•An LC–MS/MS method was established and well validated for the determination of endogenic bile acids.•A new perspective to evaluate the cholesterol lowing effect of Si-miao-yong-an decoction.•Quantitative method to clarify the possible mechanism.•A convenient and efficient method to produce blank biological matrix by activated carbon.Si-miao-yong-an decoction (SMYAD), a traditional Chinese medicine formula, significantly reduced plasma TC, LDL-c levels and increased HDL-c level in hyperlipidemia rats. Liver function test and tissue section examination indicated that SMYAD improved liver function and reduced fat accumulation in hyperlipidemia rat liver. A LC–MS/MS method was established and well validated to evaluate major bile acids derived from cholesterol metabolism through the classic neutral pathway and the alternative acidic pathway (cholic acid, chenodeoxycholic acid and their taurine and glycine conjugates) in liver and plasma. Increased total 6 bile acids concentrations in both liver and plasma were observed after oral administration of 12 g/kg/d, 24 g/kg/d and 36 g/kg/d of SMYAD in a dose dependent manner which contributed to eliminate of cholesterol. Cholic acid, taurocholic acid and glycocholic acid act as the main products of bile acid classic neutral synthesis pathway and show sharp increase (p < 0.01) after treatment of SMYAD at dosage of 24–36 g/kg/d. For liver samples, taurocholic acid level act as the largest growth section, while in plasma samples, cholic acid act as the largest growth section after SMYAD treatment, compared with Model group. By contrast, the main products of alternative acidic pathway (chenodeoxycholic acid and its glycine and taurine conjugates) show no significant increase after treatment of SMYAD. In conclusion, the cholesterol lowing effect of SMYAD may be related with the accelerated transformation of cholesterol into bile acids through the classic neutral pathway.

•It’s the first time that the in vivo multiple absorbed and metabolic components of Guizhi Fuling Capsule has been comprehensively revealed.•The in vitro Guizhi Fuling Capsule composition study took powerful supplement information to the previous study.•The identification of metabolites was achieved using UPLC/Q-TOF-MS/MS coupled with MetaboLynx software.Guizhi Fuling capsule (GFC), a prestigious traditional Chinese medicinal (TCM) prescription, is efficiently used to treat primary dysmenorrhea in the clinical practice. It’s significant to explore the metabolic fate of multiple components in vivo which are responsible for the pharmacological effects but not fully investigated. A rapid and high-throughput method using ultra performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS/MS) was established for systematic investigation on GFC, including GFC chemical compositions, and their absorption and metabolism in rat plasma, urine, uterus and brain after oral administration of GFC. A total of 102 nonvolatile GFC phytochemistry components were identified based on the accurately measured mass value, fragmentation pattern and retention behavior. Compared to the previous GFC study, additional 47 different GFC components were detected. Furthermore 21, 9, 4 and 3 prototype compounds were separately observed in plasma, urine, uterus and brain samples with the support of in vitro GFC study. While 29, 33, 10 and 8 metabolites were also identified with the assistance of the MetaboLynx tool in these biological samples. The result indicated that the developed method was suitable for the components identification even in the complex matrix. The chemical and metabolic profiling of GFC provided an abundant substance foundation for the extensive GFC research, especially for the pharmacodynamic mechanisms research.Download high-res image (203KB)Download full-size image

Larotaxel, a new taxane compound prepared by partial synthesis from 10-deacetyl baccatin III, is active against tumors. In this research, a selective LC–MS method was developed and validated for the study of degradation kinetics of larotaxel, which was carried out in aqueous solutions with different pH (1.5, 3.0, 5.0, 6.5, 7.4, 9.0, 10 and 11.0) and temperature (0, 25, 37 and 45 °C). The linear range was 0.5–25 μg/mL, the intra- and inter-day precisions were less than 7.0%, and accuracy ranged from 97.4–104.5% for each analyte. The observed rate obtained by measuring the remaining intact larotaxel was shown to follow first-order kinetics. The activation energies for degradation were 126.7 and 87.01 kJ/mol at pH 1.5 and 11, respectively. Although larotaxel was stable in pH 5, 6.5 and 7.4 buffers at 37 °C for 24 h during our study, increasing or decreasing the pH of the solutions would decrease its stabilities. Moreover, three main degradation products in alkaline condition were separated by HPLC and identified by Q–TOF–MS. The three degradation products were confirmed as 10-deacetyl larotaxel, 7, 8-cyclopropyl baccatin Ⅲ and 10-deacetyl-7, 8-cyclopropyl baccatin Ⅲ.

