| Abri Herba（Jigucao） and Abri mollis Herba（Maojigucao） originate from the dried plants of Abrus cantoniensis Hance（Leguminosae） and Abrus mollis Hance（Leguminosae） without pods, respectively. They are mainly cultivated in Guangdong and Guangxi provinces of China. Abri Herba is included in Pharmacopoeia of the People’s Republic of China, while Abri mollis Herba is included in Guangdong Chinese Materia Medica Standards. However, it is reported that they have the similar pharmacological activities, such as cleanup heat detoxification, dampness and activating blood circulation to dissipate blood stasis. They are also both used as one of the main ingredients of Guangzhou herbal tea and has been used to prepare soup by people in villages for a long time. In clinical practice Abri mollis Herba is often used as Abri Herba.In this study, a simple, sensitive and selective HPLC–MS/MS method has been developed and validated for the simultaneous quantification of fifteen flavonoids, two phenolic acids and three alkaloids in Abri Herba and Abri mollis Herba, In addition, the method has been successfully applied to differentiate 15 batches of Abri Herba and 27 batches of Abri mollis Herba stems. Furthermore, a comparison of the contents among stems, roots and leaves from the same strain in 7 batches of Abri mollis Herba and 4 batches of Abri Herba has also been performed. This method not only can be effectively applied for the quality control and evaluation of Abri Herba and Abri mollis Herba, but also provided basis for the rationality evaluation of Abri mollis Herba instead of Abri Herba.In order to further evaluate the rationality of that Abri mollis Herba can be used as Abri Herba and provide a useful tool for clinical application of the formulation of Chinese medicine, the process of drug in vivo were investigated. The LC-MS/MS methods were developed to analyze 4 flavonoids and 2 alkaloids in rat plasma, and major components in rat urine and bile after oral administration of Abri Herba and Abri mollis Herba, respectively. The methods were applied to studies on pharmacokinetics, urinary and biliary excretion of Abri Herba and Abri mollis Herba in rats, respectively. Part one Comparation of main constituents in Abri Herba and Abri mollis Herba by LC-MS/MSObjective: To establish a high-performance liquid chromatography and tandem mass spectrometry（HPLC-MS/MS） method for the simultaneous analysis of fifteen flavonoids, two phenolic acids and three alkaloids in Abri Herba and Abri mollis Herba. Multiple plots and hierarchical cluster analysis（HCA） were used to observe the batch-to-batch variations and species authentication for Abri Herba and Abri mollis Herba stem. Furthermore, a comparison of these contents among stems, roots and leaves from the same strain has also been performed. It is to provide the basis of their substitutability and rational use in clinical practice preliminary.Methods: Chromatographic separation was performed on a ZORBAX SB-C18 column（4.6 × 250 mm, 5 μm） with a mobile phase of methanol（A）, acetonitrile（B） and 0.5‰ acetic acid in water（C） using gradient elution. The mobile phase flow rate was maintained at 1.0 mL/min, and the injection volume was 10 μL. A single-factor experiment was used to optimize the extracting conditions. To classify Abri mollis Herba and Abri Herba, a method called “average linkage between groups” of HCA was performed on the analyzed 20 components from the stems of 42 samples. Multiple-reaction monitoring（MRM） mode was used for quantification. The operation conditions were as follows: ion spray voltage,-4500 V（negative ESI mode） and 5500 V（positive ESI mode）; turbo spray temperature, 650 oC; curtain gas（CUR）, 1.72 × 105 Pa; the interface heater was turned on; collision gas, medium; nebulizer gas（gas 1） and heater gas（gas 2）, 4.14 × 105 and 4.48 × 105 Pa, respectively; entrance potential（EP）, 10/-10 V; and collision cell exit potential（CXP）, 5/-5 V. Nitrogen was used in all cases.Results: The linearity of analytical response was good with correlation coefficients r2 value greater than 0.9904 for all the compounds in the concentration range. The precision, stability, LOD and LOQ of the method were good for the 20 components. The average recoveries（n=9） of these components were in the range of 94.3–108.2%. The obtained results show that Abri mollis Herba and Abri Herba present the same 20 components analyzed in the study, although with differences in their absolute content. But the relative quantity of the 20 ingredients almost have no difference. By comparing the contents of the 20 components among stems, roots and leaves obtained from a same sample of Abri mollis Herba or Abri Herba, the results were found to be as follows: leaf > stem > root, in Abri mollis Herba, and leaf > root > stem, in Abri Herba. In Abri mollis Herba. The contents of schaftoside and isoschaftoside were the highest in leaves, but the contents of abrine and hypaphorine were the highest in roots.Conclusion: The LC-MS/MS method was sensitive, selective and suitable for the simultaneous analysis of the fifteen flavonoids, two phenolic acids and three alkaloids in Abri Herba and Abri mollis Herba. It was suitable for the reliable quality control of Abri mollis Herba and Abri Herba. It preliminarily illustrated the substitution between Abri mollis Herba and Abri Herba has certain rationality by comparing the content of the main chemical components in the two herbs. Part Two Simultaneous determination of four flavonoids and two alkaloids