Analysis of Total Aflatoxins in Spices and Dried Fruits
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1 J. Fd Hyg. Safety Vol. 5, No., pp. 65~7 (00),QWTPCN QH (QQF *[IKGPG CPF 5CHGV[ Available online at Ì Ý Œ Î Ä æö  ÂÞ Ä ý  µý* t t sƒ t wsƒ t t w t t t t Analysis of Total Aflatoxins in Spices and Dried Fruits Young-Woon Kang, Tae-Yong Cho, Hee-Ra Park, Keum-Soon Oh, and Dong-Sul Kim* Food Contaminants Division, Food Safety Evaluation Department, Seoul, Korea Hazardous Substances Analysis Division, Daegu Regional KFDA, Daegu, Korea Food Import Division, Korea Food & Drug Administration, Seoul, Korea (Received December 5, 009/Revised February 3, 00/Accepted February 4, 00) ABSTRACT - We used fluorescence detector to analyse total aflatoxins (G) with TFA (Trifluoroacetic acid) derivation method and PHRED (Photochemical reactor enhanced detection) method. PHRED method was superior in reproduction and convenience, but TFA derivation method was superior in selectivity and sensitivity. The recovery rate of aflatoxin B were more than 80%, and G was more than 70%. The detection limit of B, B and G were respectively 0.05, 0.05, 0. and 0. µg/kg. Confirmed method was used to analyse total aflatoxins in total 36 items as 9 kinds 37 dried fruits and 0 kinds 79 spices. By the result, Aflatoxins were detected in 7 dried fruits (9.7%) and in 87 spices (48.6%). Key words : Aflatoxin, spices, dried fruit õ Ê v m w q Aspergillus flavus Asp. parasiticus w w ). v m B, M M v m B ƒ w. v m M v m B w ü. t,, t. x 960 q g sw 00,000 e öƒ s š,» ³ w e w Turkey X disease *Correspondence to: Dong-Sul Kim, Food Contaminants Division, 94 Tongil-ro, Eunpyung-gu, Seoul, -704, Korea Tel : , Fax : dongsul@korea.kr š. z 96 g q A. flavus l w v m w š z ƒ w. v m ü cytochrome P-450 z w y xk (aflatoxicol) v m M, Q, P y š z s v m M, M. wr v m cytochrome P-450 z w aflatoxicol v m B-8,9-epoxide DNA ww j š ). x v m B ùkû DNA v m B-DNA ƒ (aflatoxin B-DNA adducts) w ùkû. Bx y w v m w w y w š3,4), m 987 (International Agency for Research on Cancer, IARC) p m y w Group w 5)
2 66 Young-Woon Kang, Tae-Yong Cho, Hee-Ra Park, Keum-Soon Oh, and Dong-Sul Kim w w v m yw w ƒ š š v m M w š šw 6). Bx v m x š šw. B x û š šwš 7). v m w w t ƒœ w»» t w t p ƒ w v m k w. Ì Ý ßá Ì v m w w xw, l mw w š, w t ³ yw - 0 C þ k w o w. Ý v m B G t t Sigma-Aldrich Inc.(St. Louis, USA) w w. m p k HPLC (Merck Co., USA) w, yùp (Wako Co., Japan) Trifluoroacetic acid(tfa) (Sigma Co., USA) p w. Ç Ý æö é v m e AflaTest (Vicam Co., USA) ey e (Immunoaffinity column) w, j m v Shosedo Nanospace SI-, x Ÿ» 303 Fluor Detector (Shiseido Co., Tokyo, Japan) w. w j m v f xÿ» ew Photochemical Reactor Enhanced Detection (PHRED) 54 nm v (50V, 50Hz, 8Watt) ƒ š w xÿ ù n w g PTFE (Polytetrafluoroethylene) ¼ 5 m, ü 0.5 mm (AURA Industries Inc., New York, USA) w. v m B G m ƒ ƒ 00 µg/ml w v m yw t wš m p.0~50 ng/ml ƒ w v m ywt wš w t w w. Ä æö v m 70% k w z wš v m p ww monocolonal antibody š gel w k ey e w z» w v m B G trifluoroacetic acid (TFA) y (hydration) k xÿ» w š, TFA y PHRED w w w. w 5 g ³» š % yùp w 70% k 00 ml 5 š ³ yw z (Whatman No.4)w. ƒ z w. 0 ml 00 ml v j wš 0% Tween 0 30 ml ƒw k. z (Whatman GF/A)w w. 0 ml ey f (Immunoaffinity column) w m k. 0 ml w š m p 3mL k. Ð m TFA w y 50 C o wš p v 0. ml ƒw œ 5 e k z m p Á y (:8, v/v) 0.8 ml ƒw x w. PHRED w y 50 C wš» m o p ml ƒw x w. HPLC v m w xÿ»ƒ HPLC wš f Shiseido UG-0(4.6 mm 50 mm, 0.5 µm) w. TFA PHRED y HPLC Table. y w v m ywt ƒ 5 ng/g ƒw z w z mw.
