Microsoft Word - 991-999 20151112014王聪-二校

7 3 Vol. 7 No. 3 2016 3 Journal of Food Safety and Quality Mar., 2016-9 王 聪, 梁 瑞 强, 曹 进 *, 王 钢 力, 丁 宏, 张 庆 生 (, 100050) 摘 要 : 目 的 9 - 方 法,, -, ACQUITY UPLC BEH C 18,,, 9 结 果 9, r 0.99; 0.226~23.5 μg/l, 1.79~70.6 μg/l, 结 论 9,, 关 键 词 : ; ; Determination of 9 kinds of fat-soluble vitamins in healthy foods by liquid chromatography-mass spectrometry WANG Cong, LIANG Rui-Qiang, CAO Jin *, WANG Gang-Li, DING Hong, ZHANG Qing-Sheng (National Institutes for Food and Drug Control, Beijing 100050, China) ABSTRACT: Objective To establish a method for the simultaneous determination of fat-soluble vitamins in healthy foods by liquid chromatography-mass spectrometry (LC-MS). Methods The samples were extracted by ultrasonic in organic solvents, then determined by LC-MS. Quantitative ions of standards in external standard method were used as analysis of quantification. The chromatography and mass spectrometry was optimized using an ACQUITY UPLC BEH C 18 column. Nine kinds of fat-soluble vitamins added in health foods were determined qualitatively and quantitatively based on the information of molecular ion mass, specific fragment ions and chromatographic retention. Results The linearity of 9 kinds of fat-soluble vitamins was good with r>0.99. Limits of determination of 9 kinds of vitamins were 0.226~23.5 μg/l, and limits of quantitation were 1.79~70.6 μg/l. Precision, accuracy and repeatability was all satisfied. Conclusion This method is simple and reproducible, which can be applied in analysis of fat-soluble vitamins in healthy foods. KEY WORDS: healthy food; fat-soluble vitamin; liquid chromatography-mass spectrometry * 通 讯 作 者 :,, E-mail: caojin@nifdc.org.cn *Corresponding author: CAO Jin, Professor, Institute for Food and Cosmetics Control, National Institutes for Food and Drug Control, Beijing. No.2, Tiantan Xili, Beijing 100050, China. E-mail: caojin@nifdc.org.cn

992 7 1 引 言, A D E K,, α- β- γ-, β- [1] β- A, A A A, A 1 ( ) A 2 ( ), A 1, A 2, A A [2] 1 A A, A [3] D 1,25- D, D 2 ( ) D 3 ( ) E,, α- β- γ- δ- 4 [4],, α β γ δ [5] K 2- -1,4-, K K 1 K 2,,,, 1-3 [6-15] - (liquid chromatography-mass spectrometry, LC-MS),,, -,,, 9,,, 2 材 料 与 方 法 2.1 仪 器 与 材 料 - (1290 Infinity, 6460 Triple Quad LC-MS, Agilent Technologies); 1/1 (AL204, Mettler Toledo); 1/10 (XP205, Mettler Toledo) (, Fisher); (, Fisher); (, ) ( MT4S153068, ); AD ( 10244002, ) A( LGA0031, 95%, ); A ( 31010, 98.3%, Dr. Ehrenstorfer GmbH); D 2 ( 23417, 100%, Accu Standrad); D 3 ( 40916, 99.9%, Dr. Ehrenstorfer GmbH); E( 23414-02, 100%, Accu Standrad); E ( 41203, 98.5%, Dr. Ehrenstorfer GmbH); K 1 ( 41107, 99.9%, Dr. Ehrenstorfer GmbH); K 2 ( LC10207, 99.9%, Supelco); β- ( SLBG6787V, 95%, Sigma) 2.2 实 验 方 法 2.2.1 色 谱 条 件 ACQUITY UPLC BEH C 18 (2.1 mm 100 mm, 1.7 μm), 35, 0.5 ml/min, : 5 μl, A A D 3 E E K 1 K 2 β- 1, D 2 100% B 1 A B (min) 表 1 Table 1 流 动 相 及 梯 度 洗 脱 条 件 Gradient elution procedures A( : =10:90, V:V)% B( : =50:50, V:V)% 0.00 15.0 85.0 3.00 0.0 100.0 8.50 0.0 100.0 8.51 15.0 85.0 10.00 0.0 85.0

