Simultaneous Determination of Vitamin A and E in Infant Formula by Rapid Extraction and HPLC with Photodiode Array Detection

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(1)KOREAN J. FOOD SCI. TECHNOL. Vol. 36, No. 2, pp. 189~195 (2004). ©The Korean Society of Food Science and Technology. ³ºÂ» 5 PDA-HPLCö ~ BªF % jæ" A, E~ ÿªC #÷ò*Á8ëÁnËÁÚ' Î·Fë() %\². Simultaneous Determination of Vitamin A and E in Infant Formula by Rapid Extraction and HPLC with Photodiode Array Detection Byung-Man Kwak*, Ki-Woong Lee, Jang-Hyuk Ahn, and Un-Young Kong Research and Development Center, Namyang Dairy Products Corporation Rapid and simple method was developed for simultaneous determination of vitamins A and E contents in infant formula. Vitamins A and E were extracted by PDA-HPLC with reversed phase column using organic solvent, and their contents in Certified Reference Material (CRM) and infant formula were determined and compared with results of Food Standards Codex and AOAC method for evaluation of developed method. Vitamins A and E contents in CRM determined by developed method were within certified range of standard values. Developed method has great advantages of simple and rapid sample preparation and simultaneous determination of vitamins A and E by PDA-HPLC using reversed phase column. Key words: rapid extraction, PDA-HPLC, simultaneous determination of the vitamin A and E. B. . þÏj ;ï~º O»(8-12) Þ'b ÒÏ> ® . $, jïG;(13), Ö¾ "V& Î²ö ~ &> ª O»(14) ê >Ú ®b¾, þO» Ç~ * ô ²º>º 6 ® . Mulry (15)f ® 7ö Ò~º jæ" A WîÚ¢ chloroform-ethanol-water bÏ ºÂ~ HPLC ªC~ º O»j ~&b, Qian" Sheng(16)f ò 7ö Ò ~º jæ" A, D 5 E¢ aceton-CHCl3 bÏ ºÂ~ jæ" Af E¢ reversed phase column" normal phase columnj Ï~ HPLC '' ªC~& . Rushing (17)f ÿb~ ò¢ r¢Ò &>ª 5 êû»j ÒÏ 16*~ *¾Ò ";j ö jæ" A, E¢ ºÂ ê normal phase column" HPLC¢ Ï~ jæ" Af Eö & ÿªC O»j ~&b, Barnettf Fricks(18)º Î² &>ª¢ Ï~ æÏW jæ"j ªC~& . f Îv ¢~ &>2ËöB VÞV ÏÒ»¾ Ï ÏÒ»b ;ï~& . æÏW jæ" f 'V F~ & >2Ëj &æ ®V r^ö, HPLCö ~~ ªC 1² "«b ''~ Wªj Bê'b ¦Â~² B . B Þ/ ®*O» ö ~ jæ" A~ ªCf isopropyl alcohol C ºÂ~ ç

(2) "öB ªÒ ê ;7¦ÂV ªC~, jæ" E~ ªCöº Öb «ºÂ~ Bç

(3) "ö ~ ªÒ ê ;7¦ÂV ªCj ~ê >Ú ® . >ö AOAC O»ö ~ jæ" Af E~ ªCf hexane-isopropyl alcohol « ºÂ~ Bç

