6 8 Vol. 6 No. 8 2015 8 Journal of Food Safety and Quality Aug., 2015 王德伟 *, 游景水, 黄远英 (, 519040) 摘要 : 目的 (RP-HPLC) 方法 (ph 4.6), RP-HPLC, ph4.6, C 18, A 0.1%, B 0.09%(V:V) 90%(V:V),, 214 nm, 0.7 ml/min 结果 10 500 mg/l, 0.9999, 98.0%, RSD=0.8% (n=9) 结论,, 关键词 : ; ; ; Rapid determination of lactoferrin by reversed-phase high performance liquid chromatography WANG De-Wei *, YOU Jing-Shui, HUANG Yuan-Ying (By-Health Co., Ltd., Zhuhai 519040, China) ABSTRACT: Objective To establish a method for the rapid determination of the content of lactoferrin in raw material milk and dairy products by reversed-phase high performance liquid chromatography (RP-HPLC). Methods The RP-HPLC method was used in this method. According to the characteristics that lactoferrin precipitated at the isoelectric point (ph 4.6), lactoferrin was dissolved and separated by acetate buffer with ph 4.6. A C 18 column was used for rapid separation of protein, with the mobile phase A of 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B of 90% (V:V) acetonitrile solution with 0.09% trifluoroacetic acid (:V). The detection wavelength was 214 nm and the flow rate was 0.7 ml/min. Results Lactoferrin showed a good linearity between 10~500 mg/l and the correlation coefficient was 0.9999. The average recovery rate was 98.0%, with RSD of 0.8% (n=9). Conclusion The method was wide used, rapid, simple and accurate, it could be used in the determination of lactoferrin in lactoferrin raw material, milk powder, milk and health products. KEY WORDS: lactoferrin; reversed-phase high performance liquid chromatography; rapid determination; dairy 1 引言 (lactoferrin, LF) 80 kda, ( ) [1,2],, * 通讯作者 :,, E-mail: 790007532@qq.com *Corresponding author: WANG De-Wei, Licensed Pharmacist, By-Health Co.,Ltd., Zhuhai 519040, China. E-mail: 790007532@qq.com
8, : 3089 [3-8] 2012 3 30 GB 14880-2012, [9] [10-12] [13] [14-17] C 4,, ZORBAX 300 SB-C 18,,,,,, 2 材料与方法 2.1 仪器与试剂 Agilent 1260 (, ); KQ-500E ( ); XW-80A ( ); XP-205 ( Mettler Toledo ) (, ); (, ); (, ); (, ); (Sigma; : 081M7021V; : 85.0%) 20130401S 20130402S 20130402S ( ) 2.2 色谱条件 : ZORBAX 300 SB-C 18 (2.1 mm 50 mm, 3.5 μm); : 0.7 ml/min; : 25 ; : 20 µl; : 214 nm; A: 0.1%(V:V) ; B: 0.09%(V:V), 90% ; 2.3 溶液的制备 2.3.1 ph 4.6 醋酸盐缓冲液 : 18 g,, 9.5 ml, 1000 ml, ph 4.6 Table 1 表 1 流动相洗脱梯度表 Mobile phase elution gradient tables (min) A B 0 80 20 10 20 80 12 80 20 15 80 20 2.3.2 标准储备液 : 10.0 mg 10 ml,,,, 2~8 2.3.3 标准工作液 : 0.1 1 2 4 5 ml 10 ml,, 2.3.4 供试品制备 : 0.25 g, 50 ml,, ph 4.6,,, 5000 r/min 10 min,, 0.45 μm,, 20 μl 2.4 线性试验 0.1 1.0 2.0 4.0 5.0 ml 10 ml,,,, (X), (Y),, 2.5 精密度实验 6, 2.3.4,, RSD(%) 2.6 加标回收实验 0.25g 9, 50 ml, 3, 3, 3 4.0 6.0 8.0 mg,, ph 4.6,,, 5000 r/min 10 min,, 0.45 μm,, 20 μl
3090 6 3 结果与讨论 3.1 标准曲线确证结果 Y=18607.8X+249.3 r=0.9999 10 500 mg/l 3.2 检出限和定量限 LOD LOQ (S/N) LOD S/N 3, LOQ S/N 10 3.2.1 检出限 0.02 mg/ml 1~100 ml, 0.2 μg/ml, LOQ=3 0.2 μg/ml 3.2.2 定量限 0.05 mg/ml 1~100 ml, 0.5 μg/ml, LOD=3 0.5 μg/ml 3.3 精密度实验 2, 6 (RSD) 1.0%, 表 2 精密度实验结果 (n=6) Table 2 Result of precision experiment (n=6) (%) (%) RSD(%) 2.622 2.602 2.618 2.620 0.6 2.645 2.611 2.624 3.4 回收率实验 3 3, 3 98.0%, (RSD) 1.0% 3.5 实际样品的测定 2.3.4,,, 1,, Table 3 表 3 加标回收率实验 (n=9) Result of standard addition recovery experiment (n=9) (g) (g/100 g) (mg) (%) (%) RSD(%) 1 0.2367 4.329 4.09 98.9% 2 0.2325 4.440 4.33 97.7% 3 0.2397 4.303 4.11 98.1% 4 0.2154 5.343 6.01 97.6% 5 0.2014 5.398 5.77 97.0% 98.0 0.8 6 0.2207 5.333 6.13 97.7% 7 0.2043 6.523 8.07 98.8% 8 0.2012 6.488 8.02 97.0% 9 0.231 6.421 8.19 98.9%
