主题：【讨论】三聚氰胺检测结果

谨慎是必要的，这些结果关系到千家万户，关系到企业声誉与生存。
但明知不合格还掩盖事情的真相就是不可饶恕的。

楼主的前处理及仪器条件方法可否告知？下周我也想做一下我家现在有的几种奶粉。多谢

原文由 watericecat 发表:
楼主的前处理及仪器条件方法可否告知？下周我也想做一下我家现在有的几种奶粉。多谢

这是我们公司几年前做的一篇资料，已经和美国FDA申请标准方法，正在审核中，具体见下和附件


一种同时测定三聚氰胺和三聚氰酸的方法
          －LC-MS及戴安Acclaim混合基质柱WAX-1
Leo Wang, Stacy M. Henday, Xiaodong Liu, Mark Tracy, and William C. Schnute, Dionex Corporation, Sunnyvale, CA, USA


摘要
由于宠物食品被三聚氰胺和三聚氰酸污染，当三聚氰胺和三聚氰酸同时存在时会形成一种不溶结晶物，这种物质可能影响到肾脏功能，最近的研究发现很多宠物的的死亡和健康问题是由此引起的，受污染的麦子，稻米和玉米可能被制成人类食物例如面包，意面，婴儿食品等。所以对原材料包括可疑性动物组织中三聚氰胺和三聚氰酸的监测就十分重要。
现在对三聚氰胺和三聚氰酸的定量方法包括气质（GC-MS）和液质（LC-MS）。GC-MS的缺点是需要进行费时费力衍生化，而报道的LC-MS方法需要很长时间的梯度运行及柱子清洗。
本文介绍一种灵敏、简单和高通量的LC-MS方法，使用稳定的同位素标记的内标物（ISTD）来定量，可同时测定三聚氰胺和三聚氰酸。。
使用戴安公司最新开发的混合基质柱WAX-1来分离被分析物。此柱子利用疏水和离子交换混合作用机理来达到特殊的选择性和保留。色谱运行时间仅用8分钟，MSQ质谱检测器确保选择性和灵敏度。对于两种物质（2-200 ng/ml）的线性都能达到R2>0.9995。方法检出限（MDL），三聚氰胺为3.97 ng/ml，三聚氰酸为3.32 ng/ml。
通过宠物食品提取物以及生物基质：猪肉和鱼肉组织的提取物来验证方法的可靠性。宠物食品用快速、简单和自动的提取方法处理。生物样品来自美国FDA，并按规定方法处理。

仪器
戴安Summit HPLC系统：
P680 泵
ASI-100　自动进样器
TCC-100 柱温箱
UVD-340 UV-Vis　检测器

戴安MSQ　Plus单四极质谱检测器，ESI源
Chromeleon 6.8色谱管理软件

色谱条件
分析柱：戴安公司Acclaim 混合基质柱WAX-1（150×2.1mm,5um）
流速：等度90%乙腈/10% 20mM pH4 醋酸铵缓冲液（v/v）, 0.25 ml/min
柱温：20℃
进样量：5ul

MSQ质谱条件
分析模式：SIM带极性切换
锥孔电压：50 V
间隔时间：0.5 S，对于所有SIM通道
探针温度：500℃
扫描：4 SIM全程扫描，两个不同极性的全扫描（100-400 m/z,250 ms），见表1。

方法开发
色谱优化
流动相组成：在HILIC模式下，高有机物浓度的流动相容易使极性物质保留更长，并在ESI条件的MS上有更大的响应。然而，在WAX-1柱上的分离由于有弱阳离子的交换，对流动相中离子强度和PH的改变很敏感。为了保持方法的重现性，需要合适的缓冲容量，所以选择10% pH4的醋酸铵。

缓冲液pH
当三聚氰胺和三聚氰酸同时存在于不同ph条件的不同基质（有机、离子或混合物）中，越是在疏水的环境下保留时间越长。这可以用离子交换机理来解释。pH对保留的影响效果可见图2。

