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Xiao-Yao-San-Jia-Wei (XYSJW) is a commonly prescribed formulation for depression and anorexia in the Jiang Su Province Hospital of Chinese Medicine. Unfortunately, the proper dosage of this formulation is still unclear due to its limited chemical and pharmacokinetic profiles. Thus, in the present study, a sensitive, precise, and rapid procedure for the identification of absorbed compounds (Cs) in the plasma of depressed rats together with a pharmacokinetic analysis was established with the help of ultra-flow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UFLC-Q-TOF MS/MS) and ultra-flow liquid chromatography coupled with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC-QQQ MS/MS). Based on the characteristic fragmentation, neutral loss, mass defect filter, relevant literature and reference standards, 225 Cs in the XYSJW extract and 20 Cs in the plasma of the depressed rats were tentatively recognized

Oligosaccharides in Atractylodes macrocephala Koidz. (AMK) have a wide range of clinical effects, especially in terms of improving immunity. In this study, the oligosaccharide fingerprint of AMK was first established by high-performance liquid chromatography coupled with evaporative light-scattering detection. This method was confirmed to be accurate and reliable. Twenty-six batches of samples from four regions were detected, and the oligosaccharide fingerprint similarity of AMK was evaluated (> 0.900). Second, the oligosaccharide profile of AMK was analyzed using high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry. Fourteen inulin-type oligosaccharides with a degree of polymerization of 2–15 were identified, including sucrose, 1-kestose, and nystose. Finally, two representative compounds, 1-kestose and nystose, were selected for quantification analysis. The established method had good regression equations, precision, repeatability, and stability. The average contents of 1-kestose and nystose were 0.688% and 0.827%, respectively. This study provides valuable information on the quality evaluation and discrimination of different varieties of AMK.

Guizhi-Fuling capsule (GZFL), a well-known herbal remedy, has been widely used to treat primary dysmenorrhea (PD). Hence, systematic identifying multiple active ingredients and the involved mechanism is essential and urgently needed for GZFL.This study was planned to assess the pharmacokinetics of GZFL in rats, and identify whether these GZFL-derived absorbed components (ACs) contribute to the efficacy of source herbs and relevant mechanism.The in vivo pharmacokinetic profile of 11 phytochemicals and 13 metabolites in healthy and PD rats were evaluated using liquid chromatography with mass spectrometry (LC-MS/MS). Whereafter, the introduced contribution strategy assessed ACs' effect (doses = their contents in GZFL) in PD rats with the mechanism.The pharmacokinetic profiles of prototypes and metabolites differed in healthy and PD rats. As a main proxy of GZFL, 11ACs exerted an anti-PD effect (improvement of indexes for writhing latency, writhing time, PGFAs a paradigmatic example, 11ACs contributed an average of 113.55% to GZFL in terms of anti-PD efficacy, providing an approach to rapidly, accurately and consistently identify the bioactive components and their pathway from herbs.

Carboxylesterases (CESs) and Histone deacetylases (HDACs) are regarded as important signaling enzymes highly associated with the development and progression of multiple cancers, including hepatocellular carcinoma (HCC). In this work, a near-infrared (NIR) fluorescent probe named Lys-HXPI was designed and synthesized, which linked a hemicyanine dye and 6-acetamidohexanoic acid via an ester bond. Lys-HXPI displayed a remarkable increase with a NIR emission at 720 nm, a low detection limit (10 nM) for HDAC1, HDAC 6, CES1 and CES2, as well as a high selectivity for the target enzymes over other relevant analytes. Furthermore, Lys-HXPI was used to image endogenous target enzymes in living cells, tumor-bearing nude mice and tissue slices. The ability of Lys-HXPI to simultaneous image CESs and HDACs was demonstrated with RT-qPCR and the confocal imaging in Hep G2 and MDA-MB-231. Taking advantage of NIR emission, the probe was also successfully applied to imaging Hep G2 tumor mice and tissue slices. Lys-HXPI is expected to be useful for the effective detecting of CESs and HDACs in complex biosystems.

In this study, the chemical profile of Ling-Gui-Zhu-Gan Decoction (LGZGD) which was prepared by the method recorded in Synopsis of Golden Chamber was analyzed by LC–QTOF MS. A total of 89 compounds were identified, including flavonoids, pentacyclic triterpenes, glycosides, organic acids and others. Among them, nine major active components with high response value in chromatogram were selected for quantification, including liquiritin apioside, liquiritin, isoliquiritin apioside, isoliquiritin, coumarin, liquiritigenin, cinnamic acid, cinnamaldehyde and glycyrrhizic acid. A quantitative analysis of multi-components by single marker (QAMS) was applied to simultaneous determination of nine major components in LGZGD. Using liquiritin as the internal reference, the relative correction factors (RCFs) were calculated to determine the contents of other eight compounds. A series of methodological study and robustness study were carried out, which proved that the RCFs between liquiritin and others were stable. In addition, compared with the results calculated by external standard method (EMS), there were no remarkable differences between these two methods with absolute relative errors less than 3.12%. The developed QAMS method was successfully applied to preparation process analysis. In conclusion, this QAMS method was convenient, accurate and feasible enough for evaluating the quality of LGZGD.

