Normal Rat Plasma Research Articles

Identification of the metabolites in normal and AA rat plasma, urine and feces after oral administration of Daphne genkwa flavonoids by LC-Q-TOF-MS spectrometry

Daphne genkwa Sieb. et Zucc., as a traditional oriental herb, has been widely distributed and employed in China. The major bioactive components in D. genkwa are flavonoid compounds, which showed pharmacological activities such as anti-inflammatory, analgesic, anti-tumor and immunomodulatory activities. In this study, we analyzed total flavonoids in D. genkwa and their metabolites in normal and adjuvant arthritis (AA) rat plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). A total of 4 metabolites in plasma, 9 metabolites in urine and 15 metabolites in feces were characterized respectively by LC-Q-TOF-MS technology in normal rat. And 9 of the metabolites were observed in the AA rat urine, while there was no prototype drug or its metabolites detected in plasma and fecal samples. The metabolic pathway mainly involves hydroxylation, methylation, glucuronide, sulfate conjugation, oxidation and reduction, during the phase I and phase II biotransformation pathway. All the information gained here will be greatly helpful in elucidating the potential biological and pharmacological mechanism of flavonoid in D. genkwa, thus providing new ideas for drug development.

Reduced systemic exposure and brain uptake of donepezil in rats with scopolamine-induced cognitive impairment

1. Donepezil (DPZ) is an acetylcholinesterase (AchE) inhibitor used in the mild to moderately severe Alzheimer’s disease. Among its major metabolites, 6-O-desmethyl DPZ (6-DDPZ), 5-O-desmethyl DPZ (5-DDPZ) and DPZ N-oxide, the anti-AchE activities of 5-DDPZ and DPZ N-oxide have never been clearly identified before. Besides, there is no report on simultaneous determination of DPZ and its three metabolites in the brain, thus their uptake in hippocampus and cortex are unknown. Therefore, the current studies are proposed aiming to: (1) investigate the anti-AchE activities and brain uptake of DPZ and its three metabolites and (2) compare their pharmacokinetics and brain uptake between normal and scopolamine-induced rats.2. DPZ and its three metabolites demonstrated anti-AchE activities with the IC50 in the order of DPZ (7.20 × 10−2 μM), 6-DDPZ (1.14 × 10−1 μM), 5-DDPZ (4.03 × 10−1 μM) and DPZ N-oxide (1.61 μM). They were also evenly distributed in the brain and retained much longer in the brain than that in plasma in normal rats.3. Compared to normal rats, Cmax, AUC0→24h and AUC0→∞ of DPZ were reduced by 52.0%, 31.2% and 30.1%, respectively; Tmax of DPZ and its three metabolites were prolonged and their brain uptake were decreased in scopolamine-induced rats, suggesting the potential reduced absorption of DPZ.

Comparative Pharmacokinetics of Nootkatone in a RAT Model of Chronic Kidney Disease VERSUS Normal Controls

In this study, a simple, sensitive, and rapid analytical method was developed by ultra-performance liquid chromatography (UPLC) coupled with tandem quadrupole mass spectrometry (MS) for the determination of nootkatone in normal and chronic kidney disease (CKD) rat plasma using clarithromycin as internal standard. After sample preparation by simple liquid–liquid extraction, chromatography was performed on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 mm particle size) using gradient elution with the mobile phase composed of acetonitrile and water acidified with 0.1% (v/v) formic acid. Detection was achieved by electrospray ionisation MS under the multiple selective reaction monitoring mode. The linear range was 0.01‒500 ng/mL with the square regression coefficient (r) of 0.9975. The lower limit of quantification was 0.01 ng/mL. The intra- and inter-day precision was under 5% and the stability accuracy was between 3.6 and 7.0%. The average recoveries from spiked plasma samples were >83% and matrix effect was over 81%. The developed method was successfully applied to the pharmacokinetic study of nootkatone in normal and CKD rats after an oral administration of 50 mg/kg nootkaone. The results showed the cmax and area under curve of nootkaone were greatly decreased, meanwhile Vd/F and t1/2 were markedly increased in CKD rats. The pharmacokinetic characteristics of nootkatone in rats were significantly altered in CKD rats.