(4) "ö ~ ªÒ ê spectrophotometerf UV/ VIS ¦ÂV¢ ÒÏ~ ªC .. BªFº · '·Wª ~ '·²Ëï(1) V &ö ºÚ BB ® . BªF 7~ '« ' ·Wª j ªC~ 'Wj 6 > ®ê '«. þO» ®*(2) 5 AOAC(3)ö ²B>Ú ®b, Ç îßWb þ"; 7~ &æ ^B6j Ö~V * · þO» > ® . 7 ß® jæ"f ®n; zbB Öz>V £ Ö ², _f ¶Fj Ò~&j ãÖ 2Z>V £V r^ö ïj ªC~V *~ ³ ò *¾Ò "; º >, ïj º þæ þÖ"ö & ¸f ;{ W, Ò*W 5 n;Wj áV *~ þO»ê *~¢ (4-18). BªF 7~ jæ" ïj G;~V *Bº Ç. î¦8 Obîj B ê ªC~¶ ~º jæ"j ª Ò¢ . jæ" A f E~ ãÖº ®*O»(6) 5 AOACO»(7)ö ²B>Ú ®º ©" ? r¢Ò &>ªf Ú-Ú ºÂ»j Ï~ æWB Wî¾ *ª ~ O bîj ** B ê jæ"j jWÏ ºÂ~ *Corresponding author : Byung-Man Kwak. Research and Development Center, Namyang Dairy Products Co., Ltd., 160, Bongan-ri, Janggi-myun, Gongju-Si, Chungcheongnam-do, 314-914, Republic of Korea Tel: 82-41-857-1551 Fax: 82-41-857-7933 E-mail: [email protected] 189.

(5) ®"²æ B 36 ² B 2 ^ (2004). 190. V¢B jæ" Af E~ ïj G;~V *~ ò *¾ Òf VVªCO»j '' ª~ >¯~V r^ö K" * ô ²ºB . jæ" Af E~ ªCj ÿ¢ ò *¾Òf VVªCO»b >¯ K" *j » > ®º 6 ® . öBº Vö B &¦ª~ r¢Ò &>ª» ö V Ë*~ ò *¾Ò& jò »O»bB jæ" Af E¢ ÿö ºÂ~º ³ ò *¾Ò O»j BB~ , &>2Ë 'V jæ" Af E¢ 1² "«~ ç