Background: Ilexsaponin A1, one of the most representative triterpene saponin components in the roots of I. pubescens, showed its effects in anticoagulation and antithrombosis, attenuating ischemia-reperfusion-induced myocardial, angiogenesis and inhibiting phosphodiesterase. Objective: Reveal the key intestinal bacterial strains responsible for ilexsaponin A1 metabolism, and clarify their metabolic behavior. Methods: An accurate and sensitive LC-MS/MS method for the determination of “ilexsaponin A1 in General Anaerobic Medium (GAM) broth” was established and systematically validated. Then it was applied to screen and study the metabolic potential of the intestinal bacterial strains in an anaerobic incubation system. Results: Quantitation of ilexsaponin A1 could be performed within an analytical run time of 14.5 min, in the linear range of 2 - 2000 ng/ml. Enterobacter sakazakii, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium catenulatum, and Bifidobacterium angulatum were identified to have a potential effect to metabolize ilexsaponin A1 to different extents; and further bacterial metabolic studies were performed to clarify their metabolic capacity and behavior. Conclusion: This paper contributes to a better understanding of the intestinal bacterial metabolism of ilexsaponin A1 and provides scientific evidence for its clinical application. Additionally, the importance of intestinal bacterial strains in the disposition of natural products was also highlighted.

Histone deacetylase 8 (HDAC8) has been used as a therapeutic target for many cancers as it is highly expressed in neuroblastoma cells and breast cancer cells. HDAC8-selective fluorescent probes need to be urgently developed. Herein, two novel fluorescent probes, namely NP-C6-PCI and AM-C6-PCI, based on the conjugation of 1,8-naphthalimide with a highly selective inhibitor of HDAC8 (PCI-34051) were reported. Compared with PCI-34051 (KD = 6.25 × 10−5 M), NP-C6-PCI (KD = 8.05 × 10−6 M) and AM-C6-PCI (KD = 7.42 × 10−6 M) showed great selectivity toward HDAC8. Two fluorescent probes exhibited high fluorescence intensity under λex = 450 nm and a large Stokes shift (100 nm). NP-C6-PCI was selected for cell and tissue imaging due to the similarity in the bioactivity of NP-C6-PCI with PCI-34051. The ability of NP-C6-PCI to target imaging HDAC8 in SH-SY5Y and MDA-MB-231 tumor cells was demonstrated. Furthermore, NP-C6-PCI was applied to imaging SH-SY5Y tumor tissue slices to indicate the relative expression level of HDAC8. Therefore, this HDAC8-selective fluorescent probe can be expected for applications in HDAC8-targeted drug screening as well as in pathologic diagnoses.

There have been few comprehensive studies on the holistic chemical composition of Spatholobi Caulis (SC) and consequently, the information is lacking for the in-depth study of the major constituents. SC is a kind of widely used traditional Chinese medicine with its xylem and phloem alternately arranged in 3-10 rings, but the relationship of phloem ring number and the quality remains unclear. In this study, the characterization of the major constituents in SC was analyzed by ultra-fast liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry (UFLC-Triple TOF-MS/MS), and the content of 19 flavonoids in SC with different phloem ring numbers was simultaneously determined by ultra-fast liquid chromatography coupled with triple quadrupole-linear ion trap tandem mass spectrometry (UFLC-QTRAP-MS/MS). Correlation analysis was performed to evaluate the quality of SC with different phloem ring numbers according to the content of 19 flavonoids. Results showed that 50 constituents in SC were identified and the fragmentation pathways of different types of compounds were preliminarily deduced by the fragmentation behavior of the 50 constituents. In addition, the content of flavonoids increased with phloem ring number, which demonstrated that the content of flavonoids in SC was positively correlated with the number of phloem rings. Our research will contribute to the variety identification and quality evaluation of SC, and provide a scientific basis for evaluating the quality of medicinal materials based on its appearance and characteristics.

Ethnopharmacological relevance Zhi-zi-chi decoction (ZZCD) is used for treating depression as an effectively traditional Chinese medicine. Until now, studies on pharmacological research of ZZCD have mostly been centered in pharmacokinetic level. Little was known about its pharmacological mechanism of relieving depression. Aim of the study This study was to evaluate the effect of ZZCD on relieving depression via behavioral tests, serum metabolomics and signaling target expression analysis on chronic unpredictable mild stress (CUMS) model mice. Materials and methods The CUMS exposure lasted 7 consecutive weeks. The mice were administrated with ZZCD for the last 3 weeks. Behavioral tests were applied and a serum metabolomics method based on UFLC/Q-TOF-MS with multivariate statistical and global metabolic network analysis was performed to identify relevant metabolites and pathways. Finally, the protein expressions in mouse hippocampi were determined by western blot to verify the metabolomics deduction. Results Behavioral parameters were visibly changed after modeling, while high and medium dosage groups showed status improvement compared to the model group. Seventy six metabolites were identified as potential biomarkers from the metabolomics profiles in C18 and HILIC systems. In addition, 9 significant pathways related to changed biomarkers were conducted. The pathways were closely connected by some key targets, which were significantly reduced in the model group compared with those in control group, while ZZCD treated groups showed corrections after 3-week administration. The results revealed that the anti-depression efficacy of ZZCD might be associated with PKA-CREB-BDNF-TrkB-PSD-95 pathway influenced by metabolic changes, verifying the pathway annotation speculation. Conclusion This study demonstrated that ZZCD had a positive treatment effect on CUMS depression model mice. Metabolomics results revealed the holistic and interconnected metabolic changes of ZZCD in CUMS mice. The metabolic pathway annotation suggested that the anti-depression mechanism of ZZCD might be related to signaling pathway in brain. PKA-CREB-BDNF-TrkB-PSD-95 signaling expression was a verification and complement to the metabolomics results.

