HPLC Analysis of Six Active Components of Caulis Lonicerae Using a Phenyl-1 Column

Transcription

1 HPLC Anlysis of Six Active Components of Culis Lonicere Using Phenyl- Column Xu Qun, Hung Xiongfeng, nd Jeffrey Rohrer Thermo Fisher Scientific Inc., Shnghi, People s Repulic of Chin Thermo Fisher Scientific Inc., Sunnyvle, CA, USA Appliction Note 99 Key Words Phrmcopoei of the People s Repulic of Chin (PPRC), Trditionl Chinese Medicine, Nturl Iridoid Glycosides, Nturl Orgnic Acids, Flvonoids A Introduction Culis Lonicere (Figure ), the dried rttn of Lonicer jponic (Cprifolicee or honeysuckle fmily), is n importnt trditionl Chinese medicine used for the tretment of such ilments s cute fever, hedche, respirtory infection, nd epidermic diseses. The mjor ctive components in Culis Lonicere re lognin, sweroside, chlorogenic cid, cffeic cid, rutin, nd gluteolin. Their structures re shown in Figure. The Phrmcopoei of the People s Repulic of Chin (PPRC) 00 regultes Culis Lonicere with different highperformnce liquid chromtogrphy (HPLC) methods for the determintion of lognin (using phenyl sttionry phse) nd chlorogenic cid (using C8 phse), respectively. Therefore, the PPRC qulity control (QC) protocols for Culis Lonicere re inconvenient (requiring two methods) nd indequte (determining only two trget components). Although there re other methods to determine sweroside, cffeic cid, rutin, gluteolin, nd up to three other minor ctive components of Culis Lonicere on C8 sttionry phse using HPLC, 5 these methods require long seprtion times ( 5 min) nd hve insufficient pek resolution etween lognin nd sweroside. Figure. Culis Lonicere: A) Lonicer jponic, nd B) dried rttn. B Gol To develop n efficient nd comprehensive HPLC QC method for the nlysis of Culis Lonicere. This method must seprte the six min ctive components (lognin, sweroside, chlorogenic cid, cffeic cid, rutin, nd gluteolin).

2 Experimentl A Thermo Scientific Acclim Phenyl mm, µm column (P/N 07969) ws chosen for this seprtion ecuse its phenyl groups re structurlly similr to the phenyl groups nd romtic structures contined in the compounds of interest. The seprtion of Culis Lonicere smple cn e completed within Chlorogenic Acid 0 min, nd the resolution etween lognin nd sweroside in 5.5 min using 0.% formic cid/cetonitrile moile phse. Cffeic Acid Lognin Rutin Sweroside Equipment Thermo Scientific Dionex UltiMte 000 HPLC system including: DGP-600 Dul Ternry Rpid Seprtion (RS) Pump System SRD-600 Integrted Solvent nd Degsser Rck WPS-000TRS Well Plte Smpler, Thermosttted TCC-000RS Thermosttted Column Comprtment DAD-000RS Diode Arry Detector (with 5 µl seminlyticl flow cell) Thermo Scientific Dionex Chromeleon 6.80 SR9 Chromtogrphy Dt System (CDS) softwre or higher Kudos SK00LH Ultrsonic Genertor, Kudos Ultrsonic Instrumentl Co., Shnghi, Chin Regents nd Stndrds Deionized (DI) wter, 8. MΩ cm resistivity Acetonitrile (CH CN), HPLC grde, (Ct.#AC600000), Fisher Chemicl Methnol (CH OH), HPLC grde, Ct.# AC ), Fisher Chemicl Formic cid (FA), nlyticl grde, SCRC, Chin Lognin, sweroside, chlorogenic cid, cffeic cid, rutin, nd gluteolin, purity 98%, Reserch Center of Stndrdiztion of CTM, Shnghi, Chin Smple Preprtion The Culis Lonicere smple ws purchsed from the Reserch Center of Stndrdiztion of CTM, Shnghi, Chin. Accurtely weigh 0. g of smple powder nd plce in 0 ml volumetric flsk. Add 0 ml methnol/wter (:, v/v). After 5 min in n ultrsonic th, cool to room temperture. Filter it through 0. μm memrne (Millex-LH ) prior to injection. Gluteolin Figure. Structures of the six mjor ctive components of Culis Lonicere.

