•A novel and simple LC–MS/MS method for simultaneous separation and quantification of five ephedrines in dietary supplements with three different matrices was established.•Four unique chromatographic columns suitable for analysis of alkaline substances were investigated for separation of five ephedrines.•Method validation was investigated, especially for matrix effects and expanded uncertainty.•More than 500 samples were assayed using this method, and data for different matrices to study dietary supplements are presented.Ephedrines in dietary supplements can arise from herbs or illegal adulteration so a liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed for separation and quantification of ephedrine, pseudoephedrine, norephedrine, norepseudoephedrine, and methyl-ephedrine, some of which are isomer pairs of pseudo-structures. This method includes liquid–liquid extraction of ephedrines from three typical dietary supplement matrices—solid, liquid, and oil—as well as liquid chromatographic separation. After liquid chromatographic separation, ephedrines are qualitatively and quantitatively analyzed using triple quadrupole mass spectrometry with positive electrospray ionization in multi-reaction monitoring (MRM) mode. Ephedrine recoveries in a solid matrix ranged from 53.3–91.5%, in a liquid matrix from 56.4–102.3%, and in an oil matrix from 51.7–01.2%. Linearity ranges were 10–1000 ng/g in solid and oil matrix and 1–100 ng/ml in liquid matrix. Accuracy was −11.5–16.3%. Intra-day and inter-day variation are less than 5.9% and 7.3%, respectively. Expanded uncertainty of quantification is less than 0.123 ng/g in a solid matrix, less than 0.139 ng/ml in a liquid matrix, and less than 0.158 ng/g in an oil matrix. Data collected for more than 500 routine samples are presented and discussed.

•36 new 3-piperazinyl coumarins have been designed and synthesized.•All of the compounds have been evaluated for neuroprotective activity in vitro.•Compound 20 showed significant neuroprotection in vitro and in MCAO rats.•Mechanistic studies indicated that 20 exhibited antiapoptosis of neurons.•Pharmacokinetic evaluation indicated that 20 could cross the BBB of rats.Neuroprotection strategies are of great importance in the treatment of ischemic brain injury. Screening of our in-stock coumarin derivatives identified compound 1 as exhibiting neuroprotective activity. Subsequently, a structural optimization was carried out, which led to the discovery of the potent compound 20. This compound significantly attenuated the damage in a cell line derived from a pheochromocytoma of the rat adrenal medulla induced by oxygen–glucose deprivation in vitro. Furthermore, compound 20 exhibited clear neuroprotection in middle cerebral artery occlusion rats by reducing infarct size and brain-water content, improving neurological function, and suppressing neuronal loss and neuropathological changes in the cortex and hippocampus. Pharmacokinetic evaluation indicated that compound 20 could penetrate the blood–brain barrier of rats.A series of novel coumarin derivatives were designed and synthesized. The most effective compound 20 showed significant neuroprotection in MCAO rats.

The chiral separations of four pharmaceutical racemates which contain N-alkyl groups were satisfactorily resolved using SBE-β-CD as a chiral mobile phase additive (CMPA) in a RP-HPLC system (the resolution is 2.701 for ondansetron hydrochloride, 1.996 for sulpiride, 1.293 for clenbuterol hydrochloride and 0.816 for omeprazole). In addition, the effects of different parameters such as CD type and CD concentration were investigated. The separation mechanism arises through the combination of several potential interactions, including electrostatic interactions as well as hydrogen bonding interactions and hydrophobic inclusion interactions, which allow for the SBE-β-CD–drug complexation with strong stereoselectivity and stability. The resolution also relates to the number and location of N atoms in the enantiomers. This method will be applicable to the isolation of various types of biologically important enantiomers containing N-alkyl groups.The chiral separations of four pharmaceutical racemates which contain N-alkyl groups were satisfactorily resolved using SBE-β-CD as a chiral mobile phase additive (CMPA) in a RP-HPLC system. In addition, the effects of different parameters such as CD type and CD concentration were investigated.

FR429 is an ellagitannin with a potential antitumor activity, isolated and purified from Polygonum capitatum Buch.-Ham.ex D.Don, which is a traditional Miao-nationality herbal medicine in Guizhou and Yunnan of China. Our preliminary result of pharmacology study has indicated that the antitumor activity of FR429. However, the metabolism of FR429 has not been reported yet. In this study, LC–ion trap-time of flight mass spectrometry (LC–IT-TOF/MS) was used to characterize unpredictable metabolites of FR429 biotransformed by intestinal bacteria in vitro. Total thirteen metabolites were detected and characterized via comparisons of their accurate molecular masses and fragment ions of each MSn stage with those of the parent drug, and four of them were also elucidated by NMR. The results demonstrated that FR429 could be transformed by intestinal bacteria in vitro, mainly via hydrolysis and reduction reaction. This work provided a basis for the further study on the biotansformation of FR429 in vivo.