(6) "b ''~ jæ"j ªÒÎ ê PDA(photodiode array) ¦ÂV ÿö ¦Âb ³~ ÎN'. þO»j B~¶ .. Òò 5 O». þÒò. þ Òòº þÖ" ¦Ãj * Ã&bî(CRM: Certified Reference Material)" 7öB 6 >º retinol palmitate& FB ÚÖ NÒ~ BªF Af retinol acetate& FB >«Ö NÒ~ BªF B 2«j «~ ÒÏ~& . Ã &bî CRMf jæ" A¢ trans-retinolB 5.84Û 0.68 mg/kg F~ jæ" E¢ α-tocopherolB 271Û25 mg/ kg, δ-tocopherolB 20.19Û1.69 mg/kg, γ-tocopherolB 75.01 Û5.07 mg/kg(β-tocopherol ) F~ ®º Infant Formula SRM 1846(National Institute of Standard & Technology, Gathersburg, MD, USA)¢ ÒÏ~& . $, òB jæ" A¢ 5.4 mg/kg(retinol palmitate ;), jæ" E¢ α-tocopherolB 66 mg/kg F>ê BB BªF Af jæ" A¢ 5.4 mg/ kg(retinol acetate ;), jæ" E¢ α-tocopherolB 62 mg/kg ¢ F>ê BB BªF B¢ ÒÏ~& . ;W 5 ;ï ªCj *~ ÒÏB jæ" Af jæ" E ~ &®(RM: Reference Material)b all trans-retinol palmitatef all trans-retinol acetateº SimgaÒ(St. Louis, MO, USA) ¦8 «~&, DL-α-tocopherol, γ-tocopherol, δ-tocopherol f SupelcoÒ(Bellefonte, PA, U.S.A)öB «~ ÒÏ~& . <~ £b HPLCÏ H2O, methanol, absolute ethanol, isopropyl alcohol, methylene chlorideº MerckÒ(Darmstadt, Germany)öB HPLC grade¢ «~ ÒÏ~&b phenolphthalein, pyrogallol, 85% potassium hydroxidef anhydrous sodium sulfateº ß/£j ÒÏ~& . .B>º EASY pure system(Barnstead, Dubuque, IA, USA)ö ~~ 18.0 MΩ>& b ;BB bj ÒÏ~& . Î&²> þ Î&²> þf FVÏ jæ" Af E¢ ³~² º Â ê ''~ Wª j ÿö ;WÁ;ï~º ~. þ O»" ®*O» 5 AOAC O»j jv~& . Î&²> þj * òº NIST~ SRM 1846öB { B jæ" A ;(retinol palmitate)f jæ" E ;¢ b~ 100 mg/L ³ê B &Ï 1 mLj Î& ê Î& ~æ pf òf ÿ¢~² ¾Ò~ ;ï~& . Ö"8j Ï~ Î&~æ p~j ãÖ~ Ö"~¢ Î& Ö"~ö B ¦ ê Î&ïj V&b ²>Nj r j Ï~ ê Ö~& . (A1−A2)/A3Ü100; VöB A1f jæ" &Ïj Î&®. j r~ Ö"8, A2º jæ" &Ïj Î&~æ pf ãÖ ~ Ö"8, A3º jæ" &Ï Î&ï. ³ºÂ» ò Z²º SRM, BªF A 5 B¢ '' £ 1 gO 0.1 mgræ ;&® ~ 50 mL screw-capped extraction tubeö '' I dimethyl-sulfoxide 1 mL Î& 5 N2 gas Ïê~ capping ê, voltex mixer¢ Ï~ 1ª* ¾ b~& . bê £ 5ª* O~ ê ethanol 10 mL ~ vortex mixer¢ Ï~ 1ª* ¾ b~& . bB tube¢ SORVALL evolution RC centrifuge(Kendro, newtown, CT, USA)ö B 13,000 rpm, 10ª* öªÒ ê 7*[ Ïj ~ 0.2 µm membrane filter "~ HPLCö "«~& . ,. þ Ú~ ~ãf Nê 23Û2oC, Ûê 50Û5%öB >¯~ & . ³ºÂ»j * RÏ~ B HPLC grade~ methanolj Ï~ all trans-retinol palmitate, all trans-retinol acetate, α-tocopherol, γ-tocopherol, δ-tocopherol '' 1 mL 7 1, 5, 10 µgO >ê B~& . r¢Ò &>ª» jæ" A¢ ªC~V * ®*»~ ò *¾Òº ò SRM 1846, BªF A 5 B¢ '' £ 1 gO ;&® j ''~ .ï ¦z2¢Êö Iî . ò G;f 0.1 mgr æ G;~& . öêR 30 mL 5 10% pyrogallol-ethanol 1 mL ¢ &~ ¾ Df ê >Öz¢Ï(9 10) 3 mL¢ & ~ ~ï'Vö ¦O~ j>»(95oC) 7öB 30ª* j*z 8 . ³® ï'~ Nb ê b 30 mL¢ & .ï ªzrVö Tâ . 2¢Êº b 10 mL a ÚB 30 mL CFöH af ê .ïªzrVö If ê ¾ z. Ú b~ O~ ê b[j êê~ .ïªzrVö Tâ. . ~[ b[ö CFöH 30 mL 2² ºÂ~& . ºÂ ~ Îf öH[j Îv ~ b 10 mL 1², Ú 50 mL Ob ¾¦*î. æ£~ ¯f ï ìÚî rræ > ~ 4-5² >^~& . ªzrV 7öB bj Ïª® ªÒ CFöH[j ~ Z>Ö¾Þj & î>~ . ï2¢Êö Tâ . Ú Z>Ö¾Þj CFöH 10 mL Ob 2² a, af j ~ 2¢Êö &~& . Ïj 40oCöB 6{³»~, 6{bö isopropyl alcohol 10 mL¢ & ÏÎ ê, 0.2 µm membrane j8 " Ïj HPLCö "«~& . $ jæ" E¢ ªC~V * ®*»~ ò*¾Òº çV~ O»(jæ" A¢ ªC~V * ò *¾Ò)" ÿ¢~² ê¯~&b¾, 6{³» ê «Ï ¢ hexane 10 mL¢ & ÏÎ ê, 0.2 µm membrane j8 " Ïj HPLCö "«~& . AOAC O»~ ò*¾Òº ò SRM 1846, BªF A 5 B¢ '' £ 1 gO ;&® j ''~ .ï ¦z2¢Ê ö Iî . ò G;f 0.1 mgræ G;~& . &Ï 2¢ Êöº H2O 10 mL, 1% pyrogallol-ethanol 20 mL, 10.5 M KOH 5 mL¢ &~, ò 2¢Êöº 1% pyrogallol-ethanol 30 mL, 10.5 M KOH 5 mL¢ &~ ¾ Df ê 70oCöB 25 ª* j*z8 . ³® ï'~ .ï ªzrVö TV ''~ 2¢Ê¢ b 30 mL aÚ . . ºÂÏ hexane-methylene chloride(3 + 1, v/v) 30 mLj &~ ¾ b.