Exploration of the hepatoprotective chemical base of an orally administered herbal formulation (YCHT) in normal and CCl4-intoxicated liver injury rats. Part 1: Metabolic profiles from the liver-centric perspective

Ethnopharmacological relevanceYin-Chen-Hao Tang (YCHT), derived from “Treatise on Febrile Diseases” in ancient China, has been a very popular hepatoprotective three-herb formula in China and Japan, although its chemical base remains unclear. Aim of this studyAs the first step in revealing the hepatoprotective chemical base of YCHT, we aimed to clarify the absorbed ingredients and associated metabolic pathways for orally dosed YCHT in both normal and liver injury rats from a liver-centric perspective. Materials and methodsWith the aid of 10 reference compounds, the absorbed ingredients and generated metabolites were systematically characterized by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF) in the portal vein plasma (the plasma before hepatic disposition) – liver – systemic plasma (the plasma after hepatic disposition), following oral administration of YCHT in normal and CCl4-induced liver injury rats. ResultsA total of 38 compounds with six chemical structures, consisting of 10 prototypes and 28 metabolites generated through 9 biotransformations, were absolutely or tentatively identified, and 25 compounds were first reported on YCHT treatments. Among them, 8 compounds were absolutely confirmed by comparing with standard substances, and some had published hepatoprotective activities. Compared with the 35, 15, and 29 compounds identified in the portal vein plasma, liver, and systemic plasma of normal rats, respectively, the corresponding numbers of characterized compounds were 37, 13 and 29 in the liver injury rats. ConclusionsSulfation and glucuronidation were the predominant biotransformations, and intestinal metabolism, prior to hepatic metabolism, occurred for most compounds. CCl4-induced liver injury caused only slight changes in the metabolic profiles of rats orally administered YCHT. These results provided the precondition for further quantitative analysis and pharmacodynamic screening of compounds in YCHT.

Acute toxicity and antiproliferative and procoagulant activities of fractions derived from Thymus satureioides of the Moroccan High Atlas

This study aimed to determine the effects of crude extract (CE) and fractions derived from the Moroccan endemic plant Thymus satureioides (T. satureioides) on inflammation, coagulation and viability of a cell cancer line. The crude extract and fractions from the leaves of T. satureioides were analysed using HPLC and examined for their antioxidant capacity using mineral and biological substrates. The anti-inflammatory activity of the extracts was evaluated using croton oil- and carrageenan-induced oedema. The effect on blood coagulation in normal rat plasma was also determined using prothrombin time (PT) and activated partial thromboplastin time (APTT) assays. Furthermore, the oral acute toxicity and antiproliferative effects on the MCF-7 cell line of the CE were evaluated. The major identified polyphenol in the CE and polar fractions was rosmarinic acid. No mortalities due to acute toxicity were observed at 5 g/kg. All plant preparations protected the cell membranes of erythrocytes against oxidative damage and showed high scavenging activity, as measured using DPPH and FRAP. Moreover, the hydrophobic fractions demonstrated significant inhibitory effects against croton oil-induced oedema. The methanol fraction (60 mg/kg) significantly reduced oedema induced by carrageenan during the first phase. During the APTT and PT assays, the CE and fractions caused plasma coagulation even at low concentrations. An antiproliferative assay (MTT) showed a significant decrease in the number of viable cells in culture medium supplemented with CE at different concentrations. These results suggest that extracts from the leaves of T. satureioides constitute a valuable source of anti-inflammatory and anti-cancer metabolites.

Enhanced Anti-diabetic Effect of Berberine Combined With Timosaponin B2 in Goto-Kakizaki Rats, Associated With Increased Variety and Exposure of Effective Substances Through Intestinal Absorption.