Semen Strychni has been widely used as a traditional Chinese herb medicine, but its clinical use was limited for its potential neurotoxicity and nephrotoxicity. This study aimed to investigate S. Strychni-induced neurotoxicity and the neuro-protective effect of Paeonia lactiflora based on monitoring nine potential neurotoxicity biomarkers in rat serum and brain tissue. A sensitive liquid chromatography-tandem mass spectrometry method was developed and validated to monitor serotonin, tryptophan, dopamine, tyrosine and glutamate in serum and five brain regions (prefrontal cortex, hippocampus, striatum, cerebellum and hypothalamus). Analytes were separated on a CAPCELL CORE PC column (150 mm × 2 mm, 2.7 μm) with a gradient program of acetonitrile-water (0.2 % formic acid) and a total runtime of 7.5 min. In addition, enzyme-linked immunosorbent assay was conducted to determine four kinds of protein (tryptophan hydroxylase, tyrosine hydroxylase, endogenous brain-derived neurotrophic factor and nerve growth factor). Results demonstrated that the administration of S. Strychni could cause certain endogenous substances disorder. These analytes were found significantly changed (p < 0.05) in serum (except glutamate) and in certain tested brain regions in S. Strychni extract group. Pretreatment of P. lactiflora could significantly reverse the S. Strychni-induced neurotoxicity and normalize the levels of such endogenous substances. The study could be further used in predicting and monitoring neurotoxicity caused by other reasons, and it was expected to be useful for improving clinical use of S. Strychni through pretreatment with P. lactiflora.

In this study, we investigated the protective effect of total glycosides of paeony against Semen Strychni-induced neurotoxicity and discussed some probably mechanisms. Levels of estrone, estradiol, estriol and growth hormone in male rats’ serum were determined by ELISA, levels of ATP and substances associated with energy metabolism in rats’ brain were determined by HPLC and levels of progesterone was determined by a UPLC-MS/MS method. The results showed that neurotoxicity induced by Semen Strychni could cause a significant decrease (p < 0.05, compare to the blank group) in secretion of estrogens and GH and disorder brain energy metabolism at the same time. While, rats with total glycosides of paeony pre-protection (orally administrated with total glycosides of paeony for 15 days before administrating Semen Strychni extract) showed a much better condition in the secretion of hormones and brain energy metabolism, and showed no significant changes in most of those associated substances when comparing to the blank group. Our study indicated that total glycosides of paeony have neuroprotective effects on Semen Strychni-induced neurotoxicity. It could recover the disordered hormone secretion and improve the brain energy metabolism. Total glycosides of paeony is potential to be further used in clinic to protect against neurotoxicity induced by other reasons.

A simple, rapid and sensitive method based on an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) has been developed and validated for the determination of pimavanserin in rat plasma. The analyte was extracted by protein precipitation with methanol and separated on an ACQUITY BEH C18 column (100 × 2.1 mm, 1.7 µm; Waters, USA), with an isocratic elution of acetonitrile-water containing 10 mM ammonium acetate (70:30, v/v), at a flow rate of 0.2 mL/min for 2.5 min. The analyte and clarithromycin (the internal standard) were detected and quantified in positive ion mode using multiple reaction monitoring transitions at m/z 428.2 → 223.0 for pimavanserin and m/z 748.5 → 589.5 for clarithromycin. Relative coefficient (r) for the calibration curve was more than 0.9980. The intra-day and inter-day precisions (relative standard deviation, RSD%) were less than 13.3% and 10.5%, respectively, and the accuracy (relative error, RE%) was within ± 11.5%. The analytical method was successfully applied to a routine pharmacokinetic study of pimavanserin in rats after oral administration at the dose of 10 mg/kg.

The present study was designed to investigate the influence of the pretreatment of piperazine ferulate on pharmacokinetic parameters of methotrexate in methotrexate-induced renal injury rats. A simple and efficient high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) method was developed to determine methotrexate in rat plasma. Methotrexate and syringic acid (internal standard) were extracted from rat plasma samples by protein precipitation with acetonitrile. The analysis was performed on a CAPCELL PAK C18 column (150 mm × 4.6 mm, 5 µm) with acetonitrile and 5 mmol/l ammonium acetate aqueous (10:90, v/v). The linear range was 5.0 × 10−2 to 100.0 µg/ml for methotrexate. Other parameters were all within the acceptance criteria. The validated method was successfully applied the pharmacokinetic study of methotrexate between two methotrexate treated groups (with and without the pretreatment of piperazine ferulate). Compared with the methotrexate treated alone group, the pharmacokinetic parameters in the methotrexate with the pretreatment of piperazine ferulate group showed significantly lower MRT(0-t), MRT(0-∞) and T1/2. Results suggested that methotrexate can be rapidly eliminated, cleared or metabolized in rat blood, which might be related to the pretreatment of piperazine ferulate. The method provided deeper insights into rational clinical use of methotrexate with the pretreatment of piperazine ferulate on cancer patients with renal dysfunction.A HPLC-MS method had been developed and applied to investigate the influence of pretreatment of piperazine ferulate on pharmacokinetic parameters of methotrexate. The changes in the pharmacokinetic behavior indicated that the pretreatment of piperazine ferulate might exert the protective effect on methotrexate-induced renal injury.Download high-res image (42KB)Download full-size image

