| Fraxini Cortex is originated from the dried bark of Fraxinusrhynchophylla Hance, Fraxinus chinensis Roxb., Fraxinus szaboanaLingelsh. or Fraxinus stylosa Lingelsh.. As recorded in ChinesePharmacopoeia(2010 edition), it is an important traditional Chineseherbal medicine commonly used for the treatment of gout, arthritis,diarrhea and bacillary dysentery in clinic. Coumarins which are regardedas the major bioactive constituents in Fraxini Cortex, possess anti-cancer,anti-inflammatory and anti-viral effect, and are usually used to treatprimary hyperuricemia with renal dysfunction in clinic. Up to now, thepharmacokinetic study of Fraxini Cortex is limited to one or twoingredients, the pharmacodynamic mechanism of multi-componentinteraction is ignored, and it is difficult to reflect the real pharmacokineticcharacteristics of Fraxini Cortex in vivo. Moreover, there is no report onthe excretion kinetics in vivo of Fraxini Cortex. Similarly, the metabolitestudies of Fraxini Cortex were focused on the metabolism after oraladministration of single component to rats, and only the metabolite of afew composition was studied, other biologically active ingredients wereneglected. In this study, high performance liquid chromatography tandemmass spectrometry(HPLC-MS/MS) techniques was used to simultaneousdetermination of multi-component after oral administration of FraxiniCortex to rats and study the pharmacokinetics and excretion dynamics ofFraxini Cortex in vivo. In addition, we identify the metabolite in the urine,bile and plasma of rats after oral administration of Fraxini Cortex. Thisstudy provides the basis for the clinical use of Fraxini Cortex.Qinpijiegu capsule is a classical Tibetan medicine composed ofFraxini Cortex, Fritillaria Cirrhosa, Pyrethrum Tatsienense and Keel. Itdisplays good effects in analgesia and callus growth comparing withShangkejiegu tablet, a traditional medicine for treatment of bone injury,and is widely used for the treatment of bruises, sprains bones, andbruising swelling in China. In this study, a rapid high performance liquidchromatography coupled with tandem mass spectrometry(HPLC-MS/MS)method was developed for the simultaneous determination of fiveconstituents in Qinpijiegu capsule. This novel developed method wasapplied to evaluate three batches of Qinpijiegu capsule. This study hadsignificant importance for the quality control of QJC.Part one The pharmacokinetic studies of Fraxini Cortex basing onHPLC-MS/MS technologyObjective: 1 To establish a high performance liquid chromatographytandem mass spectrometry(HPLC- MS/MS) method for the simultaneousdetermination of 9 coumarins in rat plasma after oral administration ofFraxini Cortex extract, and study the pharmacokinetic of nine coumarinsin vivo. 2 To develop a HPLC-MS/MS method for the quantification of 8coumarins in rat urine and bile after oral administration of Fraxini Cortexextract, and study the excretion kinetics.Methods: 1 The preparation of Fraxini Cortex aqueous extract:Dried Fraxini Cortex(200 g) was immersed in 10-fold volumes of water(1:10, w/v) for 30 min, and decocted for 2 h for 4 times. Thereafter, thedecoction was filtered with gauze. The four decoctions were combinedand concentrated to 100 m L with a final concentration(2 g/m L) ofFraxini Cortex.Rats were given Fraxini Cortex extract(10 g/kg). 2 Thecollection and preparation of plasma, urine and bile sample: The plasmasamples within 24 hours, urine samples within 96 hours and bile sampleswithin 24 hours were collected after single oral administration of FraxiniCortex extract(10 g/kg) to rats. The plasma samples were pretreated by aone-step direct protein precipitation with methanol. The urine and bilesamples were pretreated by using the same pretreatment method asplasma. 3 Detection conditions: The chromatographic separation wascarried out on a Purospher STAR LP RP-C18(250 mm × 4.6 mm, 5 μm)column with a gradient mobile phase consisting of methanol and water(containing 0.05% acetic acid). The flow rate was 0.8 m L/min.Nodakenin was used as an internal standard(IS). All analytes and IS werequantitated through electrospray ionization(ESI) in negative ion multiplereaction monitoring(MRM) mode. The conditions of MS analysis weredesigned as follow: the spray voltage, 5500 V; the turbo spraytemperature, 650 ℃; nebulizer gas(gas 1), 60 psi; heater gas(gas 2), 65psi; collision gas, medium; the curtain gas(CUR), 25 psi. The monitoringion pair of 9 analytes were as follow: aesculin m/z 339.1/177.0, aesculetinm/z 177.0/133.0, fraxin m/z 369.0/207.0, fraxetin m/z 207.0/191.9,scopoletin m/z 191.0/175.9, isoscopoletin m/z 191.0/175.9,6-hydroxy-7,8-dimethoxy coumarin m/z 221.1/159.1,8-hydroxy-6,7-dimethoxy coumarin m/z 221.1/159.1, umbelliferone m/z160.9/133.0. In addition, the type and concentration of additives, and thecomposition of mobile phase, which affect the separation conditions,were investigated in the study. 