Objective: Inspired by the traditionally clinical application of herb pair Zhimu-Huangbo to treat diabetes, a combination of plant ingredients, timosaponin B2 (TB-2) and berberine (BBR), was evaluated for their anti-diabetic efficacy and cooperative mechanisms.Methods: The efficacy and pharmacokinetics of orally administered TB-2 (33.3 mg/kg/day), BBR (66.7 mg/kg/day), and TB-2+BBR (100 mg/kg/day) were evaluated in spontaneously non-obese diabetic Goto-Kakizaki (GK) rats, and metformin (200 mg/kg/day) was used as a positive control. The comparative exposure of the parent drugs, timosaponin A3 (TB-2 metabolite), and M1–M5 (BBR metabolites) was quantified in the portal vein plasma (before hepatic disposition), liver, and systemic plasma (after hepatic disposition) of normal rats on single and combination treatments. Cooperative mechanism of TB-2 and BBR on intestinal absorption and hepatic metabolism was investigated in Caco-2 cells and primary hepatocytes, respectively.Results: After a 6-week experiment, non-fasting and fasting blood glucose levels and oral glucose tolerance test results showed that TB-2+BBR treatments (100 mg/kg/day) displayed significantly anti-diabetic efficacy in GK rats, comparable to that on metformin treatments. However, no significant improvement was observed on TB-2 or BBR treatments alone. Compared to single treatments, combination treatments led to the increased circulating levels of BBR by 107% in GK rats. In normal rats, the hepatic exposure of BBR, timosaponin A3, and M1–M5 was several hundred folds higher than their circulating levels. Co-administration also improved the levels in the plasma and liver by 41–114% for BBR, 141–230% for TB-2, and 12–282% for M1–M5. In vitro, the interaction between TB-2 and BBR was mediated by intestinal absorption, rather than hepatic metabolism.Conclusion: Combining TB-2 and BBR enhanced the anti-diabetic efficacy by increasing the in vivo variety of effective substances, including the parent compounds and active metabolites, and improving the levels of those substances through intestinal absorption. This study is a new attempt to assess the effects of combined plant ingredients on diabetes by scientifically utilizing clinical experience of an herb pair.

Established UPLC-MS/MS procedure for multicomponent quantitative analysis in rat plasma: A contrastive pharmacokinetics study of Qiangshen tablet in normal and kidney yang deficiency syndrome models

Qiangshen tablet, an important prescription consisting of 14 kinds of Chinese herbal medicines, has been used for decades to treat kidney yang deficiency syndrome (KYDS) in China. Qiangshen tablet has been recorded in ChP (2015 edition) and possesses the effect of strengthening yang, invigorating qi and tonifying kidneys. In this research, a simple, reliable and specific method was established for simultaneous determination of stachydrine, psoralen, isopsoralen, morroniside, paeoniflorin and loganin in normal and KYDS rat plasma after intragastric administration of a Qiangshen tablet suspension by UPLC-MS/MS. Protein precipitation (PP) by acetonitrile and liquid-liquid extraction (LLE) by ethyl acetate - n-butanol (1: 1, v/v) were used for pretreatment of plasma samples. Chromatographic separation of two IS (Internal Standard) and six analytes was achieved using an ACQUITY UPLC® BEH C18 column (2.1 × 100 mm, 1.7 μm). The mobile phase consisted of 0.1% formic acid aqueous solution (solvent A) and acetonitrile (solvent B) with a gradient scheme. Multiple reaction monitoring (MRM) mode with positive and negative ion source switching was applied to perform the mass spectrometric analyses. This method has been validated with good linearity (r ≥ 0.9942) and acceptable precision and accuracy (RSD ≤ 11%, RE from −4.8% to 7.7%). The mean recovery values of the analytes and IS were all ≥68.28%, and the matrix effects ranged from 94.4% to 101.7%. The stability of the IS and analytes was measured throughout the experiment. The results showed significant differences between the pharmacokinetic traits of the analytes in the normal and KYDS groups, suggesting that pharmacokinetic procedures involving these analytes could be modified in cases of KYDS.