3 Results nd Discussion Effect of Column Temperture on the Seprtion of Culis Lonicere Active Components The effect of column temperture ws evluted for the seprtion of lognin, sweroside, chlorogenic cid, cffeic cid, rutin, nd gluteolin using n Acclim Phenyl- column. Figure A shows the seprtion of the stndrds on the column, with temperture chnging from 5 5 C in five degree steps. As expected, incresing the column temperture decresed retention time, nd seline seprtions of the stndrds were chieved in the temperture rnge. However, the investigtion of smples shown in Figure B demonstrted tht chnging column temperture could cuse some other compounds to interfere with detection of the nlytes. For exmple, incresing column temperture could improve the resolution of lognin (Pek ), nd decrese tht of sweroside (Pek ). Therefore, n opertion temperture t 0 C ws chosen s compromise etween speed nd resolution. Tle lists the clculted pek purity mtch fctors (the corresponding vlue for 00% purity is 000) for lognin, sweroside, chlorogenic cid, nd cffeic cid (the four compounds found in the smple extrct), demonstrting the good seprtion for the smple t column temperture of 0 C. Method Performnce (Reproduciility, Linerity, nd Detection Limits) The method reproduciility ws estimted y mking consecutive injections of Culis Lonicere smple mixed with lognin, sweroside, chlorogenic cid, cffeic cid, rutin, nd gluteolin stndrds. Excellent RSDs for retention time nd pek re were otined, s shown in Tle. Column: Acclim Phenyl- (.6 50 mm, µm, P/N 07969) Moile Phse: A - 0.% formic cid queous (v/v), B - cetonitrile Grdient: - 0 min, 7% B; 0 min, 7 0% B; 6 min, 0 5% B; 6 0 min, 5% B Flow Rte:.0 ml/min Inj. Volume: 5 µl Detection: UV, 6 nm Scns t Different Column Temperture:. 5 C Peks:. lognin. 0 C. sweroside c. 5 C. chlorogenic cid d. 0 C. cffeic cid e. 5 C 5. rutin 6. gluteolin,00 mau mau e d c B e d Tle. Clculted c pek purity mtch fctors.* Minutes Figure. Chromtogrms of A) mixture of stndrds (0 μg/ml ech), nd B) Culis Lonicere smple on n Acclim Phenyl- column t different column tempertures. 5 6 A Tle. Clculted pek purity mtch fctors.* Anlyte 0% Height of Pek Front 50% Height of Pek Front 50% Height of Pek Til 0% Height of Pek Til Lognin Sweroside Chlorogenic Acid Cffeic Acid Note: * The corresponding vlue for 00% purity is 000. Tle. Reproduciility for pek retention time nd re. Anlyte Retention Time RSD Pek Are RSD Lognin Sweroside Chlorogenic Acid Cffeic cid Rutin Gluteolin

4 Clirtion linerity for the six compounds ws investigted y mking five consecutive injections of mixed stndrd prepred t seven different concentrtions,.0,.0, 5.0, 0, 0, 50, nd 00 mg/l. The externl stndrd method ws used to estlish the clirtion curve nd to quntify the mounts of the six ctive components in smples. Tle reports the dt from the clirtion s clculted y the Chromeleon CDS softwre. MDLs were clculted using the single-sided Student s test method (t the 99% confidence limit). Using seven consecutive injections of Culis Lonicere smple mixed with lognin, sweroside, chlorogenic cid, cffeic cid, rutin, nd gluteolin stndrds, the testing determined the stndrd devition vlues for clculting MDLs, lso reported in Tle. Tle. Method linerity dt nd MDLs. Anlyte Regression Eqution r Rnge (mg/l) MDL (mg/l) Lognin Y = 0.09 X Sweroside Y = X Chlorogenic Acid Y = 0.99 X Cffeic Acid Y = 0.6 X Rutin Y = X Gluteolin Y = X mau Minutes 70.0 A B C D % Column: Acclim Phenyl mm, µm Moile Phse: Acetonitrile, 0.% formic cid queous (v/v) Grdient: Acetonitrile, - 0 min, 7%; 0 min, 7 0%; 6 min, 0 5%; 6 0 min, 5% Flow Rte:.0 ml/min Inj. Volume: 5 µl Temperture: 0 C Detection: UV, 6 nm Scns:. Culis Lonicere smple. mixture of stndrds, 0 µg/ml Peks: UV spectr:. lognin. sweroside. chlorogenic cid. cffeic cid 5. rutin 6. gluteolin A. lognin stndrd (Pek ) A. Pek of smple B. sweroside stndrd (Pek ) B. Pek of smple C. chlorogenic cid stndrd (Pek ) C. Pek of smple D. cffeic cid stndrd (Pek ) D. Pek of smple A 6.6 B C 7. D % nm nm nm nm Figure. Chromtogrms nd UV spectr of ) Culis Lonicere smple, nd ) mixture of stndrds (0 µg/ml ech).

