Yu-Tse Wu

Determination of melamine in rat plasma, liver, kidney, spleen, bladder and brain by liquid chromatography–tandem mass spectrometry

In this study, we describe a method for the analysis of melamine in rat plasma, liver, kidney, spleen, bladder, and brain using trichloroacetic acid precipitation with mixed-mode cation-exchange solid-phase extraction and hydrophilic interaction chromatography coupled to tandem mass spectrometry detection. Method validation was investigated completely, including linearity, precision, accuracy, matrix effect, extraction recovery, and carryover for the determination of melamine. The method exhibited a good linear range covering 20-500 ng/mL, and the overall precision ranged from 1.6 to 16.3%, with the accuracy varying from -7.9 to 15.1%. The mean matrix effects of melamine in rat plasma, liver, kidney, spleen, bladder, and brain ranged from 66.2+/-6.7 to 95.5+/-13.2%, and the mean recoveries for melamine varied from 79.8+/-8.2 to 113.0+/-9.6%. Rat kidney showed the highest level among the organs (192.5% of the plasma melamine level), and the average concentration of melamine in the brain was only 7.5% of the plasma melamine concentration. This work has pointed out that even with the application of two popular preparation procedures (acid precipitation and solid-phase extraction) of melamine, the matrix effect in analyzing biological samples still exists in certain kinds of matrices.

Pharmacokinetics of gastrodin and its metabolite p-hydroxybenzyl alcohol in rat blood, brain and bile by microdialysis coupled to LC-MS/MS

Gastrodin is a pharmacologically active substance isolated from Gastrodia elata Blume with sedation, anti-convulsion and anti-epilepsy activities. A rapid and sensitive liquid chromatography technique coupled to tandem mass spectrometry (LC-MS/MS) system was developed to determine gastrodin and its metabolite p-hydroxybenzyl alcohol (HBA) in rat blood, brain and bile collected using microdialysis technique. The analytes were separated using a reversed phase column (4.6 mm x 150 mm, 5 microm). The mobile phase for column separation was 30% methanol with a flow rate of 0.6 mL/min. As a post-column addition, 1% ammonium hydroxide solution (in methanol) was additionally pumped via a T-connection using a chromatographic pump (BAS PM-80, USA) at a flow rate of 0.2 mL/min after the column separation. A LC-MS/MS system equipped with a negative electrospray ionization (ESI) source in multiple reaction monitoring (MRM) mode was used to monitor m/z 285.0-->122.9 and m/z 123.0-->105.0 transitions for gastrodin and HBA, respectively. The lower limit of quantification (LLoQ) for gastrodin and HBA were 0.5 and 2 ng/mL, respectively. The calibration curves were linear over the range of 0.5-5,000 ng/mL and 2-1,000 ng/mL for gastrodin and HBA with a coefficient of determination >0.995, respectively. This selective and sensitive method is useful for the determination of gastrodin and HBA and in the pharmacokinetic studies of these compounds.

Biological analysis of herbal medicines used for the treatment of liver diseases

Herbal medicines have been used to treat liver disorders for thousands of years in the East and have now become a promising therapy internationally for pathological liver conditions. Biological analysis of hepatoprotective herbs is an important issue from the pharmacokinetic perspective in developing new therapeutic managements for liver disease. The biological analysis focuses on the pretreatment methods, separation and quantification of herbal medicines in biological samples. We have compiled and discuss the biological analytical method of six herbal medicines for liver protection containing Silybum marianum(silymarin), Glycyrrhiza glabra, Scutellaria baicalensis, Schisandra chinensis, Salvia miltiorrhiza and Astragalus membranaceus. This review provides a convenient reference for researchers to reduce time-consuming method optimization.

