| The herb of Rabdosia is a perennial herb widely distributed in south and south-east Asia.They have been used as a local folk medicine in China for treatment of enteritis, jaundice, hepatitis, laryngopharyngitis, lepromatous leprosy and ascariasis. In the past few years, the chemical research of the herb of Rabdosia has become a popular domain for its variety of bioactivities. Phytochemical studies on the herb of Rabdosia revealed that it contained diterpenoids, phenolic acids, flavonoids, triterpenoids, volatile oils and so on. Modern pharmacological and clinical studies have also indicated that some chemical compositions in the herb of Rabdosia possessed anti-tumor, anti-inflammatory,anti-microbial, anti-catastrophe and anti-oxidation effects. Previous study found that the herb of Rabdosia contained a large number of diterpenoids which are active constituents. LC-MS technology has demonstrated its value in analyzing complex mixtures and has become a powerful tool in the online structural characterization and quantitation of various natural compounds owing to its high sensitivity, good separation efficacy and our considerable knowledge regarding its structure. In the present study, HPLC-MS was performed to analyze chemical components and pharmacokinetics in the herb of Rabdosia. First, a novel sensitive and selective HPLC-ESI-MS method was developed and validated to simultaneously determinate and identify constituents in Isodon serra samples. Second, A ranpid analysis method for epinodosin, epinodosinol, nodosin, oridonin, lasiokaurinol, lasiokaurin and rabdoternin A in rat plasma by liquid chromatography–electrospray ionization mass spectrometry was firstly developed and validated to analyze plasma samples and its application to pharmacokinetic study of the seven analytes after oral administration of Isodon serra extract.Part one Rapid determination of 27 components in Isodon serra by LC-ESI-MS-MSObjective: To develop a novel qualitative and quantitative method using high performance liquid chromatography coupled with tandem mass spectrometry for simultaneous analysis of 27 components including eighteen diterpenoids, six phenolic acids and three flavonoids in Isodon serra an important traditional Chinese medicine.Methods: First, an information-dependent acquisition (IDA) method was employed to trigger product ion scans above the MRM signal threshold so that the 27 components could be identified through enhanced product ion (EPI) scans. Second, multiple-reaction monitoring (MRM) was employed in positive and negative mode at the same time in single analysis process and validated to simultaneously determinate and identify 27 constituents in 45 batches of Isodon serra. The instrument operated using electrospray ionization source in positive and negative mode simultaneously. The ion spray voltage was set to 5500V and -4500V, respectively. The turbo spray temperature was maintained at 500°C. Nebulizer gas (gas 1) and heater gas (gas 2) was set at 40 and 50 psi, respectively. The curtain gas was kept at 25 psi and interface heater was on. Nitrogen was used in all cases. The precursor-to-product ion pairs of 27 analytes were sodoponin (426.4/331.3), effusanin A (349.3/331.3),enmein (363.3/281.3),lasiodonin (365.3/347.3),epinodosinol (382.3/347.3),oridonin (365.3/347.3), nervosanin B (384.4/349.3), serrin B (377.4/359.3), isodonoiol (407.4/329.2), shikokianidin (491.4/329.2), rabdosinate (535.4/295.3) ,epinodosin (361.2/287.1),nodosin (361.1/257.1),ponicidin (361.2/299.1),enmenol (365.2/347.2), lasiokaurin (405.2/58.9), lasiokaurinol (407.3/58.9), Protocatechuic aldehyde(136.9/107.9),Salicylic acid(136.9/92.9),ferulic acid ( 193.0/133.9 ), caffeic acid ( 179.3/135.3 ), chlorogenic acid(353.1/191.0),rosmarinci acid(359.0/160.9),quercetin(301.1/151.0), isorhamnetin(315.1/300.3),rutin(609.5/300.1). The chromatographic separation was performed on a Diamonsil C18 column (250 mm×4.6 mm, 5μm, Dikma, USA), and the column temperature set at 25°C. The mobile phase consisted of (A) methanol (containing 0.1% formic acid) and (B) 0.1% aqueous formic acid. The flow rate was 1.0 mL/min.Results: The linear