Ruichen Guo

Metabolism and Disposition of Tribendimidine and Its Metabolites in Healthy Chinese Volunteers

AbstractBackground: Tribendimidine is a new anthelmintic agent synthesized by Chinese scientists. It is a broad spectrum agent with high activity against parasites. However, its disposition and metabolism remain unknown. Objective: To investigate the metabolism, disposition, and metabolites of tribendimidine in healthy human volunteers. Methods: Twelve healthy Chinese volunteers were chosen after clinical assessment of health status and laboratory tests. They received single oral doses of tribendimidine 400mg enteric-coated tablets. Blood and urine samples were collected at scheduled timepoints. Samples were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometric (LC-MS) and high performance liquid chromatography (HPLC) methods, respectively. Results: Tribendimidine was rapidly and completely broken down to p-(1-dimethylamino ethylimino) aniline (dADT) and terephthalaldehyde (TPAL). Furthermore, dADT was partially transformed to acetylated dADT, and TPAL completely transformed to terephalic acid (TPAC). The main pharmacokinetic parameters (± SD) of dADT were as follows: elimination half life (t1/2) 4.74 ± 1.80 h; elimination rate constant (Ke) 0.16 ± 0.06 h−1; apparent volume of distribution (Vd/F) 12.23 ± 8.69L • kg−1; apparent total clearance of the drug from plasma (CL/F) 1.63 ± 0.58L • h−1 • kg−1; area under the plasma concentration-time curve (AUC) from time 0 to time 24 hours (AUC24) 4.29 ± 1.88 μg • mL−1 • h; AUC from time zero to infinity (AUC∞) 4.45 ± 1.81 μg • mL−1 • h; maximum plasma drug concentration (Cmax) 0.64 ± 0.27 μg • mL−1; and time to Cmax (tmax) 4.20 ± 0.71 h. A total of 35.28% dADT and 28.50% TPAC were excreted through the urine within 24 hours after tribendimidine administration. Conclusion: These results reveal the disposition, metabolism, and main metabolites of tribendimidine in healthy Chinese volunteers.

Determination of salbutamol in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry and its pharmacokinetic study

A sensitive and selective liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed and validated for the determination of salbutamol in human plasma and urine, and successfully applied to the pharmacokinetic study of salbutamol in Chinese healthy volunteers after inhalation of salbutamol sulfate aerosol. Salbutamol and the internal standard (IS) acetaminophen in plasma and urine were extracted with ethyl acetate, separated on a C(18) reversed-phase column, eluted with mobile phase of acetonitrile-ammonium acetate (5 m m; 30:70, v/v), ionized by positive ion pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor → product ions of m/z 240.2 → 148.1 for salbutamol and 152 → 110 for the IS. The lower limits of quantitation of salbutamol in human plasma and urine by this method were 0.02 and 1 ng/mL, respectively. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision and several stabilities were validated for salbutamol in human plasma and urine. In conclusion, the validation results showed that this method is robust, specific and sensitive, and can successfully fulfill the requirement of clinical pharmacokinetic study of salbutamol in healthy Chinese volunteers.

Simultaneous determination of lercanidipine, benazepril and benazeprilat in plasma by LC–MS/MS and its application to a toxicokinetics study

We aim to develop a rapid, simple, sensitive and specific LC-MS/MS method for the simultaneous quantification of lercanidipine, benazepril and benazeprilat in plasma. It is performed on the Agilent 6410 LC-MS/MS under the multiple-reaction monitoring (MRM) mode with electrospray ionization. Gliclazide was used as the internal standard (IS). Analytes and IS were extracted from plasma by solid-phase extraction. The reconstituted samples were chromatographed on a Diamond C₁₈(150 mm × 4.6 mm, 5 μm) column. The mobile phase was composed of 0.1% acetic acid-acetonitrile (50:50, v/v), with gradient flow rates: 0.6 mL/min (0-4.55 min); 4.55-4.65 min, 1 mL/min; 1 mL/min (4.65-9.5 min); 9.5-9.6 min, 0.6 mL/min; 0.6 mL/min (9.6-10 min). Method validation demonstrated that the method was of satisfactory specificity, sensitivity, precision and accuracy in linear ranges of 1-2000 ng/mL for lercanidipine, 1-2000 ng/mL for benazepril and 1-1600 ng/mL for benazeprilat, respectively. The precision (RSD%) was better than 15, and the lower limit of quantitation was identifiable and reproducible at 1 ng/mL for the three analytes. The plasma samples were stable after being stored for more than 60 days and after two freeze-thaw cycles (-20 to -25 °C). It is demonstrated that this method was successfully applied to samples from a toxicokinetics study of a compound of lercanidipine and benazepril in beagle dogs.

