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. /,,*/,+. (,*+*) 205 HPLC 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, * Nagayama, Tama-shi, Tokyo,*0 **,/ 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 -+*+ and the residual standard deviation of, in the concentration range of +. 12** 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,. These results suggest that the proposed method was useful for analyzing total astaxanthin content in food supplemented with esterified astaxanthin from Haematococcus sp. (Received Oct. +0,,**3 ; Accepted Feb. /,,*+*) Keywords : astaxanthin, astaxanthin esters, cholesterol esterase, enzymatic hydrolysis : AST, Fig. + AST HPLC HPLC LCMS AST AST AST AST HPLC AST AST AST / HPLC AST HPLC +. 0 AST,*0 **,/ * Corresponding author ogawaram@jfrl.or.jp AST 1

2 206 /1 /,*+* / 26 E,,**.12 nm 2 UV-,//* HPLC **+. *,., -. gml, gml, gml Fig. + Structure of astaxanthin AST AST 2 : WS WS AST **0. g BHT *-. g / ml + /* 0* / / 2* ml AST 3 +.,** ml,. gml WS AST **+., HPLC AST *-. AST **+. *,., - **,. **2.. gml WS AST **+/. 3 Z, rac. mesoast +- AST Z, rac. mesoast CaroteNature, HPLC 32 trans-ast, 3 cis-ast +-cis- AST - AST ao,, 0ootertoo.o AST BHT HPLC +* 2, : +2 VV ao /* gml Pseudomonas HPLC A sp. 1 : - VV ao - HPLC B Schizophyllum commune **/. M ph 1*. Escherichia coli recombinant 0+. g 3** ml ph 1.* Bovine pancreas + *** ml Mucor miehei Candida antarctica Phycomyces nitens - **/. M ph 1*. 0* unitsml, AST HPLC HPLC : LC-+* ATvp, : SPD- trans-ast 3/ +* AVvp, : SPD-M+* Avp + H- NMR LC MS APCI LC : alliance,03/ : AST +* mg QUATRO micro Waters +** gml + H-NMR Inova /** /** MHz, Varian PERSONAL-++

3 27 : 207.1* nm, -** nm 0** nm - AST HPLC AST AST AST AST + AST AST ao BHT - /* gml. +,*., gml Fig., AST trans AST cis-ast HPLC HPLC **/. M 0 ml, AST +* ml 0** l -0 units HPLC,** gml LC-+* ATvp -1 SPD-+* AVvp -* +2* : SPD-M +* *.*+ g +* Avp +* ml AST Luna Silica - m f.. 0 mm+/* mm - phenomenex.* -* HPLC A A /* gml ao HPLC Fig., 2, : +2 VV. B /* gml ao 1 : -VV AST +,. mlmin 3 Fig., Outline of procedure for enzyme hydrolysis of esterified-astaxanthin and subsequent HPLC analysis

4 208 /1 /,*+* / 28 Table + Samples used in the recovery tests Fig., Sample Spiked level Extraction +,* Capsule containing Dissolve with 2** mg/ +** g AST HPLC pigments oil chloroform Tablet containing Grind with chloroform- +/* mg/ +** g vitamin B* and C methanol (, : + ) Sonication with Drink containing chloroform-methanol AST AST 3 vitamin C and +41 mg/ +** g (, : + ) and partition liquid saccharide +., with water *+. g * Vitamin B, B, B, and B +, 0 +, AST ++ -* +2* - AST HPLC A B, HPLC / + AST - HPLC LC - HPLC A - A, *2. ml LCMS APCI WS AST AST AST - Table + AST AST /* *.*+ gml AST - gml AST HPLC A +/ 2** mg+** g +/* mg+** g AST +. 1 mg+** g SN 0 3 AST E,,**.12 nm AST AST, : +, : + AST *+. *3. gml, : *2 -,. units - AST, 1 + AST AST AST AST - 1 RSD,

5 29 : 209 Fig. - E# ect of antioxidants on stability of astaxan- Fig.. E# ect of ethoxyquin on astaxanthin stability thin solutions stored at,* during enzyme reaction Astaxanthin Ca., g/ml solution(mobile phase) including, g/ml free astaxanthin acetone solution was added /* g/ml antioxidant was stored in a,* freezer. into enzyme reaction mixture in Fig., and shaken in a (a) Concentration of total astaxanthin -1 water bath. (b) Ratio of trans-astaxanthin to total astaxanthin (a) Recovery of total astaxanthin ( trans-astaxanthin and cis-astaxanthin) (b) Ratio of trans-astaxanthin to total astaxanthin Fig. - Fig. - a AST,*. +* Fig. - b cis-ast Fig.. a trans-astrans-ast cis-ast. / Fig.. b. AST AST 32.. trans, 0 - AST AST Fig.. Table, Pseudomonas fluorescens trans-ast cis-ast P. fluo. CE AST AST - P. fluo. CE

