Microsoft Word 王汉卿 二校 出1817

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1 8 5 Vol. 8 No Journal of Food Safety and Quality May., δ 13 C 王汉卿 1, 吕宁 2, 张鸿伟 2, 郝鹏飞 2, 翟丽娜 2, 孟慧琴 2, 梁成珠 (1., ; 2., ) 1, 2* 摘要 : 目的, --(gas chromatography-combustion-isotope ratio mass spectrometry, GC-C-IRMS), - 方法样, C 18, GC-C-IRMS 结果 δ 13 C , δ 13 C , 52.9%, 22.7 μg/kg, 5%(n=6) 结论 δ 13 C,, 22.7 μg/kg 关键词 : ; ; -- Determination of δ 13 C of progesterone in milk powder by gas chromatography-combustion-isotope ratio mass spectrometry WANG Han-Qing 1, LV Ning 2, ZHANG Hong-Wei 2, HAO Peng-Fei 2, ZHAI Li-NA 2, MENG Hui-Qin 2, LIANG Cheng-Zhu 1,2* (1. College of Food Science and Engineering, Ocean University of China, Qingdao , China; 2. Technical Center for Inspection & Quarantine of SDCIQ, Qingdao , China) ABSTRACT: Objective Based on the separation and purification of high performance liquid chromatography (HPLC), gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was established for determining the stable carbon isotope ratio of progesterone in milk powder, and gas chromatograph mass spectrometry (GC-MS) was established for determining the residues of progesterone in milk powder. Methods The samples were dissolved in water and extracted with acetonitrile. Then the lipid substances were removed by liquid-liquid extraction with acetonitrile saturated n-hexane. The purification was carried out on a C 18 solid phase extraction (SPE) column. The extracts were purified and concentrated by HPLC and detected by GC-C-IRMS. Results Average δ 13 C value of endogenous progesterone was and average δ 13 C value of exogenous progesterone was The recovery was 52.9%, the limit of detection was 22.7 μg/kg and the relative standard deviations (RSDs) were less than 5% (n=6). Conclusion There is a significant difference in δ 13 C value between endogenous and exogenous progesterone, and the method can distinguish endogenous and exogenous progesterone. However, only when the 基金项目 : (2014IK088) Fund: Supported by Science and Technology Project of National Bureau of Quality Inspection(2014IK088) * 通讯作者 :,, liangcz@163.com *Corresponding author: LIANG Cheng-Zhu, Professor, Technical Center For Inspection & Quarantine of SDCIQ, Qingdao , China. liangcz@163.com

2 residues of progesterone in powdered milk are more than 22.7 μg/kg, it can be detected by GC-C-IRMS. KEY WORDS: progesterone; milk powder; gas chromatography-combustion-isotope ratio mass spectrometry 1 引言,,, 176,, 235 [1], [2],,, [3], [4],,,,, [5,6], - (gas chromatography-mass spectrometry, GC-MS) [7-11] (liquid chromatographytandem mass spectrometry, LC-MS/MS) [12-14],,,, ( 13 C/ 12 C),, (isotope ratio mass spectrometry, IRMS) IRMS 3, (elemental analyzer, EA), CO 2, CO 2 13 C 12 C,, CO 2, CO 2 13 C 12 C, CO 2,,,, - -(gas chromatograph-combustion- isotope ratio mass spectrometry, GC-C-IRMS), -(gas chromatography-mass spectrometry, GC-MS), GC-MS GC-C-IRMS GC-C-IRMS, (high performance liquid chromatography, HPLC), GC-C-IRMS 2 材料与方法 2.1 仪器与试剂 Trace GC Ultra ( Thermo Fisher ); Al 3000 Trace GC Ultra ( Thermo Fisher ); ConFlo IV ( Thermo Fisher ); Delta V ( Thermo Fisher ); Precision CIR 19 ( Thermo Fisher ); Cerex 48 ( Cerex ); CR22GIII ( Hitachi ); Reax control ( Biocote ); C 18 (2 g, 6 ml, Agilent ) (, Merck ); ( 99.8%, Sigma ); ( 99 %, Dr. Ehrenstorfer GmbH ); Milli-Q ( Millipore ) 2.2 试验方法 溶液配制 : 10 mg( 0.1 mg), μg/ml, -20,, :,,,, GC-MS 样品前处理方法 (1) 5 g ( 0.1 g), 50 ml, 10 ml 1 min, 20 ml, 4 g, 10 min, r/min 10 min, 3 ml, (2) 3 ml, 1 min,,, 1 C 18 -SPE

3 5, : -- δ 13 C 1819 (5 ml 5 ml C 18 -SPE ), 5 ml 10%(V:V),, 10 ml SPE, 40, 1 ml, 0.22 μm, (3) 40, 0.1 ml, 30 μl, 1 min, min, 40, 1 ml, GC-MS GC-MS 条件 (1) : HP-5MS (30 m 0.25 mm, 0.25 μm); : He, 1.00 ml/min, ; :, : 1 μl; : 300 ; : 80 2 min, 40 /min 272, 2 min, 2 /min 280, 5 min (2) : (electron ionization, EI); : 290 ; : 230 ; : 150 ; -: 280 ; : 70 ev; (SCAN) (selected ion monitoring, SIM)SIM : m/z 147, 293, 425, 510, m/z GC-C-IRMS 样品前处理方法 (1) 10 5 g ( 0.1 g), 2.2.2, (2) 0.1 ml, HPLC (3) HPLC ml, 40, 1 ml, 1 min, 0.22 μm, 2 ml, 40, 0.1 ml, GC-C-IRMS GC-C-IRMS 条件 (1) (2)-, 1030 ; min, 1030, 200 s; δ 13 C, CO 2 ; m/z 44, 45, 46 ; HPLC 条件 : ZORBAX SB-C 18 (4.6 mm 250 mm, 5 μm); : 30 ; : A, B ; : 60%A, 1 ml/min; 100 μl; : (photodiode array detector, DAD ), : 242 nm 3 结果与分析 3.1 萃取溶剂的选择,,,,, 20 g, 40 ml, min,, 90, 3, 2, 3.2 GC-MS 衍生条件优化,,,, GC-MS,, (heptafluorobutyric anhydride, HFBA) [15],, 1 1 [15] Fig. 1 Derivatization reaction of progesterone [15], 100 ng 100 μl 30 μl, 80.7, 60 70, 2, 20 min,, 70, 40 min 3.3 净化条件优化,, Captiva ND