•(−)-(1S)-15-Hydroxy-18-carboxycembrene, the first cembrane-type diterpenoid found in the family Euphorbiaceae, isolated from Euphorbiae pekinensis Radix, was identified to be nephrotoxic using HEK 293T cells.•The diterpene time- and dose-dependently decreased cells proliferation.•Sensitive and robust UFLC–MS/MS method was developed and fully validated to investigate the in vivo behavior in rats of the diterpene.•The toxicokinetics parameters indicate the diterpene reached Cmax quickly and slowly eliminate, which indicated the toxicity of the diterpene may occur quickly and is likely to accumulate in tissues and organs.(−)-(1S)-15-Hydroxy-18-carboxycembrene, the first cembrane-type diterpenoid found in the family Euphorbiaceae, isolated from Euphorbiae pekinensis Radix, was identified to be nephrotoxic using HEK 293T cells. Tests on cell morphology, cell viability and biochemical markers about oxidation stress were carried out using inverted microscope, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and commercial kits respectively, which proved the diterpene time- and dose-dependently decreased cells proliferation. Besides, a sensitive and robust UFLC–MS/MS method was developed and fully validated to investigate the in vivo behavior in rats of the diterpene after oral administration of Euphorbiae pekinensis Radix extracts at a dosage of 9 g/kg. The method showed a good linearity in tested range (3–1500 ng/mL) with acceptable accuracy and precision. The recovery of the diterpene was more than 85% and the matrix effect was within ±20%. The toxicokinetics parameters indicate the diterpene reached Cmax quickly and slowly eliminate. The study proved the newly found diterpene was one of the nephrotoxic substances of Euphorbiae pekinensis Radix and revealed its toxicokinetics behavior.

⿢11 Semen Strychni alkaloids (SAs) were identified in cell lysate samples by HPLC-Q Exactive hybrid quadrupole Orbitrap.⿢Decreased levels of tyrosine and tyramine were observed only in combination with increased levels of SAs in cell lysates.⿢The HILIC-ESI-MS/MS method was suitable for the simultaneous analysis of exogenous and endogenous compounds.A Previous metabolomics study has demonstrated that tyrosine metabolism might be disrupted by treating with Semen Strychni on the cell nephrotoxicity model. To investigate the relationship between Semen Strychni alkaloids (SAs) and endogenous tyrosine, tyramine under the nephrotoxicity condition, an HILIC-ESI-MS/MS based analytical strategy was applied in this study. Based on the established Semen Strychni nephrotoxicity cell model, strychnine and brucine were identified and screened as the main SAs by an HPLC-Q Exactive hybrid quadrupole Orbitrap mass system. Then, a sensitive HILIC-ESI-MS/MS method was developed to simultaneously monitor strychnine, brucine, tyrosine and tyramine in cell lysate. The analytes were separated by a Shiseido CAPCELL CORE PC (150 mm ÿ 2.1 mm, 2.7 μm) HILIC column in an acetonitrile/0.1% formic acid gradient system. All the calibration curves were linear with regression coefficients above 0.9924. The absolute recoveries were more than 80.5% and the matrix effects were between 91.6%⿿107.0%. With the developed method, analytes were successfully determined in cell lysates. Decreased levels of tyrosine and tyramine were observed only in combination with increased levels of SAs, indicating that the disturbance of tyrosine metabolism might be induced by the accumulation of SAs in kidney cell after exposure of Semen Strychni. The HILIC-ESI-MS/MS based analytical strategy is a useful tool to reveal the relationships between the toxic herb components and the endogenous metabolite profiling in the toxicity investigation of herb medicines.

•We successfully prepared and evaluated kaempferol–phospholipid complex.•A UHPLC–MS/MS method was developed and fully validated to determine the concentration of KP in vivo.•The comparison of bioavailability between kaempferol and kaempferol–phospholipid complex were carried out.As one of the dietary flavonoids, kaempferol (KP) has been well known to show strong anti-oxidative effect along with other biological properties. However, the oral bioavailability of KP is relatively low due to its poor solubility. In this study, we intended to increase the solubility and bioavailability of KP by preparing kaempferol–phospholipid complex (KP–PC). The KP–PC's physicochemical properties were characterized in terms of infrared spectroscopy (IR), differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD), water/n-Octanol solubility and in vitro dissolution. KP–PC exhibited higher solubility and dissolution rate than KP, indicating a significant improvement in hydrophilicity. A UPLC–ESI–MS/MS method was developed and validated for the determination of KP in Sprague-Dawley (SD) rat plasma, so as to investigate the oral bioavailability of KP–PC versus KP. Results showed that Cmax and AUC0–48 h of KP from the complex (Cmax: 3.94 ± 0.83 μg/mL, AUC0–48 h: 57.81 ± 9.43 mg/L h) were higher than that of KP (Cmax: 1.43 ± 0.21 μg/mL, AUC0–48 h: 13.65 ± 3.12 mg/L h). This research indicated that phospholipid complex (PC) might be one of the suitable approachs to improve the oral bioavailability of KP and other poor-solubility flavonoids.