6 210 /1 /,*+* / 30 Table, 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 +,* - **+. *,., -. gml / AST HPLC - r * Fig. 1 AST AST - Fig. / E# ect of enzyme reaction time on the yield of +. 12** mg+** g free astaxanthin Table - 1 Esterified-astaxanthin in Haematococcus oil were hydrolyzed with cholesterol esterase by the procedure described *+ AOAC International AOACI in Fig., +* + 3, +*/ *.*+ 2/ ++* RSD, +, P. fluo. CE AST, AST AST AST 1 AST Fig. / - -* 3* 3* RSD, Table. RSD +.,. Horwitz ++ HPLC , AOACI RSDr Fig. 0 +, *+. - AST Fig. 0 a AST Fig. 0 b AST AST, HPLC

7 31 : 211 /, Table / HPLC LCMS AST Fig. 0 Fig. 2 3 HPLC cis-ast - **+. gml AST HPLC 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). Fig. 1 Typical calibration curve for trans-astaxanthin Table - Recovery of astaxanthin spiked to health food samples Sample Capsules Tablets Drink Spiked Recovery ( ) RSD (mg/ +** g) +, -. / 0 1 Av ( ) 2** 334/ ** *, ** +4** +/* +*, +*+ +*, +*+ +*- +** +*+ +*+ * /43 324, 3/4, 314, / , Table. Inter-day repeatability of the method Astaxanthin (mg/ +** g) RSD +, -. / 0 1 Av4 ( ),2-4*,124.,2343,2/4.,214,,204.,2*4-, , Tablets containing esterified-astaxanthin from Haematococcus were analyzed on three di# erent days

8 212 /1 /,*+* / 32 AST SN +* +0.* SN+* SN- ; ***0. gml ; ***,. g ml + g +* ml + *.**0 mg+** g + +** *.**0 gml+* ml *.**0 mg+** g + g + *** **,. mg+** g Table / Astaxanthin value on di# erent HPLC conditions Astaxanthin (mg/ +** g) Sample HPLC HPLC condition A* condition B** A/B Astaxanthin oil Capsule A Capsule B Capsule C Capsule D Capsule E Capsule F Capsule G Tablet Powder A Powder B Mean /*2- / ,. +*/4. +-4,0,,3* -, *,,*4,- 2-*410 /*/- / , * ,,-,0 --/4* 114..,*4/ *+ *433 +4*- *43. *433 *432 *431 *432 *431 *432 *432 *432 * For chromatographic conditions see Fig 4,, mobile phase A4 ** For chromatographic conditions see Fig 4,, mobile phase B4 Fig. 2 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).

9 33 : 213 Fig. 3 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.. AST AST AST AST AST -3 Fig. +* AST AST ph1 AST Pseudomonas fluorescens -1 +,* HPLC AST AST **+.. gml AST * AST AST, *+

10 214 /1 /,*+* / 34 Fig. +* Application of the proposed procedure to esterifiedastaxanthin from krill Esterified-astaxanthin was extracted from krill powder with chloroform-methanol, purified by partition, and subjected to the enzyme hydrolysis, HPLC LCMS HPLC SN ***0. mg +** g *.*, mg+** g AST AST AST LCMS + Vecchi, M., Glinz, E., Meduna, V. and Schiedt, K., HPLC separation and determination of astacene, semiastacene, astaxanthin, and other keto-carotenoids. J. High Resolut. Chromatogr. & Chromatogr. Commun., +*, -.2-/+ ( +321)., Sommer, T.R., Potts, W.T. and Morrissy, N.M., Utilization of microalgal astaxanthin by rainbow trout ( Oncorhynchus mykiss). Aquaculture, 3., 1322 ( +33+ ). - Schüep, W. and Schierle, J., Determination of stabilized, added astaxanthin in fish feeds and pre-mixes. In Carotenoids, Vol. + A, eds. Britton, G., Liaaen-Jensen, S. and Pfander, H., (Birkhäuser), pp.,1-,10 ( +33/ ).. Miao, F., Lu, D., Li, Y. and Zeng, M., Characterization of astaxanthin esters in Haematococcus pluvialis by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Anal. Biochem., -/,, (,**0). / Schiedt, K. and Liaaen-Jensen, S., Isolation and analysis. In Carotenoids, Vol. + A, eds. Britton, G., Liaaen-Jensen, S. and Pfander, H., (Birkhäuser), pp. 2+ +*2 ( +33/ ). 0 Yuan, J.P. and Chen, F., Purification of trans-astaxanthin from a high-yielding astaxanthin ester-producing strain of the microalga Haematococcus pluvialis. Food Chem., 02, (,***). 1 Jacobs, P.B., LeBoeuf, R.D., McCommas, S.A. and Tauber, J.D., The cleavage of carotenoid esters by cholesterol esterase. Comp. Biochem. Physiol., 1, B, +/1 +0* ( +32, ). 2 Bull. Jap. Soc. Sci. Fish.,,.,*3,+/ +3/2 3 Tsukuda, N. and Amano, K., Studies on the discoloration of red fishes. Content of carotenoid pigments in eighteen species of red fishes. Bull. Jap. Soc. Sci. Fish., -,, / ( +300). +* AOAC Guideline for single laboratory validation of chemical methods for dietary supplements and botanicals (,**,). ++ +, O$ cial method of analysis of AOACI. (Appendix E). /. -/0-0+,**1,+ +* +0,,, /

平成26年度化学物質分析法開発報告書

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