Determination of Carotenoids in Spear Shrimp Shells (Parapenaeopsis hardwickii) by Liquid Chromatography WAN-CHIN LIN,JOHN-TUNG CHIEN, AND BING-HUEI CHEN* Department of Nutrition and Food Science, Fu Jen University, Taipei, Taiwan 242 The objectives of this study were to develop a high-performance liquid chromatography method for analysis of carotenoids in spear shrimp shells (Parapenaeopsis hardwickii) and to compare the extraction efficiency of carotenoids by supercritical carbon dioxide (SCD) and solvents. Results showed that the most appropriate HPLC method was accomplished by employing a Cosmosil 5C18-AR-II column and a mobile phase of methanol-dichloromethane-acetonitrile (90:5:5, v/v/v) (A) and water (100%) (B) with the following gradient elution: 92% A and 8% B in the beginning, decreased to 4% B in 9.5 min, 1% B in 26 min, 0% B in 35 min, maintained for 25 min, and returned to 92% A and 8% B in 61 min. All-trans-astaxanthin and its two cis isomers, as well as five astaxanthin monoesters and 11 diesters were resolved within 60 min with a flow rate at 2 mL/min and detection at 480 nm. Astaxanthin diesters were found to contain 12 fatty acids, of which palmitic acid and stearic acid constituted a large portion, whereas astaxanthin monoesters were found to contain 10 fatty acids with arachidonic acid dominating. Solvent extraction could generate a higher content of trans- astaxanthin and astaxanthin esters, while SCD extraction could produce greater levels of 9-cis- astaxanthin and 13-cis-astaxanthin. KEYWORDS: Spear shrimp shell; astaxanthin; HPLC INTRODUCTION Astaxanthin, a naturally occurring carotenoid, which is mainly present in seafood products, especially in shells of lobster and shrimp, represents an important biological compound. Some studies have demonstrated that astaxanthin may possess strong antioxidant activity (1, 2) and exert a protective effect against chronic diseases such as cancer (3, 4). Several authors reported that the antioxidant activity of astaxanthin was higher than -carotene and R-tocopherol (1, 3, 5, 6). However, astaxanthin is susceptible to isomerization and oxidative degradation, because of the presence of long-chain conjugated double bonds (7, 8). As the formation of cis isomers of astaxanthin or its derivatives may possess different biological activity, it is important to learn about the carotenoid profile in crustacea such as shrimp shell. The traditional method used for extraction of carotenoids is often carried out by using solvents such as methanol and dichloromethane (7); however, this may create a safety problem. In recent years, supercritical carbon dioxide (SCD) has been used for extraction of carotenoids instead of solvents (9, 10). However, the extraction efficiency of astaxanthin and its derivatives remains uncertain. Two high-performance liquid chromatography (HPLC) methods have been developed to separate various astaxanthin and its derivatives (11, 12); however, the resolution needs to be improved. In Taiwan, more than 35000 tons of shrimp were produced in 2002, and about 20000 tons of shrimp shell wastes were obtained (13). Most shrimp shell wastes were used for the feed industry, the production of enzyme for meat tenderization, and the processing of chitin products (14, 15). As shrimp shell waste is a rich source of astaxanthin, it would be a great advantage to the health food industry if astaxanthin could be recovered as a functional ingredient. The objectives of this study were to compare the extraction efficiency of carotenoids in spear shrimp shells as affected by solvent and SCD extraction and to develop an HPLC method for their determination. MATERIALS AND METHODS Materials. A total of 30 kg of spear shrimp shells (Parapenaeopsis hardwickii) were obtained from a fishery market located in Keelung and were cleaned and freeze-dried prior to grinding. A total amount of 7.5 kg of dried shrimp shell powder was obtained and stored at -70 °C before analysis. All-trans-astaxanthin standard with a purity of 98% was from Sigma Co. (St. Louis, MO), and internal standard all-trans- -apo-8′-carotenal was from Fluka Chemical Co. (Buchs, Switzerland). Fatty acid methyl esters, including dodecanoic acid methyl ester (C12: 0), tetradecanoic acid methyl ester (C14:0), pentadecanoic acid methyl ester (C15:0), hexadecanoic acid methyl ester (C16:0), hexadecenoic acid methyl ester (C16:1), heptadecanoic acid methyl ester (C17:0), eicosanoic acid methyl ester (C20:0), eicosapentaenoic acid methyl ester (C20:5), and docosahexaenoic acid methyl ester (C22:6) were from Nu-Chek-Prep. Co. (Elysian, MN), and stearic acid methyl ester (C18: 0), oleic acid methyl ester (C18:1), and linoleic acid methyl ester (C18: 2) were from Sigma Co. The derivatization reagent 2,2-dimethoxypro- * To whom correspondence should be addressed. Tel: 886-2-29053626. Fax: 886-2-29021215. E-mail: nutr1007@mails.fju.edu.tw. 5144 J. Agric. Food Chem. 2005, 53, 5144-5149 10.1021/jf050664y CCC: $30.25 © 2005 American Chemical Society Published on Web 06/07/2005