Comparative study on the excretion of vitexin‐4′′‐O‐glucoside in mice after oral and intravenous administration by using HPLC

The aim of the present study was to characterize the excretion of pure vitexin‐4”‐O‐glucoside (VOG) in mice following oral and intravenous administration at a dose of 30 mg/kg. A sensitive and specific HPLC method with hespridin as internal standard, a Diamonsil C₁₈ column protected with a KR C₁₈ guard column and a mixture consisting of methanol–acetonitrile–tetrahydrofuran–0.1% glacial acetic acid (6:2:18:74, v/v/v/v) as mobile phase was developed and validated for quantitative analysis in biological samples. VOG could be excreted as prototype in excreta including urine and feces after both routes of administration, and the cumulative excretion of VOG was 24.31 ± 11.10% (17.97 ± 5.59% in urinary excretion; 6.34 ± 5.51% in fecal excretion) following oral dosing and 5.66 ± 3.94% (4.78 ± 3.13% in urinary excretion; 0.88 ± 0.81% in fecal excretion) following intravenous dosing. The results showed that the elimination of VOG after the two routes was fairly low, which meant that VOG was metabolized as other forms and the elimination after oral dosing was almost 4.3‐fold that after intravenous dosing. For both routes of administration, VOG excreted as prototype in urine was much more than that in feces, nearly 2.83‐fold for oral administration and 5.43‐fold for intravenous administration, which should be attributed to enterohepatic circulation. Taken together, renal excretion was the dominant path of elimination of VOG for oral and intravenous administration in mice and biliary excretion contributed less. Copyright © 2013 John Wiley & Sons, Ltd.     

Pharmacokinetics of 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐D‐glucoside in rat using ultra‐performance LC‐quadrupole TOF‐MS

2,3,5,4′‐Tetrahydroxystilbene‐2‐O‐β‐D‐glucoside (THSG) from Polygoni multiflori has been demonstrated to possess a variety of pharmacological activities, including antioxidant, anti‐inflammatory and hepatoprotective activities. Ultra‐performance LC‐quadrupole TOF‐MS with MS Elevated Energy data collection technique and rapid resolution LC with diode array detection and ESI multistage MSⁿ methods were developed for the pharmacokinetics, tissue distribution, metabolism, and excretion studies of THSG in rats following a single intravenous or oral dose. The three metabolites were identified by rapid resolution LC‐MSⁿ. The concentrations of the THSG in rat plasma, bile, urine, feces, or tissue samples were determined by ultra‐performance LC‐MS. The results showed that THSG was rapidly distributed and eliminated from rat plasma. After the intravenous administration, THSG was mainly distributing in the liver, heart, and lung. For the rat, the major distribution tissues after oral administration were heart, kidney, liver, and lung. There was no long‐term storage of THSG in rat tissues. Total recoveries of THSG within 24 h were low (0.1% in bile, 0.007% in urine, and 0.063% in feces) and THSG was excreted mainly in the forms of metabolites, which may resulted from biotransformation in the liver.     

Simultaneous stereoselective analysis of naftopidil and O‐desmethyl naftopidil enantiomers in rat feces using an online column‐switching high‐performance liquid chromatography method

A novel online column‐switching chiral high‐performance liquid chromatography method was developed and validated for the simultaneous determination of naftopidil (NAF) and its O‐desmethyl metabolites (DMN) enantiomers in rat feces. Direct and multiple injections of supernatant from rat feces homogenate were allowed through the column‐switching system. Analyte extraction was performed on the Capcell Pak mixed‐functional column by acetonitrile–phosphate buffer (pH 7.4; 10 mm ; 8:92, v/v) flowing at 1 mL/min. Separation of NAF and DMN enantiomers was achieved on the Chiralpak IA column by methanol–acetonitrile–acetate buffer (pH 5.3; 5 mm ; 45:33:22, v/v/v) flowing at 0.5 mL/min. The analytes were measured with a fluorescence detector at 290 nm (λ_ex) and 340 nm (λ_em). The validated method showed a good linearity [22.5–15,000 ng/mL for (+)‐/(?)‐NAF; 35–25,000 ng/mL for (+)‐/(?)‐DMN] and the lowest limits of quantification for NAF and DMN enantiomers were 22.5 and 35 ng/mL, respectively. Both intra‐ and inter‐day variations were <10%. The assay was successfully applied to the fecal excretion of NAF and DMN enantiomers in rat after single oral administration of (±)‐NAF. Nonstereoselective excretion of (+)‐ and (?)‐NAF was found in feces, while stereoselective excretion of (+)‐ and (?)‐DMN was observed with higher excretion levels of (+)‐DMN, indicating that there may exist stereoselective metabolism for NAF enantiomers. Copyright © 2014 John Wiley & Sons, Ltd.     

