三聚氰胺检测方法 GC-MS法
(Adapted from FDA/ORA Forensic
目的:
本程序规定了针对麦麸和动物食品中三聚氰胺成分检测的样品制备和气相色谱-质谱联用（GC-MS）测定方法。 This procedure is designed to screen the TMS derivative of a methanol extract of the sample and it incorporates instrumental parameters which permit 10.5 minute run times.
范围:
本程序适用于干麦麸和动物食品。本程序包括从甲醇中提取可溶于或部分可溶于甲醇的化合物。由于三聚氰胺在甲醇中溶解度有限, 因此本方法只作为定性分析用.
职责:
分析者有责任注意到本程序的任何更改.
专有名词定义:
TMS--三甲基硅烷(貌似?)
安全措施:
工作环境含有化学药品和高压气体，使用一般公认的安全措施.
设备:
Agilent安杰伦 5975型 GC-MS系统，
试剂:
甲醇: HPLC 色谱纯级
Pyridine(嘧啶): 经A.C.S. 检定
BSTFA with 1% TMCS: bis(trimethylsilyl)trifluoroacetamide with 1%
Trimethylchlorosilane（三甲基氯硅烷） (e.g. Sylon BFT, Supelco)
Trimethylchlorosilane（三甲基氯硅烷） (e.g. Sylon BFT, Supelco)
质控原理:
A blank should be run at the onset of each analysis and then randomly throughout the analysis if there is suspicion of carryover. A fortified sample (see Part C) should be analyzed with each set of samples to demonstrate effective system performance.
程序 (程序细节):
本方法使用GC程序不分流进样或分流进样（1：20）中任一种。 通常，为了获得必须的检测灵敏度而采用不分流进样模式。The split mode is suitable where sample availability is not limited and the recommended amount can be prepared (~
A. 甲醇萃取:
未知的麦麸/动物食品样品：称取大约
B. Trimethylsilyl (TMS) 衍生:
Melamine is detected as the 3-TMS derivative with a nominal molecular weight of 342 amu. 3-TMS 衍生物在GC-MS谱图上，保留时间大约在7.1分钟 (见附图).
空白对照: 移取40μL 甲醇萃取液到自动进样器瓶中，
已知样品: 一般情况下，进行已知浓度物品的分析 (比如，标准品), 需用pyridine/BSTFA适当的稀释到浓度不大于100 μg/ml（100 ppm），计算好稀释度后，转移适当量的标准溶液到自动取样器瓶中，
未知样品: 移取40μL 步骤A中得到的甲醇萃取液到自动进样器瓶中，
C. Sample Fortification:
本方法成功应用于检出湿/干动物食品合成物 (图 2) 和麦麸(图 3)中含有的三聚氰胺。基于HPLC-UV的结果, 不分流进样时，本方法能检出低于0.01%(重量)水平的三聚氰胺。（FDA官方样品，using ~
Uncontaminated wheat gluten and pet food composites were spiked with various levels of melamine and taken through the method using ~
大意：检测定性离子为342，327，171，99
D. 标准品准备:
三聚氰胺能溶于甲醇，微溶于甲醇/水混合液。在超声30 分钟条件下，三聚氰胺标准品在甲醇/水（50/50）混合液中能溶解至1.5mg/ml。 如果想得到更高浓度的三聚氰胺溶液, dimethyl sulfoxide (DMSO，二甲基亚枫) may be used as a primary solvent followed by methanol or acetonitrile for serial dilutions.
Note: Due to the presence of water in the standard solution, dry down times for the melamine standard on the Reacti-Vap will be increased.
E. 仪器参数:
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仪器：
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Agilent GC 6890N + 5975质谱 带7683B 系列自动取样器
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检测器：
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Agilent Mass Selective Detector (MSD) model 5975i
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软件：
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Enhanced Chemstation version D.02.00.275; Wiley 7th Edition and NIST 02 MS Library
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柱子：
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J&W DB-5 聚苯基甲基硅氧烷（5%苯基）
内径：
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载气：
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氦
流速：1.3 ml/cm.