N1 N2 bond in molecules containing 1, 2, 3-triazoles framework is weakened and readily cleaved when N1 is attached to a strong electron-withdrawing group. However, ticagrelor, a pyrimidine-fused triazole without a strong electron-withdrawing group on N1 site still went through isomerization reaction via N1 N2 bond rupture in the presence of acid to produce an impurity T3. Its structure was precisely obtained via NMR spectral analysis, MS spectroscopy and X-ray crystallography. The optimal condition of the isomerization reaction was systematically studied. Moreover, the isomerization mechanism was investigated and it is likely that ticagrelor and its analogues go through isomerization reaction when the 4-NH group and electron-deficiency ring exist in their structures. Besides, the steric hindrance of the substituent on 4-NH has an effect on the reactivity of equilibrium. Under mimic in vivo condition, the isomerization reaction of ticagrelor also generated, indicating ticagrelor may be not stable in stomach.

A rapid, sensitive, and accurate ultra flow liquid chromatography tandem mass spectrometry (UFLC-MS/MS ) method was developed and validated for simultaneous quantitation of glycyrrhetic acid and puerarin in plasma derived from healthy and alcoholic liver injury rats. Plasma samples from healthy and model rats were deproteinated with methanol using liquiritin as an internal standard. Chromatography separation was performed by a Waters BEH (ethylene-bridged hybrid) C18 column (2.1 × 50 mm; 1.7 μm) using a gradient elution from acetonitrile and water (containing 0.1% formic acid) and at a flow rate of 0.4 mL/min. Quantitation was performed on a Triple Quad 4500 tandem mass spectrometer coupled with an electrospray ionization source in negative multiple reaction monitoring mode. Specificity, carryover, dilution integrity, recovery, linearity, precision and accuracy, matrix effect, and stability were within acceptable limits. The newly established method was successfully applied to a pharmacokinetics study to investigate glycyrrhetic acid and puerarin in healthy and alcoholic liver injury rats.

Liguzinediol (2,5-dihydroxymethyl-3,6-dimethylpyrazine, LZDO) is a potential agent for the low-risk treatment of heart failure. 2-N-acetylcysteine-LZDO (2-NAC-LZDO) and 2-cysteine-LZDO (2-Cys-LZDO) are major LZDO metabolites found in the pharmacokinetic studies of rats and beagle dogs. To elucidate the biotransformation pathway and related enzymes, an incubation system with 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as a cofactor and N-acetylcysteine (NAC) as a trapping agent was established using liver cytosol. An ultra-flow liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UFLC-QTOF-MS) method was used to identify the major metabolites. 2-NAC-LZDO could be detected among four species (humans, monkeys, dogs, and rats) and is the dominant metabolite in human liver cytosol (HLC). The sulfotransferase (SULT) inhibitors 2,6-dichloro-4-nitrophenol (DCNP) and quercetin at a concentration of 1 μM, suppressed 2-NAC-LZDO formation in HLC by 87 and 46%, respectively. This result suggested that sulfotransferase was involved in 2-NAC-LZDO formation. The metabolism of LZDO in different species indicated that SULT activity in dogs, rats, and monkeys was higher than that in humans. Further SULT phenotyping revealed that SULT1A1 is the predominant enzyme involved in the sulfation of LZDO. The underlying mechanism for the biotransformation of LZDO was demonstrated. The potential pathway is via the sulfation of LZDO to form sulfate, and the spontaneous cleavage of the sulfate group to generate highly reactive electrophilic cations, which can bind to NAC to form the major metabolites.

As the most important marker component in Carthamus tinctorius L., hydroxysafflor yellow A (HSYA) was widely used in the prevention and treatment of cardiovascular diseases, due to its effect of improving blood supply, suppressing oxidative stress, and protecting against ischemia/reperfusion. In this paper, both an in vitro microsomal incubation and an in vivo animal experiment were conducted, along with an LC-Q-TOF/MS instrument and a 3-step protocol, to further explore the metabolism of HSYA. As a result, a total of 10 metabolites were searched and tentatively identified in plasma, urine, and feces after intravenous administration of HSYA to male rats, although no obvious biotransformation was found in the simulated rat liver microsomal system. The metabolites detected involving both phase I and phase II metabolism including dehydration, deglycosylation, methylation, and glucuronic acid conjugation. A few of the metabolites underwent more than one-step metabolic reactions, and some have not been reported before. The study would contribute to a further understanding of the metabolism of HSYA and provide scientific evidence for its pharmacodynamic mechanism research and clinical use.