Synthesis and biological activity of stable and potent antitumor agents, aniline nitrogen mustards linked to 9-anilinoacridines via a urea linkage

To improve the chemical stability and therapeutic efficacy of N-mustard, a series of phenyl N-mustard linked to DNA-affinic 9-anilinoacridines and acridine via a urea linker were synthesized and evaluated for antitumor studies. The new N-mustard derivatives were prepared by the reaction of 4-bis(2-chloroethyl)aminophenyl isocyanate with a variety of 9-anilinoacridines or 9-aminoacridine. The antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro without cross-resistance to taxol or vinblastine and showed potent antitumor therapeutic efficacy in nude mice against human tumor xenografts. It also showed that 24d was capable of inducing marked dose-dependent levels of DNA cross-linking by comet assay and has long half-life in rat plasma.

Herb–drug interaction of Andrographis paniculata extract and andrographolide on the pharmacokinetics of theophylline in rats

Herb-drug interaction has become a serious problem since herbal medicine is extensively used in the modern world. This study investigates effects of Andrographis paniculata extract (APE) and its major component, andrographolide (AG), on the pharmacokinetics of theophylline, a typical substrate of cytochrome P450 1A2 enzyme, in rats. After APE or AG pretreatment for 3 days, on the fourth day rats were administered theophylline via femoral vein cannula. The blood theophylline levels were monitored by microdialysis sampling combined with HPLC-UV. The results indicated that the clearance of theophylline was significantly increased and the area under concentration-time curve (AUC) was reduced in both AG and APE pretreated groups at low-dose theophylline administration (1mg/kg). The elimination half-life (t(1/2beta)) and mean residence time (MRT) of theophylline were shortened by 14% and 17%, respectively, in the AG pretreated group when high-dose theophylline (5mg/kg) was given. However, theophylline accumulated in rat of the group with APE pretreatment. This phenomenon suggests that some other herbal components contained in APE may interact with theophylline and retard its elimination when theophylline was administered at a high dose. Our results suggest that patients who want to use CYP1A2-metabolized drugs such as caffeine and theophylline should be advised of the potential herb-drug interaction, to reduce therapeutic failure or increased toxicity of conventional drug therapy.

Measurement of free hydroxytyrosol in microdialysates from blood and brain of anesthetized rats by liquid chromatography with fluorescence detection

Hydroxytyrosol [4-(2-hydroxyethyl)-1,2-benzenediol] is a well known natural polyphenolic component with antioxidative effects from olive oil and an aglycone of acteoside. In order to examine the in vivo metabolism of acteoside to hydroxytyrosol and the distribution of hydroxytyrosol in the blood and brain, microdialysis coupled to a liquid chromatographic system was developed to evaluate the pharmacokinetics of free-form hydroxytyrosol in rat blood and brain. Probes were implanted in the jugular vein and the brain hippocampus for blood and brain sampling purposes. Hydroxytyrosol in the microdialysis samples was separated by a reversed-phase C18 column and eluted with a mobile phase containing acetonitrile - 2% acetic acid (pH 2.6) (12:88, v/v), using a flow rate for the mobile phase of 1 mL/min. Fluorescence detection for hydroxytyrosol was set at 281 nm and 316 nm for excitation and emission wavelengths, respectively. Hydroxytyrosol and endogenous interference could be resolved within 10 min by the developed chromatographic method. The results indicated that acteoside was metabolized immediately to hydroxytyrosol in vivo and eliminated rapidly from the blood, and hydroxytyrosol could enter the brain. The blood-to-brain distribution ratio was defined by dividing the area under concentration versus time (AUC) ratio of AUC(brain)/AUC(blood), which represents the AUC for brain and blood. The results suggested that the P-glycoprotein was not involved in the brain efflux transport of hydroxytyrosol.

Oral bioavailability, urinary excretion and organ distribution of melamine in Sprague-Dawley rats by high-performance liquid chromatography with tandem mass spectrometry.