4 The pharmacokinetics parameterscalculation: The area under plasma concentration-time curve(AUC0-t), thearea under the plasma concentration-time curve to time infinity(AUC0-∞),maximum plasma concentration(Cmax), time to reach the maximumplasma concentration(Tmax) and elimination half-time(t1/2) werecalculated on each individual analyte with the non-compartment model.Cmax and Tmax were obtained directly from the plasma concentration-timeplots. T1/2 was calculated using the following equation: T1/2= 0.693/k, inwhich k was determined by linear regression analysis of the logarithmictransformation of the last four data points of the curve. AUC0-t wascalculated with ladder-shape calculation method and AUC0-∞ wascalculated according the follow equation: AUC0-∞= AUC0-t + Ct/k, inwhich Ct was the concentration of the last point in plasmaconcentration-time profiles.Results: 1 Method validation: The calibration curves were linearover the investigated concentration range, with all correlation coefficientshigher than 0.9971. The intra- and inter-day RSD were no more than19.1% and the relative errors were within the range of-10.3% to 8.8%.The average extraction recoveries for all compounds were between 74.3%-101.9%. Pharmacokinetic results: 9 coumarins achieved their maximumplasma concentrations within 1 h, demonstrating rapid absorption fromthe gastrointestinal tract. However, there was great difference in theirelimination behavior. The elimination rate in vivo of glycosides wasfaster than aglycone, which may be caused by the biotransformation ofglycoside into aglycone. Scopoletin and isoscopoletin,6-hydroxy-7,8-dimethoxy coumarin and 8-hydroxy-6,7-dimethoxycoumarin were two pairs of isomers, which possessed the similarabsorption behavior, but there was great difference in their eliminationbehavior. 2 Method validation: This method was fully validated in termsof the sensitivity, specificity, accuracy, precision(intra- and inter-day),matrix effect, recovery as well as the stability of the analyte under variousconditions, and the results satisfied the requirements of biological samplemeasurement. Excretion study: The excretion study showed that 8coumarins were excreted completely within 32 h in urine, while within 18 h in bile. Less than 10% of most coumarins were excreted in the form ofprototype, while 19.26% of aesculetin was excreted in the form ofprototype, the phenomena might result from the hydrolysis of glycosidesmediated by gastrointestinal bacteria after oral administration, and mostaesculin was biotransformed into aesculetin. 8 coumarins excreted in theform of the bile were less than 2%, indicating that the excretion rate of 8coumarins in the form of prototype was low.Conclusions: A high performance liquid chromatography-tandemmass spectrometry(HPLC-MS/MS) method was developed for thedetermination of coumarins in rat plasma, urine and bile. The validatedmethod was successfully applied to pharmacokinetic study and excretionkinetics study of coumarins in rat after oral administration of FraxiniCortex aqueous extract, among which the pharmacokinetics of sixcoumarins including fraxin, fraxetin, isoscopoletin,6-hydroxy-7,8-dimethoxy coumarin, 8-hydroxy-6,7-dimethoxy coumarinand umbelliferone were firstly studied. In addition, this was the firstreport on the excretion kinetics study in vivo of Fraxini Cortex. Thisstudy would be valuable for deeply studies on the process of FraxiniCortex in vivo later and providing certain references to clinicalapplication of Fraxini Cortex.Part two Identification of metabolites in plasma, urine and bile ofrats by HPLC-QTOF-MS/MSObjective: To establish a sensitive and effectiveHPLC-QTOF-MS/MS method for the identification of metabolites inplasma, urine and bile of rats after oral administration of Fraxini Cortex.Methods: The plasma samples within 24 h, urine samples within 72 h and bile samples within 24 h were collected after oral administration ofFraxini Cortex extract(10 g/kg) to three male SD rats. Biological sampleswere extracted with ethyl acetate, and repeated three times; thesupernatant was separated out and merged, then evaporated to drynessunder a gentle stream of air at room temperature. Then the residue wasreconstituted with methanol and vortex-mixed briefly. Chromatographicconditions: a Purospher STAR LP RP-C18(250 mm × 4.6 mm, 5 μm)column was used for separation, temperature: 25 ℃, the mobile phase:methanol(A)- aqueous solution with 0.05%acetic acid(B) with gradientelution, the elution procedure is as follows: 0-5 min, 5-20%(A); 5-20 min,20%(A); 20-40 min, 20-40%(A); 40-75 min, 40-95%(A), columntemperature: 30 ℃, flow rate: 1.0 m L/min, the injection volume was 10μL. HPLC-QTOF-MS technology with TOF-MS-IDA-8MS/MS scanswas used for detection; both the positive ion mode and negative ion modewere used for data collection. Metabolite pilot 1.5 and Peak View 2.0software were used for data analysis. According to the regular pattern ofmass spectral fragmentation of simple coumarins in Fraxini Cortex andbiotransformation of the parent compound that may occur in vivo, thechemical structures of compounds were identified.Results: 39 metabolites were characterized in rat after oraladministration of Fraxini Cortex, among which 22 compounds