In the present study, the chemical compositions of Atractylodes macrocephala Koidz. (AMK) were analyzed systematically and influence of sulfur fumigation on the chemical profiles was evaluated by ultrafast flow liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UFLC-QTOF-MS) combined with multivariate statistical analysis. 52 components were detected from non-fumigated AMK (NF-AMK) and 28 components were newly produced after sulfur fumigation, out of which 59 major peaks were identified. The concentrations of 20 compounds significantly decreased and 37 compounds obviously increased. The potential structural transformation mechanism of terpenoids was explored to illustrate the correlation of the components contents before and after sulfur fumigation. Eight sulfur-containing/dehydrated-integrated atractylenolides that evolved from the NF-AMK were screened out as potential characteristic chemical markers to examine the post-harvest handling procedures of commercial AMK with excessive sulfur fumigation and maintain consistent quality.

Valine catabolism is known to be essential for cancer cells but the detailed mechanism remains unclear. This study is to explore the critical roles of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) in colorectal cancers (CRC) and to develop a new therapy returning valine metabolism homeostasis. High HIBCH expression was first confirmed to correlate with poor survival in patients with CRC, which was then linked to the increased cell growth, resistant apoptosis, and decreased autophagy in CRC cells. The functions of HIBCH in CRC were dependent on its mitochondrial localization. High HIBCH level was further demonstrated to promote the metabolism of tricarboxylic acid cycle as well as oxidative phosphorylation in CRC cells. Based on above findings, we further discovered a novel valine catabolism inhibitor SBF-1. The pharmacological blockade of HIBCH mitochondrial localization with SBF-1 resulted in decreased cancer cell growth and increased autophagy, collectively contributing to the antitumor effect both in vitro and in vivo. Moreover, anti-VEGF therapy with bevacizumab increased HIBCH level in CRC cells, which in turn caused the resistance to the therapy. The interference with HIBCH function by SBF-1 significantly increased the antitumor efficacy of bevacizumab and led to a robust survival benefit. The present study identified HIBCH as a critical enzyme of valine catabolism in CRC progression and resistance to anti-VEGF therapy. We also provided a novel HIBCH inhibitor SBF-1, which highlighted the combined therapy using valine catabolic inhibitor along with anti-VEGF drugs, to control progression of CRC.

Systematic comparison of active ingredients in Sojae semen praeparatum (SSP) during fermentation was performed using ultra-fast liquid chromatography (UFLC)&ndash, TripleTOF MS and principal component analysis (PCA). By using this strategy, a total of 25 varied compounds from various biosynthetic groups were assigned and relatively quantified in the positive or negative ion mode, including two oligosaccharides, twelve isoflavones, eight fatty acids, N&ndash, (3&ndash, Indolylacetyl)&ndash, dl&ndash, aspartic acid, methylarginine, and sorbitol. Additionally, as the representative constituents, six targeted isoflavones were sought in a targeted manner and accurately quantified using extracted ion chromatograms (XIC) manager (AB SCIEX, Los Angeles, CA, USA) combined with MultiQuant software (AB SCIEX, Los Angeles, CA, USA). During the fermentation process, the relative contents of oligoses decreased gradually, while the fatty acids increased. Furthermore, the accurate contents of isoflavone glycosides decreased, while aglycones increased and reached a maximum in eight days, which indicated that the ingredients converted obviously and regularly throughout the SSP fermentation. In combination with the morphological changes, which meet the requirements of China Pharmacopoeia, this work suggested that eight days is the optimal time for fermentation of SSP from the aspects of morphology and content.

Curcumin (CUR) is a natural yellow pigment from turmeric with extensive bioactivities. However its relatively poor solubility limited its absorption and bioavailability. In this study, a novel series of CUR-peptide conjugates were designed and synthesized as PepT1-mediated transport drugs and their solubility, cellular uptakes and anti-tumor activities were evaluated. Ten compounds showed better water solubility than CUR due to the dipeptide moiety. Compared with CUR, compound 5e exhibited the slightly better activity and 5d showed the similar activity with CUR. Besides, compounds 5d and 5e performed higher cellular uptakes in Caco-2 cell and dose-dependently inhibited by the addition of PepT1 typical substrate glycylsarcosine (Gly-Sar). Compound 5d and 5e have improved the absorption of CUR by PepT1-mediated without affected the activity. These new dipeptide conjugates of CUR may serve as promising lead compounds for future drug development.