Semen Strychni has anti-tumor, analgesic and anti-inflammatory angiogenesis effects, but the clinical use of Semen Strychni is limited by its potential nephrotoxicity. To investigate Semen Strychni nephrotoxicity and the protective effect of Radix Glycyrrhizae, a stable, parallel and repeatable cell metabolomics strategy was developed in this study. After treatment with Semen Strychni, cell morphology was changed, cell viability was decreased and 8 biochemical indexes were altered. Then the developed cell models were successfully applied for a cell metabolomics study. The Semen Strychni group samples were completely separated from the blank group samples in the score plots of PCA and PLS-DA models. Finally, a total of 10 putative biomarkers and 24 related metabolic pathways were screened. Among the 24 metabolic pathways, the taurine and nitrogen metabolic pathways were believed to have the most importance and significance, respectively. Based on the results, the possible mechanisms of Semen Strychni nephrotoxicity might be cellular component disruption, oxidative damage, metabolic waste accumulation and the disturbance of energy and ion transport systems. Meanwhile, the Radix Glycyrrhizae treatment group showed similar behaviors to the blank group in all assays, indicating the great protective effect of Radix Glycyrrhizae against Semen Strychni nephrotoxicity. This cell metabolomics strategy might contribute to investigating the possible nephrotoxic mechanisms of herb medicines and clinical therapies of protective herbs.

Strychnos alkaloids (SAs) are considered to be the main toxic constituents in the traditional Chinese medicine, Semen Strychni. However, a general method for rapid identification of SAs in Semen Strychni and relative metabolites in serum has been time-consuming and difficult to develop due to the high similarity of their structures and physicochemical properties. Hence, this study was conducted to allow rapid and reliable characterization of the alkaloids in SAs extract, the constituents absorbed into blood as well as their metabolites in serum after oral administration of SAs extract based on high performance liquid chromatography coupled with a Q Exactive high-resolution benchtop quadrupole Orbitrap mass spectrometer. The method we developed in the present study only required 16 min with high accurate multiple-stage mass spectrometry (MSn) data. A total of 14 strychnos alkaloids, 5 of which were absorbed into serum, were detected and identified. Moreover, 22 metabolites in serum, including 19 phase I and 3 phase II metabolites were observed and characterized. Results demonstrated that the use of the Q Exactive hybrid quadrupole-Orbitrap mass spectrometer proved to be a very feasible and efficient approach for the rapid separation and identification of the complex constituents in the extract and relative metabolites in vivo. In summary, our study will provide a deep insight into the toxic substances of Semen Strychni and deliver beneficial information for further understanding the in vivo behavior of strychnos alkaloid analogues.

•A LC-MS method was developed for determination of depression biomarkers.•The method assisted in objective clinical diagnosis of depression.•The method was applied to the antidepressant effect of Zhi-Zi-Hou-Po decoction.•The method was based on the holistic view of biosystem.•The method provided insight into therapeutic effect and mechanism of prescriptions.Zhi-Zi-Hou-Po decoction (ZZHPD), a traditional Chinese medicine (TCM) formula, has been used for treatment of depressive-like symptoms for centuries. However, studies of its antidepressant effect and mechanism are challenging, owing to the complex pathophysiology of depression and complexity of ZZHPD with multiple constituents acting on different metabolic pathways. The present study was designed to develop a liquid chromatography-mass spectrometry (LC-MS) method for simultaneous determination of depression biomarkers: tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), indole-3-acetic acid (IAA), hippuric acid (HA), phenaceturic acid (PA), creatinine (Cr), glutamic acid (Glu), succinic acid (SA) and γ-aminobutyric acid (GABA), as well as to study the antidepressant effect and potential mechanism of ZZHPD based on holistic view of an organism. The analysis was performed on a CAPCELL PAK C18 column with methanol and 0.01% formic acid in water using gradient elution. The method showed a good linearity (r2 > 0.99) with the other validation parameters were within acceptance range. The results demonstrated Trp, Phe, Tyr, IAA, HA, Cr, Glu and SA levels were significant lower in model group, while PA and GABA were significant higher than those in control group. The rats with ZZHPD treatment showed a tendency of bringing the levels of all these biomarkers to normal except Cr and Glu. It could be a powerful manner to provide mechanistic insights into the therapeutic effects of complex prescriptions and further understand pathophysiology of depression to assist in clinical diagnosis.

•This is the first report for the thorough studies of the pharmacokinetics and tissue distribution profiles of DAT-230 in rats.•The plasma sample preparation procedure was one-step precipitation of proteins.•The mean pharmacokinetic parameters of t1/2 and AUC0–12 were 1.1 ± 0.4 h and 861.0 ± 281.2 ng h/mL, respectively.A rapid, sensitive and high-throughput ultra-performance liquid chromatography method with tandem mass spectrometry (UPLC–MS/MS) has been developed for the determination of DAT-230 in rat plasma, urine, feces and tissues (heart, liver, spleen, lung, kidney, stomach, intestine and brain). The biological samples were prepared by protein precipitation, and separation was achieved on an ACQUITY™ UPLC BEH C18 column (50mm × 2.1 mm, 1.7 μm) with a mobile phase that consisted of methanol – 0.2% formic acid water (80:20, v/v) at a flow rate of 0.2 mL/min. The MS/MS ion transitions were monitored at m/z 372.17 → 357.17 for DAT-230 and m/z 406.08 → 345.16 for COH-203 (internal standard, IS). The calibration curve was linear in the range of 0.1–5200 ng/mL for all biological matrices (r2 ≥ 0.996), and it had the same value for the lower limit of quantification. The validated method was successfully applied to the pharmacokinetics, tissue distribution and excretion study after intravenous administration of a 5 mg/kg dose of DAT-230 to healthy Sprague-Dawley rats. The mean pharmacokinetic parameters of t1/2 and AUC0–12 were (1.1 ± 0.4) h and (861.0 ± 281.2) ng h/mL, respectively. Tissue distribution results indicated that DAT-230 exhibited rapid distribution and high liver, kidney, spleen, stomach and intestine uptake; these organs were indicated as the major target organs of the drug. In total, 5.3% of the administered DAT-230 was excreted in an unconverted form in urine, feces and bile, which implies that DAT-230 was excreted primarily in the form of metabolites.