UPLC-Q-TOF/MS-based metabolic profiling comparison of four major bioactive components in normal and CKD rat plasma, urine and feces following oral administration of Cornus officinalis Sieb and Rehmannia glutinosa Libosch herb couple extract

Cornus officinalis-Rehmannia glutinosa herb couple is widely used herb medicine in clinical practice to treat chronic kidney disease (CKD). However, the in vivo integrated metabolism of its main bioactive components in CKD rats remains unknown. In this study, UPLC-Q-TOF/MS technique combined with Metabolynx™ software, was developed and successfully applied for analysis of metabolic profiles of the bioactive components of the herb couple in normal and CKD rat biological samples. Main parent components of the herb couple extract such as loganin, morroniside and catalpol were absorbed into the blood circulation of the normal and CKD rats. Another parent component acteoside was almost completely degraded. Seventeen metabolites involved in the in vivo metabolism processes were tentatively identified. These metabolites indicated that loganin was mainly metabolized to the demethylated product, and morroniside was firstly deglycosylated to the aglycone and the latter was subsequently demethylated and acetylated. Additionally, hydrogenation and deglycosylation were the principal metabolic reactions of catalpol; while O-glucuronide and O-sulphate conjugates were observed as major metabolites for methylated caffeic acid and hydroxytyrosol released from acteoside. Compared with the normal group, the CKD rat showed lower conversion capability. Few kinds and minor amounts of the metabolites appeared in the CKD rat samples. While considerable amounts of the parent compounds were detected in the CKD plasma. This will help maintain a high blood drug concentration which might be beneficial for the treatment of CKD. The proposed method could develop an integrated template approach to analyze screening and identification of the bioactive components in plasma, urine and feces after oral administration of herb medicines. Additionally, this investigation might provide helpful chemical information for further pharmacology and active mechanism research on herb medicines.

Buspirone Nanovesicular Nasal System for Non-Hormonal Hot Flushes Treatment.

The aim of this work was to design and characterize a new nanovesicular nasal delivery system (NDS) containing buspirone, and investigate its efficiency in an animal model for the treatment of hot flushes. The presence of multilamellar vesicles with a mean size distribution of 370 nm was evidenced by transition electron microscopy (TEM), cryo-scanning electron microscopy (Cryo-SEM), and dynamic light scattering (DLS) tests. Pharmacodynamic evaluation of the nasal treatment efficacy with the new system was carried out in ovariectomized (OVX) rat—an animal model for hot flushes—and compared with other treatments. We found that the nasal administration of a buspirone NDS resulted in a significant reduction in tail skin temperature (TST). This effect was not observed in the control buspirone-treated groups. Buspirone levels in the plasma and brain of nasally-treated normal rats were quantified and compared with those of rats that had received oral administration by a LC-MS/MS assay. A significantly higher bioavailability was achieved with the new treatment relative to an oral administration of the same drug dose. No pathological changes in the nasal cavity were observed following sub-chronic nasal administration of buspirone NDS. In conclusion, the data of our investigation show that buspirone in the new nanovesicular nasal carrier could be considered for further studies for the development of a treatment for the hot flushes ailment.

Thrombin generation test: A reliable tool to evaluate the pharmacodynamics of vitamin K antagonist rodenticides in rats