离子强度
戴安的WAX-1混合机制柱由于弱阳离子交换模式能为极性物质提供特殊的选择性    。极性越大的化合物的流动相离子强度的依赖越大。如图3所示，通过将流动相的总离子强度从2.75mM增加到11.0mM，三聚氰酸的保留时间可以从12.42分钟减少到7.40分钟。
三个色谱因子的综合考虑可以为方法优化提供更大的适应性，并能控制三聚氰酸的保留时间，大概在6到15分钟的范围。

质谱检测优化
质谱检测的优化和色谱的优化相结合。图4是最优化分离度、灵敏度和高通量方法的校准标准色谱图。


方法验证
线性和校正曲线
美国FDA的校正标准，稳定的元素标记物（三聚氰胺－15N3和三聚氰酸－13C3）作为内标物（ITSD）。这些标准物用来做标准曲线，计算线性。从2ng/ml到200ng/ml的范围内的线性非常好，可见图5，图6。

方法检测限（MDL）
    方法检测限通过对校正标准物7次重复进样，以及下面方程式来计算：
　　　MDL=tgg% ×　S｛n=7｝   
t在99%的置信区间（tgg%，n=7=3.143）,S为标准偏差。三聚氰胺和三聚氰酸的MDL分别为3.97 ng/ml和3.92 ng/ml。

生物样品的方法验证
色谱图
从美国FDA获得的猪和鱼组织提取物（控制和加标样）用FDA的标准来处理。所有的生物样品挥发干后用流动相重溶。图7是控制样品（空白猪肉样品的提取物，提取前ISTD，三聚氰胺或三聚氰酸加标）的SIM色谱图。

重现性
由于多次进样会对色谱造成影响，未知化合物的积累造成干扰。这些干扰物需要额外计算，例如梯度运行或柱子清洗，因此验证方法的重现性是非常重要的。在这个研究中，通过重复进样三聚氰胺和三聚氰酸加标样品来验证方法重现性。结果见表2。对三聚氰胺的保留时间和量的重现性都非常好。三聚氰酸保留时间的重现性有一点变换，可能是样品处理中残留的甲酸的影响。


结论
一种简单、快速的LC-MS方法可同时检测三聚氰胺和三聚氰酸。
三聚氰胺和三聚氰酸的保留时间和整个分析周期都令人满意。
验证了方法重现性。
质谱检测器的检出限<5 ng/ml。
线性极佳，在2到200 ng/ml的范围内R2>0.9995。
这个方法已用于分析美国FDA处理的生物样品。   