3, : - 9 993 2.2.2 质 谱 条 件,,, 45 psi, 4500 V, 6 L/min, 10 μa D 2 200 300, 8 350 500 2 2.2.3 对 照 品 溶 液 制 备 K 1 : K 1 10 mg, 100 ml, 5.0 ml,, ; β- : β- 5 mg, 50 ml,, ; A A D 2 D 3 E E K 2 : 10 mg, 100 ml,, A A E 0.50 ml, E K 2 0.25 ml, K 1 0.20 ml, D 2 0.40 ml, β- D 3 2.50 ml 10 ml,,,, 2.2.4 供 试 品 制 备 : 20, 1.0 g 100 ml 3 ml 10 ml, 10 min,,,, 0.22 μm, : 20,, 0.1 g 100 ml, 5 ml 10 ml,,, 0.22 μm, 2.2.5 空 白 实 验 9 2.2.6 标 准 工 作 曲 线 的 建 立,,, 2.2.4,, A A E: 0.010 0.020 0.050 0.10 0.20 0.50 μg/ml; E K 2 : 0.0050 0.010 0.025 0.050 0.10 0.25 μg/ml; K 1 : 0.0040 0.0080 0.020 0.040 0.080 0.20 μg/ml; D 2 : 0.0080 0.016 0.040 0.080 0.16 0.40 μg/ml; β- D 3 : 0.050 0.10 0.25 0.50 1.00 2.50 μg/ml Table 2 表 2 选 择 反 应 监 测 的 优 化 条 件 The retention time, qualitative ion pair and quantitative ion pair of vitamins /min /V /V A 1.750 269/93; 269/81 269/93 135 22; 22 A 2.335 269/93; 269/81 269/93 135 22; 22 D 2 1.634 397/107; 397/69 397/69 135 18; 35 D 3 3.796 385/367; 385/259 385/367 135 5; 5 E 4.200 431/165; 431/137 431/165 135 5; 40 E 4.575 473/207; 473/165 473/207 100 8; 30 K 1 4.925 451/187; 451/57 451/187 135 20; 30 K 2 3.559 445/187; 445/81 445/187 135 20; 40 β- 7.397 538/177; 538/121 538/177 150 10; 20

