HiHigh h PPerformance f CapillaryC ill ElectrophoresisEl t h i TandemT d TimeTi -off-fliflight ht MMass SSpectrometry t t ffor FiFingerprint i t StStudy d off BiBio-activeti AlkaloidsAlk l id FFrom ChiChinese TTraditional diti l MdiiMedicines ASMS 2011 ZhengxiangggZhang,g, Tao Bo, , JianqiuJqMi,,g Agilent Technologies, g , No.3, , Wang gJg Jing Bei Lu,,C Chao Yang g District, , Beijing, j g, 100102, , ChinaC WP 122

ItIntroduction d ti EitlExperimental RltResultsRl andd DiDiscussion i RltResultsRltResults andanddd DiscussionDiiDiscussionDii

Alkaloids in CChinese Traditional Medicines (C(TCMs) ) are Separation conditions Data processing DbDatabase establishmentbli h bdbased on accurate mass CE-MS ffor ththe activeti alkaloidslk l id iin ffour TCM DtbDatabase searchinghi ittimportant bibio-activeti components,t whichhi h hhas attractedtt t d The mass spectrometer was operated in MS mode at two much attention. Developing high-throughput and high- An alkaloid database based on accuracyy mass was The conditions of HPCE were optimizedp with respectp to The active alkaloids in the TCM samples were screened by Separation was performed on the Agilent 7100 HPCE using spectra/second/d. MlMolecular l ffeature extractioni algorithmlih running buffer and sheath liquid components for obtaining selective fingerprint technology is crucial to control TCM established for alkaloid rapid screening and fingerprint usingg the CE-TOF combined with the database searchingg acetate ammonium buffer (pH 2-8) as running buffer. The wasas usedsed to remoremove e the backgrobackground nd noisenoise, a compocompound nd list theh hihigh h-efficientffi i andd ffast separationi . ThThe typicalilCE-TOF qualityqy. Highg performancep capillarypyelectrophoresisp (HPCE)() analysis in the TCM samples. The MS data was processed after the MFE, which was shown in Figure 4 (Rhizoma sheathhthliliquid id flflowing i speedd was 4µL/minL/ i . AlidApplied voltagelt was contracted and the related chemical formulas were electropherograms were shown in Figure 3. Based on is a robust separation technique for complicated system bby theh MFE algorithmlihto remove theh bkbackground diions andd Coptidis as example) . was set at 20 kV and capillary temperature was set at 25oC. calculated based on accuracy mass and isotope ratio contracting a compound list. By searching the database accurate mass and database searching, the fingerprint due to high-efficiency for complex samples and has been analysis was achieved for the TCMs from different origins. The effective capillary length was 80 cm. calculation. The MFE is crucial for the TOF data processingpgwith the mass accuracy filter below 10 ppm, bio-active widely used for analyzing the TCMs. Time of flight (TOF) for the complex samples and is an automatic and high- mass spectrometryttwithith ultralt -hihigh h mass resolutionlti playsl a alkaloids were screening for four kinds of TCMs. The typical throughput tool. Figure 1 demonstrates the workflow of active alkaloid compoundsp in the database was shown in crucial role in compound identification since the accurate MS conditions MFE ddata processingi . mass of precursor ions can be provided. In this studystudy, a full Figure 2. workflow of alkaloid fingerprintgp analysisy in four TCMs was MMass system t : AAgilent il t 6224 TOF exhibited by the HPCE-TOF, involving database Formula Mass Compound name establishment based on accurate mass, data processing Ion source: ESI # Formula Mass Cpd (Mass Feature Extract, MFE) and database search. C10H9NO3 191.0582 Noroxyhydrastinine C19H13NO4 319.0844 Coptisine chloride C19H17NO4 323.1157 Stylopine、tetrahydropalmatine Nebulizer gas: N2 C19H21NO4 327.147 d-isoboldine C19H21NO4 327.147 yhscs C19H21NO4 327327.147 147 l-tetrahtetrahydrocoptisine drocoptisine Polarity: positive mode C19H21NO4 327.147 dl-tetrahydrocoptine C20H15NO4 333.1001 dihydrosanguinarine C20H17NO4 335.1163 Berberine Nebulizer pressure: 1010.0 0 psi C20H19NO4 337.1314 Dehydronantenine C20H19NO4 337337.1319 1319 Columbamine o C20H19NO4 337.1319 Dehydrocorydalmine Dryingygg gas temperature: p 130 C C20H21NO4 339339.147 147 TthdTetrahydraoberberine/Canadine bbi /Cdi C20H21NO4 339.147 Nantenine C20H23NO4 341.1627 corydalmine Drying gas flow rate: 4.0 L/min C20H23NO4 341.1627 Isocorypalmine/tetrahydrocolumbamine C20H23NO4 341.1627 d-qjhysj C20H23NO4 341341.1627 1627 Lauroscholtzine SScan mode:d fullfll scan C20H23NO4 341.1627 thaliporphine C20H23NO4 341341.1627 1627 d-ezqfjfj C20H23NO4 341.1627 yhsjs Mass range: 100-3000 m/z C20H23NO4 341.1627 Coryaline H C20H23NO4 341.1627 Coryaline I C20H25NO4 343.1783 szcj Internal mass calibration: Calibration by two reference ions C20H17NO5 351351.1106 1106 oxoglaucine C21H21NO4 351.147 qqhysj C21H21NO4 351351.147 147 PlPalmatine ti C20H19NO5 353.1263 C21H25NO4 355.1783 Coryaline K Sample preparation C21H25NO4 355.1783 Tetrahydropalmatineyp Figureg 4. Database searchingg of active alkaloids from C21H25NO4 355.1783 Corybulbine Rhizoma Coptidis. C21H25NO4 355355.1783 1783 yhn C21H25NO4 355.1783 All alkaloid standards and four TCMs (Rhizoma Coptidisp , C21H25NO4 355355.1783 1783 yhfjhfj Greater Celandine Herb, Medcinal Evodia Fruit and Rhizoma C22H23NO6 357.1525 saulatine C20H23NO5 357.1576 a-allo- Corrdalis) for this study were obtained from the Chinese C22H23NO4 365.1632 dehydrocorydalinyy Pharmaceutical University. The samples were extracted by C22H24NO4 366.1705 qqyhsjs Conclusions C20H17NO6 367367.1056 1056 (+)- diffdifferent t ratioti off watert andd methanolth l. C21H23NO5 369.1576 homochelidonine C22H27NO4 369369.194 194 CdliCorydaline C22H27NO4 369.194 Coryaline Figure 3. CE-MS electropherograms of Rhizoma Coptidis, Based on the accurate mass and database searching by the FiFigure 1. MFE fifunction ffor theh ddata processingi off TCM C23H19NO5 389.1263 eqxgj Greater Celandine Herb and Medcinal Evodia Fruit from HPCE-TOF, The excellent selectivity, high-throughput and C30H36N2O5 504.2624 Coryaliney J samples the upper to the bottombottom, respectively. goodd sensitivityiti it off ththe fifingerprint i t analysisliwas achievedhi d ffor Figure 2. The typical active alkaloid compounds in the the TCMs from different origins. database.