没有液质，那提供给大家气质方法，等会提供液相方法。
GC-MS Screen for the Presence of Melamine, Ammeline, Ammelide and Cyanuric Acid (Version 2.1) 2007This method for Melamine, Ammeline, Ammelide and Cyanuric Acid should be regarded as interim.  Because of the need to rapidly provide this information, the method has not undergone the rigorous internal and external validation required for an official method.  The performance of the method may change when different equipment and supplies are used or when different sample matrices are encountered.  The user should validate the performance of the method in their laboratory and pay particular attention to the recommended quality control elements.  Users should document with their results the version or date of the method used.
PURPOSE:
This procedure was developed by PRLNW and FCC to screen various matrices for the presence of melamine and some related compounds at the established minimum reporting level (MRL) of 10 µg/g and above using gas chromatography/mass spectrometry.  Samples are extracted using a mixture of acetonitrile/water/diethylamine and the analytes are subsequently converted to trimethylsilyl derivatives for analysis. 
SCOPE:
This procedure has been evaluated using dry protein materials (wheat gluten, rice protein, corn gluten, and soy protein), wet and dry pet foods, and dry animal feeds.  It is anticipated that the method will also be applicable to a variety of other matrices. 
RESPONSIBILITY:
It is the responsibility of the analyst to note any modifications to or deviations from this procedure in the documentation associated with the analysis.
DEFINITIONS AND ACRONYMS:
TMS – trimethylsilyl
DEA – diethylamine
SAFETY CONSIDERATIONS:
Accepted safety measures should be employed when working with chemicals and pressurized gases.  It is advisable to work in a fume hood whenever the procedure calls for the use of diethylamine and pyridine.  Use caution with diethylamine as it is a volatile strong base and can cause chemical burns.
EQUIPMENT AND SUPPLIES:
1.    Agilent 5975i GC-MS system equipped with a 30 m Agilent DB5-MS capillary column (or equivalent) or other mass spectrometer system. 
2.    Laboratory Centrifuge capable of applying 5000 g to 50 mL centrifuge tubes.
3.    Pierce Reacti-therm / Reacti-Vap Sample Preparation System or other device suitable for evaporating solutions to dryness.
4.    50-mL polypropylene centrifuge tubes with screw caps. 
REAGENTS AND STANDARDS:
1.    Diethylamine (DEA), SigmaUltra, Sigma Chemical Co.
2.    Pyridine, Certified A.C.S. Reagent, Fisher Scientific
3.    Extraction Solvent:  10 / 40 / 50 : DEA / Water / Acetonitrile
Prepare a solution which consists of 10 parts (by volume) diethylamine, 40 parts water and 50 parts acetonitrile.  Store in the dark.  The solution turns yellow with time.
4.    Silylating Reagent:  BSTFA with 1% TMCS: bis(trimethylsilyl)trifluoroacetamide with 1% Trimethylchlorosilane (e.g. Sylon BFT, Supelco)
5.    2,6-Diamino-4-chloropyrimidine, CAS 156-83-2, 98%:  Cat. C33204-5G,  Aldrich 
For use as an internal standard.  Prepare a stock solution at 1.0 mg/mL in pyridine.
For adding to sample extracts, dilute the stock solution in pyridine to a concentration of 5.0 µg/mL.
Use of 2,6-Diamino-4-chloropyrimidine (DACP) as an internal standard is still undergoing evaluation.  It is up to each laboratory to verify that their internal standard does not contain any of the analytes as impurities.  The Aldrich standard has been found to be free of any analyte impurities.
6.    Melamine, CAS 108-78-1, Cat. 240818-5G,  Aldrich
Prepare a stock solution of 1.0 mg/mL in a mixture of 20 / 80 : DEA / H2O  (v/v)
7.    Ammelide, CAS 645-93-2, Cat. A0645, TCI America
Prepare a stock solution of 1.0 mg/mL in a mixture of 20 / 80 : DEA / H2O  (v/v)
Brief sonication may be required to solubilize this standard.
8.    Ammeline, CAS 645-92-1, Cat. A0676, TCI America
Prepare a stock solution of 1.0 mg/mL in a mixture of 20 / 80 : DEA / H2O  (v/v)
A modest amount of sonication may be required to solubilize this standard. 
9.    Cyanuric Acid,  CAS 108-80-5, Cat. C0459, TCI America
Prepare a stock solution of 1.0 mg/mL in a mixture of 20 / 80 : DEA / H2O  (v/v)
Store stock solutions of standards in the refrigerator to retard hydrolysis.  It has not been established how rapidly the solutions degrade but the potential does exist.  Stock standards have sat on the benchtop at room temperature for three weeks and shown no evidence of degradation. 