Genkwa flos, a traditional Chinese medicine, displays severe hepatotoxicity when it is excessively or chronically used in its raw form. It has been proven that the chloroform extracts within Genkwa flos are responsible for its hepatotoxicity. Note that the vinegar process procedure may weaken the toxicity and enhance the therapeutic effects. This study was conducted to investigate a quality control method of the chloroform extracts of Genkwa flos and to identify the potential hepatotoxic ingredients using HL-7702 cells. A LC-MS method was developed and fully validated to simultaneously determine three flavonoids (apigenin, genkwanin and hydroxygenkwanin), three lignans (syringaresinol, medioresinol and matairesinol) and two diterpene esters (yuanhuacine and genkwadaphnin) in the chloroform extracts. With satisfactory linearity, precision, repeatability, stability and recovery, the developed method was then applied to compare the content changes of the eight compounds in raw and processed herbs. After processing, the content of flavonoids increased, the lignans did not obviously change, while the diterpene esters decreased. Compared with the blank control group, the HL-7702 cells treated with the two diterpene esters exhibited obvious morphology change, viability decrease and increase in the level of hepatic marker enzymes in cell culture supernatant. The study showed a comprehensive quantitative method to simultaneously determine eight compounds of the chloroform extracts from Genkwa flos, and they indicated that yuanhuacine and genkwadaphnin could be two of the potential hepatotoxic substances of the herb.

A simple and robust method of liquid chromatography-mass spectrometry coupled with an electrospray ionization interface was developed for the simultaneous determination of sixteen representative ingredients including seven iridoids, four soy isoflavones, two flavonoids, one gardenia yellow pigment, one monoterpene glycoside, and one organic acid in different composition of Gardenia jasminoides fruit (GJF) and Fermented Soybean (FS) (4:0, 2:1, 1:1, 1:2, 0:4), which are two component herbs of Zhi-zi-chi decoction. Chromatographic separation was accomplished on a Gemini C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) using gradient elution with the mobile phase consisting of methanol and 0.1% aqueous formic acid. The analytes were quantified without interference in the selected ion monitoring mode with positive electrospray ionization. All calibration curves show good linearity (r > 0.9994) within test ranges. The precisions and repeatability were evaluated and relative standard deviation values were less than 3.0%. The recoveries were between 96.5 and 103.7%. The validated method was successfully applied to a comparative analysis of sixteen constituents. The results demonstrate that there are significant differences in the contents of compounds in different proportions, and the combination of GJF and FS (2:1) was considered as the optimum ratio of dosage of Zhi-zi-chi decoction.

•LC–MS method was developed for simultaneous determination of five iridoids in brain.•Microdialysis coupled with brain tissue homogenate method was applied to the brain study.•The observations could contribute to the study of antidepressant.Zhi-zi-chi Decoction has been clinically utilized for the treatment of depression for more than thousand years. In order to investigate the possible bioactive components that could pass through the blood brain barrier (BBB) and the mechanism of antidepressant, a sensitive LC–MS method was developed to detect the ingredients (geniposide, scandoside methyl ester, gardenoside, deacetyl asperulosidic acid methyl ester and genipin-1-β-gentiobioside) in rat brain microdialysates and tissue homogenates samples (hippocampus, hypothalamus, premotor cortex, striatum, oblongata and cerebellum). Method development and validation are described in terms of calibration curves, extraction yield, lower limit of quantification (LLOQ), precision, accuracy, intra- and inter-day variability, which are in accordance with the requirements. Microdialysis in hippocampus demonstrated that the five iridoids possessed complete pharmacokinetic process while brain tissue homogenate method testified the distribution regularity in brain. The work clarified that the five iridoids, as antidepressant ingredients, could pass through the BBB, distribute targeted and possess complete pharmacokinetics in brain. These observations, along with the large database of rat brain microdialysates and tissue homogenates data, could enable future efforts aimed to improve our understanding of the relationship between bioactive ingredients and clinical therapy of depression.