(7) ³ºÂ» 5 PDA-HPLCö ~ BªF 7 jæ" A, E~ ÿªC. ~ O~ ê b[j ªÒ H2O-ethanol(3 + 2, v/v) 30 mL¢ 3² >~ >^~& . ç 20 mL¢ ~ N2 gas ³ » ê jæ" Aº hexane-isopropyl alcohol(100+0.25, v/v) 5 mL, jæ" Eº hexane-isopropyl alcohol(99.92+0.08, v/v) 5 mL C~ HPLCö "«~& . r¢Ò &>ª»j * RÏ~ B ò 7~ jæ" A~ ïªCj * ÒÏB jæ" A &Ïf all trans-retinol palmitate $º all trans-retinol acetate ¢ j*z* retinol *~ ê µg* ~Ö~& . ¢ zêR C~ '' 1 mL 7 '' 1, 5, 10 µg F>ê C~ ÒÏ~& . $ jæ" Eº α-tocopherol, γtocopherol, δ-tocopherol¢ methanol C~ 1 mL 7 '' 1, 5, 10 µg F>ê &j~& . 88ªC ~ þO»ö ~ jæ" Af E~ ªCf HPLC (Alliance 2690 Separation Module, Waters, USA)j ÒÏ~& .

(8) "f XTerra C18 column(4.6Ü250 mm, 5 µm, Waters, USA) j ÒÏ~&b, ÿçf methanol : H2O(93 : 7, v/v, isocratic mode) bj Ï~ ªÒ~& . ¦ÂVº 474 fluorescence detector(;7¦ÂV, Waters, USA)~ excitation" emission 2Ë j '' 340, 460 nmb ;~ ªC~&b, $ photodiode array detector 996(Waters, USA)j ÒÏ~ 200-800 nm ~ º*öB scanning~ ªC~& . ®*O»b þ jæ" A~ ªCf XTerra C18 column(4.6Ü250 mm, 5 µm, Waters, USA)j Ï~ methanol : H2O(95 : 5, v/v, isocratic mode) bj Ï~&b, fluorescence detector¢ Ï~ excitation" emission 2Ëj '' 340, 460 nmb ;~ ªC~& . jæ" E~ ªCf µPorasil column(4.6Ü250 mm, 5 µm, Waters, USA), n-hexane : isopropanol(98 : 2, v/v), F³ 0.5 mL/min isocratic mode fluorescence detector¢ ÒÏ~ excitation" emission 2Ë j '' 298, 325 nm ;Î ê G;~& . AOAC O»b þ jæ" A~ ªCf µ-Porasil column (4.6Ü250 mm, 5 µm, Waters, USA), n-hexane : isopropyl alcohol (100 + 0.25, v/v, isocratic mode), spectrophotometer~ G; 2Ë 325 nm, UV/VIS detector~ G;2Ëf 336 nmöB G;~ & . jæ" E~ ªCf µ-Porasil column(4.6Ü250 mm, 5 µm, Waters, USA), n-hexane : isopropanol(99.92 + 0.08, v/v, isocratic mode), UV/VIS detectorº 280 nmöB G;~& . HPLC Ï º 4.5 µm membraneb "~ .r2êûV îV ê ÒÏ~& . , VV Ú~ ~ãf Nê 23 Û2oC, Ûê 50Û5%öB >¯~& .. Ö 5 V PDA-HPLC¢ Ï Î&²> þ Ö Î&²> þö ÒÏB NIST SRM òº FVÏ jæ " Af E¢ ³~² ºÂ ê ''~ Wª j ÿö ; WÁ;ï~º ~ þ O»" ®*O» 5 AOAC O»b ª~ Table 1ö ªCÖ"¢ ¾æÚî . jæ" A 5 E~ ïj ªCö ®ÚB ' þO»~ ² > þÖ"º þ ~ ò *¾Ò Ëj, ªCVV, ~ã ~ Nö V¢ ² N& ®j > ®Æb¾, ²~. 