HPLC-MS Analysis of Phenols in Longan (Dimocarpus longan Lour ：在龍眼酚的高效液相色譜-質(zhì)譜分析（龍眼

1、HPLC-MS Analysis of Phenols in Longan (Dimocarpus longan Lour.) PeelSomkit Jaitrong1, John A. Manthey2, Nithiya Rattanapanone1 and Elizabeth A. Baldwin21Chiang Mai University, Chiang Mai, Thailand 2USDA/ARS Citrus and Subtropical Products Laboratory, Winter Haven, FL Biology of Longan FruitFamily: S

2、apindaceaeScientific names: Euphoria longana Lam. Euphoria longan Strend. Nephilium longana Camb. Dimocarpus longan Lour. (Batten, 1986; Choo and Ketsa 1991)Somatic chromosomes: 2n = 20 (IBPGR, 1986).Fruit Characteristics Non-climacteric fruit Harvesting time: when skin become yellowish brown and th

3、eir flesh reaches optimal eating quality0.05.010.015.020.0Total phenolic content (mgGAE/g FW)Reverse relationship between total phenolic content and specific activity of PPOTotal phenolic content (mg GAE/g FW)Specific activity of PPO (unit/mg protein)0500010000150002000025000300000681114Days after s

4、torageSpecific activity of PPO (unit/mg protein)0681114Days after storageObjectives To evaluate the main classes of phenols in longan peel. To evaluate the changes in phenolic compounds that occur during the chilling injury response. Freeze-dried longan peelPeel extracted solutionFractionation of th

5、e phenols by size-exclusion P2 (Biogel) chromatographyEllagic acid 80% aqueous methanolUnknownsFlavonoids 1 (early eluting)Flavonoids 2 (later eluting)Flow chart of FractionationHPLC of P2 Column Fractions Illustrating the 4 Main Classes of Phenols in Longan Peelmin1015202530mAU0100200300400 DAD1 B,

6、 Sig=320,4 Ref=550,20 (ALLTECHMR21_609.D) 6.340 7.059 9.109 10.439 11.735 13.142 13.925 15.448 15.723 17.728 18.541 19.766 20.695 22.108 22.742 23.305 23.602 24.067 25.063 25.780 26.179 26.508 27.006 27.312 27.657 27.961 28.402 28.803 29.446 30.115 30.664 31.168 31.525 31.862 32.059 33.059 33.371 33

7、.696 33.945 34.390min1015202530mAU20406080100120140 DAD1 B, Sig=320,4 Ref=550,20 (ALLTECHMR21_613.D) 6.037 6.833 7.167 7.535 8.426 9.059 9.736 10.634 11.883 13.072 14.078 14.466 16.032 17.262 17.666 19.175 21.062 23.218 25.439 26.544 27.111 27.364 27.756 28.040 28.567 29.035 29.752 30.120 33.031min1

8、015202530mAU020406080 DAD1 B, Sig=320,4 Ref=550,20 (ALLTECHMR21_611.D) 8.797 16.153 18.677 19.589 21.134 21.835 24.405 24.685 25.622 26.102 26.663 27.368 28.827Fraction # 27Fraction # 32Fraction # 54Flavonoids glycosideEllagic acids (EA)UnknownUnknownEAEAEAQQQQKQKQuercetin (Q)Kaempferol (K)UV/MS of

9、Ellagic acid conjugates100150200250300350400450500550600650700750800m/z0100%0100%303129121147 153177275219201257247285304611465305435345407355369477595577533519545757658649664741729686779795 799210254357M-H+Rhamnose (146)Rhamnose (146)Glucose (162)Ellagic acidUV/MS of Quercetin Glycosides0100%0100%6

10、111951011511431732392223012834653044493253454263693883994665195125255595686106126136336956657276977877467547932102553540100%0100%0100%0100%303195146129101151239217301287304449359317400384431521479501667609543577631 661692706781741752799Mar 20 2006 40 777 (26.008)2102563481951011511431733012392172834

11、65304327 345363388463415 432466487595518549591661611658635717687779731 749792210255354Mar 20 2006 40 186 (21.313)303303M-H+M-H+M-H+glucoseglucoserhamnoserhamnoseQuercetin+rhamnoseQurcetin+glucoseQuercetin+glucose+rhamnoseUV/MS of Kaempferol Glycosides100150200250300350400450500550600650700750800m/z0

12、100%0100%0100%0100%287239195133101151173217283419288289383327 344 365393420441493457647609503579513574551645677757722709767798Mar 20 2006 40 810 (27.108)x2210266345Mar 20 2006 40 212 (24.303)287239195101133 151177227283449288289373327355388423432450471692533487502541587570675665635626789709758745799

13、Mar 20 2006 40 726 (24.308)x2210265345M-H+M-H+glucosexyloseKaempferol+xyloseKaempferol+glucoseHPLC of P2 Column Fractions Illustrating the 4 Main Classes of Phenols in Longan Peelmin1015202530mAU0100200300400 DAD1 B, Sig=320,4 Ref=550,20 (ALLTECHMR21_609.D) 6.340 7.059 9.109 10.439 11.735 13.142 13.