994 7 2.2.7 检 出 限 和 定 量 限, 3 10, 3, 10 2.2.8 精 密 度 及 稳 定 性, 9, 3, RSD, 12 h 表 3 9 种 脂 溶 性 维 生 素 浓 度 (μg/ml) Table 3 Concentration of 9 vitamins(μg/ml) A 0.050 0.20 0.40 A 0.050 0.20 0.40 D 2 0.040 0.16 0.32 D 3 0.25 1.0 2.0 E 0.050 0.20 0.40 E 0.025 0.10 0.20 K 1 0.020 0.080 0.16 K 2 0.025 0.10 0.20 β- 0.25 1.0 2.0 2.2.9 回 收 率, 9, 3, RSD 2.2.10 含 量 测 定 及 重 复 性,, 6 AD, 3 结 果 与 分 析 3.1 空 白 实 验 9 ( ) 3.2 标 准 工 作 曲 线 的 建 立 9 1 9 4 9, r 0.996 表 4 9 种 脂 溶 性 维 生 素 线 性 关 系 及 线 性 范 围 Table 4 Regression equation of 9 kinds of vitamins ( ) (μg/ml) (μg/ml) (μg/ml) A Y=3516.8X+13.8 0.0100~0.502 Y=2605.6X-5.6 0.0100~0.502 Y=2808.3X+0.8 0.0100~0.502 A Y=4910.2X+23.8 0.0105~0.523 Y=3187.7X+9.9 0.0105~0.523 Y=3396.5X+8.4 0.0105~0.523 D 2 Y=1386.9X+6.5 0.00812~0.406 Y=2768.5X-6.7 0.00812~0.406 Y=2637.5X-5.7 0.00812~0.406 D 3 Y=517.5X+20.3 0.0706~3.53 Y=333.1X-6.4 0.0578~2.89 Y=415.9X+3.1 0.0578~2.89 E Y=458.9X+1.3 0.0113~0.565 Y=685.7X-1.2 0.0219~0.548 Y=305.2X-2.2 0.0226~0.565 E Y=2035.7X+19.3 0.00583~0.292 Y=1202.5X+6.4 0.0117~0.292 Y=1387.1X+10.3 0.00583~0.292 K 1 Y=7119.9X+2.5 0.00424~0.212 Y=4094.4X-3.2 0.00425~0.212 Y=4802.2X-2.3 0.00425~0.212 K 2 Y=2141.9X+8.1 0.00537~0.269 Y=1272.6X-7.4 0.0107~0.269 Y=1236.6X-2.6 0.0107~0.269 β- Y=344.2X-2.5 0.0550~2.75 Y=358.5X-8.9 0.0507~2.54 Y=331.9X-11.9 0.0507~2.54

第3期 王 聪, 等: 液相色谱-质谱联用法测定保健食品中 9 种脂溶性维生素 995 注: (1)~(9)分别为维生素 A 维生素 A 醋酸酯 维生素 K2 维生素 D3 维生素 E 维生素 E 醋酸酯 维生素 K1 维生素 D2 β-胡萝卜 素 图中横坐标为时间, 纵坐标为丰度 图1 3.3 9 种脂溶性维生素的液相色谱-质谱提取离子色谱图 Fig. 1 MRM of 9 kinds of vitamins 检出限 定量限 3.4 精密度与稳定性 分别以 3 倍信噪比 10 倍信噪比时的样品浓度 片剂中维生素 A 及其醋酸酯 维生素 D2 维生 为检出限 定量限, 结果见表 5 9 种维生素的检出 素 D3 维生素 E 及其醋酸酯 维生素 K1 维生素 限为 0.226~23.5 μg/l, 定量限为 1.79~70.6 μg/l K2 胡萝卜素的混合标准工作溶液室温放置 12 h 的

996 7 RSD 9.4% 8.5% 3.3% 6.3% 2.6% 7.2% 2.6% 7.1% 2.1%; 9 12 h RSD 2.2% 3.0% 1.3% 3.8% 4.3% 6.8% 4.1% 4.4% 2.4%, 12 h 9 0.9% 4.7% 3.3% 7.9% 6.3% 5.3% 4.1% 1.3% 2.5%; 9 3.0% 2.9% 2.2% 6.3% 4.9% 10.5% 5.3% 7.6% 4.6%; 9 5.6% 9.4% 5.4% 5.9% 2.2% 7.6% 2.8% 9.9% 14.6%; 9 2.2% 7.7% 4.2% 4.8% 5.9% 4.5% 4.2% 6.8% 12.7%, 15% 3.5 回 收 率 实 验, 6 7, 80%~120%,, 3.6 含 量 测 定 及 重 复 性, 8 9, 表 5 9 种 脂 溶 性 维 生 素 检 测 限 定 量 限 (μg/l) Table 5 Limits of determination and quantitation of 9 kinds of vitamins(μg/l) A 3.34 10.0 3.34 10.0 A 3.48 10.5 3.48 10.5 D 2 4.06 8.12 0.812 8.12 D 3 23.5 70.6 23.5 70.6 E 3.76 11.3 0.226 1.13 E 0.583 1.94 1.94 5.83 K 1 0.708 2.12 0.708 2.12 K 2 0.537 1.79 1.79 5.37 β- 18.3 55.0 5.50 18.3 表 6 9 种 脂 溶 性 维 生 素 回 收 率 试 验 结 果 ( 片 剂 )(%) Table 6 Recovery experiments of tablets (%) RSD A 94.0 94.9 88.7 93.8 98.6 97.2 99.8 103.0 101.6 4.6 A 98.1 100.9 92.6 100.5 97.7 96.3 102.3 100.8 98.8 3.0 D 2 98.9 102.9 101.0 98.8 100.6 99.0 99.6 102.9 99.9 1.6 D 3 109.1 116.7 117.0 106.9 100.1 99.2 106.7 106.3 103.4 5.9 E 104.0 102.5 105.8 102.7 111.7 106.8 104.2 97.7 97.1 4.3 E 105.6 98.7 96.3 98.3 105.3 98.8 104.8 110.6 97.8 4.8 K 1 111.2 98.9 99.8 99.8 104.1 99.4 107.7 107.6 103.8 4.3 K 2 110.4 100.9 114.7 97.6 96.4 99.6 102.2 105.1 97.9 6.1 β- 90.7 94.0 89.5 92.7 94.1 91.3 99.2 101.5 95.3 4.2