继续：
QC ELEMENTS:
A 1:1 (v/v) mixture of Sylon BFT and pyridine should be run at the onset of each analysis and then occasionally throughout the analysis to show that there is no carryover.
A method blank which consists of 20 mL of extraction solvent taken through the entire procedure including the addition of the internal standard should be evaluated to make sure that there is no contamination from the reagents, the nylon filter or the containers.
Condition the system at the beginning of each sample set by making two injections of the high standard (see below).  These injections will not be used for semi-quantitation. 
A low standard, which consists of each analyte at 0.10 µg/mL, should be analyzed at the beginning of the sample set to show that the necessary sensitivity is being attained by the instrument. 
A high standard at 1.0 µg/mL of each analyte is analyzed at the beginning of the sequence and after all samples in the batch have been injected to provide a basis for semi-quantitative evaluation and to demonstrate whether the amount of drift during the analysis of the set of samples is tolerable.
A control sample which is representative of the type of samples which are being analyzed is fortified with each analyte (viz. melamine, ammelide, ammeline and cyanuric acid) at a level of 10 µg/g.  Analysis of this spiked control sample must indicate that the compounds are present which serves to demonstrate effective system performance at the desired reporting level.  See Sample Fortification, below.
PROCEDURE:
This procedure should be used with the gc operating in the splitless mode for greater sensitivity.
A.  Extraction Procedure
Weigh out approximately 0.5 g of a representative portion of the sample into a 50mL polypropylene centrifuge tube.
Add 20 mL of extraction solvent (10:40:50 DEA : H2O : Acetonitrile).
Mix well to thoroughly wet the entire sample.
Sonicate for 30 minutes.
Centrifuge for 10 minutes at 5000 g (or better).
Filter a portion of the supernatant using 0.45µm nylon filter discs (a two-stage or molecular weight cutoff filter may be used for difficult extracts).  For example, a 2 mL filtered portion will allow 200 µL for the derivatization step and additional filtrate in the event that the derivatization needs to be repeated or if further work is necessary.
B.  TMS-Derivatives
Sample Extracts: 
Transfer 200 µL of filtrate from Step A to a gc vial.
Note:  A smaller aliquot may be used provided that the necessary sensitivity level (10 µg/g of sample) is achieved.  Reducing the amount of matrix present improves the general performance of the evaporation/derivatization step and saves wear and tear on the instrument.
Evaporate to dryness at 70°C (a low flow stream of dry air or nitrogen may be used).
Note:  Taking the filtrate completely to dryness is a critical step in the derivatization process.  The presence of water prevents formation of TMS derivatives of the analytes.  If the internal standard response is much lower than usual (less than 30%), there may have been problems associated with the derivatization step.  In addition, if the vial warms significantly to the touch after addition of the derivatization reagents, residual water was present and a new aliquot of filtrate must be prepared.
Add 200 µL pyridine.
Add 200 µL Sylon BFT.
Add 100 µL of the internal standard solution at 5.0 µg/mL in pyridine.  This produces a concentration in the extract of 1.0 µg/mL.  If you are not using the internal standard, then add 100 µL of pyridine.
Shake well or vortex to mix.
Incubate at 70oC for 45 minutes.
Ready for Injection.
             Note:  If insoluble material is observed at the bottom of the vial after 45 minute              incubation, transfer liquid portion to another gc vial or filter before analysis.
C.  Instrument Parameters
gc Conditions:
Column                                         30m DB-5MS 5% phenyl 95% dimethyl-polysiloxane                                                                            
                                          ID: 0.25mm     Film Thickness: 0.25 microns
Inlet Temperature                     280oC
Detector Temperature         290oC
Injection Mode                         Splitless
Injection Volume                       1 µL
Carrier Gas Flow                     He at 35 cm/sec (constant flow)
Oven Program                     75oC (hold 1 minute) to 320oC at 15oC/minute (hold 2.67 min) for a total run time of 20 min.
Note:  Alternate gc conditions may be used provided that adequate resolution is obtained between the target analytes.  Any such deviations from the method must be noted in corresponding documentation.  With small volume liners, some peak splitting has been observed under the above conditions.  Using a higher starting temperature (100oC) alleviated the problem. To help overcome interferences, significant additional resolution may be obtained by decreasing the ramp to 4oC/min over the interval 150oC to 200oC, which will shift the retention times.
MS Conditions (Full Scan Mode):
Tune                                              Autotune (to maximize sensitivity across mass range)
                                          A +306V multiplier bump may be added after Autotuning
Acquisition parameters         EI; scan mode, 50-450 amu
Sampling Rate                        2 (scan rate at ~3.6 scans/sec)
Threshold                                     100
Filament Delay                        6 minutes
MS Temp                                    230°C (Source); 150°C (Quad)
If the sensitivity which is required to detect analyte spikes at 10 µg/g in the matrix cannot be achieved in full scan mode, use selected-ion monitoring (SIM) parameters below.
MS Conditions (SIM Mode):  Select three or four ions to track:  M, M+2, M-15 and another
Group    Start Time a
(min)    
Ions b
        M c    M + 1    M + 2    M - 15    Other    Other
Urea / Biuret from
di- and tri-TMS
derivatives of urea d    6    276
(tri-tms)
204
(di-tms)              189
(di-tms)
261
(tri-tms)    171
    