3 Analysis of Total Aflatoxins in Spices and Dried Fruits 67 Table. Comparison of operating conditions of HPLC for aflatoxins derivatives by TFA and PHRED Item TFA PHRED Instrument Shiseido Nanospace SI-0 Shiseido Nanospace SI-0 Detector Fluorescence Ex. 360 nm/em. 450 nm Fluorescence Ex. 365 nm/em. 435 nm Column Shiseido UG-0 (4.6 mm 50 mm, 0.5 µm) Shiseido UG-0 (4.6 mm 50 mm, 0.5 µm) Mobile phase 5% Acetonitrile Acetonitrile : Methanol : Water (5:30:55) Flow rate.0 ml/min.0 ml/min Fig.. Calibration curve of aflatoxin B and G by HPLC-FLD (TFA). Ä á Ð ŠŒ Ý á TFA v m w TFA v m yw t 0., 0.5,.0,.0, ng/ml PHRED.0,.0, 5.0, 0.0, ng/ml ƒƒ w HPLC w. TFA (R )ƒ v m B G , , , ùkûš PHRED w v m B G.0000, 0.999,.0000,.0000 yw ùkü (Fig., Fig. ). á m v m z» w t z d w ù v m B œ, z z 80% ùkü ù G 70% w z ùkü mw, Immunoaffinity column 0 ml 0 ml ƒw w k ( 3%) immunoaffinity column w G ƒ w. k» w 0 ml 30 ml ƒw ( k 7%) w, t wš 70% z ùkü. w. y, z, v m z 60% w z w v m ww q. z j» w 8) O'Riordan % Tween 0 w w z ùkü. w œ p z ƒ
4 68 Young-Woon Kang, Tae-Yong Cho, Hee-Ra Park, Keum-Soon Oh, and Dong-Sul Kim Fig.. Calibration curve of aflatoxin B and G by HPLC-FLD (PHRED). Table. Recoveries of aflatoxins in various foods (n = 3) Item Recovery(%) ± Standard deviation B B G G Blank 98.9 ± ± ± ± 5. Dried mango 8.4 ± ± ± ±.4 Dried figs 83.7 ± ± ± ± 5.3 Dried berries 8. ± ± ± ± 3.0 Dried apricot 86. ± ± ± ± 4.5 Dried prune 86. ± ± ± ± 5.4 Dried kiwi 85. ± ± ± ±.6 Dried papaya 84.0 ± ± ± ±.9 Raisins 86.3 ± ± ± ± 3.5 Dried persimmon 79.3 ± ± ± ± 3. Red pepper 85.7 ± ± ± ± 3.3 Red pepper powder 83.9 ± ± ± ±.0 Shredded redpepper 9.0 ± ± ± ± 3.6 Paprika powder 8. ± ± ± ± 3.0 Spice formula(dadaeki) 85.0 ± ± ± ±.9 Ginger 8. ± ±.4 9. ± ± 4.8 Turmeric powder 8.5 ± ± ± ± 5. Curry powder 80. ± ± ± ± 4.6 Nutmeg 80. ± ± ± ± 3.3 Pepper 80.7 ± ± ± ±.4 ww z w. Table» ü w z t z ùkü v m B G 80% 70% z ùkü. á j j HPLC/FLD w w (LOD) signal/ noise(s/n) ƒ 3 w š w (LOQ) S/N ƒ 5 w w. TFA y v m B G w ƒ
5 Analysis of Total Aflatoxins in Spices and Dried Fruits 69 Table 3. Detection limit and quantitation limit of aflatoxins by HPLC/FLD Analyte TFA PHRED LOD(µg/kg) LOQ(µg/kg) LOD(µg/kg) LOQ(µg/kg) Aflatoxin B Aflatoxin B Aflatoxin G Aflatoxin G Fig. 3. Comparison of chromatograms of derivatized flatoxins standard by TFA (a) and PHRED (b). Fig. 4. Chromatogram