•A uHPLC–MS/MS method was developed for the pharmacokinetic study of Shixiao San.•Simultaneous determination of five free and total (free and conjugated) flavonoids. The validated method was successfully applied to a comparative pharmacokinetic study.•The pharmacokinetic profiles in the normal and hyperlipidemic rats were elucidated.A simple and rapid ultra-high performance liquid chromatography–tandem mass spectrometry (uHPLC–MS/MS) method has been developed for the simultaneous determination of five free flavonoids (amentoflavone, isorhamnetin, naringenin, kaempferol and quercetin) and their total (free and conjugated) forms, and to compare the pharmacokinetics of these active ingredients in normal and hyperlipidemic rats. The free and total forms of these flavonoids were extracted by liquid–liquid extraction with ethyl acetate. The conjugated flavonoids were deconjugated by the enzyme β-Glucuronidase and Sulfatase. Chromatographic separation was accomplished on a ZORBAX Eclipse XDB-C8 USP L7 column using gradient elution. Detection was performed on a 4000Q uHPLC–MS/MS system from AB Sciex using negative ion mode in the multiple reaction monitoring (MRM) mode. The lower limits of quantification were 2.0–5.0 ng/mL for all the analytes. Intra-day and inter-day precision were less than 15% and accuracy ranged from −9.3% to 11.0%, and the mean extraction recoveries of analytes and internal standard (IS) from rat plasma were all more than 81.7%. The validated method was successfully applied to a comparative pharmacokinetic study of five free and total analytes in rat plasma. The results indicated that the absorption of five total flavonoids in hyperlipidemia group were significantly higher than those in normal group with similar concentration–time curves.

•It is the first report for bioavailability study of larotaxel in rats.•It is the first method for determination of larotaxel and its metabolites.•A simple and rapid LC–MS/MS method was developed and validated.A sensitive and reliable high-performance liquid chromatography–mass spectrometry (LC–MS/MS) method was developed and validated for determination of larotaxel (LTX) and its active metabolites (M1, M2 and M3) in rat plasma. The analytes were extracted by one-step protein precipitation and separated on a Capcell pak C18 column (2.0 mm × 100 mm; 2 μm; Shiseido) using methanol–water as mobile phase at a flow rate of 0.2 mL min−1 in gradient mode. The method was validated over the concentration range of 2.5–1250 ng mL−1 for LTX and 1.0–500 ng mL−1 for M1, respectively, while M2 and M3 were monitored semi-quantitatively and quantified as M1 equivalents. Intra- and inter-day accuracy and precision were within the acceptable limits of less than 15% at all concentrations. Coefficients of correlation (r) for the calibration curves were more than 0.99 for all analytes. The quantitation method was successfully applied for simultaneous estimation of LTX and its metabolites in a pharmacokinetic study after oral administration at different doses of 10, 20, and 40 mg/kg and intravenous administration at the dose 10 mg/kg to Wistar rats, respectively. The results indicated that larotaxel has linear pharmacokinetic characteristics in rats after oral administration and its absolute bioavailability in rats was 12.24%.

Quercetin (QT) is a natural flavonoid with various biological activities and pharmacological actions. However, the bioavailability of QT is relatively low due to its low solubility which severely limits its use. In this study, we intended to improve the bioavailability of QT by preparing quercetin-phospholipid complex (QT-PC) and investigate the protective effect of QT-PC against carbon tetrachloride (CCl4) induced acute liver damage in Sprague-Dawley (SD) rats. The physicochemical properties of QT-PC were characterized in terms of infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), powder X-ray diffraction (XRPD) and water/n-octanol solubility. FTIR, DSC and XRPD data confirmed the formation of QT-PC. The water solubility of QT was improved significantly in the prepared complex, indicating its increased hydrophilicity. Oral bioavailability of QT and QT-PC was evaluated in SD rats, and the plasma QT was estimated by HPLC-MS. QT-PC exhibited higher Cmax (1.58 ± 0.11 vs. 0.67 ± 0.08 μg/mL), increased AUC0–∞ (8.60 ± 1.25 vs. 2.41 ± 0.51 mg/L h) and t1/2z (7.76 ± 1.09 vs. 4.81 ± 0.87 h) when compared to free QT. The greater absorption of QT-PC group suggested the improved bioavailability. Moreover, biochemical changes and histopathological observations revealed that QT-PC provided better protection to rat liver than free QT at the same dose. Thus, phospholipid complexation might be one of the suitable approaches to improve the oral bioavailability of QT and obtain better protective effects against CCl4 induced acute liver damage in SD rats than free QT at the same dose level.Download high-res image (163KB)Download full-size image

A novel study using LC–MS (Liquid chromatography tandem mass spectrometry) coupled with multivariate data analysis and bioactivity evaluation was established for discrimination of aqueous extract and vinegar extract of Shixiao San. Batches of these two kinds of samples were subjected to analysis, and the datasets of sample codes, tR-m/z pairs and ion intensities were processed with principal component analysis (PCA). The result of score plot showed a clear classification of the aqueous and vinegar groups. And the chemical markers having great contributions to the differentiation were screened out on the loading plot. The identities of the chemical markers were performed by comparing the mass fragments and retention times with those of reference compounds and/or the known compounds published in the literatures. Based on the proposed strategy, quercetin-3-O-neohesperidoside, isorhamnetin-3-O-neohespeeridoside, kaempferol-3-O-neohesperidoside, isorhamnetin-3-O-rutinoside and isorhamnetin-3-O-(2G-α-l-rhamnosyl)-rutinoside were explored as representative markers in distinguishing the vinegar extract from the aqueous extract. The anti-hyperlipidemic activities of two processed extracts of Shixiao San were examined on serum levels of lipids, lipoprotein and blood antioxidant enzymes in a rat hyperlipidemia model, and the vinegary extract, exerting strong lipid-lowering and antioxidative effects, was superior to the aqueous extract. Therefore, boiling with vinegary was predicted as the greatest processing procedure for anti-hyperlipidemic effect of Shixiao San. Furthermore, combining the changes in the metabolic profiling and bioactivity evaluation, the five representative markers may be related to the observed anti-hyperlipidemic effect.

