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1 5 1 Vol. 5 No Journal of Food Safety and Quality Jan., 2014 β- 郑菲菲 1, 陶保华 1, 赖世云 1, 黄百芬 2, 任一平 (1., ; 2., ) 2* 摘要 : 目的 β- 方法 2 ml 50%, 70 15min, : (1:1,v/v),, C 30, 450 nm, 结果 β- 0.2~4.0 μg/ml, : R 2 >0.9990; 80%~90%, 6 RSD <5% 结论 β- 关键词 : ; ; ; β- Simultaneous determination of lutein and β-carotene in infant formulas by high pressure liquid chromatography ZHENG Fei-Fei 1, TAO Bao-Hua 1, LAI Shi-Yun 1, HUANG Bai-Fen 2, REN Yi-Ping 2* (1. Zhejiang Beingmate Research Institute, Hangzhou , China; 2. Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou , China) ABSTRACT: Objective To establish a rapid method for simultaneous determination of lutein and β-caortene in infant formulas by high pressure liquid chromatographic. Methods The homogenized samples were added with 2 ml 50% potassium hydroxide solution, saponified at 70 for 15 min then extracted with petroleum ether and diethyl ether followed by wash and concentration. The lutein and β-carotene were separated by an C 30 column and detected by the UV detector at a wavelength of 450 nm. Results The method showed a good linearity over the range of 0.2~4.0 μg/ml with R 2 > The recoveries at three spiked levels were between 80%~90%. The relative standard deviation was less than 5%. Conclusion This method is simple, rapid and accurate for simultaneous determination of lutein and β-carotene in infant formulas. KEY WORDS: high pressure liquid chromatography; infant formulas; lutein; β-carotene 1 引言 (lutein) : α-, : C 40 H 56 O 2, : ,,,,, [1],,, * 通讯作者 :,, renyiping@263.com *Corresponding author: REN Yi-Ping, Professor Senior Engineer, Zhejiang Provincial Center for Disease Prevention and Control, No.3399, Bingsheng Road, Binjiang District, Hangzhou310051, China. renyiping@263.com

2 228 5,,,,, [2] β- (β-carotene), C 40 H 56,,,,, α-, β, γ-, β-, [3] β-,,, ( N 2 ), β- A,,,, [4,5] β-,, β- β-,, β-,, β- [2-16] β- ;, ; [6-17] β- ODS C 30, β- [18] ;,, β-, β-,, [11], [11] 3.2 μg/100 g, β- 0.8 μg/100 g, GB β- [12] [11-22] β-, 5~10, 50 ml ( ), β-, 2 材料与方法 2.1 材料与试剂试剂 ( : 98%, ); β- ( : 97%, Fluka); (Merk ), (Sigma, 99%), (, ), (, ), (, ), (, ), (, ), (,, h, ), 2,6- (, ); 1 β- Fig. 1 The structures of lutein and β-carotene

3 1, : β 仪器与设备 Ultimate-3000RS, ( DIONEX ); Allegra 64R ( Beckman Coulter ); 50 ml PP ( Labcon ); N-EVAP112 ( Organomation Associates ); : YMC Carotenoid (4.6 mm 250 mm 3 μm)( YMC ); Milli-Q ( Millipore ); UV ( ); QL-861 ( ); SHA-B ( ); BSA224S-CW ( Sartorius ) 2.2 样品前处理皂化 : 1 g( g) 50 ml, 5 ml 0.5 g 10 ml 0.1% BHT 2 ml 50%,, 70 ±1 15 min, 提取 : 20 ml : (1:1, v/v), 90 s, 7000 r/min 1 min 50 ml, 20 ml, 2,, 7000 r/min 1 min 25 ml (, 0.5 g ), 40 ±2 2.0 ml : (1:1, v/v), 0.22 μm 2 ml, (10 ) 2.3 标准溶液配制及校准标准储备液配制 : β- 10 mg, 0.1% BHT, 25 ml, 400 μg/ml : (1:1, v/v) 20 μg/ml : (1:1, v/v) 20 μg/ml, 标准溶液浓度校准 : β- 1 ml 10 ml, ( ) 10 ml ( ),, A, X( A 4 1) : X = 10 1% E1cm 标准溶液纯度校准 :, [22] β-, β- 2.4, C 30,,, 2.4 分离检测参考条件 : YMC Carotenoid C 30 (4.6 mm 250 mm, 3 μm); : 30 ; : 20 μl; : 10 ; : 450 nm; : A: B: ; : 0 ~1 min A% 100%, 1 ~2 min A% 100%~93%, 2 ~3 min A% 93%~85%, 3 ~6 min A% 85%~75%, 6 ~10 min A% 75%~60%, 10 ~14 min A% 60%~20%, 14 ~16 min A% 20%, 16 ~17 min A% 20~100%, 17~25 min A% 100%; 1 ml/min; 25 min 3 结果 3.1 色谱条件优化色谱柱的选择 Carotenoids(C 30 ),, Table 1 表 1 标准品吸光值及测定条件 Absorption and determination conditions of standards E 1% λ (nm) 1% 1cm E λ (nm) 1cm (lutein) β- (β-carotene)

