Application of HPLC‐LTQ Orbitrap MS for metabolic profiles of Polygonum multiflora extract in rats

The root of Polygonum multiflorum (PM) is an important Chinese herbal medicine for treatment of various diseases. Extensive pharmacological studies have been conducted and demonstrated that it shows a wide range of bioactivities. Meanwhile, a considerable number of hepatotoxicity cases owing to oral administration of PM have been reported and have attracted great attention. However, the limited knowledge regarding the metabolism of PM restricts the deeper studies on its pharmacological/toxicological mechanism and therapeutic material basis. The present study aimed to develop a high‐performance liquid chromatography coupled with a linear ion trap–Orbitrap hybrid mass spectrometry method for separation and identification of metabolites in rat urine and plasma after oral administration of PM. Based on the proposed strategy, metabolism profiles of PM in rats were proposed for the first time and 43 metabolites were characterized or tentatively identified. Phase II metabolism, such as glucuronidation and sulfation, are the principal pathways of the main components. These findings will be beneficial to further understanding of the pharmacological mechanism and pharmacodynamic material basis of PM.     

Analysis of deprenyl metabolites in some body compartments of rats using GC-MSD

Summary Deprenyl metabolites were analyzed in various organs of rats after chronic oral treatment. The investigation was carried out by gas chromatography combined with MSD after derivatization of the metabolites. When (−)-deprenyl (selegiline) was administered to rats higher ratios of methamphetamine to amphetamine were found in the kidney, and the heart at 1 and 5 h after treatment, than after (+)-deprenyl administration. Both deprenyl and its demethylated metabolite were also detected; however, their level was generally lower than that of either methamphetamine or amphetamine. Coadministration of verapamil with selegiline did not essentially alter the major metabolic pathway of deprenyl metabolism.     

Determination and tissue distribution studies of dantrolene sodium with hydroxypropyl‐β‐cyclodextrin in rat tissue by HPLC/MS/MS

The established analytical method for determining the concentration of dantrolene sodium (Da) in rat tissues by HPLC/MS/MS technique was successfully applied to tissue distribution studies of Da in rats. Tissue homogenate samples were pretreated by protein precipitation with pre‐cooled methanol. Chromatographic separation was achieved on an Acquity HPLC column (Kromat Universil XB‐C₁₈, 2.1 × 150 mm, 3 μm). Mass spectrometry was conducted with an electrospray ionization interface in negative ionization mode and multiple reaction monitoring was used for quantitative analysis. The results showed that Da was rapidly and widely distributed in tissues and reached the maximum concentration within 0.5 h in all tissues after oral administration of Da–hydroxypropyl‐β‐cyclodextrin (DHC). It was then metabolized by liver and finally excreted from kidney,which indicated that DHC inclusion complex has better absorption and higher oral bioavailability than Da. The results also provided evidence for the safety and effectiveness of drug clinical application.     

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.     

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).     

Determination of isoniazid concentration in rabbit vertebrae by isotope tracing technique in conjunction with HPLC

Medications compounded with isoniazid (INH) are usually applied to surgical sites at the completion of surgery to locally kill postoperative residual tubercle bacilli. However, the distribution and elimination of INH in the vertebrae in vivo are not known. In this study, isotope tracing was used in conjunction with high‐pressure liquid chromatography (HPLC) to address this. INH and technetium‐99 m‐labeled INH were applied to the vertebrae of rabbits. After 2 and 6 h, osseous tissues containing INH, as determined by radionuclide imaging, were collected for detection with HPLC. The results showed that INH mainly stayed around the vertebrae 6 h after its application and did not permeate widely into the blood or other organs, except for the kidneys. The standard deviations of INH concentrations in the technetium‐99 m‐INH group were approximately four‐fold smaller than those in the INH group. This method of coupling isotope tracing and HPLC can effectively limit experimental error during sample collection, allowing accurate and reliable identification of the concentration levels of INH in osseous tissues in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Biodistribution study of free and microencapsulated 6‐methylcoumarin in Wistar rats by HPLC

A sensitive, specific and reproducible HPLC method has been developed and validated for the quantitative determination of 6‐methylcoumarin (6MC) in plasma and other tissues in Wistar rats. A C₁₈ column was used with UV detection at 321 nm and a gradient system consisting of methanol‐deionized water was used as mobile phase. The retention time for 6MC was 14.921 min and no interfering peaks were observed for any of the matrices. Linear relationships (r² > 0.997) were obtained between the peak height ratios and the corresponding biological sample concentrations over the range 0.4–12.8 µg/mL. Precision and accuracy were evaluated; the coefficient of variation and the relative error for all of the organs were <2 and 7%, respectively. The limit of quantitation was 0.20 µg/mL for the heart and 0.30 µg/mL for the other tissues evaluated. This HPLC method was successfully used in the determination of 6MC in the biodistribution study after administration of 200 mg/kg of both 6MC‐free and 6MC‐loaded polymeric microparticles. In this study, extensive 6MC was found, in both free and microencapsulated forms, in all the organs tested. The 6MC‐free showed a range of between 1.7 and 11.5 µg/g, while the microencapsulated 6MC showed concentrations of between 6.35 and 17.7 µg/g, suggesting that 6MC improved absorption rate. Copyright © 2014 John Wiley & Sons, Ltd.     