High-performance liquid chromatography with tandem mass spectrometry (HPLC/MS/MS) was used to determine melamine oral bioavailability (BA) and urinary excretion. Organ distribution after a 14-day consecutive oral melamine administration (100 mg/kg/day, once a day) was also evaluated. A noncompartmental model was utilized to obtain pharmacokinetic parameters. According to the results, the BA of melamine was estimated to be 98.1%. Approximately 63% of administered melamine was recovered in urine within 96 h after a single oral administration (100 mg/kg). The bladder had the highest melamine concentration of all the organs after a 14-day consecutive oral administration of melamine, and almost no melamine was found in the rat brain. This result indicated that the oral absorption of melamine was almost complete and urinary excretion was the major route for its elimination. Repeated exposure to high-dose melamine may result in only slight accumulation in organs.

Analysis of biliary excretion of icariin in rats.

Icariin is a bioactive herbal ingredient isolated from Epimedii Herba. This study evaluates the distribution of icariin in rats by microdialysis sampling and high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Microdialysis probes were simultaneously placed in the jugular vein, brain striatum, and bile duct of each anesthetized rat for sampling after the administration of icariin (dose=10 or 20 mg/kg) via the femoral vein. The role of P-glycoprotein (P-gp) on icariin distribution was assessed by pretreatment with cyclosporine (CsA, dose=20 mg/kg). This study is the first report of the biliary excretion of icarin in rats, defined as the blood-to-bile distribution (k value), calculated by dividing the area under the concentration-time curve (AUC) of icariin in bile by that in blood (k=AUCbile/AUCblood). The k values were 19.0±5.9 and 18.8±3.8 at the doses of 10 and 20 mg/kg, respectively. The decreased biliary excretion of icariin due to pretreatment with CsA was evidenced by the reduced k values (18.8±3.8 vs 9.9±1.9, p=0.005). This work demonstrates that biliary excretion is the major elimination pathway for icariin disposition and that transporters, such as P-gp, might be related to icariins biliary excretion.

Pharmacokinetics and Urine Metabolite Identification of Dehydroevodiamine in the Rat

This study investigates the oral bioavailability and characterizes urine metabolites of dehydroevodiamine (DeHE), one of the bioactive alkaloids isolated from the fruit of Evodia rutaecarpa . A freely moving rat model coupled with an automated blood sample system was used to evaluate the pharmacokinetics of DeHE. High-performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectrometry were applied to determine DeHE and its metabolites. The averaged oral bioavailability of DeHE (100 and 500 mg/kg) in the freely moving rats was approximately 15.35%. Cumulative fecal and urinary excretions of unchanged DeHE were 6 and 0.5%, respectively, after a single oral dose (500 mg/kg) of DeHE. The protein binding of DeHE in rat plasma was 65.6 ± 6.5%. Six metabolites, including five DeHE-O-glucuronides and one DeHE-sulfate, were identified after oral administration. The structures of two glucuronide conjugates, DeHE-10-O-glucuronide (M3) and DeHE-11-O-glucuronide (M4), and one sulfate conjugate, DeHE-12-sulfate (M6), were assigned. The findings indicate that the oral bioavailability of DeHE was much higher than that of evodiamine, and hydroxylation and conjugative metabolism were essential for the urinary elimination of DeHE.

Herb―drug interaction of silymarin or silibinin on the pharmacokinetics of trazodone in rats

Silymarin, one of the most popular herbal medicines, has been widely used for its hepatoprotective effects. This study investigates the effects of repeated dose of silymarin and its major ingredient, silibinin, on the pharmacokinetics of the antidepressant trazodone. Treatment groups included vehicle control group, concomitant silymarin at 1.0g/kg dose, and four 7-day repeated dose induction groups of 0.5 and 1.0g/kg silymarin and 0.175 and 0.35g/kg silibinin. Microdialysis coupled with high performance liquid chromatography (HPLC) was used to simultaneously monitor blood and bile concentrations of trazodone in the rats. Results indicate that pretreatment with an extremely high dose of 1.0g/kg silymarin significantly decreases trazodones area under concentration curve (AUC), distribution half-life (t(1/2,alpha)), elimination half-life (t(1/2,beta)), and mean residence time (MRT). In conclusion, the present study finds no marked effects of silymarin and silibinin on the pharmacokinetics of trazodone under normal daily doses and the relative safety of taking the herb with trazodone.