Vitamin K antagonist rodenticide pharmacodynamics (PD) is studied in rodents with traditional laboratory tests. We wondered if thrombin generation test (TGT) could add value.Difethialone (10 mg/kg) was administered per os to 97 OFA-Sprague Dawley rats. PD was studied over a 72 h-period using the Calibrated Automated Thrombogram on platelet poor plasma before and after intoxication (3 female and 3 male rats for each 13 time points) and TGT parameters were compared with the prothrombin time (PT) and vitamin K dependent factor activities previously reported.Following intoxication, preliminary tests evidenced rapid and full inhibition of thrombin generation triggered with 5 or 20 pM human recombinant tissue factor. To study the evolution of TGT parameters following difethialone intake, we adapted the test by complementing intoxicated rat samples with pooled normal rat plasma (3/1, v/v). Adapted TGT confirmed the known higher procoagulant basal level in females compared to males through higher endogenous thrombin potential (ETP) and peak height (PH) (p < 0.0001 and p = 0.0003, respectively). An exponential model fitted well the PH and ETP decay after intoxication. In contrast to PT, the decreases were observed immediately following VKA intake and had comparable time to halving values: 10.5 h (95% CI [8.2; 13.6]) for ETP and 10.4 h (95% CI [7.8; 14.1]) for PH. The decrease of FVII and FX preceded that of PH, ETP and FII while FIX decreased later on, contributing to the severe hypo-coagulability.We demonstrated that TGT performed in samples of intoxicated rats complemented with normal plasma is a reliable tool for evaluation of VKA rodenticide PD in rats.

Comparative pharmacokinetics of triterpenic acids in normal and immunosuppressed rats after oral administration of Jujubae Fructus extract by UPLC-MS/MS

The fruit of Ziziphus jujuba (Jujubae Fructus) has been used as food and crude drug for thousands of years. Although several chemical and biological studies have revealed triterpenic acid as the main bioactive constituent of Jujubae Fructus responsible for immune-regulatory activity, only few pharmacokinetic studies have been conducted. To comprehend the kinetics of triterpenic acids and promote their curative application, a sensitive and efficient ultra-performance liquid chromatography coupled with mass spectrometry method (UPLC-MS/MS) was established. UPLC-MS/MS was applied for the simultaneous determination of ceanothic acid, epiceanothic acid, pomonic acid, alphitolic acid, maslinic acid, betulinic acid, and betulonic acid in normal and immunosuppressed rat plasma samples. After sample preparation, chromatographic separation was performed on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) with acetonitrile: methanol (1:1, v/v) and 0.5% ammonium acetate in water as mobile phase. The established method was validated and found to be specific, accurate, and precise for the seven triterpenic acids, and was successfully applied for the pharmacokinetic study of rat plasma samples. The results showed that the pharmacokinetic parameters (Cmax, Tmax, AUC0–t, AUC0–∞, and CLz/F) in the plasma samples of immunosuppressed rats were significantly different from those in normal rats, and might provide an insight for the clinical usage of triterpenic acids from Jujubae Fructus.

Active phytochemicals of Pueraria tuberosa for DPP-IV inhibition: in silico and experimental approach

BackgroundWe had earlier reported that the extract of Pueraria tuberosa significantly inhibits DPP-IV enzyme, resulting in glucose tolerance response in rats. In this study, we have explored the active phytochemicals responsible for this potential. The results have been validated in both fasting and postprandial states in the plasma of normal rats and also in fasting blood and intestinal homogenates of diabetic models.MethodsPueraria tuberosa water extract (PTWE) was administered to normal Charles Foster rats for 35 days and to diabetic model (65 mg/kg bw) for 10 days. After treatments, oral glucose tolerance test (OGTT) and insulin was done for 90 min, and the changes in the levels of GLP-1, GIP, and DPP-IV activities were monitored in fasting and postprandial states. In the case of the diabetic model, DPP-IV activity was measured in intestinal homogenate and basal insulin in plasma. The components of PTWE were analyzed via HPLC-MS based on their chemical formula, molecular mass, and retention time. Using the molecular docking study, we have selected the top five components having strong binding energy with DPP-IV.ResultsThe increase in secretion of GLP-1 and GIP was significantly higher in the postprandial state when compared to fasting condition. GLP-1 plasma concentration increased by 5.8 and 2.9 folds and GIP increased by 8.7 and 2.4 folds in PTWE and control rats, respectively. In contrast, the postprandial decrease in DPP-IV specific activities was recorded at 2.3 and 1.4 folds. The response in OGTT and insulin was also consistent with these changes. In comparison to diabetic controls, PTWE-administered rats showed decreased DPP-IV activity in the intestine, leading to enhanced basal insulin concentration. Through molecular docking, we found Puerarone and Robinin to be the most potential phytochemicals of PTWE for DPP-IV inhibition. Binding energy (kcal/mol) and dissociation constant (pM) of Robinin with DPP-IV protein were found to be 7.543 and 2,957,383.75, respectively. For Puerarone, it was 7.376 and 3,920,309, respectively.ConclusionsThus, this study provides the novel active components that contribute to the DPP-IV inhibitory property of PTWE.