Cancer is the leading cause of death all over the world. Among the chemotherapy drugs, taxanes play an important role in cancer treatment. 6258-70 is a new semi-synthetic taxane which has a broad spectrum of antitumor activity. A fast and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method was developed for quantification of 6258-70 in rat plasma and tissues in this paper. After extraction by liquid-liquid extraction method with methyl tert-butyl ether, the samples were separated on a Kinetex C18 column (50 mm×2.1 mm, 2.6 µm, Phenomenex, USA) within 3 min. The method was fully validated with the matrix effect between 87.7% and 99.5% and the recovery ranging from 80.3% to 90.1%. The intra- and inter-day precisions were less than 9.5% and the accuracy ranged from −3.8% to 6.5%. The reliable method was successfully applied to the pharmacokinetics and tissue distribution studies of 6258-70 after intravenous administration in rats. The pharmacokinetic results indicated that the pharmacokinetic behavior of 6258-70 in rats was in accordance with linear features within tested dosage of 1 to 4 mg/kg, and there was no significant difference between the two genders. The tissue distribution study showed that 6258-70 had an effective penetration, spread widely and rapidly and could cross blood-brain barrier. The results of pharmacokinetics and tissue distribution may provide a guide for future study.

A simple and high throughput method was developed and validated for simultaneous determination of valproic acid and its two toxicant ene-metabolites, 2-enevalproic acid and 4-enevalproic acid in epilepsy patient plasma using liquid chromatography–tandem mass spectrometry. Probenecid was used as internal standard and solid-phase extraction was selected for sample preparation. A chromatographic separation was performed on an Agilent Poroshell SB-C18 column (50 mm×4.6 mm i.d., 2.7 μm) by an optimized gradient elution at a flow rate of 0.9 mL/min. The total run time was 7 min. Electrospray ionization was used in negative ion mode by multiple reaction monitoring of the precursor-to-product ion transitions at m/z 143.0→143.0 for valproic acid, m/z 140.9→140.9 for 2-enevalproic acid and 4-enevalproic acid for their poor fragments, and m/z 283.9→239.9 for probenecid. The results showed good linearity of valproic acid, 2-enevalproic acid and 4-enevalproic acid in their respective linear ranges. The correlation coefficients were more than 0.998. The intra- and inter-day precision of the assay was less than 11.0% and the accuracy ranged from 2% to 12%. This analytical method was successfully applied to assay plasma concentrations of valproic acid and its two ene-metabolites in epilepsy patient plasma and used for therapeutic drug monitoring.

Ethnopharmacological relevanceGenkwa Flos, a classical traditional Chinese medicine, is used for the definite antitumor activity and tends to be taken overdose or long term in these years. While the excessive application can result in damage to liver and kidney. In this study, the indicative roles of seven potential biomarkers were evaluated to investigate hepato-nephrotoxicity in the early stages after oral administration of Genkwa Flos for 14 days.Materials and methodsHistopathology, serum biochemistry and seven potential biomarkers in serum or urine from male Sprague–Dawley rats were monitored. Hepatic and renal tissues were histopathologically examined to identify specific changes occurring. Routine serum biochemical parameters were tested by using standard clinical laboratory methods. Seven biomarkers including cholic acid, taurine, 5-oxoproline, hippuric acid, uric acid, 3-indoxyl sulfate and kynurenic acid were detected by a developed LC–MS method.ResultsThe histopathological alterations and the increased levels of serum biochemistry were detected on the 8th day after Genkwa Flos treated. The seven analytes were also found significantly changed in Genkwa Flos treated group, especially cholic acid, taurine, 5-oxoproline and hippuric acid which were changed on the 2nd or 4th day.ConclusionsAlthough serum biochemistry and histopathology suggested that Genkwa Flos was responsible for the hepato-nephrotoxicity that occurred following the ingestion of this medicinal herb, evaluation of these biomarkers might be more beneficial for the early detection of liver and kidney injuries. This study could be further used in hepatic and renal failures caused by other reasons in the following research works.Download high-res image (199KB)Download full-size image