RATIONALE As a herbal plant used in traditional Chinese medicine, Lamiophlomis rotata (Benth.) Kudo mainly displays its pharmacological effect by promoting blood circulation and hemostasis, dispelling wind, and acting as an analgesic. To identify the components contained in L. rotata, global detection and structural elucidation of both target and non-target components in the medicinal material was performed. METHODS L. rotata was ultrasonically extracted with methanol. Separation and analysis were achieved using liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS). A three-step protocol which included (1) potential components screening, (2) collection of qualitative information, and (3) database searching and structural elucidation was used for target and non-target identification. RESULTS A total of 42 components were tentatively identified, which included 12 iridoids (2 aglycones and 10 glucosides), 11 flavonoids (4 aglycones and 7 glucosides), and 13 phenylethanoid glycosides. Moreover, components of L. rotata extract belonging to the three main structural categories could be well separated in a 3D point plot according to their retention times, mass defects and degrees of unsaturation, facilitating the structural classification and identification in the subsequent studies. CONCLUSIONS The results provide a reasonable picture of the components contained in L. rotata extract and promote the further pharmacodynamic and/or pharmacokinetic characterization of this medical material, meanwhile demonstrating the utility of a universal methodology for the systematical study of herbal medicines. Copyright © 2016 John Wiley & Sons, Ltd.

The current widely utilized polymer or C8, C18 end-capped material-based sorbents for solid-phase extraction could not capture alkaloids well only based on "like dissolves like" principle. In this paper, a layer-by-layer functionalized porous Zinc sulfide nanospheres-based solid-phase extraction (SPE) combined with liquid chromatography time-of-flight/mass spectrometry (LC-TOF/MS) and gas chromatography-mass spectrometry (GC-MS) was developed for the specific enrichment and identification of alkaloids from complex matrixes, Crinum asiaticum var. sinicum crude extracts. The functionalized porous Zinc sulfide nanospheres were prepared by the amidation reaction of poly-(acrylic acid) (PAA) homopolymer with amino groups onto the porous ZnS nanospheres. Tandem LC-TOF/MS spectrometry presented that the almost all of the twenty-three main peaks in elution fraction from the SPE could be inferred as alkaloids with ion of mass according to the nitrogen rule and hit formula with Peak View1.2@software from AB SCIEX, and seven alkaloids including two new found chemical entities were directly identified from their GC-MS spectra and retention indices. We believe that this SPE protocol can also be utilized in the future to selectively enrich alkaloids from extracts of other plant species.

Although some physiological and pathological function parameters of hepatitis and liver cancer have been investigated in relation to hispidulin (5,7,4'-trihydroxy-6-methoxyflavone), the changes of small metabolites in biofluids have been reported rarely. Recent research has shown that metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UHPLC-QTOF/MS) coupled with multivariate statistical analysis provides a good understanding of hispidulin effects on mice vaccinated intraperitoneally with H22 tumor cells. Twenty-five potential biomarkers, up- or down-regulated (P

Flavonoids, including flavones, isoflavones, flavanones, chalcones, and isoflavans, have long been recognized as the main active ingredients in licorice. A method combining liquid chromatography with photodiode array detection and quadrupole time-of-flight mass spectrometry was developed to characterize components in three Glycyrrhiza species, and to expound the characteristic fragmentation behaviors in the positive ion mode. Based on the fragmentation patterns of reference compounds, a total of 39 compounds, including 37 flavonoid aglycones and two coumestans, were identified or tentatively identified. Besides, some common features, such as H2 O, CO, and CH2 O2 losses, together with retro-Diels-Alder fragmentation, were observed in these compounds. Furthermore, diagnostic fragmentations of C-ring cleavages and UV absorption on the skeleton groups were observed to structurally characterize flavonoid aglycones. In addition, typical losses of different substituent groups were detected: Neutral losses of 56 (C4 H8 ) and 68 Da (C5 H8 ) were yielded from a prenyl chain; neutral losses of 42 (C3 H6 ), 54 (C4 H6 ), and 70 Da (C4 H6 O) were generated by a pyran ring. Particularly, neutral losses of 18 (H2 O), 16 (CH4 ), 112 (C8 H16 ), and 98 Da (C7 H14 ) predicted a hydroxyl, a methoxyl, double prenyl chains, and a prenyl chain with a pyran ring, respectively.

A rapid and sensitive UFLC–MS/MS method was developed for the simultaneous determination of liguzinediol and its four primary metabolites (M1, M2, M3, and M4) in rat plasma using antipyrine as internal standards. The analytes were separated on an XR-ODS column (50 mm × 2.0 mm, 2.2 μm) using 0.1 % formic acid–methanol gradient elution at a flow rate of 0.5 mL·min−1. The detection was performed on a triple quadrupole tandem mass spectrometer in a multiple reaction monitoring mode with positive electrospray ionization. Method validation was performed as per the Food and Drug Administration guidelines. The resulting calibration curves offered satisfactory linearity (r > 0.9993) with the set ranges. The limits of quantification for liguzinediol, M1, M2, M3, and M4 were 20, 20, 21, 27, and 10 ng mL−1, respectively. The recovery rates in different matrices ranged from 91.2 to 114.1 %, and the inter-day and intra-day precisions were all less than 13.4 % for the target analytes. After validation, this method was successfully applied to further study pharmacokinetics profiles of liguzinediol and its metabolites after intravenous administration of 10 mg·kg−1 in male and female rats.