Pharmacokinetic and metabolism studies of bavachinin through ultra‐high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Bavachinin (BVC), one of the main bioactive prenylated flavonoids derived from Psoralea corylifolia Linn, has a wide variety of pharmacological effects, such as antiangiogenic, antitumor, antiallergic, anti‐inflammatory and antibacterial activities, especially as a pan‐peroxisome proliferator‐activated receptors agonist. A rapid and sensitive method for quantifying BVC in rat plasma was developed and validated through ultra‐high‐performance liquid chromatography coupled with electrospray‐ionization tandem mass spectrometry. Furthermore, a complete metabolic investigation of BVC was performed through ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. In the pharmacokinetic analysis, BVC exhibited rapid oral absorption (T_max = 0.68 ± 0.21 h), good elimination (T_1/2 = 2.27 ± 1.63 h) following oral administration and poor absolute bioavailability (5.27%). Moreover, 11 metabolites of BVC in plasma, urine, bile and feces were characterized. The main metabolic pathways of BVC involved isomeriszation, glucuronidation, sulfonation, hydroxylation, methoxylation and reduction. In conclusion, the present study provides a sensitive quantitative method with a lower limit of quantification of 1 ng/mL and an improved comprehension of the physiological disposition of BVC.     

Studies on pharmacokinetics,body distribution,plasma protein binding rate,and excretion of 1-methyl hydantoin in rats in vivo

In this paper, plasma concentration, plasma protein binding rate, body distribution, and excretion of both oral and intravenous administration of rats were determined by high performance liquid chromatography (HPLC) combining with UV detector. The blood drug concentration of oral and intravenous administration was summarized. The bioavailability of T_1/2 was approximately 0.75?hr. At the meanwhile, the bioavailability was about 18.84?±?2.21%. The plasma protein binding rate of 1-methyl hydantoin was about 24.36?±?0.93%, belonging to low protein binding drug. The result shows that 1-methyl hydantoin can be rapidly distributed in various organs and tissues and quickly eliminated within 6?hr without accumulation in the organs. Its discharge from the urine and feces was 16.58?±?4.48% and 3.37?±?0.71%, respectively. All of the results showed that the recovery rate, liner relationship, specificity, stability, and precision of the method were good. The study also proved that 1-methyl hydantoin has been eliminated quite faster in rats.     

Identification and quantitative analysis of physalin D and its metabolites in rat urine and feces by liquid chromatography with triple quadrupole time‐of‐flight mass spectrometry

Physalin D is known to show extensive bioactivities. However, no excretion study has elucidated the excretion of physalin D and its metabolites. This study investigates the excretion of physalin D and its metabolites in rats. Metabolites in rat urine and feces were separated and identified by liquid chromatography with triple quadrupole time‐of‐flight mass spectrometry. Furthermore, a validated high‐performance liquid chromatography with tandem mass spectrometry method was developed to quantify physalin D, physalin D glucuronide, and physalin D sulfate in rat feces and urine after the intragastric administration of physalin D. The analyte showed good linearity over a wide concentration range (r  > 0.995), and the lower limit of quantification was 0.0532 μg/mL and 0.226 μg/g for urine and feces, respectively. Nine metabolites, including five phase I and four phase II metabolites, were identified and clarified after dosing in vivo. Only 4.0% of the gavaged dose, including physalin D and its phase II metabolites, was excreted in urine, whereas 10.8% was found in feces in the unchanged form. The results indicate that the extensive and rapid metabolism may be the main factors leading to the short half‐life of physalin D. These results can provide a basis for further studies on the structural modification and pharmacology of physalin D.     