4 230 5 β-, [18-22] HPLC C 18 C 30 [11-22],,, C 18,, β-, C 30, 2 2, 1: 1 min, β-, ; 2 3:,, β- ; 4:, β-,, 流动相的选择优化 GB β- [12] : : (3:12:85) ; GB/T [7] : (85:15),, β- 100%, β- ( 2.4), β-, 定容液和进样体积的确定 : (1:1, v/v) 2.2, β-, ;, : (1:1, v/v), 20 μl, 20 μl, : (1:1, v/v), 20 μl 3.2 皂化条件的优化皂化时间和温度的优化,,, 1 g 50 ml, 5 ml, 0.5 g, 10 ml 0.1% BHT, 2 ml 50%, , min β-, 4 Fig. 2 2 C 30 Comparison of resolution with different specifications of C 30 column : 1, 2 β-, 3

5 1, : β- 231 Fig. 3 图 3 Chromatogram of standard and sample Fig. 4 4 β- (n=3) Effect of saponification conditions on amount of lutein and β-carotene (n=3)

6 , β ,, 70 80, 70, β ,,, min, β- ;, β-, 70, 15 min 皂化加碱量选择 1 g 50 ml, 5 ml, 0.5 g, 10 ml 0.1% BHT, ml 50%, min, β-, 5 5, 2 ml,, β- 2 ml,, β- 1 ml 50%,,, 1 ml, 2 ml 50% 3.3 线性与检出限回收率与精密度线性关系 : β-, μg/ml, 2.4,, 灵敏度 : β- 2.2, 0.05 μg/ml β-, 3 (S/N), 10 (S/N), 回收率 : 3 β-, 6, 2.2 β- 80%~90%, RSD 5%, 实际样品测定 [7] 6 (n=3),, 3, Fig. 5 5 β- (n=3) Effect of alkali concentration on the burst size of lutein and β-carotene (n=3)

7 1, : β- 233 Table 2 表 2 叶黄素和 β- 胡萝卜素的线性检出限加标回收率和重复性 Linear, correlation, coefficient, LODs, LOQs, recoveries and RSDs of lutein and β-caortene (μg/100g) % (n=6) RSD% (n=6) R 2 LOD(μg/100g) LOQ(μg/100g) β Y=0.4168X Y=0.4560X Table 3 表 3 样品测定结果 The determination results of samples (n=3) lutein (μg/100 g) β-carotene (μg/100 g) +4 86± ± ± ± ± ± ±6.7 90± ± ± ±7.1 89± ( ) 51± ± ±3.4 79± ± ± ± ± ±1.7 75± ± ± 结论 β-, β-,, β- 5 致谢 DSM( ) 参考文献 [1],. [J]., 2005, (5): Zhu HX, Zheng JX. A review: structure, distribution, properties and physiological functions of lutein [J]. China Food Addit, (5): [2] Brendon DG, Harvey EI. Liquid chromatographic method for the determination of lutein in milk and pediatric formulas [J]. Inter Dairy J, 2008, 18: [3],,,. β- [J]., 2007, 26(3): Hu XJ, Zhang LF, Wang XL, et al. β-carotene oxidation products and its inhibition on tumorous cells [J]. J Food Sci Biotechnol, 2007, 26(3): [4] Su Q, Kevin GR, Nicholas DH, et al. Review carotenoids separation methods applicable to biological samples [J]. J Chromatogr B, 2002, 781(26): [5],,. β- [J]., 2007, 9(6): Yan XH, Wang ZW, Wang ZN. Application and advancement of β-carotene [J]. Food Drug, 2007, 9(6): [6] GB [S].