The dried flower of Carthamus tinctorius L. (honghua) is a widely used traditional Chinese medicine in clinics to treat coronary heart disease, hypertension, and cerebrovascular disease due to its functions of ameliorating circulation and removing blood stasis. Hydroxysafflor yellow A (HSYA) is an active marker component of honghua. In this paper, ultra-flow liquid chromatography coupled with quadrupole-time-of-flight mass-spectrometry (UFLC-Q-TOF/MS) was established and successfully applied to the detection and identification of the metabolites in bile, urine, plasma and feces samples of normal and model rats with orally administrated HSYA. A total of 8 metabolites were observed in normal rats, while 7 metabolites were detected in model rats. The distribution of metabolites in the plasma, bile, urine and feces of normal and model rats had obvious differences. The major in vivo metabolic pathways for HSYA included hydroxylation, hydroxylation+methylation, acetylation and glucuronidation, and there were also dehydration, hydrogenation, hydration, and hydroxylation+glucuronidation. All of these metabolites were reported for the first time, and these results are valuable and important for the understanding of the metabolic process and therapeutic mechanism of HSYA and some other pigments in honghua.

A sensitive ultrafast liquid chromatography coupled with triple quadrupole mass spectrometric (UFLC-MS/MS) method for the quantification of anhydrosafflor yellow B (AHSYB), a major active water-soluble pigment from Carthamus tinctorius, in rat plasma has been developed and validated. Sample preparation was achieved by protein precipitation of plasma with four volumes of methanol. Rutin was used as the internal standard (IS). The analytes were separated using a C18 column with an 8min gradient elution, followed by mass spectrometric detection using negative electrospray ionization (ESI(-)) in multiple reaction monitoring (MRM) mode. The method was linear in the concentration range of 25-10,000ng/mL for AHSYB. Intra-day and inter-day precision variation was less than 6.5%. The relative error of accuracy was within ±9.4%. The mean recovery of AHSYB was higher than 70.9%. The established method was successfully applied to the pharmacokinetic study after intravenous (2.5mg/kg) and oral (30mg/kg) dosing of AHSYB in normal rats. And the pharmacokinetic properties of AHSYB in rats with acute blood stasis and the differences between normal and acute blood stasis syndrome rats were also investigated. The results showed that the compound was poorly absorbed (∼0.3%) and the AUC0-t, AUC0-∞ and F were all significantly lower (P

An HPLC method was employed to create an impurity profile for liguzinediol as an active pharmaceutical ingredient (API), which resulted in the identification of two related impurities. Therefore, in order to improve the quality control of the liguzinediol-API, we identified and then developed a method for quantifying the two impurities (impurity-1 and impurity-2) by LC-TOF-MS-MS and then chemically synthesized them for further studies. Based on spectral data from IR, MS, (1)H and (13)C NMR, the structures of impurity-1 and impurity-2 were characterized as 2-hydroxymethyl-3,6-dimethylpyrazine and 2-hydroxymethyl-3,5,6-trimethylpyrazine, respectively. We further validated the method according to the International Conference on Harmonization guidelines to demonstrate the sensitivity, precision, linearity, accuracy and stability of the method described. In addition, the potential mechanisms underlying formation of impurity-1 and impurity-2 in the liguzinediol-API are discussed in detail.

Leaves of Platycladus orientalis have been used as blood cooling and homeostatic therapy for thousands of years in traditional Chinese medicine. Emerging evidences of modern pharmacology have proved flavonoids as the key elements responsible for the efficacies. However, there has been no report on pharmacokinetic study of the flavonoids from Platycladus orientalis leaves extract. In this study, a sensitive and rapid ultra-flow liquid chromatography-tandem mass spectrometry method was established and validated for the simultaneous determination of amentoflavone, afzelin, hinokiflavone and quercitrin in rat plasma. The four flavonoids and luteolin (internal standard, IS) were recovered from rat plasma by methanol–ethyl acetate (v:v, 50:50). Chromatographic separation was performed on a C18 column with gradient elution. Our results showed that the recoveries from spiked control samples were more than 85% for all analytes and IS. The relative standard deviations of intra-day and inter-day precision were within 15% while the REs ranged from −6.6% to 8.0%. The validated method in this study was successfully applied to pharmacokinetic study in healthy rats after oral administration of P. orientalis leaves extract.

Lophatherum gracile Brongn. (L. gracile) has been used for food and medicine in China for thousands of years. Flavonoids are one of the main bioactive components of this herb. A reliable method was established using liquid chromatography with tandem mass spectrometry to determine the concentrations of seven flavonoids. The separation was performed using a C18 column at 40°C and the mobile phase was a mixture of methanol and 0.3% formic acid (v/v) in water in gradient elution mode at a flow rate of 1 mL/min. Quantification was performed through tandem mass spectrometry with negative electrospray ionization and multiple reaction monitoring at m/z [M-H]−. The method was validated using calibration curves, limits of detection and quantification, precision, repeatability, stability, and accuracy. Seven compounds in eleven samples were determined, and there were significant differences in the concentrations of isoorientin, swertiajaponin, afzelin, luteolin-7-O-β-D-glucoside, vitexin, isovitexin, and swertisin. ...