with fluorescence detection of aflatoxin B and G, using photochemical reactor with light off and light on. ƒ 0.05, 0.05, 0.0, 0.0 µg/kg ùkûš w ƒƒ 0., 0., 0.4, 0. µg/kg ùkû, PHRED y B G w ƒƒ 0., 0., 0.3, 0. µg/kg ùkûš w ƒƒ 0.4, 0., 0.6, 0.4 µg/kg ùkû (Table 3). w TFA y w ƒ 5µg/ kg š (006) š w ùk ü ù v m w ƒ B G Table 4. Repeatability of detection of aflatoxins by TFA and PHRED Relative standard deviation(rsd, %) Aflatoxin B Aflatoxin B Aflatoxin G Aflatoxin G TFA PHRED G 0.0 š w 9) Cho š w ùkü. PHRED y TFA y w vj ƒ ûš vj s w w ƒ TFA y w ùkû. w S/N ƒ 5 w w S/N ƒ 0 w ƒ ù S/N ƒ 5 ƒ w w w. TFA Ð m PHRED Ð m v m UV/VIS» w ƒ û w ƒ 0 µg/kg k f ƒ w xÿ» w. ù v m B G xÿ» ƒ wù B G HPLC w xÿ (quenching) y xÿ ƒ w. w Pre column TFA y w, Post column»yw y g w. w Post column PHRED w Ÿyw w» w w wš w TFA y PHRED mw. PHRED ƒ ù vj ù resolution TFA yƒ w (Fig. 3). PHRED w xÿ y (54 nm) w B G w ù B Gƒ ƒƒ xÿ B a G a y. Fig. 4 PHRED g Ë j m ù kü. PHRED w v m ƒ j TFA y yw v f xÿ» e ew w. y x PHREDƒ yw TFA y w w ùkû (Table 4). PHRED y w v m TFA y w û ù vj e ƒ TFA y w TFA y x y x ƒ w.
6 70 Young-Woon Kang, Tae-Yong Cho, Hee-Ra Park, Keum-Soon Oh, and Dong-Sul Kim Table 5. Incidence (positive/analysed samples) and range of aflatoxins levels in dried fruits Range of AFs levels (µg/kg) Incidence Type of food (Average) No. % AFB AFB AFG AFG AFs Dried mango 0/6 0.0 Dried figs 3/ (0.00) (0.09) (0.039) Dried berries 0/6 0.0 Dried apricot 7/ Dried prune 6/ (0.068) -0.9 (0.038) (0.5) (0.068) (0.89) Dried kiwi 0/5 0.0 Dried papaya 0/5 0.0 Raisins 0/ (0.065) Dried persimmon 3/5 0.0 Total 7/ (0.09) (0.065) (0.09) æöšœ Õ yw k» k w þ w š» w t ù t w» w. k wš» w yy w» w (9.7%) v m (Table 5). š,», j qq v m xk m ƒ y û q v m q., s v m š 0.84, µg/kg ƒƒ w» d. y v m ƒ w š t 007 RASFF(Rapid Alert for Food and Feed) v m w (53%) ƒ y lj t 0). ù 0 3 (5%) v m š µg/kg. œ 5 3 (0%) v m G š µg/kg. v m s³ 0.3 µg/kg w w ù w t,, s, y œ ƒ v w ƒ. k w v m ƒ»ù ƒ û w q w. w % q ƒ w š 4% v m. š, š ƒ 79 w v m w. š, š ƒ, š, w t, t. ƒ ( v m µg/kg, s³.58 µg/kg) (Nutmeg) ù w» w w š xw q» x v m B 0 µg/kg w ³ wš, w v m B 5µg/kg w, v m 0 µg/kg w š t w» v w. e v m s³.0 µg/kg, š 5.4 µg/kgƒ š e ƒ y s³ 0.5 µg/kg, š.3 µg/kg wì ƒ w q. š w š ƒ qv e ƒƒ v m š 5.9 µg/kg(s³.0 µg/kg),.3 µg/ kg(s³ 0.5 µg/kg) ù š, š» (s³ µg/kg w) (Table 6). w v m ƒ v w t» š ƒ w, e, y qv e q.
