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1 7 5 Vol. 7 No Journal of Food Safety and Quality May, 2016 / - 李秀英 1, 黄嘉乐 1, 邱志超 1, 何雪滢 2, 林森煜 1, 黄宝华 2, 郭新东 (1., ; 2., ) 1* 摘要 : 目的,, - (UPLC-MS/MS) 方法, 1.5 mol/l, min ph, HSS T3 UPLC 0.1% (V:V)-,, 结果 1.00~500 μg/l, (r 2 )0.998, 0.24 mg/100 g 0.63 mg/100 g, %~102%, 1.9%~4.0% 2.7% 4.1% 结论,,,, 关键词 : ; - ; ; ; Rapid determination of choline and L-carnitine in milk powder by ultra-high performance liquid chromatography tandem mass spectrometry using microwave digestion for sample preparation LI Xiu-Ying 1, HUANG Jia-Le 1, QIU Zhi-Chao 1, HE Xue-Ying 2, LIN Sen-Yu 1, HUANG Bao-Hua 2, GUO Xing-Dong 1* (1. Guangzhou Quality Supervision and Testing Institute, Guangzhou , China; 2. School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou , China) ABSTRACT: Objective To establish a simple and rapid method for determination of choline and L-carnitine in milk powder by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) using microwave digestion for sample preparation. Method The sample was extracted by microwave digestion with 1.5 mol/l HCl solution at 100 for 10 min, and then diluted by water. The sample was separated on an HSS T3 UPLC column with acetonitrile and 0.1% formic acid solution as mobile phase under gradient elution mode, then detected by tandem mass spectrometry under ESI at positive and multiple reaction monitoring (MRM) mode. Stable isotope was used as an internal standard for quantification. Result Choline and L-carnitine showed good linear relationships in the range of 1.00~500 μg/l (r 2 >0.998), and the limits of quantitation (LOQ) of choline and L-carnitine were 0.24 基金项目 : (2013PS04) Fund: Supported by the Scientific and Technological Project of Administration of Quality and Technology Supervision of Guangdong Province (2013PS04) * 通讯作者 :,, gdone@21cn.com *Corresponding author: GUO Xin-Dong, Professor, Guangzhou Quality Supervision and Testing Institute, No.38, Baqier Road, Guangzhou , China. gdone@21cn.com

2 5, : / mg/100 g and 0.63 mg/100 g respectively. The fine recoveries (93.6%~102%), the relative standard deviations (1.9%~4.0%) and RSD of inter-day (2.7% and 4.1%) of this method were also obtained. Conclusion Compared with the standard method, the proposed method is rapid, sensitive, and accurate. It shortens the sample preparation time by microwave digestion, and is suitable for the determination of choline and L-carnitine in milk powder. KEY WORDS: microwave digestion; ultra-high performance liquid chromatography-tandem mass spectrometry; milk powder; choline; L-carnitine 1 引言,,, [1,2],,,,,, GB GB [3,4], [5-9], (GB ) [8] 70 3 h, GB [9],,,,,,,, [10-12], [13-16],,,,,, - (UPLC-MS/MS) UPLC,, [17] ;,,, 2 材料与方法 2.1 仪器 1290/ ( Agilent ); Acquity HSS T3 ( Waters ); 3K15 ( Sigma ); BSA224S ( Sartorius ); Milli-Q ( Millipore ); Ethos One ( Milestone ); MS3 digital ( IKA ); MS204S ( - ); ph ( ) 2.2 材料与试剂 ( 99.6%, Fluka ) ( 99.0%, Dr.Ehrenstorfer GmbH ) - -D 3 ( 98%, Sigma-Aldrich ); -D 8 ( 99.6%, CDN ); (, CNW ); (, Merck ); (, ); 2.3 方法 溶液配制 -D 8 -D 3 1 mg/ml, 4 (5 μg/ml): 0.5 ml, 100 ml, 4 -D 8 -D 3 (10 μg/ml): 1.00 ml -D 8 -D 3, 100 ml, 4 C (1.5 mol/l): 12.5 ml 80 ml,, 100 ml (20%, m:v): 20 g 80 ml,, 100 ml 样品处理 1.0 g( g), 10 ml 1.5 mol/l,, :, 14 min 100, 10 min,, 20% NaOH ph 7.0, 100 ml