Cyanuric Acid    9    345
(100)e    346
(30)    347
(14)    330
(33)    188
(11)    
Ammelide    9.7    344
(100)    345
(30)    346
(14)    329
(50)    286
(7)    198
(28)
DACP (internal std.)    10.18    288    289    290    273    275    237
Ammeline    10.5    343
(100)    344
(30)    345
(14)    328
(115)    285
(30)    198
(27)
Melamine    11    342
(54)    343
(16)    344
(8)    327
(100)    285
(12)    197
(13)

a  Start Times may need to be adjusted based upon the retention time of standards on your system.
b  Dwell times should be adjusted to produce a cycle time of about 4 scans/sec
c  M is the Molecular Ion for the tri - TMS derivative of the compound.
d  Urea and Biuret are not formally part of the screen but they are related to the compounds of interest and may be detected
e  Percent relative abundance with respect to the most abundant ion from directly silylated standards under Standard Spectrum Autotune.  The relative abundances should be confirmed under the conditions of use by evaluating standards. 

D.  Peak Identification and Results
The approximate retention times of the tri-TMS derivatives are as follows (minutes):
Cyanuric Acid     9.2
Ammelide        10.0
DACP            10.3  (di-TMS derivative)
Ammeline        10.7
Melamine        11.2
These need to be confirmed on your system and the parameters above adjusted to fit.  Figure 1 is a standard chromatogram showing the cyanuric acid tri-TMS derivative (9.2 minutes), ammelide tri-TMS derivative (10.0 minutes), ammeline tri-TMS derivative (10.7 minutes) and melamine tri-TMS derivative (11.2 minutes).  Figures 2 through 5 represent the mass spectra of each of these compounds.  Figure 6 represents the mass spectrum of 2,6-diamino-4-chloropyrimidine di-TMS derivative.
When full scan mode is used and the matrix is even somewhat complicated, it is usually necessary to examine tracings of individual ions (extracted ion chromatograms) to observe the analytes.  The extracted ion chromatograms may be generated for each analyte (see SIM table above for appropriate ions) which will help simplify the process of confirming positive or negative results (i.e. pulling things out of the grass).  Ideally, each of these analytes and the internal standard can be entered into the data analysis portion of the Chemstation software as a target compound.  By entering the appropriate retention time, ion ratios and peak area for each compound, the software can calculate sample results automatically.  Results can be reviewed using the “QEdit” view which also provides an excellent visual display for documentation of sample results (see Figure 7 for a sample QEdit report for melamine).
In full scan mode, the criteria for identification of target analytes include the agreement of the retention times with those of the standards to within 0.05 minute.  Also, the mass spectra need to correspond to those of the standards with no significant peaks absent.  There may be additional peaks present due to overlap with other components but this should be examined carefully.
In SIM mode, the criteria for identification of target analytes include the agreement of the retention times with those of standards to within 0.05 minute.  Mass spectral confirmation of an analyte is based upon the ratios of the integrated areas for selected ions to the integrated area of the most abundant of the selected ions being tracked.  The criterion is that each ratio (as a percentage) corresponds to that observed for a standard to within 10 units.  Using melamine as an example, track the ions at 342 (M), 327 (M-15) and 343 (M+1), use the data in the table above as representative of a standard.  For a peak nominally identified as melamine based upon retention time, the ratio of the area of peak (at 11.2 min) from m/z = 342 (M) to that of the area of the peak at m/z = 327 (M-15) needs to be between 44% and 64% while that of the ratio of the peak at m/z = 343 (M+1) to that of the area of the peak at m/z = 327 (M-15) needs to be between 6% and 26%. 
E.  Sample Fortification and Mixed Standard Preparation
A Mixed Standard Spiking solution is prepared by mixing portions of the stock standard solutions with 10/40/50 DEA/Water/Acetonitrile to create a solution which is 100 µg/mL of each analyte. 
Spike Preparation
Weigh a 0.5 g portion of the matrix of interest into a 50 mL centrifuge tube.  A previously analyzed, representative “blank” matrix may be used.  If a representative matrix is not available, select one of the samples to be analyzed to use for spiking.
Add 50 µL (Low Spike) of Mixed Standard Spiking solution directly to the representative control sample and proceed with the method.  The Low Spike must be observed to declare negatives as less than 10 µg/g.  If it is not then negative samples will need to be re-analyzed.
A High Spike may also be prepared by adding 250 µL of Mixed Standard Spiking to another 0.5 g portion of the control.  The High Spike provides an additional check in the event the Low Spike is not observed.  The absence of the High Spike would serve as an indication of a major problem either with the sample preparation or the instrument.