4 Captiva ND, 5 g ( 0.1 g), 50 ml, 10 ml 1 min, 20 ml, 4 g, 10 min, r/min 10 min, 3 ml, 3 ml, 1 min,,, [1] Captiva ND 3 ml Captiva ND, [16] 2 AB, 6, A, B Captiva ND, 2.2.2, 3, A 74 %, B 59%, 2 Fig. 2 Optimization of derivatization reaction,,,, Y=1.974X-3.527, , 2~100 ng/ml, S/N=3 (limit of detection, LOD), (S/N)=10 (limit of quantitation, LOQ), LOD 0.61 μg/kg, LOQ 1.98 μg/kg GC-MS 方法回收率及精密度 3, ng/ml, , 72.4%~101.4%, 4.5%~7.9%, 3.5 HPLC 结果分析 色谱条件优化, C 18 C 8,, C 18, ZORBAX SB-C 18, 40%50%60%70%80%, 60%,,, 60%,, 60% ml/min, 3, 0.8 ml/min,, 50 ml, 1.2 ml/min, 1 ml/min HPLC 的测定及馏分收集时间的确定 GC-C-IRMS 0.1 ml, HPLC, 4 5, 4, 5, 9.8 min,, 9.5~11.5 min Fig (n=6) Comparison of two methods for lipids removal (n=6) 3.4 GC-MS 结果分析 GC-MS 线性关系及检出限 ng/ml, , Fig. 4 4 Chromatogram of progesterone standard

5 5, : -- δ 13 C δ C C/ C - C C 样品 C C PDB PDB GC-C-IRMS, HPLC HPLC, GC-C-IRMS δ 13 C GC 5 Fig. 5 Chromatogram of milk powder sample spiked with progesterone 3.6 GC-C-IRMS 结果分析 GC-C-IRMS 样品测定 δ, δ PDB(pee dee belemnite):, CO 2, CO 2, CO 2 (IAEA-600, δ 13 C= ), m/z=444546, m/z=45 13 C, m/z=44 12 CGC-C-IRMS δ 13 C, 13 C 12 C 6 δ 13 C, 6 7, δ 13 C 1 Fig. 6 6 GC-C-IRMS mass spectrogram of progesterone standard Fig. 7 7 GC-C-IRMS mass spectrogram of progesterone in milk powder sample 表 1 孕酮标准品和奶粉中孕酮在同位素质谱的保留时间 δ 13 C 值和相对标准偏差 (n=6) Table 1 Retention time, δ 13 C value and RSD of progesterone standard and progesterone in milk powder sample by GC-C-IRMS (n=6) /s δ 13 C / (RSD)/%

6 GC-C-IRMS 精密度 准确性 () 10 δ 13 C, 1, 6, 2, 6, SPSS, 2,, GC-C-IRMS, 50 μg/ml (6 ) δ 13 C, 0.5%, GC-C-IRMS GC-C-IRMS 检出限 GC-MS 83.4%, GC-C-IRMS GC-MS, GC-C-IRMS HPLC,,, GC-C-IRMS, GC-MS, 6, GC-C-IRMS 52.9% Delta V δ 13 C,, 1~10 μg/ml, δ 13 C, 6 μg/ml, 6 μg/ml GC-C-IRMS δ 13 C, 6 μg/ml( 12 ng) GC-C-IRMS, 22.7 μg/kg, 22.7 μg/kg GC-C-IRMS δ 13 C, 22.7 μg/kg GC-C-IRMS δ 13 C 3.7 小结,, C 18 -SPE,, GC-C-IRMS GC-MS GC-MS,,,,, GC-MS GC-C-IRMS,, GC-MS GC-C-IRMS GC-MS, GC-MS HPLC, HPLC, 60%, ZORBAX SB-C 18, 1 ml/min,,,,,,, (elemental analyzer, EA) (gas chromatography, GC)(liquid chromatography, LC), CO 2,, LC-IRMS δ 13 C, LC,,, LC-IRMS GC-C-IRMS GC-MS GC-C-IRMS, 2, GC-MS ng/ml, Delta V, 6 ng/μl GC-C-IRMS GC-MS, GC-C-IRMS GC-MS, GC-C-IRMS,,,, GC-C-IRMS 4 结论 HPLC GC-C-IRMS δ 13 C GC-C-IRMS, GC-MS GC-C-IRMS, GC-MS, 22.7 μg/kg GC-C-IRMS, 22.7 μg/kg GC-C-IRMS GC-C-IRMS, ,, 20 μg/kg,,, 参考文献 [1],,, [J]., 2012, (11): Luo HT, Huang XL, Wu HQ, et al. Simultaneous determination of 14 estrogens and progesterones residues in dairy by liquid chromatographytandem mass spectrometry [J]. Chin J Anal Lab, 2012, (11): [2],,, [J]., 2010, 28(11):

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