Separation and identification of norcantharidin metabolites in vivo by GC-MS method.

Norcantharidin (NCTD), the demethylated analogue of cantharidin, inhibits the proliferation of a variety of human tumor cell lines, and appears to cause the least nephrotoxic and inflammatory side effects. Although NCTD has been used to treat human cancers in China for years, there is no report regarding its metabolism up to now. This is the first report to separate and identify the main metabolites of NCTD in vivo by GC-MS using TMS derivatives. Two hydrolyzed products and five phase I or phase II metabolites were found in rat by the chromatogram comparisons of the blank with incurred biological samples. Multiple stages of fragmentation patterns were used to confirm the metabolites characterizations. The established GC-MS method can also be applied to identifying unknown metabolites of the drugs containing hydroxyl or carbonyl groups in molecular structure.

In vivo metabolism study of bergenin in rats by HPLC-QTOF mass spectrometry.

Bergenin is the major component of Ardisia creanta sims and Rodgersia sambucifolia hemsl with many biological activities. Although bergenin has been used to treat human diseases in China for man years, there is no report regarding its metabolism. This is the first report to separate and identify the metabolites of bergenin in vivo. In the study, HPLC/Q-TOF-MS/MS was used to investigate the metabolites of bergenin in vivo by analyzing the rat body fluid and feces samples. Three metabolites of bergenin were finally identified by the TIC chromatograms, and the structures were also confirmed by their MS(2) spectra.

Development and validation of a sensitive LC-MS method for the determination of promethazine hydrochloride in human plasma and urine.

SummaryWe developed and validated a sensitive and low sample volume liquid chromatographic-mass spectrometric (LC-MS) method for determination of Promethazine hydrochloride in human plasma (0.5 ml) or urine (0.1 ml). The lower limit of quantification in human plasma and urine was 1.00 ng/ml. The inter- and intra-day precisions (CV %) in both plasma and urine were lower than 10%, the mean method accuracies and recoveries from spiked plasma samples at three concentrations were more than 97%. The developed method was successfully applied to determine Promethazine hydrochloride in human plasma and urine, and proved suitable to clinical pharmacokinetic study.

Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of bergenin.

Glucuronidation plays critical role in the elimination of bergenin; however the metabolic mechanism of UDP-glucuronosyltransferases (UGTs) in the process remains to be investigated. In this study, the kinetics of bergenin glucuronidation by pooled human liver microsomes (HLMs) and 12 recombinat UGT isozymes were investigated. The glucuronidation of bergenin can be shown in HLMs with a Km value of 231.62 ± 14.08 µm and a Vmax value of 2.17 ± 0.21 nmol/min/(mg protein). Among the 12 human UGTs investigated, UGT1A1 was identified as the major isoform catalyzing the glucuronidation of bergenin [Km value of 200.37 ± 26.73 µm and Vmax value of 1.88 ± 0.26 nmol/min/(mg protein)]. The bergenin glucuronosyltransferase activities in HLMs and UGT1A1 were inhibited by phenylbutazone, estradiol and bilirubin. The results demonstrate that bergenin glucuronidation in HLMs is specifically catalyzed by UGT1A1.

Development and validation of a sensitive LC-MS method for the determination of tramadol in human plasma and urine.

