| Herba Lysimachiae and Herba Desmodii Styracifolii are importantmembers of traditional Chinese medicine (TCM) originated from the driedLysimachia christinae Hance (Primulaceae) and Desmodium styracifolium(Osb.) Merr.(Leguminosae) plants, respectively. Although the two herbsbelonged to different families are regarded as different remedies inPharmacopoeia of The People’s Republic of China, they exhibit similarpharmacological effects, such as antibiosis, antipyretic and diuretic effects.Substitution use of each other is not uncommon in clinical application. It isabsolutely necessary to compare the similarities and differences of chemicalcompositions in the two kinds of medicinal materials.In this paper, we developed and validated a simple, sensitive and reliableLC–MS/MS method for the simultaneous determination of18components inHerba Lysimachiae and Herba Desmodii Styracifolii, including15flavonoidsand3phenolic acids. In addition,16batches Herba Lysimachiae and21batches Herba Desmodii Styracifolii from different sources were comparedusing the developed method. This method can be effectively applied for thequality control and evaluation of Herba Lysimachiae and Herba DesmodiiStyracifolii.Furtherly, two LC-MS/MS methods were firstly developed and validatedto analyze4flavonoids and1phenolic acid in rat plasma and7flavonoids and1phenolic acid in rat bile after oral administration of Herba DesmodiiStyracifolii., respectively. The methods were applied to studies onpharmacokinetics and biliary excretion of Desmodii Styracifolii in rats,respectively. The results would be helpful for further investigating the action mechanism of Herba Desmodii Styracifolii and may provide a useful tool forclinical application of the formulation of Chinese medicine.Part one Comparation of main constituents in Herba Lysimachiaeand Herba Desmodii Styracifolii by LC-MS/MSObjective: To develop a LC-MS/MS method for the simultaneousanalysis of18components (15flavonoids and3phenolic acids) in HerbaLysimachiae and Herba Desmodii Styracifolii. Multiple plots and hierarchicalcluster analysis was used to observe the batch-to-batch variations and speciesauthentication for Herba Lysimachiae and Herba Desmodii Styracifolii.Methods: Analytes were detected using a hybrid quadrupole linear iontrap mass spectrometer that was equipped with an electrospray ionizationsource. The electrospray ionization source was operated in the negative andmultiple-reaction monitoring (MRM) modes using the following conditions:ion spray voltage,-4500V; the turbo spray temperature,650°C; curtain gas(CUR),1.72×105Pa and interface heater was turned on; collision gas, medium;nebulizer gas (gas1) and heater gas (gas2),4.14×105and4.48×105Pa. Theprecursor-to-product ion pairs of18analytes were152.9/108.9forprotocatechuic acid,353.1/191.0for chlorogenic acid,193.0/133.9for ferulicacid,300.9/150.9for quercetin,609.0/299.9for rutin,284.9/92.8forkaempferol,271.1/150.9for naringenin,315.0/150.9for isorhamnetin,255.0/118.9for liquiritigenin,269.0/116.9.0for apigenin,285.0/132.9forluteolin,463.2/300.1for isoquercitrin,463.2/300.1for hyperin,431.2/311.1for vitexin,431.2/311.1for isovitexin,447.2/357.1for homoorientin,563.2/353.0for schaftoside,563.2/353.0for isoschaftoside, respectively.Separation was performed using a Diamonsil C18column (150mm×4.6mm,5μm) which was eluted with methanol and0.1‰acetic acid. The columntemperature was set to40°C. The mobile phase flow rate was maintained at1.0mL/min and the injection volume was10μL. The method was successfullyapplied to differentiate16batches of Herba Lysimachiae and21batches ofHerba Desmodii Styracifolii.Results: The linearity of analytical response was good with correlation coefficients of greater than0.993for all the compounds in the concentrationrange. The precision, stability, LOD and LOQ of the method were good forthe18components. The average recoveries (n=9) of these components were inthe range of95.2–108.6%with RSD values between0.9%and4.6%. Theresults demonstrate that Herba Lysimachiae and Herba Desmodii Styracifoliialmost have the same components, but the content of each component isdifferent. Also, herba Lysimachiae samples from various sources weresignificantly