High Standard
Dilute the Mixed Standard Spiking Solution to 10 µg/mL.  Place 50 µL of this solution in a vial and take it through the derivatization reaction including the addition of the internal standard.  This produces a High Standard at a concentration of 1.0 µg/mL of each analyte.
Low Standard
Dilute the Mixed Standard Spiking Solution to 1 µg/mL.  Place 50 µL of this solution in a vial and take it through the derivatization reaction including the addition of the internal standard.  This produces a Low Standard at a concentration of 0.10 µg/mL of each analyte.
F. Reporting
In the event that the analytes were observed in the representative control sample that was fortified at 10 µg/g, and no analyte signals were observed in the samples at levels which approach those in the fortified control, then the samples are not contaminated with melamine, ammeline, ammelide or cyanuric acid at levels in excess of 10 µg/g.
When it is close (within 50%), consider preparing additional portions from those samples along with a couple of additional fortified controls.
If it is clear that one or more of the analytes are present in the samples (based on the identification criteria above) and at levels in excess of 10 µg/g, then a semi-quantitative estimate may be obtained by comparing analyte responses to those from standards as described below.
G.  Obtaining a Semi-quantitative Estimate of Target Analytes
Choose an ion to use for estimating the amount of the target analyte (usually the molecular ion).  Using the High Standard  (1.0 µg/mL) which was run at the beginning of the set of samples and the High Standard that was run at the end of the set of samples,  calculate the average peak area for the selected ion.  Apply the following formula:
Concentration in Derivatized Test Sample Extract (µg/mL)  =  Area (Test Sample) / Average Area (Standard)  X  1.0 µg/mL.
Then,  Concentration in Sample (µg/g) =  Concentration in Derivatized Test Sample Extract (µg/mL)  X  0.50 mL  X  20.0 mL / 0.20 mL X  1 / Sample Wt.  (g)
If the internal standard areas are very different (> 20%) between the test sample and the standard then adjust the area of each analyte response by dividing by the integrated area of the internal standard (from its molecular ion).  Use these ratios in place of the integrated areas above.  Consider re-analysis if the difference is extremely large since that may indicate a partially clogged syringe or other instrumental problem.
Finally,  if the signal is more than 25 X larger than the signal from the standard,  then prepare a new extract using 0.25 g of sample and 40 mL of the extraction solvent since the solubility limit for ammeline and ammelide in 10/40/50 DEA/water/acetonitrile is being approached.
As a general rule-of-thumb, the closer the analyte signal from the unknown is to the analyte signal from the standard, the better the estimate of the unknown concentration.  So if more accurate numbers are required, prepare additional standards and re-analyze the samples in question.