191. Table 1. Recoveries(%) of each methods for the determination of vitamin A and E Treatment Vitamin Vitamin A Vitamin E. Direct1). Food standards codex2). AOAC3). 94.57Û4.734) 98.66Û5.14. 94.37Û4.52 95.47Û4.09. 94.85Û5.11 95.23Û4.96. 4). 1). Photodiode array detector Florescense detector 3) UV/VIS detector 4) The values are meanÛS.D. of 3 replications. 2). Fig. 1. PDA-HPLC chromatogram of vitamin A and E.. þ öB >¯ Ö"º Table 1öB { > ®º ©" ? ³ºÂ/PDA-HPLCªCO»¾ ®*O» 5 AOAC O»~ jæ" Af E~ Î&²>N þöB ¸f ²>Nj & . Þ, jæ" E~ ãÖ ~ þO» V ® *O» 5 AOACO»ö j ² ¸² G;>îb¾, 3&æ O» Îv B FÒ Ö"¢ ¾æÚî . PDA-HPLC¢ Ï jæ" A,E ªÒ 5 ¦Â Fig. 1f þöB &®b ÒÏB 'V æÏW jæ"j XTerra C18 ç columnöB PDA detector¢ Ï~ ªC Ö" . ³ ºÂ»ö ~ C18 ç column 5 PDA detector¢ Ï~ ªC~º ãÖ ''~ jæ" Ò ;¢ & ªC > ®î . PDA detector¢ Ï~ ª C Ö" retinol, retinol acetate, δ-tocopherol, γ-tocopherol, DL-α-tocopherol, retinol palmitate~ Bb Îv ªÒ>º ©j { > ®î . $, Fig. 2öB { > ®º ©¾" ;7¦ÂV¢ ÒÏ~º ãÖöê jæ" Aº retinol, retinol acetatef retinol palmitate~ ¦ÂBB PDA detector¢ Ï Ö"f ÿ¢ ¦ÂBB¢ { > ®î ..

(9) 192. ®"²æ B 36 ² B 2 ^ (2004). Fig. 2. FLD-HPLC chromatogram of vitamin A.. Fig. 3. FLD-HPLC chromatogram for saponificated vitamin A.. ¾, ®*O» 5 AOACO»~ jæ" A ªCf ~ ³ºÂ»" Ò òö B retinol-acetate $º retinol palmitate~ ester ;¢ KOHf ÖzB pyrogallol¢ Î&~ j>»çöB j*z* Fig. 3" ? retinol ; *~>Ú ¦Â >V r^ö, òö >Ú ®º j æ" A~ ;& ÚÆ ; Ò~ºæ r > ì º 6 ® . Þ, ®*O» 5 AOACO»~ jæ" E ªCO»f. Bç

(10) "j ÒÏ~ jæ" E~ WîÚ j ''~ WW ªb ªÒ~ ¦Â . ¦ÂBBº α-tocopherol, γtocopherol, δ-tocopherol~ BB Fig. 4 (a)f ?f chromatogramj "î . ¾, öBº ~¾~ ç

(11) "b jæ" A, E¢ ÿö ªÒ~&V r^ö(Fig. 1) Fig. 4 (b)f ?f BB& Bb ¦Â >º 6 ç~ . ¯, ®*O» 5 AOACO»~ ãÖf Ò jæ" E~ ¦Â BB& B ç>. Fig. 4. FLD-HPLC chromatogram of vitamin E by normal-phase column (a) and reversed-phase column (b)..