The metabolisms of five xanthones isolated from a Tibetan medicinal herb Halenia elliptica D. Don, including 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1), 1-hydroxy-2,3,4,7-tetramethoxy-xanthone (HM-2), 1-hydroxy-2,3,4,5-tetramethoxy-xanthone (HM-3), 1,7-dihydroxy-2,3,4,5-tetramethoxy-xanthone (HM-4) and 1,5-dihydroxy-2,3-dimethoxy-xanthone (HM-5), were studied in rat liver microsomes in vitro. High performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC–ESI-IT-TOF) was applied for identification of metabolites of five xanthones mentioned above and 1H NMR was used to elucidate the major metabolites. The structures of thirteen metabolites were identified and seven of them had not been reported before. Moreover, xanthone isomers herein could be distinguished by difference of fragmentation behaviors with increase of stages or relative abundances. The results indicated that in vitro metabolic transformation of HM-1, HM-2, HM-3, HM-4 and HM-5 occurred mainly at 2-, 4-, 5-, 7-carbonic positions on their structures of parent drugs. The metabolites could be new vasoactive substances. This work will provide a basis for study on the structure–activity relationships of these xanthones and their derivatives from Tibetan herbal in the next work.Highlights► LC–ESI-IT-TOF and 1H NMR were used for identification of xanthones’ metabolites. ► Structures of thirteen metabolites were identified, seven had not been reported. ► In vitro metabolic transformation occurred mainly at C-4, C-2, C-5 and C-7. ► Metabolites of xanthones via characteristic metabolism could be vasoactive. ► A basis for structure–activity relationship study of xanthones would be provided.

Metabolism study has been carried out on 1-hydroxy-2,3,5-trimethoxyxanthone (HM-1) and 1-hydroxy-2,3,4,7-tetramethoxyxanthone (HM-2), which are two biologically active ingredients isolated from the Tibetan herb, Halenia elliptica D. Don., in rat liver microsomes in vitro. A method of high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMSn-ESI-IT-TOF) was applied to analyze metabolites of HM-1 and HM-2 on line, and five metabolites were identified containing 1,5-dihydroxy-2,3-dimethoxyxanthone (HM-5), 1,7-dihydroxy-2,3,4-trimethoxyxanthone (HM-9), 1,4, 7-trihydroxy-2,3-dimethoxyxanthone (HM-10), 1,4-dihydroxy-2,3,7-trimethoxyxanthone (HM-11) and 1,2-dihydroxy-3,4,7-trimethoxyxanthone (HM-12). Among these metabolites, HM-9, HM-11, and HM-12 were isomers mutually. The results indicated that HM-1 and HM-2 occurred Phase I metabolic reaction of demethylation in rat microsomes in vitro.

A method of high performance liquid chromatographic separation of clausenamide enantiomers with chiral-AGP (α1-acid glycoprotein) stationary phases has been established. The absolute configurations of (−)clausenamide and (+)clausenamide are 3S, 4R, 5R, 6S and 3R, 4S, 5S, 6R, respectively. The present method has been used to analyze the (−)clausenamide and (+)clausenamide and its analogues such as the major metabolite and synthetic derivatives of clausenamide.

The enantiomers separation of eight pharmaceutical racemates collected in Chinese Pharmacopoeia 2010 (Ch.P2010), including nitrendipine, felodipine, omeprazole, praziquantel, sulpiride, clenbuterol hydrochloride, verapamil hydrochloride and chlorphenamine maleate, was performed on chiral stationary phase of amylose ramification by high performance liquid chromatography (HPLC) on Chiralpak AD-H column and Chiralpak AS-H column with the mobile phase consisted of isopropanol and n-hexane. The detection wavelength and the flow rate were set at 254 nm and 0.7 mL/min, respectively. The effects of proportion of organic additives, alcohol displacer and temperature on the separation were investigated. The results indicated that eight chiral drugs were separated on chiral stationary phase of amylase ramification in normal phase chromatographic system. The chromatographic retention and resolution of enantiomers were adjusted by factors, including the changes of the concentration of alcohol displacer in mobile phase, organic alkaline modifier and column temperature. It was shown that the resolution was improved with reducing concentration of alcohol displacer. When the concentration of organic alkaline modifier was 0.2%, the resolution and the peak shape were fairly good. Most racemates mentioned above had the best resolution at column temperature of 25 °C. The best temperature should be kept unchanged in the process of separation so as to obtain stable separation results.Graphical abstractEight chiral drugs were separated by two amylose ramification chiral stationary phase, Chiralpak AD-H and Chiralpak AS-H, and the separation mechanism was interpreted.Download full-size image