Tissue distribution study of periplocin and its two metabolites in rats by a validated LC–MS/MS method

Periplocin is a cardiac glycoside and has been used widely in the clinic for its cardiotonic, anti‐inflammatory and anti‐tumor effects. Although it is taken frequently by oral administration in the clinic, there have been no reports demonstrating that periplocin could be detected in vivo after an oral administration, so there is an urgen need to determine the characteristics of periplocin in vivo after oral administration. In this study, a sensitive and reliable liquid chromatography–tandem mass spectrometry method was developed and validated to identify and quantify periplocin and its two metabolites in rat tissue after a single dosage of perplocin at 50 mg/kg. The results demonstrated that periplocin and its two metabolites were detected in all of the selected tissues; periplocin could reach peak concentration quickly after administration, while periplocymarin and periplogenin reached maximum concentration > 4.83 h after administration. The tissue distribution of analytes tended to be mostly in the liver, and higher analyte concentrations were found in the heart, liver, spleen, lung and kidney, but a small amount of chemical constituents was distributed into the brain. The consequences obtained using this method might provide a meaningful insight for clinical investigations and applications.     

Determination of piceid in rat plasma and tissues by high-performance liquid chromatographic method with UV detection

A rapid, sensitive and selective HPLC method was developed and validated for determination of piceid in rat plasma and tissues. The drug was isolated from plasma and tissues by a simple protein precipitation procedure. Chromatographic separation was performed on a C(18) column with acetonitrile-water (26:74, v/v) as mobile phase. The method was successfully applied to the pharmacokinetics and tissue distribution research after oral administration of a 50 mg/kg dose of piceid to healthy male Wistar rats. The pharmacokinetic parameters showed that piceid was quickly absorbed, distributed and eliminated within 4 h after oral administration. The tissue distribution results showed that, at 10 min, the concentrations of piceid in most tissues reached peak level except in heart and testis. The highest level of piceid was found in stomach, then in small intestine, spleen, lung, brain, testis, liver, kidney and heart. The amount of piceid in testis and heart reached the peak level at 30 min. At 120 min, the amount of piceid in all tissues decreased to a low percentage of the initial concentration. Piceid was absorbed throughout the gastrointestinal tract with considerable absorption taking place in the stomach and small intestine. There was no long-term accumulation of piceid in rat tissues.     

Tissue distribution study of columbianadin and its active metabolite columbianetin in rats

Columbianadin, one of the main bioactive constituents of the roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan, has been found to possess obvious pharmacological effects in previous studies. In this study, a valid and sensitive reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was established and validated for the determination of columbianadin (CBN) and its active metabolite columbianetin (CBT) in rat tissue samples. Sample separation was performed on an RP‐HPLC column using a mobile phase of MeOH–H₂O (75:25, v/v) at a flow rate of 1.0 mL/min. The UV absorbance of the samples was measured at the wavelength 325 nm. The calibration curves for CBN were linear over the ranges of 0.5–20 µg/g for brain, testes and muscle, 1.0–10.0 µg/g for stomach and intestine, and 0.2–20.0 µg/g for heart, liver, spleen, lung and kidney. The calibration curves for CBT were linear over the ranges of 0.5–25 µg/g for stomach and intestine, and 0.1–10.0 µg/g for heart, liver, spleen, lung and kidney. The analysis method was successfully applied to a tissue distribution study of CBN and CBT after intravenous administration of CBN to rats. The results of this study indicated that CBN could be detected in all of the selected tissues after i.v. administration. CBN was distributed to rat tissues rapidly and could be metabolized to CBT in most detected tissues. Of the detected tissues, heart had the highest uptake of CBN, which suggested that heart might be one of the main target tissues of CBN. Concentrations of CBT were obviously higher in the digestive system than in other assayed tissues. The information provided by this research is very useful for gaining knowledge of the capacities of CBN and CBT to access different tissues. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis of phenolic and triterpenoid compounds in licorice and rat plasma by high‐performance liquid chromatography diode‐array detection,time‐of‐flight mass spectrometry and quadrupole ion trap mass spectrometry

