6 9 Vol. 6 No. 9 2015 9 Journal of Food Safety and Quality Sep., 2015-6 王 建 凤 1, 刘 艳 1*, 杨 一 帆 2, 贾 丽 1, 黄 华 3, 杜 振 霞 2, 范 筱 京 1, 张 经 华 (1.,, 100089; 2., 100029; 3., 100041) 1 摘 要 : 目 的 (SPE-UPLC-MS/MS) 6 方 法 0.05 mol/l, HLB,, 结 果 6(S/N=3) (S/N=10) 0.05~0.4 μg/kg 0.1~1.6 μg/kg 66.3%~110.0% 结 论, 关 键 词 : ; ; ; Determination of 6 kinds of cephalosporins in milk by solid phase extraction coupled with ultra high performance liquid chromatography-tandem mass spectrometry WANG Jian-Feng 1, LIU Yan 1*, YANG Yi-Fan 2, JIA Li 1, HUANG Hua 3, DU Zhen-Xia 2, FAN Xiao-Jing 1, ZHANG Jing-Hua 1 (1. Beijing Engineering Research Center of Food Safety Analysis, Beijing Centre for Physical and Chemical Analysis, Beijing 100089, China; 2. College of Science, Beijing University of Chemical Technology, Beijing 100029, China; 3. Beijing Municipal Center for Food Safety Monitoring and Risk Assessment, Beijing 100041, China) ABSTRACT: Objective To developed a method for the detection of 6 kinds of cephalosporins in milk by solid phase extraction coupled with ultra high performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS). Methods The samples were extracted with 0.05 mol/l phosphate buffer solution, cleaned up by HLB solid phase extraction column, and then analyzed by UPLC-MS/MS with multiple reaction monitoring (MRM) mode. Results The LODs and LOQs of 6 kinds of cephalosporins were in the ranged of 0.05~0.4 μg/kg and 0.1~1.6 μg/kg, respectively, with the mean recoveries varied from 66.25% to 109.97%. Conclusion The established method is simple, rapid and effective, and suitable for the determination of 6 kinds of cephalosporins in milk samples. KEY WORDS: solid phase extraction; ultra performance liquid chromatography-tandem mass spectrometry; cephalosporin; milk 基 金 项 目 : (Z141100002614020) Fund: Supported by Beijing Municipal Natural Science Foundation (Z141100002614020) * 通 讯 作 者 :,, E-mail: xgly36@163.com *Corresponding author: LIU Yan, Associate Researcher, Beijing Center for Physical and Chemical Analysis, No. 27, West Third Ring Road, Haidian District, Beijing 100089, China. E-mail: xgly36@163.com
9, : - 6 3381 1 引 言,, [1,2],,,, [3-5],,, [6,7], -(UPLC-MS/MS) [8-14] Li [9,10] 2 UPLC-MS/MS,,, 82.5%~98.3%, RSD 5.5% [11] 3 HPLC-MS/MS,,, 81%, RSD 12% Hou [12] UPLC-MS/MS,, 94.6%~117.1%, RSD 14%, 6 SPE-UPLC-MS/MS, 2 材 料 与 方 法 2.1 试 剂 与 仪 器 (, ); (, Optima ); (, Alfa Aesar ); (, ); (, ); (, ) UPLC XEVO-TQ MS ( Waters ); Acquity UPLC BEH C 18 (2.1 mm 50 mm, 1.7 μm, Waters ); CR22G ( Hitachi ); TGL-16G ( ); Vortex-Genie 2 ( Scientific Industries ); KQ-500DE ( ); N-EVAP112 ( Organomation ); OASIS HLB (6 cc, 500 mg, Waters ) 2.2 色 谱 质 谱 条 件 : BEH C 18 (2.1 mm 50 mm, 1.7 μm); : 0.5 mmol/l (A, ph=5) (B); : 0.2 ml/min; : 25 ; : 5 μl; : 0~2 min, 5%~10%B; 2~5 min, 10%~25%B; 5~7 min, 25%~35%B; 7~9 min, 35%~95%B; 9~11.5 min, 95%~5%B : (MRM), ESI(+) MRM 1 2.3 样 品 处 理 2.3.1 溶 液 配 制 0.05 mol/l (PBS): 11.4 g 1 L, 1 mol/l NaOH 6 mol/l HCl ph ; 0.5 mmol/l (A): 0.193 g 0.5 L, 0.45 μm, ph 5 6, 1000 mg/l, 4 - (5:95, V:V), 1 mg/l 2.3.2 样 品 提 取 (2.0±0.2) g, 6 ml PBS, 20 s, 2.3.3 样 品 净 化 6 ml, 3 ml PBS 3 ml/min 3 ml PBS 3 ml,, 12 ml, 6 ml,, 40 - (5:95, V:V) 1 ml, 0.22 μm