The total flavonoids from Persimmon leaves (PLF), extracted from the leaves of Diospyros kaki L. Dispryosl and Ebenaceae, is reported to possess many beneficial health effects. However, the oral bioavailability of PLF is relatively low due to its poor solubility. In the present study, the phospholipid complexes of total flavonoids from Persimmon leaves (PLF-PC) was prepared to enhance the oral bioavailability of PLF and to evaluate its antiatherosclerotic properties in atherosclerosis rats in comparison to PLF. A HPLC-MS method was developed and validated for the determination of quercetin and kaempferol in rats plasma to assess the oral bioavailability of PLF-PC. The effect of PLF (50 mg/kg/d) and PLF-PC (equivalent to PLF 50 mg/kg/d) on atherosclerosis rats induced by excessive administration of vitamin D (600,000 IU/kg) and cholesterol (0.5 g/kg/d) was assessed after orally administered for 4 weeks. The relative bioavailabilities of quercetin and kaempferol in PLF-PC relative to PLF were 242% and 337%, respectively. The levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), apolipoprotein A1 (ApoA1) and apolipoprotein B (ApoB) in serum were measured by an automatic biochemistry analyzer. The morphological changes of aorta were observed with optical microscopy. According to the levels of biochemical parameters in serum and the morphological changes of aorta, PLF-PC showed better therapeutic efficacy compared to PLF. Thus, PLF-PC holds a promising potential for increasing the oral bioavailability of PLF. Moreover, PLF-PC exerts better therapeutic potential in the treatment of atherosclerotic disease than PLF.Download high-res image (138KB)Download full-size image

BackgroundWater extract of the fixed combination of Gardenia jasminoides Ellis fruit, Citrus aurantium L. fruit and Magnolia officinalis Rehd. et Wils. bark, traditional name – Zhi-Zi-Hou-Po (ZZHPD) is used for treatment of depressive-like symptoms in traditional Chinese medicine for centuries.Hypothesis/PurposeThe present study aimed to explore antidepressant-like effects and potential mechanisms of ZZHPD in a rat model of chronic unpredictable mild stress (CUMS).Study designAntidepressant-like effects of ZZHPD were investigated through behavioral tests, and potential mechanism was assessed by neuroendocrine system, neurotrophin and hippocampal neurogenesis.MethodsAntidepressant-like effects of ZZHPD (3.66, 7.32 and 14.64 g/kg/day) were estimated through coat state test, sucrose preference test, forced swimming test and open-field test. Effects of ZZHPD on hypothalamic-pituitary-adrenal (HPA) axis were evaluated by hormones measurement and dexamethasone suppression test. In addition, the expression of brain-derived neurotrophic factor (BDNF) in hippocampus was measured, as well as hippocampal neurogenesis was investigated by doublecortin (DCX) and 5-bromo-2-deoxyuridine/neuronal nuclei (BrdU/NeuN).ResultsThe results demonstrated that ZZHPD significantly reversed the depressive-like behaviors, normalized the levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT), restored the negative feedback loop of HPA axis and improved the levels of BDNF, DCX and BrdU/NeuN compared with those in CUMS-induced rats.ConclusionThe above results revealed that ZZHPD exerted antidepressant-like effects possibly by normalizing HPA axis function, increasing expression of BDNF in hippocampus and promoting hippocampal neurogenesis.Download high-res image (217KB)Download full-size image

Genkwa flos, a traditional Chinese medicine, displays severe hepatotoxicity when it is excessively or chronically used in its raw form. It has been proven that the chloroform extracts within Genkwa flos are responsible for its hepatotoxicity. Note that the vinegar process procedure may weaken the toxicity and enhance the therapeutic effects. This study was conducted to investigate a quality control method of the chloroform extracts of Genkwa flos and to identify the potential hepatotoxic ingredients using HL-7702 cells. A LC-MS method was developed and fully validated to simultaneously determine three flavonoids (apigenin, genkwanin and hydroxygenkwanin), three lignans (syringaresinol, medioresinol and matairesinol) and two diterpene esters (yuanhuacine and genkwadaphnin) in the chloroform extracts. With satisfactory linearity, precision, repeatability, stability and recovery, the developed method was then applied to compare the content changes of the eight compounds in raw and processed herbs. After processing, the content of flavonoids increased, the lignans did not obviously change, while the diterpene esters decreased. Compared with the blank control group, the HL-7702 cells treated with the two diterpene esters exhibited obvious morphology change, viability decrease and increase in the level of hepatic marker enzymes in cell culture supernatant. The study showed a comprehensive quantitative method to simultaneously determine eight compounds of the chloroform extracts from Genkwa flos, and they indicated that yuanhuacine and genkwadaphnin could be two of the potential hepatotoxic substances of the herb.

A simple and robust method of liquid chromatography-mass spectrometry coupled with an electrospray ionization interface was developed for the simultaneous determination of sixteen representative ingredients including seven iridoids, four soy isoflavones, two flavonoids, one gardenia yellow pigment, one monoterpene glycoside, and one organic acid in different composition of Gardenia jasminoides fruit (GJF) and Fermented Soybean (FS) (4:0, 2:1, 1:1, 1:2, 0:4), which are two component herbs of Zhi-zi-chi decoction. Chromatographic separation was accomplished on a Gemini C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) using gradient elution with the mobile phase consisting of methanol and 0.1% aqueous formic acid. The analytes were quantified without interference in the selected ion monitoring mode with positive electrospray ionization. All calibration curves show good linearity (r > 0.9994) within test ranges. The precisions and repeatability were evaluated and relative standard deviation values were less than 3.0%. The recoveries were between 96.5 and 103.7%. The validated method was successfully applied to a comparative analysis of sixteen constituents. The results demonstrate that there are significant differences in the contents of compounds in different proportions, and the combination of GJF and FS (2:1) was considered as the optimum ratio of dosage of Zhi-zi-chi decoction.