SummaryA sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS) method was developed and validated for the determination of Tramadol in human plasma and urine. The analyte was separated on a Diamonsil C18 column with ammonium acetate( 5mmol·L−1)-methanol(50:50, v:v) adjusted PH by caustic soda at a flow rate of 0.8ml min−1, and analyzed by mass spectrometry is in positive ion mode. The ion mass spectrum of m/z were 264.1 for Tramadol and 248.0 for Tinidazole (I.S.), respectively. The weighted (1/x2) calibration curve was linear over plasma concentration range 1.00–400.00ng/ml and urine concentration range 0.01–16.00 ng/ml, with a correlation coefficient (r) of 0.9995 and 0.9997, respectively. The lower limit of quantification in human plasma was 1.00ng/ml. The inter- and intra-day precisions (CV%) in both plasma and urine were lower than 10%, the mean method accuracies and recoveries from spiked plasma samples at three concentrations ranged from 98.2 to 100.1% and 61.6 to 62.9%, respectively. The developed method was successfully applied to determine Tramadol in human plasma and urine, and provided suitable profiles for clinical pharmacokinetic study of Tramadol.

Determination of brusatol in plasma and tissues by LC–MS method and its application to a pharmacokinetic and distribution study in mice

OBJECTIVES The quassinoid brusatol, which can be isolated from Brucea javanica (L.) Merr., becomes popularly studied because of its anti-tumor activity. In order to further investigate brusatol and extend its applications, a sensitive analytical method for determination of brusatol in biological samples is essential. However, few methods had been reported until now. In this study, a highly sensitive and reproducible LC-MS method for simultaneous quantification of brusatol in mouse plasma and tissues was developed and validated. METHOD Plasma samples and tissue homogenate were extracted with diethyl ether after addition of the internal standard solution(IS). The supernatant was blown to dryness with nitrogen and residual was reconstituted with 100μl of methanol. The separation was performed on an Intersil ODS-3 column and gradient elution was conducted with the mobile phase of water and methanol (0-5min 47:53, 5-5.5min 47:53-10:90, 5.5-9min 10:90, posttime 4min 47:53) at a flow rate of 0.8mL/min. Quantification was performed in the selected ion monitoring (SIM) mode at m/z 543.2 for brusatol and 220.0 for IS (ornidazole). The method was validated by analyzing quality control plasma and tissue homogenate samples, and was applied to analyze samples obtained from mice after injections of brusatol via the tail vein. RESULTS With ornidazole as the internal standard, calibration curve of the method ranged from 10 to 320ng/ml for plasma and 10-240ng/ml for tissues. Recovery rate of brusatol from plasma and tissues were between 71.09%-94.91%. Relative standard deviation (RSD) for inter- and intra-day precision was less than 15%, and the accuracy was between 96.1%-111.8%. The pharmacokinetics and distribution study of brusatol in mice after three single doses via the tail vein were carried out based on this method. The concentration of brusatol in plasma decreased rapidly and a more than 10 fold concentration of brusatol was found as compared to that in other tissues. CONCLUSIONS This is the first reported LC-MS method for detecting brusatol in tissues and can accurately determine the concentrations of these compounds in plasma and different tissues. Further research on the metabolism of brusatol in vivo is still needed.

Construction of expression system of rabbit aldehyde oxidase cDNA for the clarification of species differences

SummaryA remarkably large species difference in cinchonidine oxidation activity catalyzed by aldehyde oxidase (AO) has been known, in particular between rabbit and monkey. As the first step in clarifying the phenomenon from the view point of structures of the active site, we attempted to construct an expression system of rabbit AO cDNA. The nucleotide sequences of cloned full-length rabbit AO cDNA were determined and confirmed to agree completely with those of genome DNA. The expression system inEscherichia coli was constructed in reference to the previously established method for monkey AO. Both expressed rabbit and monkey AO proteins correctly reproduced the remarkable species differences observed in their liver cytosols towards cinchonidine and methotrexate. Namely, the expressed rabbit AO protein showed extremely high activities than did that of monkey AO. A difference in the structure of the active site might be responsible for the substrate-dependent species difference towards the relatively bulky molecules of cinchonidine and methotrexate. The use of molecular biology techniques will be very useful to verify the hypothesis.