relationships, linearity, precision, accuracy, limit of detection and limit of quantification of the method were good for the 27 components. And we successfully applied it to analyze 45 Isodon serra samples from different sources. The results demonstrated that a number of reasons such as different localization, harvesting and collection site might contribute to the differences in the level of active constituents among various Isodon serra. These all suggested that each collecting procedure involved should be standardized in the future. In this way, the quality of Isodon serra could be assured.Conclusion: A novel sensitive and selective HPLC-ESI-MS-MS method operating both negative and positive scanning modes in single analysis process was developed and validated to simultaneously determinate and identify 27 constituents in Isodon serra samples. The method plays an important role in the quanlity control of Isodon serra.Part two A rapid analysis method for 7 diterpenoids in rat plasma by liquid chromatography-electrospray ionization mass spectrometry and its application to pharmacokinetic study of Isodon sarra extractObjective: To develop a sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS) method and validate for the simultaneous determination of epinodosin, epinodosinol, nodosin, oridonin, lasiokariurinol, lasiokaurin and rabdoternin A in rat plasma, using sulfamethoxazole as the internal standard (IS).Methods: Blood samples were collected into heparinized centrifuge tubes from the fossa orbitalis vein at 10, 30, 60, 90, 105, 120, 150, 180, 240, 300, 480 , 600, 720 min after single oral administration of Isodon serra extract (10 mL/kg). Within 30 min after blood withdrawal, the samples were centrifuged at 4,000 rpm for 10 min and the separated plasma samples were frozen in polypropylene tubes at -20°C prior to analysis. The plasma samples were pretreated by Liquid-liquid extraction with Acetic ether and chromatographic separation was performed on Diamonsil C18 column (250 mm×4.6 mm, 5μm), and the column temperature set at 25°C. A linear gradient elution of eluents A (methanol containing 0.1% formic acid) and B (0.1%, v/v aqueous formic acid) was used for the separation. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source. A novel multi-determination-periods program was executed to achieve a higher sensitivity by setting five scanning periods. The method presented here utilizes a novel determination strategy, enabling the application of positive and negative ESI-MS in a single run.The instrument was operated switching electrospray ionization source in positive and negative mode in a single run. The optimized mass transition ion-pairs (m/z) for quantitation were361.2/287.1 for epinodosin, 382.3/347.3 for epinodosinol, 361.2/257.1 for nodosin, 365.3/347.3 for oridonin, 407.3/329.1 for lasiokaurinol, 405.2/59.0 for lasiokaurin, 363.2/283.1 for rabdoternin A and 254.1/156.0 for IS The total run time was 15.50 min between injections.Results: The specificity, linearity, accuracy, precision, recovery, matrix effect and several validation results demonstrate that this method is rapid, specific and reliable. The proposed method was further applied to investigate the pharmacokinetics of all analytes after a single oral administration of Isodon serra extract to rats. The seven analytes had parallel pharmacokinetic parameters in vivo such as Tmax, k and T1/2. In general, all seven analytes were absorbed rapidly; they were detected in plasma at 10 min after oral administration and eliminated quickly at a similar rate. However, the Tmax values of epinodosinol, nodosin, oridonin, lasiokaurinol, lasiokaurin and rabdoternin A were longer than that of epinodosin. Meanwhile, the eliminate rate of oridonin was the fastest and epinodosinol was the slowest.Conclusion: A sensitive and selective LC-ESI-MS method for the simultaneous determination of epinodosin, epinodosinol, nodosin, oridonin, lasiokariurinol, lasiokaurin and rabdoternin A in rat plasma was firstly developed and validated. The proposed method showed appropriate accuracy and repeatability and was successfully applied to a plasma samples