An LC-MS/MS method for quantitation of cyanidin-3-O-glucoside in rat plasma: Application to a comparative pharmacokinetic study in normal and streptozotocin-induced diabetic rats.

A sensitive and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to determine cyanidin-3-O-glucoside (Cy-3G) in normal and streptozotocin-induced diabetic rat plasma. Chromatographic separation was carried out on a Zorbax SB-C18 (50 × 4.6 mm, 5 μm) column and mass spectrometric analysis was performed using a Thermo Finnigan TSQ Quantum Ultra triple-quadrupole mass spectrometer coupled with an ESI source in the negative ion mode. Selected reaction monitoring mode was applied for quantification using target fragment ions m/z 447.3 → 285.2 for Cy-3G and m/z 463.0 → 300.1 for quercetin-3-O-glucoside (internal standard). The calibration curve was linear over the range 3.00-2700 ng/mL (r2 ≥ 0.99) with the lower limit of quantitation at 3.00 ng/mL. Intra- and inter-day precision was <14.5% and mean accuracy was from -11.5 to 13.6%. Stability testing showed that Cy-3G remained stable during the whole analytical procedure. After validation, the assay was successfully used to support a preclinical pharmacokinetic comparison of Cy-3G between normal and diabetic rats. Results indicated that diabetes mellitus significantly altered the in vivo pharmacokinetic characteristics of Cy-3G after oral administration in rats.

Comparatively evaluating the pharmacokinetic of fifteen constituents in normal and blood deficiency rats after oral administration of Xin-Sheng-Hua Granule by UPLC-MS/MS.

Xin-Sheng-Hua Granule (XSHG), a famous traditional Chinese medicine prescription, are clinically applied for the treatment of postpartum disease through nourishing blood and promoting blood circulation. In this investigation, a multi-constituents (trigonelline, stachydrine hydrochloride, hydroxysafflor yellow A, chlorogenic acid, amygdalin, leonurine, liquiritin, ferulic acid, senkyunolide I, senkyunolide H, glycyrrhizic acid, senkyunolide A, ligustilide, butylidenephthalide and glycyrrhetinic acid) pharmacokinetic study of XSHG was conducted for the first time. These fifteen constituents in both normal and blood deficiency rat plasma were monitored by using the established and validated ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry (UPLC-TQ-MS/MS) method. The samples were prepared through removing protein from plasma with three volumes of methanol. Sufficient separation of target constituents and internal standards (chloramphenicol and clarithromycin) was obtained on a Thermo Scientific Hypersil GOLD column (100mm×3mm, 1.9μm) within a 20min gradient elution (0.1% formic acid aqueous - acetonitrile). Multiple reaction monitoring (MRM) mode was applied to monitor target analytes in both positive and negative electrospray ionization. For the fifteen selected target analytes, this method was fully validated with excellent linearity (r≥0.9925), satisfactory intra- and inter-day precisions (RSD≤11.87%), as well as good accuracies (RE, between -12.84 and 11.69). And the stabilities, matrix effects and extraction recoveries of the rat plasma samples were also within acceptable limits (RSD<15%). Compared to normal group, the pharmacokinetics of major active constituents (except liquiritin and glycyrrhetinic acid) had significant differences (P<0.05) in the model rats, indicated that several metabolite enzymes activities could be altered at disease condition.

Effects of iron supplementation on growth, gut microbiota, metabolomics and cognitive development of rat pups.

