Qualitative And Quantitative Analysis Of Fructus Sophorae And Its Pharmacokinetics Study By HPLC-MS Technologies

Fructus Sophorae or HuaiJiao, the dried ripe fruits of Styphnolobiumjaponicum (L.) Schott (Leguminosae), is a herbal ingredient used in TCM forits haemostatic properties. Pharmacological studies on Fructus Sophorae haverevealed that it contains flavonoids, alkaloids, terpenoids, Amino Acid,saccharide, phospholipids, and so on. Specifically, flavones are the maineffective components. In the past few years, the chemical research of the herbof Fructus Sophorae has become a popular domain for its variety ofbioactivities. Fructus Sophorae is a traditional Chinese medicine commonlyused as haemostatic properties. In recent years, domestic and foreign reportson the phytochemical of Fructus Sophorae gradual increaseed, whichcontained anticancer, anti-tumor, anti-obesity, antifertility action,anti-oxidation effects and the treatment of hypertension and hemorrhoids, et al.Qualitative and quantitative analysis of Fructus Sophorae and itspharmacokinetics study was almost limited. At present vary kind of medicinepreparation and healthy production of Fructus Sophorae have already beenproduced and used in clinical. On account of different sources and climaticconditions, its chemical constituents may vary substantially. Therefore, inorder to further effectively utilize it and enhance the clinical safety,simultaneous quantitative analysis of active components is more reliable andaccurate method for the quality control of traditional Chinese medicine. Inpresent study, we firstly developed an accurate and simple LC-MS-MSmethod for simultaneous determination of six major components in FructusSophorae. Simultaneous quantification of bioactive components byHPLC-MS-MS coupled with chemometric techniques would be awell-acceptable strategy to comprehensively control the quality of FructusSophorae. Because the therapeutic effects of TCMs are based on the complexinteractions of multiple ingredients, the research of the pharmacokineticstudies of multiple flavones after administration of Fructus Sophorae extractis essential to understand their role in human health. However, as far as weknow, no analytical methods have been reported for the simultaneousdetermination of these six flavonoids in biological samples. In this study, wedeveloped a rather sensitive and selective LC–MS/MS method tosimultaneously determine sophoricoside, genistin, genistein, rutin, quercetinand kaempferol in rat plasma. The method was applied to pharmacokineticsafter oral administration of Fructus Sophorae extract to rats. A sensitive andselective HPLC-MS method was developed for simultaneous determination ofthe six flavonoids in rat bile and urine and was applied to the excretionamount study of the flavonoids after oral administration of Fructus Sophoraeextract. The obtained results would be very helpful for evaluating the clinicalapplication of this herb.Part one Simultaneous quantification of six major flavonoids fromFructus Sophorae by LC-ESI-MS-MS and statistical analysisObjective: A new, sensitive and selective high performance liquidchromatography–tandem mass spectrometric method (HPLC–MS/MS) wasdeveloped for the determination of six major flavonoids includingsophoricoside, genistin, genistein, rutin, quercetin, kaempferol in FructusSophorae. Principal component analysis (PCA) and hierarchical clusteringanalysis (HCA) were used to classify and differentiate these samples.Methods: Chromatographic separation was performed on a C18columnwith linear gradient elution of methanol and0.5‰acetic acid (v/v) at a flowrate of0.8mL/min. The total run time was8.0min. The detection wasaccomplished in the negative mode using multiple-reaction monitoring(MRM). The ion spray voltage was set to4500V, and the turbo spraytemperature was kept at650℃. Nebulizer gas (gas1) and heater gas (gas2)was set at60and65arbitrary units, respectively. The curtain gas was kept at25arbitrary units and interface heater was on. The effect of origin in Fructus Sophorae on the total amount of those analytes was analyzed by PCA usingSPSS. The HCA of Samples1–30was performed using SPSS software.Results: The correlation coefficients were all higher than0.9904. TheLODs and LOQs for each compound were less than3.12ng/mL and12.5ng/mL, which showed a high sensitivity. The overall intra-and inter-dayprecisions (RSD) for the investigated components were less than2.07%and2.44%, respectively. The average recovery was in the range of96.24%–103.34%. All analytes were found to be stable with48h. The resultsdemonstrated that the quantitative difference of six active compounds wasuseful for chemotaxonomy of many samples from different sources and thestandardization and differentiation of many similar samples. The genuinemedicinal materials could be distinguished from other general samples. ThePCA and HCA further confirmed the excellent quality of genuine medicinalmaterials.Conclusion: The method is robust and specific and it was successfullyapplied to the simultaneously determination of these active components inFructus Sophorae. Simultaneous quantification of bioactive components byHPLC-MS-MS coupled with chemometric techniques would be awell-acceptable strategy to comprehensively control the quality of FructusSophorae.Part two Simultaneous determination and pharmacokinetic study of sixflavonoids from Fructus Sophorae extract in rat plasma byLC–MS/MSObjective: To establish a method to determination the six flavonoidsincluding sophoricoside, genistin, genistein, rutin, quercetin and kaempferol inrat plasma after the orally administrating of Fructus Sophorae extract,HPLC-MS was used and validated