more different from the Herba Desmodii Styracifolii samples.Conclusion: The LC-MS/MS method was sensitive, selective andsuitable for the simultaneous analysis of18constituents in Herba Lysimachiaeand Herba Desmodii Styracifolii. Thus, this method could be used for thereliable quality control of Herba Lysimachiae and Herba DesmodiiStyracifolii.Part Two Simultaneous determination of five constituents in rat plasmaby LC–MS/MS and its application to pharmacokinetic studyafter oral administration of Herba Desmodii Styracifolii extractObjective: To establish a LC-MS/MS method for the simultaneousdetermination of four flavonoids including schaftoside, isovitexin, luteolin,apigenin and one phenolic acid named ferulic acid in rat plasma. The methodwas applied to evaluate pharmacokinetics after oral administration of HerbaDesmodii Styracifolii extract to rats.Methods: Plasma samples were collected from the fossa orbitalis veinand acidified with appropriate amount of HCl, then methanol was added asprotein precipitant. The samples were analyzed with gradient elution consistedof methanol and0.1‰acetic acid using sulfamethoxazole as internal standard(IS). Separation was performed using a Diamonsil C18column (150mm×4.6mm,5μm). The column temperature was set to30°C. The electrosprayionization source was operated in the negative and MRM modes. The mobilephase flow rate was maintained at0.8mL/min and the injection volume was20μL.Results: The developed method has good precision, accuracy, stability, linearity, detection limit and quantitation. The mean extraction recoveries ofthe investigated analytes in plasma at three different concentration levels werefound to be65.3–90.3%which indicated that the protein precipitation methodwas acceptable. The observed matrix effects ranged from82.5–104.6%. Thecharacteristics of pharmacokinetics show that flavonoid glycoside (schaftosideand isovitexin) could be much quickly absorbed but much slowly metabolizedthan flavonoid aglycones (luteolin and apigenin) and phenolic acid was moreeasily absorbed into the blood and quickly metabolized than flavonoids.Conclusion: The developed method was specific, sensitive, accurate andreproducible, and suitable for the study of the analytes after oraladministration of Herba Desmodii Styracifolii extract. The results would behelpful for further investigating the action mechanism of Herba DesmodiiStyracifolii and may provide a useful tool for clinical application of theformulation of Chinese medicine.Part three Simultaneous determination of eight constituents in rat bile byLC-MS/MS and its application to biliary excretion study afteroral administration of Herba Desmodii Styracifolii extractObjective: To developed and validated a selective and sensitiveLC-MS/MS method for analysis of schaftoside, isoschaftoside, isovitexin,liquiritigenin, isorhamnetin, naringenin, apigenin and ferulic acid in rat bile.The method was applied to study biliary excretion after oral administration ofHerba Desmodii Styracifolii extract to rats.Methods: The bile samples were collected during0-1,1-2,2-4,4-6,6-8,8-12,12-24h after oral administration of Herba Desmodii Styracifolii extractto rats. A simple liquid–liquid extraction (LLE) method by ethyl acetate wasused. Separation on a Diamonsil C18column (150mm×4.6mm,5μm) wasachieved by methanol and0.1‰acetic acid with gradient elution. The columntemperature was set to40°C. The mass spectrometer was operated in thenegative and MRM modes. The internal standard (IS) was sulfamethoxazole.Results: The correlation coefficients were all higher than0.9915and themethod has good intra-, inter-day precisions and stability. The extraction recoveries of the analytes ranged from63.2to98.6%and the matrix effectvalues obtained for analytes ranged from88.3%to110.3%. In twenty-fourhours, the average percentages of the eight analytes were (0.047±0.01)%,(1.145±0.46)%,(0.007±0.004)%,(0.998±0.24)%,(0.015±0.01)%,(0.043±0.018)%,(0.010±0.006)%and (0.147±0.075)%, respectively.Conclusions: The method was simple, selective and specific, andsuitable for the bile excretion study of the eight analytes in Herba DesmodiiStyracifolii extract. |
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