6 4 Vol. 6 No. 4 2015 4 Journal of Food Safety and Quality Apr., 2015 李洪燕 1, 李秀英 1, 陈俊禧 2, 刘冬虹 1, 林森煜 1, 卢宇靖 2, 黄金凤 (1., 511400; 2., 510006) 1* 摘要 : 目的 - - (ID-UHPLC- MS/MS) 方法, HSS T3 0.1%(v:v) -,, 结果 5~1000 µg/l, (R 2 ) 0.9998; 0.02 mg/100 g, 0.07 mg/100 g, 3 93.4%~105%, (n=6) 2.3%~4.4%, (n=3) 2.72% 结论, 关键词 : ; - ; ; Determination of pantothenic acid in multivitamin supplement solution by ultra high performance liquid chromatography-tandem mass spectrometry with isotope dilution LI Hong-Yan 1, LI Xiu-Ying 1, CHEN Jun-Xi 2, LIU Dong-Hong 1, LIN Sen-Yu 1, LU Yu-Jing 2, HUANG Jin-Feng 1* (1. Guangzhou Quality Supervision and Testing Institute, Guangzhou 511400, China; 2. Guangdong University of Technology, Guangzhou, 510006, China) ABSTRACT: Objective To develop a simple and rapid method for determination of pantothenic acid in multivitamin supplement by ultra high performance liquid chromatography - tandem mass spectrometry with isotope dilution (ID-UHPLC-MS/MS). Methods The sample was extracted and purified by perchloric acid solution, and separated on an HSS T3 UPLC column with acetonitrile and 0.1% formic acid solution as mobile phase under gradient elution mode, then detected with tandem mass spectrometry under ESI at positive and multiple reaction monitoring (MRM) mode. Stable isotope was used as an internal standard for quantification. Results Pantothenic acid has a good linear relationship in the range of 5~1000 μg/l, the limit of detection (LOD) and quantitation (LOQ) of the method was 0.02 mg/100 g and 0.07 mg/100 g respectively. The fine recovery (93.4%~105%), the relative standard deviation (2.3%~4.4%, n=6) and inter-day with RSD (2.72%, n=3) of this method were also obtained. Conclusion The proposed method was successfully applied to determine pantothenic acid in multivitamin supplement. Compared with the standard method, the proposed method was sensitive, rapid, and accurate. It provides a rapid approach for the analysis of pantothenic acid in complex matrix multivitamin supplement solution. * 通讯作者 :,,, E-mail: 82309762@qq.com *Corresponding author: HUANG Jin-Feng, Senior Engineer, Guangzhou Quality Supervision and Testing Institute, Guangzhou 511400, China. E-mail: 82309762@qq.com