in rat plasma by LC-MS/MS and its application to a comparative study between the pharmacokinetics of Abri Herba and Abri mollis Herba extract after oral administrationObjective: To develop a high-performance liquid chromatography and tandem mass spectrometry（HPLC-MS/MS） method for the simultaneous analysis of 6 ingredients（hypaphorine, abrine, schaftoside, isoschaftoside, isoliquiritigenin and luteolin） in rat plasma after oral administration of Abri Herba and Abri mollis Herba extract, and the comparative study of pharmacokinetic characteristics.Methods: The rats were given a gavage of certain dose with Abri Herba and Abri mollis Herba extract. Blood samples were collected from the vein of the eye ground at different time points after a single oral administration（5mL/kg） and protein precipitation with methanol was chosen as the preparation method. Samples were separated on a ZORBAX SB-C18（4.6 × 150 mm, 5 μm） column with a Security-Guard C18（4.6 mm × 3 mm; 5 μm） column. The mobile phases consisted of methanol（A） and 0.5‰ acetic acid in water（B）were used with the following gradient elution profile: initial 0-4 min, maintained 30:70（v/v） A-B; 4-8 min, linear change from 30:70（v/v） A-B to 36:64（v/v） A-B; 8-12 min, linear change from 36:64（v/v） A-B to 95:5（v/v） A-B; 12-16 min, isocratic elution with 95:5（v/v） A-B; 16-16.1 min, return to 30% A. The flow rate was maintained at 1.0 mL/min, and the injection volume was 20 μL. The Mass spectrometric conditions was the same with part one. Using DAS 3.0 software to obtain pharmacokinetic parameters of each composition and to compare the pharmacokinetic parameters of the same ingredients.Results: The developed method has good precision, accuracy, stability, linearity, detection limit and quantitation. The linearity of analytical response was good with r2 value greater than 0.9933 for all the compounds in the concentration range. The range of intra- and inter-day RSD% was 3.7%-14.6%, RE% was 11.0%-13.5%. The extraction recovery and matrix effect were 83.5%-102.7% and 69.2%-101.0%, respectively. The pharmacokinetic results showed that 6 compositions were absorbed quickly in vivo. All ingredients obtained their maximum plasma concentrations within 3h, the flavonoids could be absorbed into the plasma significantly faster than the alkaloids and Tmax of all the four flavonoids（schaftoside, isoschaftoside, isoliquiritigenin and luteolin） was the same. However there was great difference in the elimination behavior. The elimination rate of glycosides（schaftoside and isoschaftoside） were slower than aglycone（isoliquiritigenin and luteolin） in vivo. The comparative experimental results of Abri mollis Herba and Abri Herba showed that the curve of 6 compounds were consistent. Except the time to peak of hypaphorine and abrine had a little difference, the other four were all the same.Conclusion: The method was simple, selective and specific, and suitable for the pharmacokinetic study of the six analytes in Abri mollis Herba and Abri Herba extract. The experiment results showed that the main effective components of Abri mollis Herba and Abri Herba extract had the same pharmacokinetic characteristics, which further proved the rationality of the substitution each other in clinical use. Part three Simultaneous determination of main ingredients of Abri Herba and Abri mollis Herba in rat urine and bile by HPLC–MS/MS and its application to a comparative study of their excretion kinetics after oral administrationObjective: To establish a LC-MS/MS method for the simultaneous analysis of 9 ingredients in urine and 6 ingredients in bile after oral administration of Abri Herba and Abri mollis Herba extract, and the comparative study of excretion kinetics.Methods: The rats were put in the metabolic cages after giving a gavage of certain dose with Abri Herba and Abri mollis Herba extract to collect urine. Six rats were undergone biliary intubation after giving a gavage of certain dose with Abri Herba and Abri mollis Herba extract to collect bile. The urine and bile samples were pretreated by using the same pretreatment method as plasma samples. The chromatographic condition and Mass spectrometric conditions was the same with part one. The injection volume was 10 μL.Results: In urine and bile, the linearity of analytical response was good with correlation coefficients for all the compounds in the concentration range. The range of intra-, inter-day, extraction recovery and matrix effect were all in the allowable range. In urine, the 9 ingredients were all complete drainage within 96 h. In addition to the high excretion rate of formononetin, the other ingredients had a low excretion rate. In the two herbs, the percentage of cumulative excretion of hypaphorine and formononetin had a great difference, the other components have no obvious difference. The comparation of bile excretion showed that each component in prototype excretion rate was very low and the components have great differences except the abrine in Abri mollis Herba and Abri Herba, respectivily.Conclusion: The method was simple, selective and specific for the urine excretion study of 9 ingredients and the bile excretion study of 6 constituents in Abri mollis Herba and Abri Herba extract. In the two herbs, most of the ingredients are similar in urine excretion, but there were quite different in bile. This study provided a basis for the comprehensive evaluation of the rationality of the alternative on each other. |
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