5 Smple Anlysis Chromtogrms of Culis Lonicere smple s well s the mixture of lognin, sweroside, chlorogenic cid, cffeic cid, rutin, nd gluteolin stndrds re shown in Figure. Comprison of UV spectr nd retention times llows the identifiction of lognin, sweroside, chlorogenic cid nd cffeic cid in the Culis Lonicere smple. Recoveries for ech stndrd in spiked Culis Lonicere smple rnged from 85 99%, suggesting tht the nlysis method is ccurte. The results re shown in Tle. Conclusion This work descries n HPLC method tht seline resolves six ctive components in Culis Lonicere using n Acclim Phenyl-,.6 50 mm, μm column nd 0.% formic cid/cetonitrile moile phse. This method cn e used for the qulity control of Culis Lonicere, common medicinl plnt in Chin. It is superior to the PPRC method tht mesures only two of the purported ctive components of Culis Lonicere with two seprte methods tht require long seprtion times nd hve insufficient pek resolution etween lognin nd sweroside. Tle. Anlysis results of the ctive components of Culis Lonicere References. Gou, Z.P.; Wn, D.G. Determintion of Chlorogenic Acid in Culis Lonicere y HPLC, Chin. Trditionl Ptent Med. 006, 8, 08.. Phrmcopoei Commission of the Ministry of Helth of the People s Repulic of Chin. Phrmcopoei of the People s Repulic of Chin, 9th ed.; Chin Medicl Science nd Technology Press, 00; Vol., 79.. Qin, Z.M.; Li, H.J.; Li, P.; Tng D.; Qin, S.J. Determintion of Eight Bioctive Compounds in Culis Lonicere Jponice nd Folium Lonicere Jponice y High Performnce Liquid Chromtogrphy, Chin. J. Anl. Chem. 007, 8, Qin, Z.M.; Li, H.J.; Li, P.; Chen, J.; Tng D. Simultneous Quntifiction of Seven Bioctive Components in Culis Lonicere Jponice y High Performnce Liquid Chromtogrphy, Biomed. Chromtogr. 007,, Li, H.J.; Qin, Z.M.; Li, P.; Ren, M.T.; Chen, J.; Xin, G.Z. Simultneous Determintion of Nine Bioctive Constituents of Culis Lonicere Jponice y High Performnce Liquid Chromtogrphy Coupled with Mss Spectrometry, Nturl Product Communictions 008, (5), Appliction Note 99 Anlyte Detected Added Found Recovery (%) Lognin Sweroside Chlorogenic cid Cffeic cid Rutin Not Found Gluteolin Not Found Thermo Scientific Dionex products re, designed, developed, nd mnufctured under n ISO 900 Qulity System. 0 Thermo Fisher Scientific Inc. All rights reserved. All trdemrks re the property of Thermo Fisher Scientific Inc. nd its susidiries. Specifictions, terms nd pricing re suject to chnge. Not ll products re ville in ll countries. Plese consult your locl sles representtive for detils. U.S./Cnd (87) Brzil (55) 7 50 Austri () Benelux () () 5 9 Denmrk (5) Frnce () Germny (9) Irelnd (5) Itly (9) Sweden (6) Switzerlnd () United Kingdom () Austrli (6) 90 5 Chin (85) 8 8 Indi (9) Jpn (8) Kore (8) Singpore (65) Tiwn (886) AN99_E 0/S LPN06