4, : - - 1505 KEY WORDS: pantothenic acid; ultra high performance liquid chromatography-tandem mass spectrometry; isotope dilution; multivitamin supplement solution 1 引言 B 5,,, A(Co A) (ACP), ;, ;,, [1-3],,,,,, [4-5] [6-10] - [11] [12] [13] [14-16],, 1 mg/l [6-10],,,,,, [14-16],,,,,,,,,, 2 材料与方法 2.1 主要仪器与试剂 - (Agilent 1290/6490, Agilent ); Acquity HSS T3 ( Waters ); (3K 15, Sigma ); (BSA224S, Sartorius ); (MS3, IKA ) ( 98.0%, Dr Ehrenstorfer GmbH ); -[ 13 C 6, 15 N 2 ]( 99.5%, Iso Sciences ); (, ); (, Merck ); (70%~72%, ); 2.2 标准溶液的配制 (200 μg/ml): 54.5 mg, 250 ml( 0.92), 4 (200 μg/ml): -[ 13 C 6, 15 N 2 ] 10 mg, 50 ml, 4 (0.5 mol/l): 500 ml 300 ml, 50 g,, 1 L, 2.3 样品前处理 1.0 g( 0.0001 g) 10 ml, 5 ml,, 2 min, 12000 r/min 2 min, 0.5 ml 50 ml, 25 μl,, 1 min, 0.22 μm, UHPLC-MS/MS 2.4 UHPLC-MS/MS 检测条件 : Waters HSS T3 (2.1 mm 100 mm, 1.8 μm); 0.45 ml/min; 30 ; 2 μl; A: 0.1%(v:v) ; B: 1
1506 6 表 1 流动相梯度洗脱条件 Table 1 Gradient program for PA /min A/% B/% 0.0 92 8 2.2 80 20 2.4 50 50 4.0 50 50 4.1 92 8 7.0 92 8, 86% 75%,,,, ; 0.5 mol/l (ph 3.8),, 10 min,,, 92% 89%, 0.5 mol/l, ; (MRM) ; 1.5 kv; 150 ; 14 L/min, 250 ; ( )11 L/min; 3000 V 2 3 结果与讨论 3.1 样品处理的优化 1,,, Fig. 1 Comparison of various solvent on recoveries,,,,,,,, 50%(v:v) 50%(v:v) 0.5 mol/l (ph 3.8) ( 1 ):, 1250, 68%; 50%(v:v) 50%(v:v),, 3.2 色谱与质谱条件的优化 α, γ- -β, β- -β-,, C 18, HSS T3, HSS T3 [M+H] +,, 0.1%(v:v), -, 2 表 2 泛酸及其同位素内标的定性和定量离子对 碰撞池加速电压和碰撞能量 Table 2 Qualitative and quantitative ion pairs, cell accelerator voltages and collision energy for PA and PA-Internal Standard (m/z) (m/z) (V) (V) 220.1 89.9* 9 4 220.1 72.0 17 4 -[ 13 C 6, 15 N 2 ] 224.2 94.1* 10 4 : *
4, : - - 1507 Fig. 2 2 MRM MRM chromatograms of PA and PA internal standard 3.3 方法的验证 3.3.1 线性范围 检出限和定量限, ( 100 µg/l ) UHPLC-MS/MS, (A) A i, A A i Y (X, µg/l), 5~1000 µg/l, Y 1.22X-0.0197, (R 2 ) 0.9998 ( S/N=3) ( S/N=10),,, 0.02 mg/100 g 0.07 mg/100 g 3.3.2 回收率, 40% 80% 130%, 6,, 3 3 93.4% 105%, 2.3% 4.4% 3.3.3 方法稳定性 1.3, 3, 6,, 4, 2.07%~2.52%, 2.72%, MRM 3 3.3.4 与国标方法的对比 GB/T 22246-2008 3, ( 5),,,,,,,,,, UHPLC-MS/MS,
1508 6 Table 3 表 3 泛酸加标回收试验结果 Recoveries and relative standard deviations of PA (mg/100 g) (mg/100 g) (%) (%) (%) 38.0 15.0 94.2 99.3 96.4 99.5 97.9 100 97.9 2.3 30.0 101 97.1 105 104 93.4 98.6 99.8 4.4 50.0 96.7 100 104 94.8 100 97.3 98.8 3.3 Table 4 表 4 样品中泛酸的稳定性试验结果 (n=6) Results of stability experiment of PA in samples (n=6) (mg/100 g) (mg/100 g) (%) (%) day 1 25.2 25.8 26.3 26.6 26.9 26.0 26.1 2.32 day 2 24.8 25.2 24.9 24.7 25.8 26.3 25.3 2.52 day 3 25.3 26.4 26.1 26.8 25.9 26.6 26.2 2.07 2.72 3 MRM Fig. 3 MRM chromatograms of sample Table 5 表 5 本方法与国标法检测结果的比较 Comparison of the proposed method and the standard method (mg/100 g) (mg/100 g) (mg/100 g) 1 42 36 40 2 36 23 33 3 68 56 70
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1510 6 作者简介 李洪燕, 工程师, 主要研究方向为食品营养分析检测技术 E-mail: 1105664207@qq.com 黄金凤, 高级工程师, 主要研究方向为食品及食品相关产品检测, 仪器分析技术 E-mail: 82309762@qq.com