Development of a HPLC-MS/MS Method to Determine 11 Bioactive Compounds in Tongmai Yangxin Pill and Application to a Pharmacokinetic Study in Rats

A sensitive and reliable HPLC-MS/MS method has been developed and validated for simultaneous determination of eleven bioactive compounds (rhein, emodin, stilbene glycoside, liquiritin, ononin, verbascoside, gallic acid, schisandrin, liquiritigenin, glycyrrhizic acid, and isoliquiritigenin) in rat plasma after oral administration of Tongmai Yangxin Pill. The collected plasma samples were prepared by liquid-liquid extraction with ethyl acetate after acidification. Eleven compounds were separated on a CORTECS™ C18 column with mobile phases consisting of 0.1% formic acid in deionized water and acetonitrile. The flow rate was 0.3 mL/min. The detection was performed on a tandem mass system with an electrospray ionization (ESI) source in both positive and negative ionization using multiple-reaction monitoring (MRM) mode. The calibration curves were linear over the range of 8-2000 ng/mL for glycyrrhizic acid; 4-1000 ng/mL for liquiritin; 0.8-200 ng/mL for emodin, gallic acid, ononin, schisandrin, and stilbene glycoside; 0.4-100 ng/mL for isoliquiritigenin, liquiritigenin, rhein, and verbascoside, respectively. The intra- and interday precision of the analytes were less than 9.3% and 8.5%. The intra- and interday accuracy were in the range of -14.0% to 10.3% and -6.5% to 9.6%. Meanwhile, the extraction recovery of the analytes in plasma samples ranged from 85.2% to 109.1% and matrix effect from 89.2% to 113.4%. The developed method was successfully applied to the pharmacokinetics of eleven bioactive compounds in rat plasma after oral administration of Tongmai Yangxin Pill prescription

Identification and Quality Evaluation of Raw and Processed Asarum Species Using Microscopy, DNA Barcoding, and Gas Chromatography-Mass Spectrometry

Asarum (Aristolochiaceae) is one of the common herbs used to relieve exterior syndromes. Some volatile components of Asarum which have toxic effect may cause adverse reactions such as headache, general tension, unconsciousness, and respiratory paralysis. Therefore, Asarum is normally processed to reduce such toxicity and adverse effects. The bioactive ingredients contained in different Asarum herbs vary significantly; this variation may be attributed to their differences in species, origins, or processing methods. In this study, 16 batches of Asarum herbs were collected, and their species were identified using DNA barcoding, which is a method for distinguishing plant species, coupled with microscopy. A gas chromatography-mass spectrometry (GC-MS) method for simultaneous determination of 10 compounds was established to evaluate the contents of raw and processed Asarum herbs. Multivariate analysis was then applied to compare different batches of herbs based on the GC-MS data. DNA barcoding identified the herbs as being derived from four sources, and herbs from different origins showed different microscopic features. The results demonstrated that most of the samples were clearly clustered into distinct groups that corresponded to species types. All raw and processed samples were classified by partial least squares discriminant analysis (PLS-DA) based on the 10 analyzed compounds. The findings suggested that safrole and methyleugenol with a variable importance in the project (VIP) > 1 are unique compounds that can be used to differentiate between Asarum species. Safrole, methyleugenol, and 2,6,6-trimethylcyclohepta-2,4-dien-1-one were identified as significant constituents, the presence of which can be used to differentiate between raw and processed Asarum samples. These results indicate that species and processing methods show important effects on the composition of Asarum herbs

Comparative Investigation of the Differences in Chemical Compounds between Raw and Processed Mume Fructus Using Plant Metabolomics Combined with Chemometrics Methods

Mume Fructus is a well-known herbal medicine and food with a long history of processing and application. Different processing methods impact the intrinsic quality of Mume Fructus. Thus, it is of great significance to investigate the changes in chemical components during processing (i.e., raw compared to the pulp and charcoal forms). In this study, plant metabolomics methods based on mass spectrometry detection were established to analyze the chemical ingredients of Mume Fructus comprehensively. Chemometric strategies were combined to analyze the profile differences of Mume Fructus after different processing methods. The established strategy identified 98 volatile and 89 non-volatile compounds of Mume Fructus by gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS/MS), respectively. Moreover, the orthogonal partial least squares discriminant analysis (OPLS-DA) indicated that raw Mume Fructus and the Mume Fructus pulp and charcoal were distributed in three regions. Subsequently, 19 volatile and 16 non-volatile components were selected as potential chemical component markers with variable importance in the projection using (VIP) >1 as the criterion, and the accuracy was verified by a Back Propagation Neural Network (BP-NN). To further understand the difference in the content of Mume Fructus before and after processing, 16 non-volatile chemical component markers were quantitatively determined by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS). The results revealed that, compared with raw Mume Fructus, the total content of 16 components in the pulp of Mume Fructus increased while it decreased in the charcoal. Therefore, this study used GC-MS, UHPLC-Q-TOF-MS/MS and UHPLC-MS/MS modern technology to analyze the differences in chemical components before and after the processing of Mume Fructus and provided a material basis for further research on the quality evaluation and efficacy of Mume Fructus

