25 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /1, No. /,,/,+. (,+*) 205 HPLC High Performance Liquid Chromatography Quantification of Total Astaxanth

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(1) 25 . Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /1, No. /, ,*/,+. (,*+*) !%&

(2) ". 205.

(3) HPLC ´µ¶·¸ ¹NO¸ ºP»Q R S ¼½TUV ' ()*+ . High Performance Liquid Chromatography Quantification of Total Astaxanthin in Health Foods Masako Ogawara, Yoko Ino, Mikihiko Yoshida, Takashi Hishiyama and Tomoji Igarashi Japan Food Research Laboratories, 0ῌ++ῌ+* Nagayama, Tama-shi, Tokyo ,*0ῌ**,/ ῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍ Astaxanthin is a red carotenoid pigment mostly distributed among marine organisms. Due to the multifunctional physiological benefits through its potent antioxidant activity, many products are supplemented with astaxanthin in the current market place. Although the demands for quantitative verification of astaxanthin have rapidly increased, the existence of its esterified forms in the products sometimes hampers accurate determination. In this study, high performance liquid chromatography was applied to examine their versatile quantification after enzyme-hydrolysis of esterified astaxanthin from Haematococcus sp. The activity among nine enzymes was compared using the chloroform extract of Haematococcus sp. A cholesterol esterase from Pseudomonas fluorescens gave the maximum activity at the optimized reaction condition of -1 for +,* min. In the validation study, an astaxanthin ester solution was added to three health food products to obtain an average recovery with a range of 31-+*+ῌ and the residual standard deviation of ,ῌ in the concentration range of +.1ῌ2** mg/+** g sample. A repeatability test using health food tablets containing Haematococcus extract was repeated seven times to determine precision of the method, giving a residual standard deviation of ,ῌ. These results suggest that the proposed method was useful for analyzing total astaxanthin content in food supplemented with esterified astaxanthin from (Received Oct. +0, ,**3 ; Accepted Feb. /, ,*+*) Haematococcus sp. Keywords : astaxanthin, astaxanthin esters, cholesterol esterase, enzymatic hydrolysis. ῍ῌ῏ῌ῎ :

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(121) CDEDFG H /1 I H / J ,*+* / K. 210. Table ,. 30 . Screening of enzyme. Enzyme. Origin. Manufacturer. Free Astaxanthin Concentration (mg/+** g). Cholesterol esterase. Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Schizophyllum commune Escherichia coli (recombinant) Bovine pancreas. Wako Pure Chemical Sigma-Aldrich Toyobo Toyobo Kikkoman Oriental yeast. ,*.* ,*1* ,*,* +,* ,-* ,*. Lipase. Mucor miehei Candida antarctica Phycomyces nitens. Wako Pure Chemical Wako Pure Chemical Wako Pure Chemical. ,* ,* ,*. Acetone solution of Haematococcus oil was used as substrate described in Fig4 ,4 Free astaxanthin concentrations were calculated by subtracting original free astaxanthin value from measured total astaxanthin value4. AST LMN7OP) Q* 3 RST +%&UV#- WXY= Z[\ +,* ) -ῌ ῔ῐ῕ῑῒ῏῍῎ΐ ῌ 4 . *.*+* *.,* ,* - Q] . ῌgῌml / P AST ^_ HPLC `* a/#bO 4cd) - efOcdgh* 5i?j r*.3333

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(131) .~$. D"pzK-i~ A. EF ῌ .. *.*+ ῌ g ῌ ml .UXC AST àbc HPLC. Fig. 0. HPLC chromatograms of astaxanthin in Haematococcus oil during enzyme reaction. (a) : at middle stage of enzyme hydrolysis (b) : at last stage of enzyme hydrolysis AST : Ataxanthin Ester AST : Esterified-astaxanthin HPLC conditions are described in Fig. , (mobile phase A).. Table Sample Capsules Tablets Drink. Typical calibration curve for trans-astaxanthin. Fig. 1. Recovery of astaxanthin spiked to health food samples Recovery (῍). Spiked (mg/+** g). +. ,. -. .. /. 0. 1. Av. RSD (῍). 2** +/* +41. 334/ +*, 3/43. 3340 +*+ 324,. +** +*, 3/4,. 3242 +*+ 314,. +*, +*304-. 3343 +** 3141. 3340 +*+ 314/. +** +*+ 3043. +4** *430 +4+,. Table .. Inter-day repeatability of the method Astaxanthin (mg/+** g). +. ,. -. .. /. 0. 1. Av4. RSD (῍). ,2-4*. ,124.. ,2343. ,2/4.. ,214,. ,204.. ,2*4-. ,2.4.. +4.,. Tablets containing esterified-astaxanthin from Haematococcus were analyzed on three di#erent days.

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(133) AST SῌN . Table /. Astaxanthin value on di#erent HPLC conditions. AB +* CDEFG)*HIJK +0.* KLM ! S ῌ N +* $%. ! S ῌ N - NOP Q. Astaxanthin (mg/+** g) Sample. !"# ; *.**0 ῌgῌml $% !"# ; *.**, ῌgῌ ml & '()*+,-./)012 + g. R +* ml STU + K !VW XYZ & *.**0 mgῌ+** g [\ *.**0 ῌgῌml+* ml. + +** *.**0 mgῌ+** g + g + ***. G]G ^_àV)0b cde fghijklmnop\PqrsntA] & jklm'uvSTwxyzPAn{\ P A&|}~z 4à,K !V *.*, mgῌ+** g #nz&. Fig. 2. 32 . Astaxanthin oil Capsule A Capsule B Capsule C Capsule D Capsule E Capsule F Capsule G Tablet Powder A Powder B Mean. HPLC condition A*. HPLC condition B**. A/B. /*2/2243 +1-40 .+14+ .04,. +*/4. +-4,0 ,,3* -,.41 104*, ,*4,-. /*//3.4+ +0143 ..-4, .0413 +*14+-40, ,-,0 --/4* 114.. ,*4/.. +4*+ *433 +4**43. *433 *432 *431 *432 *431 *432 *432. 2-*410. 2-.433. *432. * For chromatographic conditions see Fig4 ,, mobile phase A4 ** For chromatographic conditions see Fig4 ,, mobile phase B4. LC/MS (APCI-positive mode) total ion chromatogram of /** ng astaxanthin working standard (a) ; mass spectra for peak + (b), peak , (c), and peak - (d) Peaks ; +trans-astaxanthin, ,3cis-astaxanthin, -+-cis-astaxanthin HPLC conditions are described in Fig. , (mobile phase A, *.2 ml/min)..

(134) 789ῌ: : ;<#= >?@/A. 33 . Fig. 3. 213. LC/MS (APCI-positive mode) total ion chromatogram of astaxanthin capsule containing Haematococcus extract (a) ; mass spectra for peak + (b) and peak , (c). Peaks ; +trans-astaxanthin, ,3cis-astaxanthin, -+-cis-astaxanthin HPLC conditions are described in Fig. , (mobile phase A, *.2 ml/min). Mass spectrum for peak - couldn’t be obtained because of its lower amount.. .

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