High‐performance liquid chromatography with diode‐array detection (HPLC/DAD), time‐of‐flight mass spectrometry (HPLC/TOFMS) and quadrupole ion trap mass spectrometry (HPLC/QITMS) were used for separation and identification of several compounds in licorice and rat plasma after oral administration of the herbal extract. Three phenolic compounds and one triterpenoid in licorice extract were unambiguously identified by comparing with the standard compounds. A formula database of known compounds in licorice was established, against which the other 42 compounds were identified effectively based on the accurate extract masses and formulae acquired by HPLC/TOFMS. In order to differentiate the isomers, tandem mass spectrometry was also used. The deduced fragmentation behaviors in QITMS were used to distinguish seven groups of isomers in licorice. By means of the three detectors, 46 compounds in licorice were identified. After oral administration of the extract, 25 compounds in rat plasma were detected and identified by comparing and contrasting the compounds measured in licorice with those in the plasma samples by HPLC/TOFMS. It is concluded that a rapid and effective method based on three analytical techniques was established, which is useful for identification of multiple compounds in licorice in vitro and in vivo. The result should be very useful for the quality control and curative mechanism study of licorice. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of ecliptasaponin A in rat plasma and tissues by liquid chromatography‐tandem mass spectrometry

A sensitive, rapid and specific high‐performance liquid chromatography tandem mass spectrometry method (HPLC‐MS/MS) was developed to determine ecliptasaponin A in rat plasma and tissues after oral administration. Ginsenoside Rg1 was used as the internal standard (IS). The plasma and tissues samples were prepared by liquid‐liquid extraction with ethyl acetate and separated on an Eclipse Plus C₁₈ column (2.1 mm × 150 mm, 5 µm) at a flow rate of 0.4 mL/min using acetonitrile and water (containing 0.05% acetic acid) as the mobile phase. The tandem mass detection was carried out with eletrospray ionization in negative mode. Quantification was performed by using multiple reaction monitoring (MRM), which monitored the fragmentation of m/z 633.4→587.2 for ecliptasaponin A and m/z 859.4→637.4 for the IS. The calibration curves obtained were linear in different matrices, and the lower limit of quantification (LLOQ) achieved was 0.5 ng/mL both for rat plasma and tissues. The intra‐ and inter‐day precisions were below 15%. This method was successfully applied to pharmacokinetic study of ecliptasaponin A in rat plasma and tissues after oral administration. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

芎汤活血有效部位在大鼠体内整体吸收规律的HPLC指纹图谱研究

研究芎汤活血有效部位给药大鼠体内的整体吸收规律。将活血有效部位给大鼠灌胃后,测绘不同时间点血浆的HPLC指纹图谱,通过比较计算特征峰欧式距离,研究该复方药对的吸收规律。芎汤活血有效部位大鼠给药后,45 min药物吸收最高,1.5 h之内体内药物浓度保持在较高水平,随后变化较小。采用指纹图谱技术测定复方给药后的血浆成分,能更好地反映中药多成分在体内的吸收规律。     

Simultaneous determination of tiopronin and its primary metabolite in plasma and ocular tissues by HPLC

Tiopronin, formally 2‐mercaptopropionylglycine (MPG), is currently prescribed to treat cystinuria and rheumatoid arthritis, and its antioxidant properties have led to its investigation as a treatment for cataracts, a condition in which oxidative stress is strongly implicated. To study its accumulation in the eye, a reliable, isocratic HPLC method was developed for the determination of MPG and its primary metabolite 2‐mercaptopropionic acid (MPA) in plasma and relevant ocular tissues. This method utilizes pre‐column derivatization and fluorescence detection. The 3.5 min separation enables high‐throughput analysis, and validation experiments demonstrated that this method is suitable for evaluating ocular accumulation of MPG and MPA at concentrations as low as 66 and 33 nm , respectively. Excellent linearity was achieved over the working concentration range with R² > 0.997. Extraction recovery was reproducible within each matrix and exceeded 97%. Accuracy was within 13.3% relative error, and intra‐ and inter‐day precisions were within 6% CV and 7% CV, respectively. Sample stability was demonstrated under various storage conditions, and the use of an internal standard conferred exceptional ruggedness. This method has been successfully applied for the determination of MPG and MPA in plasma, cornea, lens and retina following intraperitoneal administration of the drug in Wistar rats.     