Development and Validation of a HPLC-MS/MS Method for Simultaneous Determination of Twelve Bioactive Compounds in Epimedium: Application to a Pharmacokinetic Study in Rats

A rapid and reliable HPLC-MS/MS method has been developed and validated for the simultaneous quantification of twelve bioactive compounds (baohuoside II, baohuoside I, sagittatoside A, sagittatoside B, magnoflorine, epimedin A, epimedin B, epimedin C, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid and icariin) in rat plasma. The collected plasma samples were prepared by protein precipitate with acetonitrile. The twelve compounds were separated on a CORTECS®C18 column (4.6 mm × 150 mm, 2.7 μm) with a gradient mobile phase system of 0.1% (v/v) formic acid and acetonitrile at a flow rate of 0.3 mL/min. All of the analytes were quantitated using electrospray ionization (ESI) in negative ion mode with selected reaction monitoring (SRM). The intra- and inter-day accuracy ranged from −5.6% to 13.0%, and the precisions of the analytes were less than 10.9%. The mean recoveries of the analytes were in the range of 60.66% to 99.77% and the matrix effect ranged from 93.08% to 119.84%. Stability studies proved that the analytes were stable under the tested conditions, with a relative standard deviation (RSD) lower than 11.7%. The developed method was successfully applied to evaluating the pharmacokinetic study of twelve bioactive compounds after oral administration of Epimedium extract in rat

Correlation between Quality and Geographical Origins of Cortex Periplocae, Based on the Qualitative and Quantitative Determination of Chemical Markers Combined with Chemical Pattern Recognition

Quality assessment of Cortex Periplocae remains a challenge, due to its complex chemical profile. This study aims to investigate the chemical components of Cortex Periplocae, including its non-volatile and volatile constituents, via liquid chromatograph–mass spectrometry (LC-MS/MS) and gas chromatography–mass spectrometry (GC-MS) assays. The established strategy manifested that Cortex Periplocae from different producing areas was determined by identifying 27 chemical markers with ultra-high-performance liquid chromatography, coupled with quadrupole tandem time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS), including four main groups of cardiac glycosides, organic acids, aldehydes, and oligosaccharides. These groups’ variable importance in the projection (VIP) were greater than 1. Simultaneously, the samples were divided into four categories, combined with multivariate statistical analysis. In addition, in order to further understand the difference in the content of samples from different producing areas, nine chemical markers of Cortex Periplocae from 14 different producing areas were determined by high performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS), and results indicated that the main effective constituents of Cortex Periplocae varied with places of origin. Furthermore, in GC-MS analysis, samples were divided into three groups with multivariate statistical analysis; in addition, 22 differential components whose VIP were greater than 1 were identified, which were principally volatile oils and fatty acids. Finally, the relative contents of seven main volatile constituents were obtained, which varied extremely with the producing areas. The results showed that the LC-MS/MS and GC-MS assays, combined with multivariate statistical analysis for Cortex Periplocae, provided a comprehensive and effective means for its quality evaluation

HPLC-MS/MS Analysis of Aconiti Lateralis Radix Praeparata and Its Combination with Red Ginseng Effect on Rat CYP450 Activities Using the Cocktail Approach

Red ginseng is often combined with Aconiti Lateralis Radix Praeparata to reduce alkaloids-related toxicity of the latter. Such herb-pairing also results in better therapeutic effect in heart failure, as compared to the singular use of either herb. The purpose of this study was to investigate the effect of Aconiti Lateralis Radix Praeparata and its combination with red ginseng on the activities of CYP450 enzymes in rats. A sensitive and reliable HPLC-MS/MS method was established and validated for the simultaneous determination of eight probe drugs, phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), dapsone (CYP3A4), 7-hydroxycoumarin (CYP2A6), bupropion (CYP2B6), and amodiaquine (CYP2C8), in rat plasma using diazepam as internal standard (IS). The chromatographic separation was performed on a Waters XBridge™ C18 column (2.1 mm × 100 mm, 3.5 μm) using a gradient elution with the mobile phase consisting of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. The method was successfully applied in evaluating the effect of Aconiti Lateralis Radix Praeparata and red ginseng on the activities of CYP450 enzymes. The pharmacokinetic results of the eight probe drugs suggested that Aconiti Lateralis Radix Praeparata may inhibit the activity of CYP2A6, CYP2C19, CYP2B6, CYP1A2, CYP3A4, and CYP2C9 enzymes in rats. Comparison between the two groups, Aconiti Lateralis Radix Praeparata combined with red ginseng and Aconiti Lateralis Radix Praeparata, indicated that red ginseng may inhibit the activity of CYP2D6 and CYP2B6 enzymes while inducing the activity of CYP1A2, CYP3A4, and CYP2C9 enzymes