J. Fd Hyg. Sfety 20(3), 141 146 (2005) j v ù t w, b, r s β-e p w Á½ Á½ Áw Á w Á z Á t t Studies on Simultneous Determintion of nd b, Pheophorbide, nd β-crotene in Chlorell nd Spirulin Products Young J Lee, So Hee Kim, Jin-Sook Kim, Jeong A Hn, He Jeom Seo, Hyo Jeong Lim, nd Soo Young Choi 4FPVM3FHJPOBM'PPEBOE%SVH"ENJOJTUSBUJPO 3FDFJWFE.BSDI"DDFQUFE4FQUFNCFS ABSTRACT A simple nd sensitive nlysis method bsed on reverse phse (RP) HPLC with UV detector ws developed for simultneous determintion of chlorophyll nd b, pheophorbide nd β-crotene in Chlorell nd Spirulin products. For dded concentrtion (50 µg/ml) of chlorophyll nd b, pheophorbide nd β-crotene, recoveries of those were 70.3, 71.6, 60.1 nd 90.5%, respectively, with reltive stndrd devitions of 2.8, 6.0, 10.6 nd 10.4%. Limit of detection nd quntifiction hd rnges of 0.1~1.0 µg/ml nd 0.2~2.0 µg/ml, respectively. Clibrtion curve ws liner with correltion coefficient of 0.995 for chlorophyll nd b, pheophorbide nd β- crotene. Results of simultneous determintion in Chlorell nd Spirulin products were showed rnges of 121.9~543.0 µg/ml for chlorophyll, 0.6~160.0 µg/ml for chlorophyll b, 19.2~60.3 µg/ml for pheophorbide nd 383.6~1713.7 µg/ml for β-crotene, respectively. b contents in Chlorell products were detected bove 30 times level to those in Spirulin products. β-crotene contents in Spirulin products were detected 2.7 times level to those in Chlorell products. Key words: chlorophyll nd b, pheophorbide, β-crotene, Chlorell, Spirulin» t w w, 90% w» w ƒ w, e š ùkû 1). š» t w x, 68%ƒ» t z ƒ ƒ š y 2).» t 1980 t» w» w w». 1991 Food for Specific Helth Use(FOSHU) Actƒ, 1994 Dietry Supplement Helth nd Eduction Act ù 1989 (DSHEA)ƒ ƒƒ 3). z tœ ü t ƒw š, 2002» t 4), 2 ³ w» t w w» Author to whom correspondence should be ddressed. ƒ w Áe Á Á Á Áy xk Áƒœw t wš. w, xw» tœ» t x 32 t w» Á³ ƒƒ ù y w xw» Á³ x w w š rák Á w w š. p,» t t j v ù t š w w sƒ. t x 5,6) w ƒ ü ey w w y w yw w y e p t w w yw w ƒwš 7). 10) ƒœ g8), j 9) š l w yw e p w ƒƒ HPLC 141
142 :PVOH+B-FFFUBM š š ù, xw j v ù t Á³, b r s w y x w x Ÿ TLC ³ ƒƒ ƒ wš HPLC»» w y v w.,» t j v ù t, b, r s β-e p w HPLC y wš w. x w 2004 1 l 2004 10 ¾ ü y l v m j v ù t ƒƒ 4 w w. w j t 97~100% w w t, v ù t 3 90~100% w t 1 51% w t w. w Wko p w š, HPLC w w Fisher HPLC w. t t w (97%), b(95%), r s (93%) Wko t, β-crotene(95%) Sigm t w. t, b, r s β-e p t t y e wš m ƒw ƒƒ 500 µg/ml w z, x w w. Á w z m š, b, r s β-e p t 0.1, 0.2, 1, 5, 10, 50, 100, 200, 400 µg/ml, b 0.08, 0.15, 0.75, 1.5, 7.5, 15, 75, 150 µg/ml, r s 0.1, 0.5, 1.0, 5.0, 10, 50, 100 µg/ml, š β-e p 1, 2, 5, 10, 50, 100, 200 µg/ml ƒƒ w š w. j v ù t w ƒ yw š w w. w w ƒ yw 5.0~7.5 µg/ml t 11) w j m» Morrison w. z w 50 µg/ml yw t 1 ml e p w w t 100 mg ƒw, 10 ml m ƒw t ƒ w d w. e p m j v ù t w w š, 100 mg ƒƒ w 10 ml m ƒw 20 q» wš (4 C) o w ew. ew w (3,000 rpm, 5 min, 4 C)w d w z e o 10 ml m š yww w w d w. d ww ƒ w m w. 1 ml m ƒw 0.45 µm Nylon filter(technochrom, Indi) w HPLC x w. HPLC m HPLC HP1100(Agilent, USA) w š, HPLC d q, b, r s β-e p d w 440 nm d w Tble 1 12). Tble 1. HPLC nlyticl condition of chlorophyll, chlorophyll b, pheophorbide, nd β-crotene Column Flow rte Detector Injection vol. Mobile phse Grdient progrm Cpcellpk C 18 120Å, 5 µm 4.6 mm I.D. 250 mm 0.8 ml/min UV 440 nm 20 µl A-Methnol : 0.5 M Ammonium cette (80:20) B-Methnol : Acetone (70:30) Time(min) A(%) B(%) Initil 100 0 5 75 25 10 50 50 15 25 75 35 0 100 45 0 100 50 100 0 60 100 0 š š m w t RP-HPLC w Ÿ» w yw t w y w (Fig. 1). y