Determination of a novel nonfluorinated quinolone,nemonoxacin, in human feces and its glucuronide conjugate in human urine and feces by high‐performance liquid chromatography–triple quadrupole mass spectrometry

Three methods were developed and validated for determination of nemonoxacin in human feces and its major metabolite, nemonoxacin acyl‐β‐ d ‐glucuronide, in human urine and feces. Nemonoxacin was extracted by liquid–liquid extraction in feces homogenate samples and nemonoxacin acyl‐β‐ d ‐glucuronide by a solid‐phase extraction procedure for pretreatment of both urine and feces homogenate sample. Separation was performed on a C₁₈ reversed‐phase column under isocratic elution with the mobile phase consisting of acetonitrile and 0.1% formic acid. Both analytes were determined by liquid chromatography–tandem mass spectrometry with positive electrospray ionization in selected reaction monitoring mode and gatifloxacin as the internal standard. The lower limit of quantitation (LLOQ) of nemonoxacin in feces was 0.12 µg/g and the calibration curve was linear in the concentration range of 0.12–48.00 µg/g. The LLOQ of the metabolite was 0.0010 µg/mL and 0.03 µg/g in urine and feces matrices, while the linear range was 0.0010–0.2000 µg/mL and 0.03–3.00 µg/g, respectively. Validation included selectivity, accuracy, precision, linearity, recovery, matrix effect, carryover, dilution integrity and stability, indicating that the methods can quantify the corresponding analytes with excellent reliability. The validated methods were successfully applied to an absolute bioavailability clinical study of nemonoxacin malate capsule. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of a novel anticancer c‐Met inhibitor LS‐177 in rat plasma and tissues with a validated UPLC‐MS/MS method: application to pharmacokinetics and tissue distribution study

LS‐177 is a novel small‐molecule kinase inhibitor employed to interrupt the c‐Met signaling pathway. A rapid and sensitive ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for determination of LS‐177 in rat plasma and tissues. The biosamples were extracted by liquid–liquid extraction with methyl tert‐butyl ether and separated on a C₁₈ column (50 × 4.6 mm, 2.6 µm) using a gradient elution mobile phase consisting of acetonitrile–0.1% formic acid water. Under the optimal conditions, the selectivity of the method was satisfactory with no endogenous interference. The intraday and interday precisions (relative standard deviation) were <10.5% and the accuracy (relative error) was from ?12.5 to 12.5% at all quality control levels. Excellent recovery and negligible matrix effects were observed. Stability studies showed that LS‐177 was stable during the preparation and analytical processes. The UPLC‐MS/MS method was successfully applied to pharmacokinetic and tissue distribution studies. The results indicated that there was no significant drug accumulation after multiple‐dose oral administration of LS‐177. The tissue distribution study exhibited significant higher uptakes of LS‐177 in stomach, intestine, lung and liver among all of the tissues. The results in pharmacokinetics and tissue distribution may provide a meaningful basis for clinical application. Copyright © 2014 John Wiley & Sons, Ltd.     

Systematic characterization of the metabolites of echinacoside and acteoside from Cistanche tubulosa in rat plasma,bile, urine and feces based on UPLC‐ESI‐Q‐TOF‐MS

Echinacoside (ECH) and acteoside (ACT), as the most and major active components of Cistanche tubulosa, were reported to possess cardioactive, neuroprotective and hepatocyte protective effects, as well as antibacterial, antioxidative effects. Recently, more studies have focused on their pharmacological activities. However, their metabolic profiles in vivo have not been sufficiently investigated. This study proposes an approach for rapidly identifying the complicated and unpredictable metabolites of ECH and ACT in rat plasma, bile, urine and feces, and systematically and comprehensively revealing their major metabolic pathways, based on powerful ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry. Plasma, bile, urine and feces were collected from rats after a single 200 mg/kg oral dose. A total of 49 metabolites were detected in rat biological samples. Through analyzing metabolites in bile samples, it was found that ECH and ACT were subjected to a marked hepatic first‐pass effect in liver. Copyright © 2016 John Wiley & Sons, Ltd.     