(12) ³ºÂ» 5 PDA-HPLCö ~ BªF 7 jæ" A, E~ ÿªC. 193. Fig. 5. PDA-HPLC spectrums and 3D chromatogram for saponificated of Infant formula SRM 1846.. Fig. 6. PDA-HPLC spectrum and chromatogram of vitamin A and E.. >² ªC>º ©j { > ®î . PDA-HPLC¢ Ï G;Ö jL PDA detector¢ Ï~ Ö"¢ ªC ãÖ Fig. 5" ?. ªC &ç peak "æö 7 >~ peak ¦Â>î . Fig. 6f &bî~ 2D, 3D chromatogram" ªC&ç peak. ~ &2Ëj ¦ï Ö"¢ & . V¢B Fig. 5~ ' ' peak ¢bîªj { ê ¢ ºÂ~ jæ" A.

(13) ®"²æ B 36 ² B 2 ^ (2004). 194. Fig. 7. Extracted chormatogram of vitamin A and E. Table 2. The values of vitamin A and E in infant formula determined under various analytical method. (unit: mg/kg). Method Sample. 4). Vitamins. Food standards codex. AOAC. PDA. Direct FLD. FLD. UV/VIS. Infant formula A. Vitamin A Vitamin E2). 05.53Û0.173) 121.3Û5.640. 5.49Û0.23 120.8Û9.340. 5.48Û0.19 110.3Û3.720. 5.55Û0.21 113.2Û5.760. Infant formula B. Vitamin A Vitamin E2). 4.45Û0.55 113.7Û3.440. 4.55Û0.38 110.8Û3.440. 4.46Û0.56 105.2Û4.940. 4.57Û0.17 103.9Û3.750. SRM 18461). Vitamin A Vitamin E2). 5.62Û0.26 358.8Û15.20. 5.63Û0.25 356.9Û15.50. 5.69Û0.12 347.9Û35.80. 5.65Û0.12 348.4Û36.50. 1). Certified concentration values of SRM 1846: vitamin A (5.84Û0.68 mg/kg), vitamin E (366.2Û31.76 mg/kg) Content of vitamin E: total α-, δ-, γ-, β-tocopherol (β-tocopherol was detemined as γ-tocopherol) 3) The values are meanÛS.D of 3 replications. 4) Means in the same column with the same superscripts are not significantly different (p<0.05) 2). f E¢ ªC~& . ³ ºÂ» 5 j*z ºÂ»öB jæ " Aº 325 nmöB, jæ" Eº 290 nmöB & >2Ëj &b, jæ" Af E peak ~ ºÂ Ö"¢ ''~ chromatogramb Fig. 7ö ¾æÚî . ~ ³ºÂ»ö ~ PDA-HPLC O»" ®* 5 AOAC O»j Ï ªC Ö"¢ Table 2ö ¾æÚî . ; ïªCj ~V * ''~ þO»öB ²æF~ ç&ê> (correlation coefficient r2 value)º 1) ³ºÂ»" 2Ë¦ÂV ö ~ þ: r2 = 0.998, 2) ³ºÂ»" ;7¦ÂV¢ Ï. þ: r2 = 0.997, 3) ®*»" ;7¦ÂVö ~ þ: r2 = 0.999, 4) AOAC ºÂ»" UV/Vis ¦ÂVö ~ þ: r2 = 0.999î . Table 2öB " > ® jæ" Af E Îv ~ ³*¾Ò ê PDA detectorö ~ G;Ö"f ;7¦ÂVö ~ G;Ö"º FÒ~ ¦ÂOö ~ Nº ~ ìrj r > ® .. jæ" A~ ãÖö PDAf FLD¢ jv þöB Bª F A~ G;Ö"º '' 5.53" 5.49 mg/kg, BªF B~ G ;Ö"º '' 4.45 " 4.55 mg/kg, NIST SRM 1846~ G;Ö "º '' 5.62, 5.63 mg/kgî . jæ" E~ ãÖö PDAf FLD¢ jv þöB Bª F A~ G;Ö"º '' 121.3" 120.8 mg/kg, BªF B~ G;Ö"º '' 113.7" 110.8 mg/kg, NIST SRM 1846~ G ;Ö"º '' 358.8, 356.9 mg/kg î . ò*¾Ò O»j *Ú' þO»*~ N¢ j v~V * þöBº jæ" A~ ãÖ ' þO»*~ N º ~ ìîb¾, jæ" E~ ãÖöº ~ ³º Â»ö ~ þ Ö"& ®*O» 5 AOACO»ö j. ² ¸² G;>î . jæ" A~ ïj ªCö ®ÚB þO»*~ jvÖ" 5 ²> þ Ö"º ' þ ~ ò *¾Ò Ëj, ªCVV, ~ã ~ Nö V¢ ² N& ®j > ®Æb¾, .

