuterol metabolism.

[Enantioselective pharmacokinetics of benproperine in healthy volunteers].

AIM: To investigate the enantioselective pharmacokinetic process of benproperine in healthy volunteers. METHODS: An enantiospecific HPLC method was developed and used to determine the plasma concentrations of each enantiomer. Six healthy Chinese male volunteers received an oral dose of 60 mg (+/-)-benproperine. The ratios of the enantiomers in plasma samples were measured on a chiral AGP column. The plasma concentration of each enantiomer was then calculated using the ratios of enantiomers and total concentration of the two enantiomers previously measured. RESULTS: The plasma levels of (-)-(S)-benproperine were always significantly higher than those of its antipode in six volunteers. The mean AUC0-t and Cmax values for (-)-(S)-benproperine were 2.18 and 2.12 times higher than those of (+)-(R)-benproperine. There was no significant difference between the T1/2 for both enantiomers, tested by paired t test (P > 0.05). Half an hour after administration of benproperine, the S/R ratio in plasma samples was as high as 3.8, and in 2 hours it drastically decreased to about 2.2, then kept on till 24 hours. CONCLUSION: Benproperine showed significant enantioselective pharmacokinetics in the human after an oral dose of the racemate.

[Study on hydroxylated metabolites of benproperine in human urine].

AIM: To investigate the hydroxylation process of benproperine in humans. METHODS: After an oral administration of 60 mg benproperine to ten healthy male volunteers, urine samples collected within 0-24 h were extracted by solid phase extraction and analyzed by liquid chromatography-ion trap mass spectrometry. A microbial transformation of benproperine combined with semi-preparative HPLC was used to get two reference substances of the hydroxylated metabolites, and their structures were then elucidated by NMR. Furthermore, the structures of conjugated metabolites were speculated based on their characteristics in MS fragmentation. RESULTS: Five hydroxylated metabolites of benproperine and some of their conjugates with endogenous glucuronic acid or sulfuric acid were found in urine of volunteers after the dose. The structures of two metabolites were identified as 4"-hydroxybenproperine and 4"'-hydroxybenproperine by comparison of HPLC retention times and mass spectra with those of authentics obtained from the microbial transformation. CONCLUSION: Hydroxylation of benproperine occurs preferentially at para-position of the alkoxyl group in the aromatic ring. The hydroxylated metabolites of benproperine in human urine mainly exist as their glucuronic acid or sulfuric acid conjugates.

[Study on major metabolites of 3H-1,2-dihydro-2-(4-methylphenylamino)-methyl-1-pyrrolizinone in rabbits].

The metabolites of a 750 mg oral dose of Z-47 [3H-1, 2-dihydro-2-(4-methylphenylamino) methyl-1-pyrrolizinone], a new anti-inflammatory and analgesic agent, in rabbit urine were separated and detected with high performance liquid chromatographic method. On basis of the chromatographic behavior of Z-47 metabolites and biotransformation pathways of drugs with partial structure of Z-47, the carboxylic derivative of Z-47 [4-(3H-1, 2-dihydro-1-pyrrolizinone-2-methylamino) benzoic acid] was proposed as a potential metabolite so that the compound was synthesized. The authentic substance was then compared with one of the metabolites by the chromatographic retention time and the ratio of their UV-absorbances at two wavelengths. The enzyme-hydrolyzed product of another metabolite was also analysed. It was consequently confirmed that the carboxylic derivative of Z-47 and its acyl beta-D-glucuronide are major metabolits of Z-47 in rabbits.

[Formulation and evaluation of a new content uniformity testing scheme by variables].

A new content uniformity testing scheme by variables for pharmaceutical preparation has been formulated in two steps: 1. The locus of the point (mu, sigma) representing pairing of the two population variables of active ingredient in individual dosage units was found by statistical probability computation and approximated to two connecting lines, one circular segment and one straight line, the equations of which contain the two sample variables mean and S; 2. Two straight lines delimiting a double sampling zone were found by weighing the two contradictory indices, high accuracy and small sample size of the sampling inspection scheme. The sample size in the initial inspection is n1 = 10, while that in the second inspection is n = n1 + n2 = 30. The 4 discriminants of the new scheme are as follows: [formula: see text] where A = [mean - 100]; S = [sigma (X - mean)2/(n - 1)]1/2; l is the tolerant content limit of a dosage unit; k1, k2, k3 and k4 are parameters that depend on l; and other symbols are self-evident. The numbers of k1, k2, k3, k4 for +/- l% = +/- 15% and d%-8, 7, 6 and 5% are given, where d is the acceptance defective in lot. The algorithms used are outlined. Some of the merits of the new scheme--accuracy, adaptability and practicability--are discussed.