An expedient reverse‐phase high‐performance chromatography (RP‐HPLC) based method for high‐throughput analysis of deferoxamine and ferrioxamine in urine

The present study was planned to optimize and validate an expedient reverse‐phase high chromatography (RP‐HPLC) based protocol for the analysis of deferoxamine (DFO) and ferrioxamine (FO) in urinary execration of patients suffering β ‐thalassemia major. The optimized RP‐HPLC method was found to be linear over the wide range of DFO and FO concentration (1–90 μg/mL) with appreciable recovery rates (79.64–97.30%) of quality controls at improved detection and quantitation limits and acceptable inter and intraday variability. Real‐time analysis of DFO and FO in the urine of thalassemic patients (male and female) at different intervals of Desferal®(Novartis Pharmaceuticals Corporation) injection revealed DFO and FO excretion at significantly (p < 0) different rates. The maximum concentrations of DFO (76.7 ± 3.06 μg/mL) and FO (74.2 ± 3.25 μg/mL) were found in urine samples, collected after 6 h of drug infusion while the minimum levels of DFO (1.10 ± 0.12 μg/mL) and FO (2.97 ± 0.13 μg/mL) were excreted by patients after 24 h. The present paper offers balanced conditions for an expedient, reliable and quick determination of DFO and FO in urine samples.     

Absorption and distribution of lupeol in CD‐1 mice evaluated by UPLC–APCI+–MS/MS

Lupeol is a dietary triterpene that shows limited water solubility, which affects its bioavailability. It is well known that poor oral bioavailability is one of the major causes of therapeutic variability. Lupeol has been reported to have multiple biological activities; however, there are no reports about its bioavailability. Therefore, the objective of this research was to evaluate the systemic bioavailability of lupeol. An experimental strategy with three groups of female CD‐1 strain mice was proposed (control, olive oil and lupeol in olive oil), at six experimental times (0.5, 2, 4, 8, 12 and 24 h) with four animals per experimental point. Mice were sacrificed for organs, urine, feces and blood collection. Lupeol was extracted from samples and analyzed by UPLC–APCI⁺–MS/MS, obtaining the pharmacokinetics parameters time to peak concentration 6.444 ± 0.851 h and peak concentration 8.071 ± 2.930 μg/mL. Study of direct digestion and absorption in various organs showed important concentrations of lupeol at earlier post‐administration times (stomach, 137.25 ± 19.94 ng/mg and small intestine, 99.00 ± 12.99 ng/mg). The main excretion route was fecal, with a peak at 12 h post‐administration (163.28 ± 9.83 μg/mg). Absorption of lupeol by the animals was better than expected despite its nonpolar nature (extent of absorption F = 0.645 ± 0.0581).     

Pharmacokinetics,protein binding and metabolism of a quinoxaline urea analog as an NF‐κB inhibitor in mice and rats by LC‐MS/MS

13–197 is a novel NF‐κB inhibitor that shows promising in vitro efficacy data against pancreatic cancer. In this study, we characterized the pharmacokinetics, tissue distribution, protein binding and metabolism of 13–197 in mice and rats. A valid, sensitive and selective LC‐MS/MS method was developed. This method was validated for the quantification of 13–197, in the range of 0.1 or 0.2‐500 ng/mL in mouse plasma, liver, kidney, lung, heart, spleen, brain, urine and feces. 13–197 has low bioavailability of 3 and 16% in mice and rats, respectively. It has faster absorption in mice with 12‐fold shorter T_max than in rats. Tissue concentrations were 1.3–69.2‐fold higher in mice than in rats at 72 h after intravenous administration. 13–197 is well distributed to the peripheral tissues and has relatively high tissue–plasma concentration ratios, ranging from 1.8 to 3634, in both mice and rats. It also demonstrated more than 99% binding to plasma proteins in both mice and rats. Finally, <1% of 13–197 is excreted unchanged in urine and feces, and metabolite profiling studies detected more than 20 metabolites in mouse and rat plasma, urine and feces, which indicates that 13–197 is extensively metabolized and primarily eliminated by metabolism rather than by excretion. Copyright © 2013 John Wiley & Sons, Ltd.     