(14) ³ºÂ» 5 PDA-HPLCö ~ BªF 7 jæ" A, E~ ÿªC. ²~ þ öB >¯ þO», ®*O» 5 AOAC O»*~ jvÖ"º BªF A~ ãÖ '' 5.53, 5.48, 5.55 mg/kg, BªF B~ ãÖ '' 4.45, 4.46, 4.57 mg/kg, NIST SRM 1846~ ãÖº '' 5.62, 5.69, 5.65 mg/kgb FÒ Ö "¢ ¾æÚî . Þ, jæ" E~ ãÖöº ~ þO», ®*O » 5 AOACO»*~ N¢ jv þöB BªF A~ G;Ö"º '' 121.3, 110.3, 113.2 mg/kg, BªF B~ G; Ö"º '' 113.7, 105.2, 103.9 mg/kg, NIST SRM 1846~ G ;Ö"º '' 358.8, 347.9, 348.4 mg/kgb ~ þO » ² ¸² G;>îb¾ G;òöB ËB ¢W". þJN¢ J~ FÒ Ö"& ÖÂ>îrj r > ®. (p<0.05). BÃ&bî NIST SRM 1846j Ï~ ~. þO»ö ~~ G; Ö"º jæ" A& 5.62 mg/kgb B Ã8 5.84Û0.68 mg/kg~ º* Ú G; >îb, jæ" E& 358.8 mg/kgbB Ã8 366.2Û31.76 mg/kg~ º* Ú G; >î . V¢B ~ ³ºÂ» ®*O» 5 AOACO» " G;8ö ² N& ®Vº ~æò BªFö Ò~º jæ" Af E~ ïj G;~V *~ 'V 2&æ~ jæ" þO»j ÒÏ~æ p ~ þO»j ÒÏ ~ ³~ ÎN' ªCj >¯~ºö ÏF > ®j ©b ÒòB .. º. £. öBº BªF 7ö '·;z¢ * Î&~º j æ" Af E~ ïG;j * 'V þO»j ÒÏ~ æ p 2&æ jæ"j ÿö ªC~º ³ªC»j *f ? >¯~& . jæ" Af E¢ FVÏ ÿö ³~ ² ºÂ~ ç

(15) "" PDA-HPLC¢ Ï~ ''~ Wª b Îv ªÒ ê ÿö ¦Â~º O»j ÒÏ~& . B&Ãbî 5 BªF¢ ò ÒÏ~ ~. þO»ö ~ G;B 8j ®*O» 5 AOACO»ö ~ G;8" jv Ö" FÒ G;Ö"¢ áj > ®î . $ ~ þO»ö ~~ >¯ BÃ&bî 7~ jæ" Af E~ ïG; Ö"º ÃB &8Ú~ Ö "¢ "î . V¢B, jæ" A $º E¢ ;z ªF, F ~ ªö FB® 7öB jæ" Af E~ ïj G; ~¶ r ;B Ëjf Kb ''~ 2&æ þO» j >¯~V& Ú[¾ *» jº ãÖ, öB >¯ þO»" ? ò *¾Ò¢ *~ ³~² > ¯ öò jî¢ ç