As a triterpene saponin, pedunculoside is one of the most abundant, representative and active components in plants of genus Ilex (Aquifoliaceae). Pedunculoside has been used to treat myocardial ischemia, ameliorate hyperlipidemia and prevent liver injury. In this paper, a systemic in vitro liver microsomes / S9 and intestinal bacteria incubation, and in vivo animal experiment were performed, using LC-Q-TOF/MS analysis and a three-step data processing protocol. As a result, Bifidobacterium adolescentis and Bifidobacterium breve were identified to potentially metabolize pedunculoside among the intestinal bacteria tested. A total of 11 metabolites were found and tentatively identified, with 6 in both microsomal and bacterial incubation systems, and 9 after rats orally administered with pedunculoside. The metabolites detected involving both phase I and phase II metabolism, mainly through deglycosylation (hydrolyzation), dehydrogenation, hydroxylation and conjugation, and some of them underwent more than one-step metabolic reactions. Most of the metabolites have not been reported before. In vitro, liver microsome and intestinal bacteria prefer to metabolize pedunculoside in totally different ways; while in vivo, intestinal tract is the most important site for the metabolism and excretion of pedunculoside, where both intestinal bacteria and the host metabolic enzymes participate in its metabolism and disposition. The importance of intestinal bacteria should be highlighted. This study would contribute to a better understanding of pedunculoside metabolism, which can provide scientific evidence for its pharmacodynamic mechanism research and prove its clinical application.

As a herbal plant used in traditional Chinese medicine, Lamiophlomis rotata (Benth.) Kudo mainly displays its pharmacological effect by promoting blood circulation and hemostasis, dispelling wind, and acting as an analgesic. To identify the components contained in L. rotata, global detection and structural elucidation of both target and non-target components in the medicinal material was performed.L. rotata was ultrasonically extracted with methanol. Separation and analysis were achieved using liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS). A three-step protocol which included (1) potential components screening, (2) collection of qualitative information, and (3) database searching and structural elucidation was used for target and non-target identification.A total of 42 components were tentatively identified, which included 12 iridoids (2 aglycones and 10 glucosides), 11 flavonoids (4 aglycones and 7 glucosides), and 13 phenylethanoid glycosides. Moreover, components of L. rotata extract belonging to the three main structural categories could be well separated in a 3D point plot according to their retention times, mass defects and degrees of unsaturation, facilitating the structural classification and identification in the subsequent studies.The results provide a reasonable picture of the components contained in L. rotata extract and promote the further pharmacodynamic and/or pharmacokinetic characterization of this medical material, meanwhile demonstrating the utility of a universal methodology for the systematical study of herbal medicines. Copyright © 2016 John WileySons, Ltd.

Flavonoids, including flavones, isoflavones, flavanones, chalcones, and isoflavans, have long been recognized as the main active ingredients in licorice. A method combining liquid chromatography with photodiode array detection and quadrupole time-of-flight mass spectrometry was developed to characterize components in three Glycyrrhiza species, and to expound the characteristic fragmentation behaviors in the positive ion mode. Based on the fragmentation patterns of reference compounds, a total of 39 compounds, including 37 flavonoid aglycones and two coumestans, were identified or tentatively identified. Besides, some common features, such as H2 O, CO, and CH2 O2 losses, together with retro-Diels-Alder fragmentation, were observed in these compounds. Furthermore, diagnostic fragmentations of C-ring cleavages and UV absorption on the skeleton groups were observed to structurally characterize flavonoid aglycones. In addition, typical losses of different substituent groups were detected: Neutral losses of 56 (C4 H8 ) and 68 Da (C5 H8 ) were yielded from a prenyl chain; neutral losses of 42 (C3 H6 ), 54 (C4 H6 ), and 70 Da (C4 H6 O) were generated by a pyran ring. Particularly, neutral losses of 18 (H2 O), 16 (CH4 ), 112 (C8 H16 ), and 98 Da (C7 H14 ) predicted a hydroxyl, a methoxyl, double prenyl chains, and a prenyl chain with a pyran ring, respectively.

Anhydrosafflor yellow B (AHSYB) is one of the major active water-soluble pigments from Carthamus tinctorius, which has been found to inhibit ADP-induced platelet aggregation and possess significant antioxidant activity. However, the metabolic fate of AHSYB in vivo remains unknown. In order to explore whether AHSYB is extensively metabolized, the metabolites of AHSYB in plasma, urine, bile, and feces samples after intravenous administration to the rats were investigated by ultra-fast liquid chromatography/quadrupole time-of-flight mass spectrometry (UFLC/Qq-TOF-MS/MS) combined with Metabolitepilot™ software. In total, AHSYB and 22 metabolites including both phase I and phase II metabolism processes were found and tentatively identified from the bio-samples. The metabolic pathways were involved in oxidation, reduction, hydroxylation, methylation, dimethylation, O-acetylation, hydrolyzation, sulfation, glucuronidaton, glutathionation and combination with glucose. The results showed that the renal and biliary routes play an important role in the clearance and excretion of AHSYB as well as hepatocyte metabolism. All of these results were reported for the first time and would contribute to a further understanding of the in vivo intermediated processes and metabolic mechanism of AHSYB and its analogs.