UHPLC‐MS method for determination of gambogic acid and application to bioavailability,pharmacokinetics, excretion and tissue distribution in rats

A sensitive ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC‐MS) method was developed for determination of gambogic acid (GA) in rat plasma, urine, bile and main tissues. GA was separated on an Agilent Zorbax XDB–C₁₈ column (50 × 2.1 mm, 1.8 µm) with gradient mobile phase at the flow rate of 0.2 mL/min. The detection was performed by negative electrospray ionization with multiple reaction monitoring mode. The calibration curves of GA were linear between 1.0 and 1000 ng/mL in rat plasma and bile and between 1.0 and 500 ng/mL in urine and tissues. The lowest limit of quantification for all matrices was 1.0 ng/mL. Both accuracy and precision of the assay were satisfactory. This validated method was firstly applied to bioavailability (BA), pharmacokinetics, excretion and tissue distribution in rats. The BAs of GA (40 and 80 mg/kg) in rats were 0.25 and 0.32%, respectively. GA was distributed extensively in rats after oral administration and exhibited the highest level in liver. GA reached the cumulative excretion amount of 25.3 ± 1.7 µg in bile and 0.275 ± 0.08 µg in urine after i.g. 80 mg/kg to rats at 24 h. The present results would be helpful for further clinical use of GA as a potential anticancer drug. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitative Monitoring of Rutin in Human Urine by Flow Injection‐Chemiluminescence Analysis

In this paper, a rapid and sensitive flow injection‐chemiluminescence (FI‐CL ) method is proposed for the quantitative determination of rutin based on the inhibitory effect of rutin on the chemiluminescence intensity from the luminol–chymotrypsin (CT ) system. The decrease of CL intensity was found to be proportional to the logarithm of rutin concentration in the range 0.1–30.0 ng/mL . A method for the quantification of rutin is proposed, with the limit of detection (LOD ) of 0.03 ng/mL (3σ). A complete analytical process including sampling and washing for rutin determination, which was conducted at a flow rate of 2.0 mL /min, could be performed completely within 30 s, yielding a sample efficiency of 120 h⁻¹. The proposed procedure was successfully applied for the determination of rutin in human urine after oral intake, with recoveries varying from 93.9 to 108.1% and relative standard derivation <4.0% (n = 5). Results showed that urine reached the maximum concentration at ~2.5 h, and the total excretion ratios were (83.5 ± 0.6) and (86.8 ± 0.7)%, respectively, for two volunteers in 8 h. The pharmacokinetic parameters, including the half‐life (1.05 ± 0.02 h), absorption rate constant (1.18 ± 0.01 h⁻¹), and elimination rate constant (0.70 ± 0.01 h⁻¹), were obtained. The possible CL mechanism of the luminol–CT –rutin reaction is discussed by FI‐CL , fluorescence, and molecular docking methods.     

Study on the pharmacokinetics and metabolism of costunolide and dehydrocostus lactone in rats by HPLC‐UV and UPLC‐Q‐TOF/MS

A method based on high‐performance liquid chromatography coupled with ultraviolet detection was developed for studying the pharmacokinetics of costunolide (Cos) and dehydrocostus lactone (Dehy) in rats after intravenous (i.v.) administration. Following i.v. administration, the maximum plasma concentrations of Cos and Dehy were observed to be 12.29 ± 1.47 and 5.79 ± 0.13 µg/mL, respectively. The bioavailability of Cos was larger than that of Dehy; however, the clearance and the volume of distribution of Dehy were much larger than those of Cos. An ultraperformance liquid chromatography/quadrupole time‐of‐flight mass spectrometry system with automated MS^E (E represents collision energy) data analysis software (MetaboLynx^TM) was used to analyze and identify the metabolites of Cos and Dehy in vivo. Four metabolites of Cos and six metabolites of Dehy were discovered from the plasma, urine and feces of rats. The main metabolic pathway of Cos was phase II biotransformation, but the main metabolic pathways of Dehy was phase ? biotransformation. Two sequential desaturations and N‐acetylcysteine conjugation were the common metabolic pathways of Cos and Dehy in rats. This information may be useful for the further development of the two drug candidates. Copyright © 2014 John Wiley & Sons, Ltd.     