(16) "" PDA-HPLCö ~ jæ" Af E¢ ÿö ªCb ÎN' ªCj ê¯ > ® j ©b ÒòB .. ^. 195. ò. 1. The Korean Nutrition Society. Recommended dietary allowances for Koreans. 7th rev. The Korea Nutrition Society, Seoul, Korea (2001) 2. Korea Food and Drug Administration. Food Standards Codex. Korean Foods Industry Association, Seoul, Korea (2003) 3. AOAC. Official Methods of Analysis of AOAC International. 17th ed. Association of Official Analytical Chemists, Gaithersbrug, MD, USA (2000) 4. Wilhelm F. Vitamins. Walter de Gruyter, New York, NY, USA (1988) 5. William Jr. OL, Don MH, Thomas WH, James IM, Edna RY, Ronald RE, Abdel-Gawad MS. Vitamin A and vitamin E content of infant formulas produced in the United States. J. Assoc. Off. Anal. Chem. 68: 509-511 (1985) 6. Korea Food and Drug Administration. Food Code, pp. 894-897, 912-915. Korean Foods Industry Association, Seoul, Korea (2003) 7. AOAC. Official Methods of Analysis of AOAC International. 17th ed. Ch. 50, pp. 1-5. Association of Official Analytical Chemists, Gaithersbrug, MD, USA (2000) 8. Alphonse FW, Lucia AM. Improved reverse phase liquid chromatographic determination of vitamins A and D in fortified milk. J. Assoc. Off. Anal. Chem. 67: 62-64 (1984) 9. Harvey EI. Simplified sapnification procedure for the routine determination of total vitamin E in dairy products, foods and tissues by high-performance liquid chromatography. Analyst. 113: 1217-1221 (1988) 10. Søren KJ. Retinol determination in milk by HPLC and fluorescence detection. J. Dairy Res. 61: 233-240 (1994) 11. Soledad AH, Sonia NR, M Teresa VN, Abel MF. Determination of vitamins A and E in infant milk formula by high-performance liquid chromatography. J. Chromatogr. A 778: 243-247 (1997) 12. Jonathan WD, Karlene RS. Determination of Vitamins A (Retinol) and E (alpha-Tocopherol) in Foods by Liquid Chromatography : Collaborative. J. AOAC Int. 85: 424-434 (2002) 13. AOAC. Official Methods of Analysis of AOAC International. 17th ed. Ch. 45. Association of Official Analytical Chemists, Gaithersbrug, MD, USA (2000) 14. Kim PZ, Kim CH. A strudy on the simultaneous analysis of fatsoluble vitamins in food stuffs and vitamin products by high performance liquid chromatography. Korean J. Chem. Soc. 33: 46-54 (1989) 15. Mary CM, Ronal HS, James RK. Isomerization of retinyl palmitate using conventional lipid extraction solvents. J. Assoc. Off. Anal. Chem. 66: 746-750 (1983) 16. Qian H, Sheng M. Simultaneous determination of fat-soluble vitamins A, D and E and pro-vitamin D2 in animal feeds by onestep extraction and high-performance liquid chromatography analysis. J. Chromatogr. A 825: 127-133 (1998) 17. Larry GR, Willie MC, Harold Jr. CT. Simultaneous analysis of vitamins A and E in rodent feed by high-pressure liquid chromatography. J. Agric. Food. Chem. 39: 296-299 (1991) 18. Stephen AB, Leroy WF. Simultaneous determination of vitamin A acetate, vitamin D2 and vitamin E acetate in multivitamin mineral tablets by high performance liquid chromatography with coupled columns. Anal. Chem. 51: 641-645 (1979) (2003j 10ú 8¢ %>; 2004j 4ú 12¢ j).

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