7 Analysis of Total Aflatoxins in Spices and Dried Fruits 7 Table 6. Incidence(positive/analysed samples) and range of aflatoxins levels in spices Type of food Range of AFs levels (µg/kg) Incidence Minimum - Maximum (Average) No. % AFB AFB AFG AFG AFs Red pepper /5 6.7 Red pepper powder 0/ Shredded red pepper Paprika powder 3/ Spice formula (Dadaeki) Š Ê (0.73) w v m 70% k w f k w immunoaffinity column w š y TFA y w PHRED y w š xÿ» w., TFA PHRED w š w ùk ü. TFA PHRED w vj ƒ w TFAƒ û ùkü. ù, PHRED w v m yw y r w š, wvjƒ TFA w y w w.» x w v m z mw, v m G z û w immunoaffinity column k 3% G f š w k 7%ƒ w š, y,, z z yy w % Tween 0 w z w. x z d w, v m B G 80% ùkü š G 70% ùkü (0.046) -.98 (0.9) (0.03) (0.003) (0.03) (.00) 0/6 0.0 /7.8 Ginger 5/ Turmeric powder / Curry powder 4/ Nutmeg 0/ Pepper 3/5 5.0 Others 8/ Total 87/ (0.95) (0.09) (0.08) (0.) (0.757) (.849) (0.049) (0.079) (0.09) -.4 (0.79) (0.03) (0.09) (0.080) (0.55) (0.0) (0.49) (0.4) (0.04) (0.86) (0.09) (0.468) (0.06) (0.088) (0.030) (0.059) (0.030) (0.060) ) -.38 (0.479) (0.09) (0.9) -.99 (0.537) (.033) (.579) (0.4) (0.363) (9.7%) v m. xk m ƒ y û q š,», j qq v m š, w, s v m š 0.84, µg/kg ƒƒ. v m ƒ š š y 0 3 (5%) v m š µg/kg. œ 5 3 (0%) v m G š µg/kg. w (48.6%) v m. ƒ ( v m µg/kg, s³.58 µg/kg) (Nutmeg) ù w» w w š xw q» x v m B 0 µg/kg w ³ w š, w v m B 5µg/kg w, v m 0 µg/kg w š t w» v w. e v m s ³.0 µg/kg, š 5.4 µg/kgƒ š e ƒ y s³ 0.5 µg/kg, š.3 µg/kg
8 7 Young-Woon Kang, Tae-Yong Cho, Hee-Ra Park, Keum-Soon Oh, and Dong-Sul Kim wì ƒ w q. š w š ƒ qv e ƒƒ v m š 5.9 µg/kg(s³.0 µg/kg),.3 µg/kg(s³ 0.5 µg/kg) ù š, š» (s³ µg/kg w). v m w l ey e (immunoaffinity column) w wš p v (TFA, trifluoroacetic acid) Ÿ y xk PHRED (Photochemical reactor enhanced detection) mw w. x r PHRED w ù w TFA w. y x v m B 80% 70% z š, v m B G ƒƒ 0.05, 0.05, µg/kg w ùkü. y x w v m w k w w w., š (9.7%) v m, w (48.6%) v m. Ù G.N. : Dietary aflatoxins and human liver cancer. IV. Incidence of primary liver cancer in two municipal populations of Thailand., Food and Cosmetics Toxicology, 0: 7-79, Sun TT, Chu YY. Carcinogenesis and prevention strategy of liver cancer in areas of prevalence. J Cell Physiol., 3: 39-44, Peers, F. Gosch X, Kaldor J, Linsell C. A., Pluijmen M. : Aflatoxin exposure, hepatitis B virus infection and liver cancer in Swaziland, Int J Cancer, 39: , International Agency for Research on Cancer (IARC) : Overall evaluations of carcinogenicity: An updating of IARC monographs volumes to 4. IARC Monogr Eval Carcinog Risks Hum, Suppl 7: -440, International Agency for Research on Cancer (IARC). Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC Monogr Eval Carcinog Risks Hum, 56: -599, Wang J.S.roopman JD. : DNA damage by mycotoxins. Mutat Res., 44: 67-8, O'Riordan M., M. G. Wilkinson : A survey of the incidence and level of aflatoxin contamination in a range of imported spices preparations on the Irish retail market, Food Chemistry, 07: , Cho, S. H., C. H. Lee, M. R. Jang, Y. W. Son, S. M. Lee, I. S. Choi, S. H. Kim, D. B. Kim : Aflatoxin contamination in spices and processed spice products commercialized in Korea, Food Chemistry, 07: 83-88, European Commission : The Rapid Alert System for Food and Feed (RASFF) Annual Report, p5, Peers, F.G. and Linsell, C.A. : Dietary aflatoxins and human primary liver cancer., Ann. Nutr. Aliment., 3: , Shank, R.C.hamarapravati, N.ordon, J.E., and Wogan,
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