3 1878 7, 10 ml,, 0.1 ml 10 ml, 0.1 ml (10 μg/ml),, 0.22 μm UPLC-MS/MS UPLC-MS/MS 检测条件 : Waters HSS T3 (2.1 mm 100 mm, 1.8μm); 0.45 ml/min; 30 ; 2 μl; A: 0.1%(V:V) ; B: 1 表 1 液相色谱条件参数 Table 1 The chromatography parameters, min A (0.1% ), % B ( ), % : ; ; (MRM) ; 1.5 kv; 150 ; 14 L/min, 250 ; ( )11 L/min; 3000 V 2 3 结果与讨论 3.1 色谱与质谱条件的优化,, C 18, HSS T3, HSS T3 [M+H] +,, 0.1%,, 表 2 胆碱和左旋肉碱的质谱 MRM 模式优化参数 Table 2 The MRM optimized parameters of choline and L-carnitine m/z (V) (V) choline L-carnitine 104.1>60.0 * > > >60.0 * 4 16 choline-d > L-carnitine-D > ,,, -, 微波水解条件的考察,,, 3, 1.5 mol/l, Fig. 1 1 MRM (50.0 μg/l) MRM chromatograms of standard solutions (50.0 μg/l)

4 5, : / 微波水解温度的选择, 70, , 10 min, NIST SRM 1849a, 150,,,, UPLC-MS/MS, ( 4 ) 1.00~500 µg/l, (r 2 ) (LOD, S/N=3),, ( 3), 0.24 mg/100g 0.63 mg/100g 3 (n=3) Fig. 3 Effect of microwave digestion time on the extraction recoveries of choline and L-carnitine (n=3) 2 (n=3) Fig. 2 Effect of microwave digestion temperature on the extraction recoveries of choline and L-carnitine (n=3) 微波水解时间的优化,,,,,,,,, 10~ min,,, 10 min, 95.9% 97.8%,,, 10 min 3.3 线性范围与检出限, ( 100 µg/l ) UPLC- MS/MS, (A) A i, A A i Y (X, µg/l) 3.4 回收率, , 6,, %~102%, 1.9%~4.0% 3.5 方法稳定性 (NIST SRM 1849a), 3 d, 6,, 5, 2.3%~2.7% 3.5%~4.6%, 2.7% 4.1%, (NIST SRM 1849a) MRM 与国标方法的对比 GB GB ,,, UPLC-MS/MS-,,, Table 3 3 Linear regression equations, linear ranges, r 2 and the method detection limits of choline and L-carnitine (μg/l) r 2 ILOD (μg/l) MLOD (mg/100 g) MLOQ (mg/100 g) choline 1.00~500 Y =0.2774X L-carnitine 5.00~500 Y = X

5 表 4 胆碱和左旋肉碱加标回收试验结果 (n=6) Table 4 Recoveries and relative standard deviations of choline and L-carnitine(n=6) (mg/100 g) (%) (%) choline L-carnitine 表 5 方法稳定性试验结果 (n=6) Table 5 Results of stability experiment(n=6) (mg/100 g) (mg/100 g) (%) (%) choline 1 day day day L-carnitine 2 day day day : 1, 109±11 mg/100 g, ±1.4 mg/100 g 4 NIST SRM 1849a MRM Fig. 4 MRM chromatograms of sample solution Table 6 表 6 本方法与国标法比较的结果 Comparison of the proposed method and the standard method (mg/100 g) GB (mg/100 g) (mg/100 g) GB (mg/100 g) NIST SRM 1849a * * : NIST SRM 1849a 109±11 mg/100 g( ) 13.6±1.4 mg/100 g