8 234 5 GB National food safety standard for using of nutritional fortification substance in foods [S]. [7] GB/T [S]. GB/T Determination of lutein in milk powder-hplc-uv method [S]. [8],,,. [J]., 2011, 36(10): Shan Y, Yang JB, E LM, et al. Determination of lutein in formula food for infant and young children by HPLC [J]. Food Sci Technol, 2011, 36(10): [9]. [J]., 2011, 9(10): Tao DL. Determination of lutein in infant formula milk powder [J]. Life Sci Instrum, 2011, 9(10): [10],,,. [J]., 2012, 33(4): Shan Y, Jiang YJ, Chen Y, et al. Extraction and determination of lutein in formulated food r infant and young children [J]. Food Sci, 2012, 33(4): [11],,,. α- β- [J]., 2012, 22(3): Huang BF, Zheng K, Ren YP, et al. Simultaneous determination of lutein, α-carotene and β-carotene level in foods for infants and young children and milk products by HPLC [J]. Chin J Health Lab Tech nol, 2012, 22(3): [12] GB/T β- [S]. GB/T Determination of β-carotene in foods for infants and young children milk and milk products [S]. [13],. - [J]., 2010, 38(6): Huang FF, Zhou JY. Determination of lutein in milk and dairy products by high performance liquid chromatography [J]. Chin Dairy Ind, 2010, 38(6): [14],,,. β- [J]., 2006, 12(1): Tu YM, Xia H, Yang J, et al. Determination of β-carotene in fortified milk powder by RP-high performance liquid chromatography [J]. Anal Test Technol Insrum, 2006, 12(1): [15],,,. β- [J]., 2010, 35(10): Shu S, Yan JC, Peng C, et al. Determination of β-carotene infant formulas by reversed-phase high-performance liquid chromatograpy [J]. Food Sci Technol, 2010, 35(10): [16],,. β- [J]., 1999, 27 (4): E LM, Zhou H, Pan BQ. Determination of β-carotene in milk powder by HPLC [J]. China Dairy Ind, 1999, 27 (4): [17] Liu Y, Xu MJ, Canfield LM. Enzymatic hydrolysis extraction and quantitation of retinol and major carotenoids in mature human milk [J]. Nutri Bio Chem, 1998, 9: [18],,,. C 30 -HPLC-PDA [J]., 2006, 27(9): Hui BD, Li J, Pei LP, et al. Separation and identification all E-lutien and E zeaxantin in foods with C 30 -HPLC-PDA [J]. Food Sci, 2006, 27(9): [19] Larsen E, Christensen LP. Simple saponification method for the quantitative determination of carotenoid in green vegetables [J]. J Agric Food Chem, 2005, 53(4): [20] Craft NE,Soares JH. Relative solubility, stability and absorptivity of lutein and β-carotene in organic solvents [J]. Am Chem Soc, 1992, 40(3): [21] Scott KJ, Finglas PM, Seale R, et al. Inter laboratory studies of HPLC procedures for the analysis of carotenoids in foods [J]. Food Chem, 1996, 57(6): [22] Bobm V, Puspitasari-Nienaber N, Ferruzzi MG, et al. Trolox equivalent antioxidant capacity of different geometrical isomers of α-carotene, β-carotene, lycopene, and zeaxanthin [J]. J Agric Food Chem, 2002, 50(1): 作者简介 ( 责任编辑 : 张宏梁 ) 郑菲菲, 助理工程师, 主要研究方向为食品检验技术 xiaodcd@163.com 任一平, 教授级高级工程师, 主要研究方向为卫生理化检验技术及食品安全检验技术 renyiping@263.net

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min 25 4 2013 8 监测技术 5 1 2 1 ( 1. 江苏省疾病预防控制中心, 江苏南京 210009; 2. 南京医科大学第二附属医院, 江苏南京 210029) 5 Phenomenex kinetex C 18 2. 6 μm 4. 6 mm 150 mm 10 mmol /L ph 2. 5 + 60 40 DAD 280 nm 5 20. 0 mg /L ~ 100 mg /L 0.