7 5 Vol. 7 No. 5 2016 5 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., 511400; 2., 510006) 1* 摘要 : 目的,, - (UPLC-MS/MS) 方法, 1.5 mol/l, 100 10 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, 3 93.6%~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 511400, China; 2. School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, 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 :,, E-mail: gdone@21cn.com *Corresponding author: GUO Xin-Dong, Professor, Guangzhou Quality Supervision and Testing Institute, No.38, Baqier Road, Guangzhou 510110, China. E-mail: gdone@21cn.com
5, : / - 1877 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 10765-2010 GB 10767-2010 [3,4], [5-9], (GB 5413.20-2013) [8] 70 3 h, GB 29989-2013 [9],,,,,,,, [10-12], [13-16],,,,,, - (UPLC-MS/MS) UPLC,, [17] ;,,, 2 材料与方法 2.1 仪器 1290/6490 - ( 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 2.3.1 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 2.3.2 样品处理 1.0 g( 0. 001 g), 10 ml 1.5 mol/l,, :, 14 min 100, 10 min,, 20% NaOH ph 7.0, 100 ml
1878 7, 10 ml,, 0.1 ml 10 ml, 0.1 ml (10 μg/ml),, 0.22 μm UPLC-MS/MS 2.3.3 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 ( ), % 0.00 95 5 2.00 70 30 3.00 95 5 4.00 95 5 : ; ; (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 * 4 18 104.1>45.0 4 20 162.1>103 4 16 162.1>60.0 * 4 16 choline-d 8 112.2>68.1 4 20 L-carnitine-D 3 165.1>103 4 16,,, -, 1 3.2 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)
5, : / - 1879 3.2.1 微波水解温度的选择, 70, 70 100 150 200, 10 min, NIST SRM 1849a, 150,,,, UPLC-MS/MS, ( 4 ) 1.00~500 µg/l, (r 2 ) 0.998 (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) 3.2.2 微波水解时间的优化,,,,,,,,, 10~100 5 10 15 20 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, 5 50 250, 6,, 4 3 93.6%~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 4 3.6 与国标方法的对比 GB 5413.20-2013 GB 29989-2013,,, 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 + 0.5413 0.9997 0.072 0.072 0.24 L-carnitine 5.00~500 Y = 0.0044X 0.0108 0.9989 0.19 0.19 0.63
1880 7 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) (%) (%) 1.20 94.1 3.2 choline 12.0 95.4 2.5 60.0 102 1.9 3.20 93.6 3.9 L-carnitine 32.0 101 2.7 150 99.9 4.0 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 2 113 115 108 109 111 110 111 2.3 day 1 109 110 106 108 113 105 109 2.7 day 3 110 109 107 112 105 106 108 2.4 L-carnitine 2 day 2 13.9 15.1 14.2 14.4 14.2 13.6 14.2 3.6 day 1 13.1 13.3 14.3 14.1 13.3 13.7 13.6 3.5 day 3 13.6 13.8 15.1 14 14.4 13.3 14.0 4.6 2.7 4.1 : 1, 109±11 mg/100 g, 2 13.6±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 5413.20-2013(mg/100 g) (mg/100 g) GB 29989-2013(mg/100 g) NIST SRM 1849a * 111 119 13.8 15.9 98.1 103 9.68 10.1 132 129 7.22 7.29 115 110 8.05 8.33 * : NIST SRM 1849a 109±11 mg/100 g( ) 13.6±1.4 mg/100 g
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