6 5, : / 结论, -,, -,, HSS T3, 0.1%,,,,, -,, 参考文献 [1],,. [J]., 2011, 34(5): Wu ZP, Gao W, Lu J. Determination of choline in milk powder for infants and young children by ion chromatography [J]. J Dairy Sci Technol, 2011, 34(5): [2],,,. [J]., 2012, 12(6): Cao WJ, Cui H, Shen BZ, et al. Determination of choline chloride, Na +, K +, Mg 2+ and Ca 2+ in milk powder by ion chromatography [J]. Mod Sci Instrum, 2012, 12(6): [3] GB [S]. GB National food safety standard-older infants and young children formula [S]. [4] GB [S]. GB National food safety standard-infant formula [S]. [5],,,. [J]., 2013, 4(6): Liu MX, Hu CX, Ren L, et al. Determination of choline in milk powder by ion chromatography [J]. J Food Saf Qual, 2013, 4(6): [6] Andrew JO, Jason RS, Thomas DN, Development and validation of a simple UHPLC-MS/MS method for the simultaneous determination of trimethylamine N-oxide, choline, and betaine in human plasma and urine [J]. J Pharma Biomed, 2015, 109: [7] Ciulu M, Solinas S, Floris I, et al. RP-HPLC determination of water-soluble vitamins in honey [J]. Talanta, 2011, 83(3): [8] GB [S]. GB National food safety standard-determination of choline in infant formula and dairy product [S]. [9] GB [S]. GB National food safety standard Determination of L-carnitine in infant formula and dairy product [S]. [10],,. - [J]., 2014, 42(11): Zhang J, Wang X, Li GM. Simultaneous determination of trace elements and major elements in food by ICP-MS using microwave digestion for sample preparation [J]. Chin J Anal Chem, 2014, 42(11): [11],,,. - [J]., 2016, 02: Liu J, Wang BH, Fu WL, et al. Determination of copper content in fresh cherry by microwave digestion-graphite furnace atomic absorption spectrometry [J]. J Food Saf Qual, 2016, 02: [12],,,. - [J]., 2016, 2: Xu GR, Qu JG, Chang Y, et al. Accurate determination of sulfur content in sediments by double focusing inductively coupled plasma mass spectrometry combined with microwave digestion and studies on related matrix effect [J]. Chin J Anal Chem, 2016, 2: [13],,,. [J]., 2006, 23(1): Li Y, Liu ZH, Liu JX, et al. Study on extraction and determination of choline in pine pollen [J]. Chin J Spect Lab, 2006, 23(1): [14],. [J]., 2012, 35(2): Zhao HX, Hao WQ. Determination of choline in formula milk powder for infants and young children [J]. J Dairy Sci Technol, 2012, 35(2): [15],,,. 11 [J]., 2015, 31(7): Miao L, Mo JL, Zhou HJ, et al. Simultaneous detection of eleven water-soluble vitamins in infant formula using liquid chromatographymass spectrometry [J]. Mod Food Sci Technol, 2015, 31(7): [16] Susanne HK, Wolfgang H, Yannick R, et al. Quantification of acetylcholine, choline, betaine, and dimethylglycine in human plasma and urine using stable-isotope dilution ultra performance liquid chromatography - tandem mass spectrometry [J]. J Chromatogr B, 2010, 878(32): [17],,,. [J]., 2015, 6(5): Qu B, Chen R, Geng SW, et al. Liquid chromatography-mass spectrometry in applications of veterinary drugs multi-class and multi-residues analysis [J]. J Food Saf Qual, 2015, 6(5): 作者简介 ( 责任编辑 : 金延秋 ) 李秀英, 工程师, 主要研究方向为食品营养分析检测技术 lixiuying@xmu.edu.cn 郭新东, 教授级高级工程师, 主要研究方向为食品及相关产品安全检测技术 gdone@21cn.com