3, : - 9 997 表 7 9 种 脂 溶 性 维 生 素 回 收 率 试 验 结 果 ( 软 胶 囊 )(%) Table 7 Recovery experiments of soft capsules (%) RSD A 98.6 100.4 95.1 102.1 95.6 99.4 103.7 91.8 91.1 4.5 A 101.0 98.3 99.2 104.7 101.6 101.5 104.2 99.5 100.2 2.2 D 2 111.5 108.5 111.8 109.2 110.5 106.7 117.1 114.9 112.1 2.9 D 3 92.7 102.3 93.2 97.2 98.0 104.2 92.5 95.7 99.7 4.3 E 101.5 92.3 106.0 86.1 94.6 94.3 102.6 111.5 90.7 8.3 E 97.4 101.6 98.2 106.1 93.0 100.5 100.9 99.7 109.8 4.8 K 1 106.1 93.2 93.3 109.1 108.3 99.5 102.0 101.3 97.3 5.9 K 2 101.3 114.3 86.6 87.0 110.0 96.5 100.2 96.8 101.5 9.2 β- 109.7 111.6 106.3 109.0 111.4 114.1 116.1 115.5 110.0 2.9 表 8 Table 8 片 剂 中 9 种 脂 溶 性 维 生 素 的 含 量 测 定 结 果 (mg/g) The content of 9 kinds of vitamins in tables(mg/g) 1 2 3 4 5 6 RSD(%) A 0.491 0.485 0.474 0.537 0.490 0.494 0.495 4.3 A 0.853 0.824 0.811 0.786 0.788 0.780 0.807 3.4 D 2 0.213 0.202 0.206 0.235 0.211 0.200 0.211 6.1 D 3 0.0132 0.0127 0.0128 0.0127 0.0136 0.0138 0.0132 3.8 E 0.284 0.297 0.294 0.294 0.284 0.299 0.292 2.2 E 49.7 49.3 51.3 47.5 53.9 47.5 49.9 4.9 K 1 0.0819 0.0769 0.0736 0.0695 0.0697 0.0666 0.0730 7.7 K 2 0.224 0.220 0.218 0.231 0.239 0.247 0.230 4.9 β- 0.264 0.263 0.243 0.260 0.250 0.257 0.256 3.2 表 9 Table 9 软 胶 囊 剂 中 9 种 脂 溶 性 维 生 素 的 含 量 测 定 结 果 (mg/g) The content of 9 kinds of vitamins in soft capsules(mg/g) 1 2 3 4 5 6 RSD(%) A 9.09 9.56 9.59 9.11 9.27 8.67 9.22 3.7 A 45.2 46.8 45.3 44.3 44.8 44.0 45.0 2.2 D 2 0.513 0.542 0.557 0.541 0.486 0.518 0.526 4.9 D 3 8.51 8.14 9.66 8.34 8.30 8.74 8.62 6.4 E 9.99 9.84 8.92 9.26 9.40 9.18 9.43 4.4 E 10.5 10.3 10.3 10.5 11.7 10.7 10.7 4.8 K 1 10.3 9.89 10.3 9.87 10.3 9.36 10.0 3.7 K 2 11.3 12.0 11.1 10.9 11.0 10.3 11.0 4.9 β- 10.6 11.3 11.2 11.1 10.5 11.4 11.0 3.5