analysis of the seven analytes after oral administration of Isodon serra extract, which maybe provide some references to the apprehension of the action mechanism and clinical application of Isodon serra.Part three Quantitative analysis of twelve diterpenoids in rat bile after oral administration of Isodon serra extract by high-performance liquid chromatography–electrospray ionization tandem mass spectrometryObjective: To develop a sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS) method and validate for the simultaneous determination of 12 diterpenoids (effusanin A, nodosin, enmein, lasiodonin, epinodosinol, oridonin, rabdosinate, epinodosin, ponicidin, lasiokaurin, lasiokaurinol, and Hebeirubesensin K) in rat bile, using using sulfamethoxazole as the internal standard (IS).Methods: Bile samples were collected during 0~1h,1~2h,2~4h,4~6h,6~8h,8~10h,10~12h,12~24h,24~36h periods after single oral administration of Isodon serra extract (10 mL/kg). The bile samples were pretreated by liquid–liquid extraction with ethyl acetate (EtOAc) and chromatographic separation was carried out on a C18 column with gradient elution. The detection of analytes was performed on a tandem mass system equipped with a turbo ion spray interface in positive and negative mode at the same time using multiple-reaction monitoring (MRM). The optimized mass transition ion-pairs (m/z) for quantitation were effusanin A (349.3/331.3), nodosin (363.3/253.1), enmein (363.3/281.3), lasiodonin (365.3/347.3), epinodosinol (382.3/347.3), oridonin (365.3/347.3), rabdosinate (535.4/295.3),epinodosin (361.2/287.1),pinodosin (361.2/363.2) , lasiokaurin (405.2/59.0) , lasiokaurinol (407.3/329.1),Hebeirubesensin K (365.2/299.2) and IS (54.1/156.0). The total run time was 15 min between injections.Results: The specificity, linearity, accuracy, precision, recovery, matrix effect and several stabilities were validated for diterpenoids in rat bile samples. The intra- and inter-day RSD were no more than 7.9% and 9.5%9.7%, respectively and the relative errors were within the range of -9.6% to 8.9%. The average extraction recoveries for all compounds were between 88.9%-108.7%. 14.93, The average percentages cumulative biliary excretion of diterpenoids (1-4, 6-10, 12 ) over the dose administered were 0.0302, 0.6361, 2.6722, 0.0165, 0.000243, 0.0371, 0.0126, 0.0032, 0.0445, 0.04727, 0.000332 and 0.0565%, while compounds 5 and 11 were less than 0.0001%.Conclusion: A sensitive and selective LC-ESI-MS method for the simultaneous determination of 12 diterpenoids (effusanin A, nodosin, enmein, lasiodonin, epinodosinol, oridonin, rabdosinate, epinodosin, ponicidin, lasiokaurin, lasiokaurinol, and Hebeirubesensin K) in rat bile was firstly developed and validated. The results showed that this method is robust, specific and sensitive and it can successfully fulfill the requirements of the excretion study of the twelve diterpenoids in Isodon serra.Part four Studies on fingerprints of Isodon serra from different area and PCA analysisObjective: To establish HPLC-PDA fingerprints of Isodon serra from different areas, to provide a new method for scientific evaluation and quality control of them.Methods: Two analysis methods (PCA and semblance analysis were applied to resolve the high performance liquid chromatographic fingerprints of Isodon serra. The chromatographic procedure was carried out with DiamonsilTM C18 (250 mm×4.6 mm, 5μm) as an analytic column and a mixture consisting of acetonitrile and 0.05% phosphoric acid in gradient as mobile phase, the temperature of column was 30?C. The detection wavelength was set at 238 nm and the flow rate was 0.8 mL/minResults: The mutual mode of PDA-HPLC fingerprints was set up, and the 19 common peaks were pinpointed. The similar degrees to the 18 batches Isodon serra of different producing areas were compared. Both of the analysis methods could be successfully used to classify these samples according to their origin.Conclusion: This method of the operation is simple, quick, accurate and can be used for the identification and quality control of Isodon serra.
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