14、925 15.448 15.723 17.728 18.541 19.766 20.695 22.108 22.742 23.305 23.602 24.067 25.063 25.780 26.179 26.508 27.006 27.312 27.657 27.961 28.402 28.803 29.446 30.115 30.664 31.168 31.525 31.862 32.059 33.059 33.371 33.696 33.945 34.390min1015202530mAU20406080100120140 DAD1 B, Sig=320,4 Ref=550,20 (AL

15、LTECHMR21_613.D) 6.037 6.833 7.167 7.535 8.426 9.059 9.736 10.634 11.883 13.072 14.078 14.466 16.032 17.262 17.666 19.175 21.062 23.218 25.439 26.544 27.111 27.364 27.756 28.040 28.567 29.035 29.752 30.120 33.031min1015202530mAU020406080 DAD1 B, Sig=320,4 Ref=550,20 (ALLTECHMR21_611.D) 8.797 16.153

16、18.677 19.589 21.134 21.835 24.405 24.685 25.622 26.102 26.663 27.368 28.827Fraction # 27Fraction # 32Fraction # 54Flavonoids glycosideEllagic acids (EA)UnknownUnknownEAEAEAQQQQKQKQuercetin (Q)Kaempferol (K)Flow chart of isolation of UnknownsTook fraction contain unknownRun on prep TLCPrep HPLCGot m

17、ain spot on TLCRun on LCMS2735468910UV and MS of interesting unknown compounds in longan peelD600 # 5D600 # 6D600 # 7100150200250300350400450500550600650700750800m/z0100%0100%0100%0100%0100%0100%3: Scan ES+ 177145137117175697201373355325321305202231277265535517499469375407465429694675657557627573609

18、591698713729775787799Apr 3 2006 18 319 (36.647)2: Scan ES+ 1771371191451613552013252192313053012493376973733575353745174994874694513854336926756576275915735536096987147317757887991: Scan ES+ 3551771371311623252191792312493093033433733566925355173744874434253854796756576275365915736096976987147597307

19、91 7974: Diode Array 2103272874: Diode Array 2103272874: Diode Array 210326287Glucose(162) Glucose(162) UnknownHydroxycinnamates3600320028002400200018001600140012001000800600cm-1%T 28462917335717001626159915081451125611241070102680136003200280024002000180016001400120010008006004045505560657075808590

20、95cm-1%T 3386292528531717162816031515127011571075893713570ElutiontimeUVMS fragmentsPossible structure14.80+),303 M-H , 465 (+glu611(+rha), 757 (+rha)Ellagic acid+glucose+ 2 rhamnose15.08303 M-H+, 435, 611, 757Ellagic acid-pentose conjugate20.37303 M-H+, 465 (+glu),611 (+rha)Quercetin+ glucose+ rhamn

21、ose21.38303 M-H+, 465(+glu ),595, 741Quercetin+glucose25.90303M-H+, 449(+rha)Quercetin+rhamnose24.28287 M-H+, 449 (+glu)Kaempferol+glucose27.14287 M-H+, 419Kaempferol-pentose conjugate26.92177, 373, 535, 697 ? (UVabsorbance 285, 326 nm)137, 177, 327, 355, 535, 697? (UV absorbance 284, 326 nm)27.4813

22、7, 177, 337, 451, 535, 69227.25? (UV absorbance 284, 325 )nmMain class of phenolics in longan peelObjectives To evaluate the main classes of phenols in longan peel. To evaluate the changes in phenolic compounds that occur during the chilling injury response.Hydrolysis 5 mL sample solution +5 mL 2N H

23、Cl (v/v) Heat at 100oC, 1 hNeutralized with KOH Concentrate by SpeedVac concentrator for 1h (to remove MeOH)Run sample through Sep-Pak C18Run on HPLC-MSEluted in MeOHThe changes of quercetin and kaempferol in longan peel stored at 5oC for 14 days0.26 mg/g DW0.06 mg/ g DWFreshAfter 14 daysQuercetin 0.26 mg/gKaempferol 0.06 mg/gPea