998 7 4 讨 论,,, 0.2 ml/min, E E β-,,,,, 0.5 ml/min,, A 328,, 269 A 286,, 269,, A,,, A, 1, E, E,,, E E β-, 3, 350 500,, D 2, D 2,,, D 2 5 结 论 - 9,,, 参 考 文 献 [1],,,. β- [J]., 2008, 28(16): 1381. Wu CS, Huang JJ, Ai XL, et al. The review of research of β-carotene [J]. Chin Hosp Pharm J, 2008, 28(16): 1381. [2]. A β- [J]., 1998, 13: 17. Lin SM. The review of research of vitamin A and β-carotene in animal nutration [J]. China Feed, 1998, 13: 17. [3] GB 14880-2012 [S]. GB 14880-2012 Standard for the use of nutritional fortification substances in foods [S]. [4]. ( 7 )[M]. :, 2010. Zha XL. Biochemistry (Seventh edition) [M]. Beijing: People s Medical Publishing House Co., Ltd, 2010. [5],,,. HPLC A E [J]., 2015, 36(3): 320 321. Jiang Bo, Hu WZ, Liu CJ, et al. Simultaneous determination of vitamin A and different configuration of vitamin E in vegetable oils by HPLC [J]. Sci Technol Food Ind, 2015, 36(3): 320 321. [6] GB/T 5009.82-2003 A E [S]. GB/T 5009.82-2003 Determination of retinol and tocopherol in foods [S]. [7] GB 5413.9-2010 A D E [S]. GB 5413.9-2010 Determination of vitamin A, D, E in foods for infants and young children, milk and milk products [S]. [8] GB/T 9695.26-2008 A [S]. GB/T 9695.26-2008 Meat and meat products-determination of vitamin A content [S]. [9] GB/T 5009.83-2003 [S]. GB/T 5009.83-2003 Determination of carotene in foods [S]. [10] GB 5413.35-2010 β- [S]. GB 5413.35-2010 National food safety standard-determination of β-carotene in foods for infants and young children, milk and milk products [S]. [11] NY 82.15-1988 β- [S]. NY 82.15-1988 Determination of β-carotene in juice [S]. [12] GB/T 9695.30-2008 E [S]. GB/T 9695.26-2008 Meat and meat products-determination of vitamin E content [S] [13] NY/T 1598-2008 E [S]. NY/T 1598-2008 Determination of tocopherol content in edible vegetable oils [S].

3, : - 9 999 [14] GB/T 5009.158-2003 K 1 [S]. 作 者 简 介 GB/T 5009.158-2003 Determination of vitamin K 1 in vegetables [S]. [15] GB 5413.10-2010 K 1 [S]. 王 聪, 博 士, 助 理 研 究 员, 主 要 研 究 方 向 为 食 品 化 学 检 测 E-mail: wangcong351@126.com GB 5413.10-2010 National food safety standard-determination of vitamin K 1 in foods for infants and young children, milk and milk products [S]. ( 责 任 编 辑 : 杨 翠 娜 ) 曹 进, 博 士, 研 究 员, 主 要 研 究 方 向 为 分 析 化 学 E-mail: caojin@nifcd.org.cn