Quantitative determination of characteristic components from compound of Lysionotus pauciflorus Maxim. by LC-MS/MS and its application to a pharmacokinetic study.

Tuberculosis of cervical lymph nodes is called scrofula in Traditional Chinese Medicine (TCM). Clinical manifestation is that unilateral or bilateral neck can have multiple enlarged lymph nodes of different sizes. Current therapeutic drugs include Lysionotus pauciflorus Maxim. tablets and compound of Lysionotus pauciflorus Maxim., which have a significant effect on tuberculosis of cervical lymph nodes. This compound is composed of three herbs, Lysionotus pauciflorus Maxim., Prunella vulgaris L. and Artemisia argyi Levl.et Vant. A selective and sensitive LC-MS/MS method was established and validated in rat plasma for the first time. Chromatographic separation was achieved on a Wonda Cract ODS-2 C18 Column (150 mm × 4.6 mm, 5 µm). The mobile phase contained 0.1% formic acid aqueous solution and acetonitrile with a flow rate of 0.8 mL/min. The detection was performed in negative electrospray ionization mode and the precursor/product ion transitions of six components and internal standard (IS) sulfamethoxazole were quantified in multiple reaction monitoring (MRM) using QTRAP-3200 MS/MS. The method fulfilled US Food and Drug Administration guidelines for selectivity, sensitivity, accuracy, precision, matrix effect, extraction recovery, dilution integrity, and stability. This proposed method was then successfully applied to a pharmacokinetic study after oral administration of 10 mL/kg compound extracts in rats. The pharmacokinetic parameters and plasma concentration-time profiles would prove valuable in pre-clinical and clinical investigations on the disposition of compound medicine.

Ultrahigh-performance liquid chromatography coupled with triple quadrupole and time-of-flight mass spectrometry for the screening and identification of the main flavonoids and their metabolites in rats after oral administration of Cirsium japonicum DC. extract.

RATIONALE: Cirsium japonicum DC., a traditional Chinese medicine, has been shown to have anti-haemorrhagic and anti-tumour effects. Pharmacological studies have demonstrated that this curative effect may be related to flavonoids. The present work aimed to screen and identify the main flavonoids and their corresponding metabolites in rats after oral administration of Cirsium japonicum DC. extract. METHODS: A rapid and simple method based on ultrahigh-performance liquid chromatography coupled with triple quadrupole and time-of-flight mass spectrometry (UHPLC/QTOF-MS) was developed for the identification of the primary absorbing components and metabolites of the principal flavonoids. The absorbing components were first characterized, followed by the selection of representative constituents. In this study, the main flavonoids, pectolinarin, linarin and pectolinarigenin, were selected as templates to identify possible metabolites. RESULTS: A total of 27 metabolites were detected in rat blood, urine and bile samples. A hydrolysis reaction was the first step for pectolinarin and linarin, followed by oxidation and reduction reactions. However, phase II metabolites for pectolinarin and linarin were not detected. The primary biotransformation routes of pectolinarigenin were identified as oxidation, reduction, hydrolysis, and glucuronide and glucose conjugation. CONCLUSIONS: The metabolic pathways of pectolinarin, linarin and pectolinarigenin were summarized. This study not only proposed a practical strategy for rapidly screening and identifying metabolites but also provided useful information for further pharmacological studies and the design of new drugs based on Cirsium japonicum DC.

Qualitative and Quantitative Analyses of Active Constituents in Trollius ledebourii.

Trollius ledebourii has been more involved in Mongolian medicine and is often used as a type of tea for heat-clearing and detoxifying in the populus. In this study, a rapid and sensitive method was established for the qualitative and quantitative analyses of the major constituents in T. ledebourii. Ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was developed for the identification of the multi-constituents in T. ledebourii. A total of 37 chemical constituents in T. ledebourii extract were unambiguously or tentatively identified, including 17 flavonoid glycosides, 6 flavones, 3 flavonols, 1 dihydroflavone, 8 phenolic acids, 1 amide and 1 triterpene. Pectolinarin, naringenin, isorhamnetin, diosmetin, protocatechuic acid, paeonol, caffeic acid and ferulic acid were first detected in T. ledebourii and the buttercup family. High-performance liquid chromatography-quadrupole ion trap tandem mass spectrometry was applied for the simultaneous determination of 11 compounds, which were either with high contents or strong bioactivities. Satisfactory linearity was achieved with a wide linear range and fine determination coefficient (r > 0.9987). The overall recoveries ranged from 98.07 to 101.2%, and the precision in terms of RSD was <0.74%. The results might provide the basis for quality control analysis of T. ledebourii.

Identification of metabolites of Helicid in vivo using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

Helicid is an active natural aromatic phenolic glycoside ingredient originating from a well-known traditional Chinese herbal medicine and has the significant effects of sedative hypnosis, anti-inflammatory analgesia and antidepressant. In this study, we analyzed the potential metabolites of Helicid in rats by multiple mass defect filter and dynamic background subtraction in ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Moreover, we used a novel data processing method, 'key product ions', to rapidly detect and identify metabolites as an assistant tool. MetabolitePilot™ 2.0 software and PeakView™ 2.2 software were used for analyzing metabolites. Twenty metabolites of Helicid (including 15 phase I metabolites and five phase II metabolites) were detected by comparison with the blank samples. The biotransformation route of Helicid was identified as demethylation, oxidation, dehydroxylation, hydrogenation, decarbonylation, glucuronide conjugation and methylation. This is the first study simultaneously detecting and identifying Helicid metabolism in rats employing UHPLC-Q-TOF-MS technology. This experiment not only proposed a method for rapidly detecting and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of Helicid in vivo. Furthermore, it provided an effective method for the analysis of other aromatic phenolic glycosides metabolic components in vivo.