BackgroundIron deficiency is common during infancy and therefore iron supplementation is recommended. Recent reports suggest that iron supplementation in already iron replete infants may adversely affect growth, cognitive development, and morbidity.MethodsNormal and growth restricted rat pups were given iron daily (30 or 150 μg/d) from birth to postnatal day (PD) 20, and followed to PD56. At PD20, hematology, tissue iron, and the hepatic metabolome were measured. The plasma metabolome and colonic microbial ecology were assessed at PD20 and PD56. T-maze (PD35) and passive avoidance (PD40) tests were used to evaluate cognitive development.ResultsIron supplementation increased iron status in a dose-dependent manner in both groups, but no significant effect of iron on growth was observed. Passive avoidance was significantly lower only in normal rats given high iron compared with controls. In plasma and liver of normal and growth-restricted rats, excess iron increased 3-hydroxybutyrate and decreased several amino acids, urea and myo-inositol. While a profound difference in gut microbiota of normal and growth-restricted rats was observed, with iron supplementation differences in the abundance of strict anaerobes were observed.ConclusionExcess iron adversely affects cognitive development, which may be a consequence of altered metabolism and/or shifts in gut microbiota.

A comparative pharmacokinetic study of three flavonoids and three anthraquinones in normal and gastrointestinal motility disorders rat plasma after the oral administration of Wei‐Chang‐Shu tablet using high‐performance liquid chromatography–tandem mass spectrometry

A simple, fast and reliable high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification and pharmacokinetic study of three flavonoids (liquiritigenin, isoliquiritigenin and formononetin) and three anthraquinones (emodin, rhein and aloe-emodin), which are the bioactive ingredients of Wei-Chang-Shu tablet found in rat plasma. After extraction by liquid-liquid extraction with ethyl acetate, chromatographic separation was achieved on an Agilent Zorbax SB-C18 column (4.6 × 150 mm, 5 μm) at a flow rate of 1 mL/min by gradient elution using 0.1% aqueous acetic acid and acetonitrile. The detection was performed using a triple quadrupole mass spectrometer equipped with electrospray ionization source in the negative ionization and selected reaction monitoring mode. Method validation was performed in terms of specificity, carryover, linearity (r >0.99), intra-/inter-day precision (1.0-10.1%), accuracy (relative error, <7.6%), stability (0.6-13.2%), extract recovery (74.9-91.9%) and matrix effect (89.1-109%). The lower limits of quantification of the six analytes varied from 0.92 to 10.4 ng/mL. The validated method was successfully applied to compare the pharmacokinetic properties of Wei-Chang-Shu tablet in normal rats and in rats with gastrointestinal motility disorders. The results indicated that there were obvious differences in the pharmacokinetic behavior between normal and model rats. This study will be helpful in the clinical application of Wei-Chang-Shu tablet.

UPLC-Q-TOF/MS-Based Metabolic Profiling Comparison of Two Major Bioactive Components and Their Metabolites in Normal and CKD Rat Plasma, Urine and Feces Following Oral Administration of Fructus Corni Extract.

Fructus Corni has been used for nourishing liver and kidney in clinical practice for many years. However, the in vivo integrated metabolism profile of its bioactive components remains unknown. In the present study, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry coupled with the MetaboLynxTM software was established for screening and identification of the metabolites of the loganin and the morroniside in normal and chronic kidney disease (CKD) rat plasma, urine and feces after oral administration of Fructus Corni extract. The results showed that 11 metabolites including taurine and glucuronide conjugates, deglycosylated, dehydrated, (de)hydroxylated, (de)methylated, acetylated products were tentatively detected in normal rat samples while only eight metabolites were obtained in the model samples. Two parent compounds were both absorbed into the blood circulation of the normal and CKD rats. However, the compounds in the CKD rat showed lower metabolic capability. Few kinds and minor amounts of the metabolites were appeared in the CKD rat plasma, urine and feces. While considerable amounts of the parent compounds were detected in the CKD plasma. This helped maintain a high blood drug concentration which might be beneficial for the treatment of CKD. These results will be helpful for the further investigation of the pharmacokinetic study of Fructus Corni in vivo.