4UVEJFTPO4JNVMUBOFPVT%FUFSNJOBUJPOPG$IMPSPQIZMMBBOEC1IFPQIPSCJEFBBOEβ$BSPUFOFÃ 143 Fig. 1. Clibrtion curves of chlorophyll (A), chlorophyll b (B), pheophorbide (C) nd β-crotene (D)., b, r s, š β-e p š Fig. 2 ùkü. š ƒ yw ü ƒ, yw 0.995 j ùkü w w. Á w z m w ƒ ƒ yw ƒ ùkû. b β-e p š r s. Morrison w w yw β-e p w (limits of detection; LOD) 0.1~1.0 µg/ml, w (limits of quntittion; LOQ) 0.2~2.0 µg/ml ùkû. ƒ t (clibrtion rnge) Tble 2. e p w wš yw t ƒw w z Tble 2. Clibrtion rnge, detection limit, quntifiction limit of chlorophyll, chlorophyll b, pheophorbide, nd β- crotene Substnce Clibrtion rnge (µg/ml) Detection limit (µg/ml) Quntifiction limit (µg/ml) 0.2-400 0.1 0.2 b 0.2-150 0.1 0.2 Pheophorbide 1.0-100 0.5 1.0 β-crotene 2.0-200 1.0 2.0 d w s³ 73% z ùkü, β- e p 90.5% ƒ š, r s ƒ ƒ û 60.1% y. t r (reltive stndrd devition; RSD) 2.8~10.6%, w yw ƒ ƒ û t r (Tble 3). j v ù t m» t ü m š j
144 :PVOH+B-FFFUBM Tble 3. Recovery of chlorophyll, chlorophyll b, pheophorbide nd β-crotene in known concentrtion MenÛSD RSD(%) 70.3Û1.94 2.76 b 71.6Û4.32 6.03 Pheophorbide 60.1Û6.36 10.59 β-crotene 90.5Û9.47 10.46 v ù t w, b, r s β- e p HPLC w ƒ y w ƒ û w r s» w 100 mg w. wš w e p yw» sww yw, Ÿw w w. w ƒ y yw» w w š, m, k, k. m w s q w» z» šƒ 13). ù» t j t s n w w œ sw m w w 14), x j v ù t m kw w. 8 Fig. 2. HPLC profile of cetone extrct from stndrd mixture (chlorophyll, chlorophyll b, pheophorbide nd β-crotene) (A), Chlorell (B) nd Spirulin (C) products. Detection ws crried out t 440 nm. Chromtogrphic conditions re described in Tble 1.
4UVEJFTPO4JNVMUBOFPVT%FUFSNJOBUJPOPG$IMPSPQIZMMBBOEC1IFPQIPSCJEFBBOEβ$BSPUFOFà 145 xw m z HPLC/PDA w yw e p w w m w w xw y w, w y yw m w š 10). j v ù t RP-HPLC j v ù t m w HPLC j m t j m w y ù, b β-e p w ƒ j (Fig. 2). HPLC j m l ƒ yw y w ù, e š w t l ƒ vj HPLC/ PDA w t ù j m w, ƒƒ w vj y w (not shown). e p yw HPLC w 1980 z l w ù15), yw 8,10,16) w x š š. HPLC j t 232.1~543.0, b 75.8~160.0, r s 37.2~60.3, š β-e p 383.6~709.4 mg/100g ùkû. š 90~100% v ù t 121.9~503.6, b 0.6~15.5, r s 19.2~30.3 š β-e p 865.2~ 1713.7 mg/100 g ùkû. 4 j t(a~d) w 4w w t 1.6~2.3 ƒ ùkù ù, v ù t(e~h) 2~25 v ù t w j t t ƒ ùkû. w j t, b ƒ 3:1 ùkù s³ w w. b w j t s³ 374.0 mg/100 g, v ù t 10.5 mg/100 g 30 w wš ùk û, β-e p w v ù t s³ 1335.4 mg/100 g j t s³ 495.0 mg/100 g s³ w 2.7. t r s w j y. š v ù Tble 4. Comprison of the quntity of chlorophyll, chlorophyll b, pheophorbide nd β-crotene contined in cetone extrcts of Chlorell nd Spirulin products. Products b Pheophorbide β-crotene A 232.1 75.8 37.2 709.4 B 543.0 143.4 60.3 454.8 Chlorell C 304.7 104.6 40.7 383.6 D 416.1 160. 57.0 432.0 E 503.6 15.5 30.3 1427.4 F 121.9 0.6 19.6 1713.7 Spirulin G 449.8 15.5 19.2 865.2 H 281.1 10.5 0.8 707.4 51% w w H 100% w w t ƒ s³ w w w ƒ ùkû (Tble 4). j v ù t» z wš w, b r s x» tœ» j v ù t» Á³ β-e p wz v ù» Á³ ƒƒ š w.» Á³ e v w x Ÿ d w w w. ù ww v w w. b y x 85% m w z dq sw k z y w w. r s x diethyl ether, w š x w. w ƒ wš z s³ 70.3% ùkû ù» Á³ x Ÿ ƒ x» Á³ w». b ³ y x ù x e š y ƒ w. r s z 60.1% ùkû ù» 2~4.» Á³ y x Ÿ w ƒ w, x j w».
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