奶粉中三聚氰胺的测定1 原理
三聚氰胺是一种三嗪类含氮杂环有机化合物，微溶于水，可溶于甲醇、甲醛、乙酸、热乙二醇、甘油、吡啶等。本方法采用20%甲醇溶剂，应用5%三氯乙酸沉淀蛋白，提取样品中的三聚氰胺，经高效液相色谱仪C18柱分离，检测波长235nm条件下进行检测。
2 试剂 使用优级纯、色谱纯试剂，色谱用水符合GB/T6682一级水的规定。
2.1 20%甲醇溶液：甲醇+水(2+8) 
2.2 5%三氯乙酸溶液：称取三氯乙酸5g溶解于100mL水中。
2.3乙腈
2.4 混合液：称取2.21g柠檬酸和1.88 g己烷磺酸钠，溶于水中，稀释至1000mL，混匀。
2.5流动相：混合液（2.4）+乙腈（2.3）=92+8。
2.6 标准储备液配制：准确称取三聚氰胺标准品0.2000g，用20%甲醇溶液（2.1）定容至100mL。三聚氰胺含量为2.00mg/ mL，于4℃冰箱内保存。
2.7三聚氰胺标准使用液的配制：准确吸取标准储备液(2.6)0.100mL；1.00mL；2.50mL；5.00mL； 10.00mL至100mL容量瓶中，并用20%甲醇溶液（2.1）定容至刻度。配制成三聚氰胺浓度分别为2.00mg/L；20.0mg/L；50.0mg/L；100mg/L；200mg/L的标准系列。
3仪器和设备
3.1高效液相色谱仪：附二级管阵列检测器（DAD）或紫外检测器（UV）
3.2超声波清洗器
3.3超速离心机
3.4涡旋混合器
3.5滤膜：0.45μm
4 分析步骤
4.1试样处理：将奶粉样品混匀，准确称取样品1～5g于50mL比色管中，加入20%甲醇溶液（2.1）溶液约35mL，震荡混匀，超声提取5min，加入5%三氯乙酸(2.2)5mL，并用20%甲醇溶液(2.1)定容至刻度，摇匀。以3000rpm/min超速离心5min。取上清液经0.45μm滤膜过滤，备用。
4.2色谱参考条件
4.2.1流动相：10mmol/L己烷磺酸钠+10mmol/L柠檬酸∶乙腈=92∶8；
4.2.2流速:1.0ml/min；
4.2.3柱温：35℃；
4.2.4检测器：二极管阵列检测器或紫外检测器，波长235nm；
4.2.5色谱柱：C18柱子250mm ×4.6 mm，5μm；
4.2.6进样量：10～20μL
4.3色谱分析：进样10～20μL标准使用液及样品处理液于高效液相色谱仪中，以保留时间及光谱定性，以样品峰面积与标准系列比较定量。
5结果计算
样品中三聚氰胺含量按下列公式进行计算。
 
式中：X—样品中三聚氰胺的含量，单位为毫克每千克（mg/kg）；
C—从曲线上查出样品中三聚氰胺浓度，单位为毫克每升（mg/L）；
V—样品中总体积，单位为毫升（mL）；
m—样品质量，单位为克（g）

6 色谱图

图1三聚氰胺标准高效色谱图（1—三聚氰胺）

图2 三聚氰胺标准品紫外吸收光谱图

图3 奶粉样品三聚氰胺液相色谱图（1-三聚氰胺）

图4 奶粉样品中三聚氰胺标准品紫外吸收光谱图
7精密度
在重复性条件下获得的两个独立测定结果的相对相差不得超过算术平均值的10%。 

非常感谢pingguwu老师和dx_application版友提供的方法，三聚氰胺现在可是热点啊。

缺少衍生化试剂，看单位其它科室有没有先。