to study the pharmacokinetics.Methods: Twelve rats were divided into two groups at random and thenwere both given single doses of Fructus Sophorae extract (2mL/kg). Bloodsamples were collected from the vein of the eye ground at0,10,25,45,60,75,90,120,180,270min (the first group) and0,1.5,3,4.5,6,8,10,12,18,24,30, 36,48,72h (the second group) after a single oral administration. The plasmasamples were pretreated and extracted by a simple liquid–liquid extraction(LLE) method by ethyl acetate. Sulfamethoxazole (SMZ) was used as internalstandard. Chromatographic conditions: Reverse-phase Diamonsil C18column(150×4.6mm,5μm) with the column temperature set at25℃. A lineargradient elution of eluents A (methanol) and B (0.5‰acetic acid; v/v) wasused for the separation. The following gradient condition was used: initial0–1.5min, linear change from35%A to75%A;1.5–6min, linear change from75%A to95%A; and6–8min, isocratic elution95%A; finally35%Amaintained for8min. The flow rate was set at0.8mL/min. Mass spectrometry:The ESI interface operated in the negative mode was used. The ion sprayvoltage was set to4500V, and the turbo spray temperature was kept at650℃.Nebulizer gas (gas1) and heater gas (gas2) was set at60and65arbitraryunits, respectively. The curtain gas was kept at25arbitrary units. Forstructural identification of each analyte, the information-dependent acquisition(IDA) method was used to trigger the enhanced product ion (EPI) scans byanalyzing MRM signals. The optimized mass transition ion-pairs (m/z) forquantitation were sophoricoside (431.1/267.9), genistin (431.1/267.9),genistein (269.0/133.0), rutin (609.2/300.0), quercetin (301.0/150.9),kaempferol (284.9/93.0), IS(252.0/155.9).Results: Full validation of the assay including specificity, linearity,accuracy, precision, extraction recovery and matrix effect was acceptable. Thecalibration curves of six flavonoids exhibited good linearity. The developedand validated method was applied to the pharmacokinetic evaluation ofFructus Sophorae in rats following oral administration. The analytes weredivided into two clusters: flavonoid glycoside (sophoricoside, genistin, rutin)and flavonoid aglycones (genistein, quercetin, kaempferol). Flavonoidglycoside could achieve the maximum plasma concentration at1h whileflavonoid aglycones could achieve the maximum plasma concentrationbetween10and12h after oral administration. The results show that theflavonoid glycoside in Fructus Sophorae were absorbed firstly, then the aglycones were absorbed. The values of the elimination rate constants kranged from0.0117to0.0201for flavonoid glycoside and from0.0011to0.0016for flavonoid aglycones, which indicated that the flavonoid aglyconeshad slower elimination rates. A double-peak phenomenon of genistein ispresented. This phenomenon may be relevant to entero-hepatic recirculation.Metabolic process such as de-glucose of sophoricoside and genistin mightpartly explains this phenomenon.Conclusion: A selective LC-ESI-MS method was developed andvalidated for the simultaneous determination of the six bioactive constituentsof Fructus Sophorae extract in rat plasma. The results showed that this methodis robust, specific and sensitive and it can successfully fulfill the requirementsof pharmacokinetic study. The results provided a meaningful basis for theclinical application of this herb.Part three Simultaneous determination and excretion study of sixflavonoids in rat bile and urine after oral administration ofFructus Sophorae extract by HPLC/electrospray ionizationmass spectrometryObjective: A selective HPLC-MS method was developed and validatedfor analysis sophoricoside, genistin, genistein, rutin, quercetin and kaempferolin rat bile and urine.Methods: Chromatographic separations were performed with an AlltechC18pre-column (4.6×7.5mm,5μm) and a Diamonsil RP-C18analytical column(150×4.6mm,5μm). The mobile phase consisted of methanol (A) and watercontaining0.1%formic acid (B). The mass spectrometer was operated in thenegative modes by monitoring the precursor–product combination in multiplereaction monitoring (MRM) mode. Internal standard was sulfamethoxazoleand the dosage of Fructus Sophorae extract was2.5mL/kg. For bile, thesamples were collected during0-2,2-4,4-6,6-8,8-12,12-24,24-30,30-36h.For urine, the samples were collected during0-4,4-8,8-12,12-24,24-36,36-48,48-60,60-72,72-96h. The six flavonoids and IS were extracted by asimple liquid–liquid extraction (LLE) method by ethyl acetate. Results: The correlation coefficients were all higher than0.9953. Theresults of the inter-and intra-day precision (<15%) and accuracy (within±15%) at QC concentrations were acceptable. The extraction recovery ofanalytes ranged from64.9%to81.5%for bile, and from66.7%to83.2%forurine. For matrix effect, the values ranged from85.7%to105.5%for bile, andfrom87.4%to103.0%for urine, which indicates no matrix effect forquantification of the target flavones in the developed method. Stability dataindicated good stability for all the analytes over four storage conditions in bileand urine. In the bile samples, the six flavonoids excreted completely inthirty-six hours and the average percentages of six flavonoids excreted were0.058%,0.712%,0.966%,0.026%,0.261%,0.680%. In the urine samples, thesix flavonoids excreted completely in ninety-six hours. The averagepercentages of six flavonoids excreted were0.049%,0.156%,0.133%,0.111%,2.763%,2.028%.Conclusion: The method is robust and specific and it can successfullycomplete the requirements of the excretion study of six major flavonoids in ratbile and urine in Fructus Sophorae. These results may offer useful informationfor clinical application of traditional Chinese medicines.