Tissue distribution of capilliposide B,capilliposide C and their bioactive metabolite in mice using liquid ‐tandem mass spectrometry

Lysimachia capillipes Hemsl (Primulaceae), a folk medicinal plant in China, showed significant anti‐tumor activities in vivo and in vitro . Capilliposide B (LC‐B) and capilliposide C (LC‐C) are the main bioactive components in this plant. To explore their tissue distribution, a reliable bioanalytical method for the quantification of LC‐B, LC‐C and their bioactive metabolite, capilliposide A (LC‐A), in mouse tissues was developed and validated. In this study, the tissue distribution profiles of the three compounds were examined after intravenous administration of pure LC‐B and oral administration of total saponins of L. capillipes Hemsl extract (LCE) for 10 days. Method validation was conducted over the curve range 10.0–5000 ng/mL for all three analytes in various tissue homogenates. The relative standard deviation of intra‐day and inter‐day precision of the QC samples was <14.7%, and the accuracy ranged from 85.9 to 114.0%. The results indicated that LC‐B was rapidly and widely distributed throughout the whole body except for muscle following intravenous administration of LC‐B. In addition, LC‐A was only in liver, intestine, lung and stomach. After oral administration of LCE, LC‐B and LC‐C were distributed into various tissues. The highest levels were observed in stomach and intestine.     

Non‐Anticoagulant Heparin Prodrug Loaded Biodegradable and Injectable Thermoresponsive Hydrogels for Enhanced Anti‐Metastasis Therapy

Metastasis is a pathogenic spread of cancer cells from the primary site to surrounding tissues and distant organs, making it one of the primary challenges for effective cancer treatment and the major cause of cancer mortality. Heparin‐based biomaterials exhibit significant inhibition of cancer cell metastasis. In this study, a non‐anticoagulate heparin prodrug is developed for metastasis treatment with a localized treatment system using temperature sensitive, injectable, and biodegradable (poly‐(ε‐caprolactone‐co‐lactide)‐b‐poly(ethylene glycol)‐b‐poly(ε‐caprolactone‐co‐lactide) polymeric hydrogel. The drug molecule (heparin) is conjugated with the polymer via esterification, and its sustained release is ensured by hydrolysis and polymeric biodegradation. An aqueous solution of the polymer could be used as an injectable solution at below 25 °C and it achieves gel formation at 37 °C. The anti‐metastasis effect of the hydrogels is investigated both in vitro and in vivo. The results demonstrated that local administration of injectable heparin‐loaded hydrogels effectively promote an inhibitory effect on cancer metastasis.     

Development of a novel ion‐pairing HPLC‐FL method for the separation and quantification of hydroxychloroquine and its metabolites in whole blood

Hydroxychloroquine (HCQ) is an old antimalarial drug that has proven to be a safe and effective treatment for systemic lupus erythematosus (SLE) and other autoimmune diseases. Since hematic concentration of HCQ is closely related to the therapeutic response, monitoring the levels of the drug and its metabolites in the blood of HCQ‐treated patients helps the clinician in the evaluation of partial or complete unresponsiveness to treatment. We developed and validated a novel ion‐pairing HPLC‐FL method for the simultaneous dosage of HCQ, and its major metabolites desethylhydroxychloroquine, desethylchloroquine and bisdesethylchloroquine, after extraction from whole blood. This methodological approach was used for the analysis of real samples obtained from patients affected by SLE and undergoing HCQ treatment. The same samples were also analyzed using a previously validated LC/MS/MS method and data obtained with the two approaches were in substantial agreement with each other. Results presented in this work indicate that this approach can be successfully used to monitor the level of HCQ and its metabolites in the blood of various categories of patients (i.e. low and high responders, or those not adhering to the therapy). Comparison of HPLC‐FL and LC/MS/MS data confirmed the efficacy of the proposed method for routine clinical analyses.     

Determination of cefixime and its metabolises by high-performance capillary electrophoresis

Cefixime (CX), an oral cephalosporin antibiotic, and its metabolites in human digestive organs were separated by various modes of high-performance capillary electrophoresis. The zone electrophoresis mode in phosphate buffer (pH 6.8) containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate gave the best separation, permitting the complete resolution of CX and all of five metabolites. On the other hand, the plain zone electrophoresis mode in phosphate buffer (pH 6.8) offered a simple procedure for the direct determination of urinary CX concentration using intact urine samples.     

Determination of protoapigenone in rat plasma by high‐performance liquid chromatography with UV detection and its application in pharmacokinetic studies

A simple and sensitive HPLC method using UV detection was developed to determine the concentration of protoapigenone in rat plasma. Chromatographic separation was conducted on a C₁₈ column with a mobile phase consisting of an acetonitrile–methanol–aqueous phase (containing 0.2% acetic acid, pH 3.0) system at a flow rate of 1.0 mL/min. The UV detector was set at 248 nm. The calibration curve was linear over the range of 0.031–10.0 µg/mL. The lower limit of quantification was 31 ng/mL. The recoveries for plasma samples ranged from 70.3 to 82.5%. The intra‐ and inter‐day accuracy and precision fulfilled the international standards. This method was successfully applied to a pharmacokinetic study of protoapigenone in rats after oral administration of protoapigenone. It was shown that protoapigenone could be absorbed rapidly after oral administration and could reach the maximum concentration within 1 h. Copyright © 2013 John Wiley & Sons, Ltd.