3382 6 Table 1 表 1 6 种 头 孢 菌 素 类 抗 生 素 的 MRM 质 谱 参 数 MS parameters in MRM mode of 6 kinds of cephalosporins (m/z) (m/z) (ev) (V) 114.0* 18 16 C 16 H 17 N 3 O 5 S 364.1 cefadroxil 158.0 8 16 174.0* 16 20 C 16 H 17 N 3 O 4 S 348.0 cefalexin monohydrate 158.0 8 20 134.1* 14 24 C 23 H 26 N 6 O 9 S 3 529.2 cefquinome sulfate 394.1 12 24 158.0* 8 20 C 16 H 19 N 3 O 4 S 350.1 cefradine 108.1 18 20 241.1* 18 34 C 20 H 25 N 5 O 7 S 2 512.2 cefetametpivoxyl 398.1 20 34 315.0* 16 26 C 16 H 15 N 2 NaO 6 S 2 419.0 cefalotin sodium 359.0 12 26 *: (Quantitative ion) 3 结 果 与 讨 论 3.1 色 谱 条 件 和 质 谱 条 件 3.1.1 质 谱 条 件 的 优 化, - (5:95, V:V) 6 1 mg/l,, 6 ESI+ ;, 2,, 1 3.1.2 色 谱 条 件 的 优 化 C 18, - - 2, 6, 1, -, 6 -,, -,, ph 3 4 5, 6, 2, ph 3 ; ph 4 ; ph 5 1. (cefadroxil) 2. (cefalexin monohydrate) 3. (cefquinome sulfate) 4. (cefradine) 5. (efetametpivoxyl) 6. 1 6 Fig. 1 Influence of different mobile phase on the responsive intensity of 6 kinds of cephalosporin compounds in milk
9, : - 6 3383 1. (cefadroxil) 2. (cefalexin monohydrate) 3. (cefquinome sulfate) 4. (cefradine) 5. (cefetametpivoxy) 6. 2 ph 6 Fig. 2 Influence of different ph of mobile phase on the responsive intensity of 6 kinds of cephalosporin compounds in milk 1. (cefadroxil) 2. (cefalexin monohydrate) 3. (cefquinome sulfate) 4. (cefradine) 5. (cefetametpivoxyl) 6. 3 ph 6 Fig. 3 Influence of different ph of selected solvent on the recoveries of 6 cephalosporins compounds in milk ph 5 ph 4 41.4%, ph 4 ph 5, ph 5 3.2 样 品 前 处 理 的 优 化 3.2.1 提 取 液 ph 值 的 优 化,,, ph, 1 mol/l NaOH 6 mol/l HCl PBS ph, ph 5 6.5 7 7.5 8.5 10, ph 6.5 PBS, ph 10 PBS 3.2.2 提 取 液 体 积 的 优 化, 6 8 10 ml PBS, 4,,,, 6 ml 1. (cefadroxil) 2. (cefalexin monohydrate) 3. (cefquinome sulfate) 4. (cefradine) 5. (cefetametpivoxyl) 6. 4 6 Fig. 4 Influence of the volume of selected solvent on the recoveries of 6 kinds of cephalosporin compounds in milk 3.2.3 淋 洗 液 体 积 的 优 化,, HLB,,
3384 6 6 ml 12 ml, ph 10 1. (cefadroxil) 2. (cefalexin monohydrate) 3. (cefquinome sulfate) 4. (cefradine) 5. (cefetametpivoxyl) 6. 5 6 Fig. 5 Influence of the volume of selected solvent on the recoveries of 6 cephalosporin compounds in milk 3.3 方 法 学 考 察 3.3.1 检 出 限 和 定 量 限,, 3(S/N=3) ; 10(S/N=10) 6 2 3.3.2 基 质 标 准 曲 线 及 线 性 范 围,,, 1~100 ng/ml, ; 2~200 ng/ml, ; 4~400 ng/ml, 6 (r 2 ) 0.994 3.3.3 回 收 率 和 精 密 度 3 2.3.1 2.3.2, 2, 66.2%~110.0%, (RSD) 15.3% 表 2 6 种 头 孢 菌 素 类 抗 生 素 在 牛 奶 基 质 中 的 检 出 限 定 量 限 添 加 回 收 率 和 相 对 标 准 偏 差 及 基 质 添 加 曲 线 线 性 相 关 系 数 的 变 化 范 围 (n=3) Table 2 LOD, LOQ, range of recovery, relative standard deviation and linear correlation coefficient of 6 kinds of cephalosporin compounds in milk (n=3) LOD(μg/kg) LOQ(μg/kg) r 2 (μg/kg) (%) 8 75.38% 0.89% cefadroxil 0.1 1.0 0.9997 80 69.20% 3.73% 160 80.27% 7.16% 8 88.79% 3.01% cefalexin monohydrate 0.4 1.6 0.9987 80 71.82% 6.46% 160 100.10% 2.18% 4 71.61% 8.30% cefquinome sulfate 0.2 0.5 0.9959 40 86.78% 15.22% 80 92.75% 4.37% 4 91.79% 3.49% cefradine 0.2 0.5 0.9940 40 83.36% 7.06% 80 92.22% 8.35% 2 67.90% 12.1% cefetametpivoxyl 0.05 0.1 0.9987 20 72.26% 3.82% 40 109.97% 3.20% 4 66.25% 8.27% cefalotin sodium 0.2 0.5 0.9967 40 67.05% 0.22% 80 89.03% 6.85% RSD (%)
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