线速度：
压力： 17.5 P
模式：恒流
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进样口：
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进样方式：不分流或分流(1:20)
进样量：1μl
进样口温度:
隔垫吹扫气流：10ml/min，2min
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GC参数：
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开始温度:
升温速率：
最终温度:
转移线温度：
程序时间：10.5 分钟
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MS参数：
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Scan Mode: Full (Electron Ionization) Filament Delay: 4.2 min (splitless); 3.8 min (split) Threshold: 100 MS Quad: 150ºC MS Source: 230ºC |
F. 峰鉴定:
The mass spectrum of the 3-TMS derivative of melamine (Figure 1) exhibits the molecular ion at m/z=342, a methyl group loss at m/z=327, and significant ions at m/z=171, 99, and 73. The standard of melamine should be prepared (and TMS-derivatized) and analyzed along with the sample preps on the same day to confirm the identification. Figures 2a and 2b are examples of a GC-MS chromatogram of melamine contaminated pet food. Additional peaks present in the TIC are consistent withTMSderivatives of phosphate and various sugars. Figure 3 is an example of a GC-MS chromatogram of melamine contaminated wheat gluten.
Updated FCC Developmental Melamine Quantitation (HPLC-UV)
April 2, 2007
样品取样/制备:
麦麸:Retsch ZM 100 离心粉碎 (
湿的动物食品: Moist chunks and gravy were blended to the consistency of a gritty pudding prior to sampling.
麦麸:Retsch ZM 100 离心粉碎 (
湿的动物食品: Moist chunks and gravy were blended to the consistency of a gritty pudding prior to sampling.
样品称重于玻璃管中后，使用 50:50 乙腈:水溶液进行萃取.
程序: 增加指出体积的抽出溶剂 (见到下面的笔记) 1. 无边帽和旋涡彻底地, 由于这一个步骤变得攻击性 (紧要关头的适当减慢三聚氰胺的分解率). Sonicate 30 分钟. 经过 2 阶段的 GMF 的榨出物的过滤器部分-尼龙 (
1 小麦麸质: 在比例方面吸取对 10 毫升的
1 潮湿的伴侣动物饲料: 在比例方面吸取对 10 毫升的 2.0
2 对于最后的稀释是有偿付能力的可能是水或 0.1 N HCl. Final dilution is necessary for compatibility with ion-pairing chromatography. We have observed some differences in behavior between the wheat gluten and pet food samples with respect to maintaining solubility during final dilution (these are most likely matrix components which fall out). 0.1 N HCl seems to help maintain solubility for final dilution of the wheat glutens.
HPLC-UV 工作条件:
色谱柱: Zorbax Rx C8 (retention is too high on C18 column)
Buffer:
流动相: 85:15乙腈缓冲液
流速: 1.0 ml/min.
进样体积: 10 ml
柱温: 40 oC (column thermostatting is necessary for ion-pair separations)
检测波长: 240 nm
收集波长: 200 – 400 nm (look for lmax near 236 nm)
保留时间: 4.2 - 4.3 min.
运行时间: 10 min.
色谱柱: Zorbax Rx C8 (retention is too high on C18 column)
Buffer:
流动相: 85:15乙腈缓冲液
流速: 1.0 ml/min.
进样体积: 10 ml
柱温: 40 oC (column thermostatting is necessary for ion-pair separations)
检测波长: 240 nm
收集波长: 200 – 400 nm (look for lmax near 236 nm)
保留时间: 4.2 - 4.3 min.
运行时间: 10 min.
Standard Preparation:
Stock standard was prepared in 76:24 acetonitrile: water. A check stock standard was prepared in 60:40 acetonitrile: water. Dilutions were made in either water or 0.1N HCl giving equivalent calibration curves.
Stock standard was prepared in 76:24 acetonitrile: water. A check stock standard was prepared in 60:40 acetonitrile: water. Dilutions were made in either water or 0.1N HCl giving equivalent calibration curves.
Figures of Merit:
Linear range: established from 1.0 – 400 mg/ml; calibration range 1.0 – 200 mg/ml used for most of work
Reproducibility: duplicate preparations agree within 0.1 - 5% relative basis
Spike/recovery (based on spiking solid melamine powder into test matrix prior to extraction): 90 -110%.
Linear range: established from 1.0 – 400 mg/ml; calibration range 1.0 – 200 mg/ml used for most of work
Reproducibility: duplicate preparations agree within 0.1 - 5% relative basis
Spike/recovery (based on spiking solid melamine powder into test matrix prior to extraction): 90 -110%.