Effects of compatibility of radix aconiti carmichaeli and pinelia ternate on pharmacokinetic of 9 aconitum alkaloids in normal and model rat plasma

Effects of compatibility of radix aconiti carmichaeli and pinelia ternate on pharmacokinetic of 9 aconitum alkaloids in normal and model rat plasma

Simultaneous Determination of Four Tanshinones by UPLC-TQ/MS and Their Pharmacokinetic Application after Administration of Single Ethanol Extract of Danshen Combined with Water Extract in Normal and Adenine-Induced Chronic Renal Failure Rats.

Salvia miltiorrhiza, one of the major traditional Chinese medicines, is commonly used and the main active ingredients—tanshinones—possess the ability to improve renal function. In this paper, the UPLC-TQ/MS method of simultaneously determining four tanshinones—tanshinone IIA, dihydrotanshinone I, tanshinone I, and cryptotanshinone—was established and applied to assess the pharmacokinetics in normal and chronic renal failure (CRF) rat plasma. The pharmacokinetics of tanshinones in rats were studied after separately intragastric administration of Salvia miltiorrhiza ethanol extract (SMEE) (0.65 g/kg), SMEE (0.65 g/kg) combined with Salvia miltiorrhiza water extract (SMWE) (1.55 g/kg). The results showed Cmax and AUC0–t of tanshinone IIA, tanshinone I, cryptotanshinone reduced by 50%~80% and CLz/F increased by 2~4 times (p < 0.05) in model group after administrated with SMEE. Nevertheless, after intragastric administration of a combination of SMWE and SMEE, the Cmax and AUC0–t of four tanshinones were upregulated and CLz/F was downregulated, which undulated similarity from the model group to the normal group with compatibility of SMEE and SMWE. These results hinted that SMWE could improve the bioavailability of tanshinones in CRF rats, which provides scientific information for further exploration the mechanism of the combination of SMWE and SMEE and offers a reference for clinical administration of Salvia miltiorrhiza.

Ultra-high performance liquid chromatography with ultraviolet and tandem mass spectrometry for simultaneous determination of metabolites in purine pathway of rat plasma.

It has been proved that the purine metabolic pathway has been implicated in various biological disorders including gout, diabetes, coronary heart diseases, and neurodegenerative diseases. The analysis of the purine metabolic pathway in organisms reveals important alterations under different physiological and pathological conditions, which contributes to the pathological study, diagnosis, and therapy of related diseases. In the present study, an ultra-high performance liquid chromatography with ultraviolet and tandem mass spectrometry (UHPLC-UV-MS/MS) method was developed for conducting the comprehensive analysis of the metabolite profiles of the purine pathway in rat plasma through a single analysis. The purine metabolites including adenosine-5'-monophosphate, guanosine-5'-monophosphate, adenosine, inosine, guanosine, inosine-5'-monophosphate, deoxyadenosine, deoxyguanosine, deoxyinosine, xanthine, hypoxanthine, and uric acid, were separated and quantified in the short running time of 10min. After rapid chromatographic separation achieved by an Agilent Zorbax SB-Aq column, high concentration of uric acid and the remaining purine metabolites at lower levels were respectively detected by ultraviolet detector and triple quadruple mass spectrometry within a single analysis. The proposed method was validated by applying charcoal-stripped plasma as a matrix and it was proved to be linear (R2>0.982), accurate (with a relative error for accuracy <±15% and the relative standard deviation for intra- and inter-run precision <11%) and reproducible (with a matrix effect ranging between 86.49% and 111.44% with a maximum RSD of 8.69%). As a result, the method was successfully applied to the quantification of the endogenous purine metabolites in rat plasma. It was found that the concentration levels of the purine metabolites may keep a physiological balance as an integrated system in normal individuals and the concentration level of uric acid in rat plasma was 64μM which was more than 200 times greater than the other purine metabolites. The established method and the measurement of the concentration of these purines in normal rat plasma may help the investigation of the action mechanisms between purine disorders and related diseases.