8, : 3091 1 Fig. 1 Chromatogram of sample 4 结论 RP-HPLC,, (ph 4.6), ph 4.6,, ph, ZORBAX 300 SB-C 18 (2.1 mm 50 mm, 3.5 μm),,,,, 参考文献 [1],. [J]., 2009, (2): 88 90. Cheng DY, Sun JH. The effect of lactoferrin on immune function [J]. Clin Pediatr J, 2009, (2): 88 90. [2],,,. [J]., 2007, (9): 208 511. Li N, Hu ZH, Pang GC, et al. Immunoregulatory function of lactoferrin [J]. Food Sci, 2007, (9): 208 511. [3],,,. [J]., 2008, (2): 48 51. Gao SX, Han YG, Su D, et al. Research progress of lactoferrin and its physiological function [J]. Chin Cattle Ind Sci, 2008, (2): 48 51. [4],,,. [J]., 2012, (10): 41 44. Sun J, Zhang H, Guo HY, et al. Research progress of antibacterial function of lactoferrin [J]. China Dairy, 2012, (10): 41 44. [5],,. [J]., 2003, (1): 143 146. Ling XP, Pang GC, Li GQ. The latest research progress of lactoferrin [J]. Food Sci, 2003, (1): 143 146. [6],,. [J]., 2006, (1): 231 235. Pang GC, Hu ZH, Chen QS. The antiviral activity of lactoferrin [J]. Food Sci, 2006, (1): 231 235. [7],. [J]., 2005, (12): 32 36. Hu YY, Zhang SW. The functional properties of lactoferrin and its application [J]. Chin Dairy Ind, 2005, (12): 32 36. [8]. [J]., 2000, (4): 25 26. Lu SD. Function and application of lactoferrin [J]. Dairy Sci Technol, 2000, (4): 25 26. [9],,.
3092 6 [J]., 2008, (3): 24 25. Du L, Ren L, Su MY. Study on detection method of lactoferrin in the infant formula [J]. Dairy Sci Technol, 2008, (3): 24 25. [10],,,. [J]., 2009, (1): 122 124. Sun GQ, Kang XH, Liu WX, et al. Determination of lactoferrin in milk product by capillary electrophoresis[j]. Food Res Dev, 2009, (1): 122 124. [11],,,. [J]., 2011, (5): 54 56. GU Y, Cheng LH, Jiang JD, et al. The mass fraction of lactoferrin detection by capillary electrophoresis in infant milk powder [J]. Chin Dairy Ind, 2011, (5): 54 56. [12],. [J]. ( ), 2009, (2): 82 84. Liu YJ, Hu XY. Determination of lactoferrin in milk by capillary electrophoresis[j]. Farm Prod Process (J), 2009, (2): 82 84. [13],. [J]., 2011, (1): 173 175. Xia M, Ying TJ. Indirect determination of lactoferrin in milk powder by atomic absorption spectrophotometry [J]. Chin J Pharm Anal, 2011, (1): 173 175. [14],,,. [J]., 2012, (7): 167 168. Wang F, Dou WY, Shi YL, et al. Determination of content of infant formula lactoferrin lactoferrin in RP-HPLC [J]. Chin Brew, 2012, (7): 167 168. [15],,,. [J]., 2009, (1): 30 34. Cheng J, Gao XJ, Liu XF, et al. Study on the method of detection of bovine colostrum lactoferrin by RP-HPLC [J]. Dairy Sci Technol, 2009, (1): 30 34. [16],. [J]., 2010, (2): 165 167. Xia M, Ying TJ. Lactoferrin content by HPLC detection in dairy products [J]. Food Sci, 2010, (2): 165 167. [17],,,. [J]., 2010, (8): 130 132. Zhang LT, Chen XX, Wei JH, et al. Detection of food additives lactoferrin RP-HPLC [J]. Food Res Dev, 2010, (8): 130 132. 作者简介 ( 责任编辑 : 白洪健 ) 王德伟, 执业药师, 主要研究方向为保健食品的质量控制 E-mail: 790007532@qq.com