ABSTRACTBerberine (BBR) has been confirmed to show extensive bioactivities for the treatments of diabetes and hypercholesterolemia in clinic. However, there are few pharmacokinetic studies to elucidate the excretions of BBR and its metabolites. Our research studied the excretions of BBR and its metabolites in rats after oral administration (200 mg/kg). Metabolites in bile, urine, and feces were detected by liquid chromatography coupled to ion trap time‐of‐flight mass spectrometry; meanwhile, a validated liquid chromatography coupled with tandem mass spectrometry method was developed for their quantifications. Sixteen metabolites, including 10 Phase I and six Phase II metabolites were identified and clarified after dosing in vivo. Total recovered rate of BBR was 22.83% (19.07% of prototype and 3.76% of its metabolites) with 9.2 × 10−6% in bile (24 h), 0.0939% in urine (48 h), and 22.74% in feces (48 h), respectively. 83% of BBR was excreted as thalifendine (M1) from bile, whereas thalifendine (M1) and berberrubine (M2) were the major metabolites occupying 78% of urine excretion. Most of BBR and its metabolites were found in feces containing 84% of prototype. In summary, we provided excretion profiles of BBR and its metabolites after oral administration in rats in vivo.

ObjectiveBerberine (BBR) clinically lowers blood lipid and glucose levels via multi-target mechanisms. One of the possible mechanisms is related to its effect on the short chain fatty acids (SCFAs) of the gut microbiota. The goal of this study is to investigate the therapeutic effect and mode of action of BBR working through SCFAs of the gut microbiota (especially, butyrate).MethodsGas chromatography (GC) was used to detect butyrate and other SCFAs chemically. The effect of BBR on butyrate production was investigated in vitro as well as in several animal systems. Microarrays were used to analyze the composition change in the intestinal bacteria community after treatment with BBR. BBR-induced change in the energy production and gene regulation of intestinal bacteria was examined in order to elucidate the underlying molecular mechanisms.ResultsWe show that oral administration of BBR in animals promoted the gut microbiota to produce butyrate, which then enters the blood and reduces blood lipid and glucose levels. Incubating gut bacterial strains in vitro with BBR increased butyrate production. Orally treating animals directly with butyrate reduced blood lipid and glucose levels through a mechanism different from that of BBR. Intraperitoneal BBR administration did not increase butyrate but reduced blood lipid and glucose levels, suggesting that BBR has two modes of action: the direct effect of the circulated BBR and the indirect effect working through butyrate of the gut microbiota. Pre-treating animals orally with antibiotics abolished the effect of BBR on butyrate. A mechanism study showed that BBR (given orally) modified mice intestinal bacterial composition by increasing the abundance of butyrate-producing bacteria. Furthermore, BBR suppressed bacterial ATP production and NADH levels, resulting in increased butyryl-CoA and, eventually, butyrate production via upregulating phosphotransbutyrylase/butyrate kinase and butyryl-CoA:acetate-CoA transferase in bacteria.ConclusionPromotion of butyrate (etc) production in gut microbiota might be one of the important mechanisms of BBR in regulating energy metabolism.

The enantiomers separation of thirteen drugs collected in Ch.P2010 was performed on chiral stationary phase of cellulose ramification (chiralpak OD and chiralpak OJ) by high performance liquid chromatographic (HPLC) methods, which included ibuprofen (C1), ketoprofen (C2), nitrendipine (C3), nimodipine (C4), felodipine (C5), omeprazole (C6), praziquantel (C7), propranolol hydrochloride (C8), atenolol (C9), sulpiride (C10), clenbuterol hydrochloride (C11), verapamil hydrochloride (C12), and chlorphenamine maleate (C13). The mobile phase consisted of isopropanol and n-hexane. The detection wavelength was set at 254 nm and the flow rate was 0.7 mL/min. The enantiomers separation of these thirteen racemates on chiralpak OD column and chiralpak OJ column was studied, while the effects of proportion of organic additives, alcohol displacer and temperature on the separation were studied. And the mechanism of some of racemates was discussed. The results indicated that thirteen chiral drugs could be separated on chiral stationary phase of cellulose ramification in normal phase chromatographic system. The chromatographic retention and resolution of enantiomers could be adjusted by factors including column temperature and the concentration of alcohol displacer and organic alkaline modifier in mobile phase. It was shown that the resolution was improved with reducing concentration of alcohol displacer. When concentration of organic alkaline modifier was 0.2% (v/v), the resolution and the peak shape were fairly good. Most racemates mentioned above had better resolution at column temperature of 25 °C. When racemates were separated, the temperature should be kept so as to obtain stable separation results.

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb–drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC50 = 1.06 μM) and CYP2C9 (IC50 = 3.89 μM), minimal inhibition on CYP3A4 (IC20 = 11.94 μM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the Ki values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12 μM, 2.00 μM and 95.03 μM, respectively. HM-1 competitively inhibited testosterone 6β-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.