Annona squamosa Linn (Annonaceae) is a commonly used and effective traditional Chinese medicine (TCM) especially in the South China. The seeds of Annona squamosa Linn (SAS) have been used as a folk remedy to treat "malignant sores" (cancer) in South of China, but they also have high toxicity on human body.To discover the potential biomarkers in the mice caused by SAS.We made metabonomics studies on the toxicity of SAS by ultraperformance liquid-chromatography high-definition mass spectrometry coupled with pattern recognition approach and metabolic pathways analysis.The significant difference in metabolic profiles and changes of metabolite biomarkers between the Control group and SAS group were well observed. 11 positive ions and 9 negative ions (P0.05) were indicated based on UFLC-QTOF-HDMS. The metabolic pathways of SAS group are discussed according to the identified endogenous metabolites, and eight metabolic pathways are identified using Kyoto Encyclopedia of Genes and Genomes (KEGG).The present study demonstrates that metabonomics analysis could greatly facilitate and provide useful information for the further comprehensive understanding of the pharmacological activity and potential toxicity of SAS in the progress of them being designed to a new anti-tumor medicine.

3,4-Divanillyltetrahydrofuran is the main active ingredient of nettle root which can increase steroid hormones in the bloodstream for many of bodybuilders. To better understand its pharmacological activities, we need to determine its pharmacokinetic profiles. In this study, a rapid and sensitive ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method has been developed for the determination of 3,4-divanillyltetrahydrofuran in the plasma of rats. Chromatographic separation was performed on a C18 column at 40°C, with a gradient elution consisting of methanol and water containing 0.3% (v/v) formic acid at a flow rate of 0.8mL/min. The detection was performed using an electrospray triple-quadrupole MS/MS via positive ion multiple reaction monitoring mode. The lower limits-of-quantification determined were 0.5ng/mL. The intra- and inter-day precision (RSD%) was found to be within 15% and the accuracy (RE%) ranged from -4.0% to 7.0%. This simple yet sensitive method was fully validated and could be successfully applied to the study on pharmacokinetics of 3, 4-divanillyltetrahydrofuran.

UFLC-QTRAP MS was used to develop a sensitive and rapid method of evaluating content variation during Semen sojae praeparatum (SSP) fermentation. It did this through the simultaneous quantification of 22 free amino acids and 16 nucleosides and nucleobases in the raw materials and processed products of SSP. The method was shown to be reproducible and accurate. The limits of detection and quantity values were 0.09-168.75 and 0.31-562.50 ng/mL for the 38 analytes, respectively. The data were examined through principal components analysis to compare the content variations. The quantitative results showed that the ingredients were properly determined in most of the samples and were converted regularly throughout the SSP fermentation process. These results correspond to the morphologic changes and principal components analysis results.

Ultra-flow liquid chromatography/quadrupole-time-of-flight mass spectrometry (UFLC/Q-TOF MS) method combined with metabolitepilotMT software was used for analysis of the metabolites of liguzinediol in dogs. Urine, bile, feces and plasma samples were collected after intravenous administration of 8 mg/kg liguzinediol to healthy dogs. Besides liguzinediol, seven metabolites were detected and identified by UFLC/Q-TOF MS method. The results showed that liguzinediol had some main metabolic pathways in dogs including oxidation, sulfation, cysteine conjugation, N-acetylcysteine conjugation and glucuronidation.

Peperomia dindygulensis, with secolignans (SLs) as major bioactive constituents, is a commonly used traditional folk medicine in mainland China for treatment of stomach, liver, mammary, and esophageal cancers. However, to date, there is no method available for the qualitative and quantitative analyses of SLs in this medicinal plant. The purpose of this study was to establish a sensitive, selective, and reproducible method for rapidly profiling, identifying, and determining SLs in the whole plant of P. dindygulensis. Ultra high-performance liquid chromatography (UHPLC) coupled with ultraviolet detector (UV) and quadrupole tandem time-of-flight mass spectrometry (Q-TOF-MS) were used for this analyses. The fragmentation behaviors of different types of SLs were described. A total of thirteen SLs, including two new derivatives, were identified or tentatively characterized in P. dindygulensis samples. In addition, seven major SLs in herbal samples from different regions in China were successfully determined. The method developed in this study is suitable for the qualitative and quantitative analyses of SLs in P. dindygulensis, and may be applicable for determining or identifying SLs from other Pepermia genus plants.

Ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/QTOF MS) was employed to investigate the in vivo metabolism of liguzinediol. Urine, bile, feces and plasma samples were collected after intravenous administration of 10 mg/kg liguzinediol to healthy rats. Altogether seven metabolites were detected and tentatively identified based on the characteristics of their protonated ions. The metabolites were mainly transformed by four main metabolic pathways including oxidation, sulfation, glycine conjugation and glucuronidation.