A novel LC‐MS/MS method for determination of tissue distribution and excretion of timosaponin B‐II in rat biological matrices

Timosaponin B‐II (TB‐II) is a natural bioactive steroid glycoside extracted from the Chinese medicinal herb Anemarrhena asphodeloides Bge. (Fam. Liliaceae). It has been demonstrated to have a good anti‐inflammatory effect and a low bioavailability (1.1%). Clinical research has focused on developing it into a completely new medicine. In this study, a rapid and sensitive analytical method based on LC‐MS/MS has been developed for the determination of TB‐II in rat biological matrices (tissues, bile, urine and feces samples). The analytes and internal standard were isolated from 100 μL samples by solid‐phase extraction and then separated using a DIKMA Inertsil ODS‐3 column (5 µm, 2.1 × 150 mm) with an isocratic mobile phase consisting of acetonitrile–0.05% formic acid (35:65) at a flow rate of 0.25 mL/min. Calibration curves (1/χ²‐weighted) offered satisfactory linearity (r² ≥ 0.990) within the test range. The accuracy, precision, recoveries and matrix effects were satisfactory in all the biological matrices examined. The assay was successfully applied to a tissue distribution and excretion study in rats. The preclinical data are useful for the design of clinical trials of TB‐II. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of an UPLC‐Q/TOF‐MS assay for the quantitation of neopanaxadiol in beagle dog plasma: Application to a pharmacokinetic study

Neopanaxadiol (NPD), the main panaxadiol constituent of Panax ginseng C. A. Meyer (Araliaceae), has been regarded as the active component for the treatment of Alzheimer's disease. However, few references are available about pharmacokinetic evaluation for NPD. Accordingly, a rapid and sensitive method for quantitative analysis of NPD in beagle dog plasma based on ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry was developed and validated. Analytes were extracted from plasma by liquid–liquid extraction and chromatographic separation was achieved on an Agilent Zorbax Stable Bond C₁₈ column. Detection was performed in the positive ion mode using multiple reaction monitoring of the transitions both at m/z 461.4 → 425.4 for NPD and internal standard of panaxadiol. All validation parameters, such as lower limit of quantitation, linearity, specificity, precision, accuracy, extraction recovery, matrix effect and stability, were within acceptable ranges and the method was appropriate for multitude sample determination. After oral intake, NPD was slowly absorbed and eliminated from circulatory blood system and corresponding plasma exposure was low. Application of this quantitative method will yield the first pharmacokinetic profile after oral administration of NPD to beagle dog. The information obtained here will be useful to understand the pharmacological effects of NPD.     

Determination of a novel paclitaxel derivative (NPD‐103) in human plasma by ultra‐performance liquid chromatography–tandem mass spectrometry

A sensitive and specific ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS‐MS) method for quantification of a newly developed anticancer agent NPD‐103 has been established. An aliquot of human plasma sample (200 µL) was spiked with ¹³C‐labeled paclitaxel (internal standard) and extracted with 1.3 mL of tert‐butyl methyl ether. NPD‐103 was quantitated on a C₁₈ column with methanol–0.1% formic acid (75:25, v/v) as mobile phase using UPLC‐MS‐MS operating in positive electrospray ionization mode with a total run time of 3.0 min. For NPD‐103 at the concentrations of 1.0, 5.0 and 10.0 µg/mL in human plasma, the absolute extraction recoveries were 95.58, 102.43 and 97.77%, respectively. The linear quantification range of the method was 0.1–20.0 µg/mL in human plasma with linear correlation coefficients greater than 0.999. The intra‐ and inter‐day accuracy for NPD‐103 at 1.0, 5.0 and 10.0 µg/mL levels in human plasma fell into the ranges of 95.29–100.00% and 91.04–94.21%, and the intra‐ and inter‐day precisions were in the ranges of 8.96–11.79% and 7.25–10.63%, respectively. This assay is applied to determination of half‐life of NPD‐103 in human plasma. Copyright © 2008 John Wiley & Sons, Ltd.