wereidentified in plasma, 36 compounds were identified in urine sample, 24 compounds was identified in bile. 13 compounds were prototype, and 26 compounds were metabolites.Conclusions: The method is sensitive, effective, and can besuccessfully applied to the structural identification of metabolites in urine,plasma and bile of rats after oral administration of Fraxini Cortexaqueous extract. This study will help to clarify the pharmacodynamicmechanism of Fraxini Cortex in vivo.Part three Study on the quality control of Qinpijiegu capsuleObjective: To establish a rapid high performance liquidchromatography coupled with tandem mass spectrometry(HPLC-MS/MS)method was developed for the simultaneous determination of fiveconstituents in Qinpijiegu capsule, a classical Tibetan prescription, andapply the proposed method to analyze three batches of Qinpijiegu capsulesamples.Methods: 1 The investigatoion of the extraction conditions:Ultrasonic extraction with methanol was optimized. Proportion ofmethanol solvent(25%, 50%, 75%, 100%) and ultrasound time(30 min,45 min, 60 min, 75 min, 90 min) were investigated to obtain the optimalextraction conditions. The second method was reflux extraction withalkalinized chloroform-methanol, an orthogonal test which contained fourvariables and three levels was employed to investigate the extractionconditions. The following four variables, each with three levels, wereoptimized in this study: the time of alkalization(factor A), its levels( 30 min, 60 min, 120 min); the ratio of methanol and chloroform(factor B),its levels(1:1, 1:2, 1:4); the volume of solution(factor C), its levels(25m L, 50 m L, 75 m L) and the extraction time(factor D), its levels(30 min,60 min, 90 min). The interaction between different variables was ignoredin this study. The total amount of components in analytes was selected asthe index for the evaluation of method. 2 Detection conditions: Theseparation of five compounds, namely aesculin, aesculetin, fraxin,peimine and peiminine was performed on a Purospher STAR LP RP-C18(250 mm × 4.6 mm,5 μm)column with linear gradient elution ofacetonitrile-0.3‰ formic acid water. Detection was carried out bymultiple-reaction monitoring(MRM) mode using electrospray ionizationin the positive and negative ion switching mode. The condition of MSanalysis were designed as follow: the spray voltage, 5500 and-4500 V;the turbo spray temperature, 650 ℃; nebulizer gas(Gas 1), 60 psi; heatergas(Gas 2), 65 psi; collision gas, medium; the curtain gas(CUR), 25 psi.Data collection and analysis were accomplished with the Analyst 1.5.2software provided by Applied Biosystems/MDS Sciex. The establishedmethod was validated with linearity, limit of detection(LOD), limit ofquantification(LOQ), precision, accuracy and stability,Results: 1 The results of investigatoion of the extraction conditions:Comparing optimal ultrasound extraction with methanol and refluxextraction with chloroform-methanol, it was found that there was nosignificant difference between two methods in content of peimine andpeiminine. While the content of aesculin, aesculetin and fraxin wereobviously increased by ultrasound extraction. Moreover, the method ofultrasound extraction was simple, fast, and easy to operate. Eventually,the samples were prepared by ultrasound extraction with 25 m L 75%methanol for 60 min. 2 Method validation: The proposed method wasfully validated with acceptable linearity(r2>0.9977), precision(RSD<7.4%), repeatability(RSD<2.5%), stability(RE<9.1%) andrecovery in the range of 89.7-121.4% with(RSD<10.8%). 3 Samplemeasurement results: Aesculin, aesculetn and fraxin were the dominantcompounds. Among them, the content of aesculin was the highest,followed by fraxin. The mean values of them were 7.96 mg/g and 6.18mg/g, respectively. Aesculetin was lower than former two coumarins withthe content of 4.48 mg/g. On the other hand, the contents of peimine andpeiminine were relatively low, and the content of peiminine(0.08 mg/g)was the lowest. In addition, there were no remarkable variations in thecontents of aesculin, peimine and peiminine in different batches, all theRSDs of batch to batch was lower than 10%. While, there were obviousvariations in the contents of aesculetin and fraxin, with the RSDs of17.4% and 16.0% respectively, which might be caused by the variationsof medicinal material and production process in different batches.Conclusions: In the present study, a HPLC-MS/MS method wasestablished for the simultaneous determination of multiple activecomponents in Qinpijiegu capsule for the first time. This novel developedmethod was applied to evaluate three batches of Qinpijiegu capsule. As aresult, the method was demonstrated to be fast, simple and sensitive. Atthe same time, the established method accurately reflected thecharacteristic of the preparation because five constituents, which workedsynergistically were simultaneously determined. It would be reliable andefficient, and had significant importance for the quality control ofQinpijiegu capsule. |
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