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ZIC®-HILIC column for rapid detection of dicyandiamide in infant milk powder
Merck Millipore Application Lab2013-01-29
1 Introduction
In January 2013, New Zealand milk and milk products were detected to contain low levels of the toxic substance Dicyanodiamide (DCD), and a ban on the sale of milk products containing DCD has been ordered. Eighty percent of China ’s imported milk powder comes from New Zealand, and more than 60% of the world ’s milk is produced in New Zealand. New Zealand dairy farmers usually spray dicyandiamide on pastures to prevent the by-product nitrate in fertilizers from flowing into rivers and lakes.
Dicyandiamide
At present, there is no corresponding food content standard and testing standard. Merck Millipore is committed to the development of analytical methods to provide customers with simple and fast solutions. Dicyandiamide is highly polar and easily soluble in water. It is easily ionized under neutral and acidic conditions. It is suitable for separation by the Zwitterionic Hydrophilic (ZIC®-HILIC) chromatography column of Merck Millipore. At the same time, dicyandiamide has strong ultraviolet absorption at 218nm, which can be quickly analyzed by HPLC-UV conditions using ZIC®-HILIC column, or trace detection can be performed by LC-MS / MS.
2 Instruments and reagents
2.1 Instruments
2.1.1 Column: ZIC®-HILIC, PEEK HPLC Column 250 x 4.6 mm, 5 μm, 200 Å (1.50458.0001)
2.1.2 HPLC: Agilent 1260HPLC (DAD detector)
2.1.3 Transferpette®S single channel pipette, Digital adjustable 500-5000ul (Brand: BR704782)
2.1.4 Microporous membrane: 0.45um, PTFE (Millipore: SLFHX13NL)
2.1.5 Pure water system: Milli-Q3 (Millipore)
2.1 Instruments
2.1.1 Column: ZIC®-HILIC, PEEK HPLC Column 250 x 4.6 mm, 5 μm, 200 Å (1.50458.0001)
2.1.2 HPLC: Agilent 1260HPLC (DAD detector)
2.1.3 Transferpette®S single channel pipette, Digital adjustable 500-5000ul (Brand: BR704782)
2.1.4 Microporous membrane: 0.45um, PTFE (Millipore: SLFHX13NL)
2.1.5 Pure water system: Milli-Q3 (Millipore)
2.2 Reagent
2.2.1 Chromatography acetonitrile: LichroSolv® Acetonitrile Gradient Grade (1.00030.4008)
2.2.2 Ammonium acetate: Ammonium acetate for analysis EMSURE® ACS, Reag. Ph Eur (1.01116.1000)
2.2.3 Formic acid: Formic acid 98-100% for analysis EMSURE® ACS, Reag. Ph Eur (1.00264.1000)
2.2.4 2% formic acid solution: take 20ml of formic acid and add it to 1000ml of ultrapure water
2.2.5 70/30 acetonitrile-formic acid solution: remove 700ml of acetonitrile (2.2.1) and mix with 300ml of formic acid solution (2.2.4)
3 Experimental methods
3.1 Sample handling
method 1:
Take 2g of infant formula milk powder in a 50mL PP tube, add 18mL of 2% formic acid aqueous solution, and seal with a lid. Shake for 60 seconds, sonicate for another 30 minutes, remove, shake for another 60 seconds, and centrifuge (4000 rpm) in a centrifuge for 20 minutes. Take out, absorb 50ul of supernatant, add 950ul of acetonitrile in a 10ml PP tube, seal with a lid, shake for 60 seconds, aspirate the solution and filter to obtain the test solution.
Method 2:
Take 2g of infant formula milk powder in a 50mL PP tube, add 20mL of acetonitrile-formic acid aqueous solution, and seal with a lid. Shake for 60 seconds, sonicate for another 30 minutes, take it out, shake for 60 seconds, let it stand, absorb the upper solution, and filter to obtain the test solution.
The spiked samples were prepared in the same way.
3.2 Test conditions
3.2.1 Chromatographic conditions
Mobile phase (v / v): A-ammonium acetate solution (10 mM), B-acetonitrile
Proportion *: A: B = 12: 88
Flow rate: 1 mL / min
Injection volume: 20ul
3.3 Test results
According to Method 1, the results are as follows:
3.3.1 Map of reference substance
(DCD concentration 1.25ug / ml)
The retention time of dicyandiamide is 4.98min, the number of theoretical plates is 13086, and the tailing factor is 1.2.
3.3.2 No milk powder detected
3.3.3 Milk powder labeling
The concentration of the standard sample added in the test solution: 5ug / ml
The concentration of the standard sample added in the test solution: 0.05ug / ml.
3.3.4 A well-known brand milk powder
No dicyandiamide was detected in the brand's milk powder.
4 Conclusion
The ZIC®-HILIC column can be used for rapid detection of dicyandiamide. The processing method is simple, the chromatographic peak shape is good, the method reproducibility is good (RSD <2%), the current relationship between the detection concentration of 0.5-10ug / ml is good (R = 0.999), the recovery rate of the sample is 95.6-101.5% between. According to the method 1, the LOD is 1.3ug / g milk powder. According to the method 2, the LOD can reach 0.07ug / g.
The column can also be used for the simultaneous detection of melamine, dicyandiamide, cyanuric acid, metformin, urea, etc., such as:
In addition, LC-MS / MS uses the following chromatographic conditions:
Column: ZIC®-HILIC (5 µm, 200Å) PEEK 150x2.1 mm
Mobile phase: A: 95: 5 ACN: 0.1% formic acid / 10 mM ammonium formate in H2O
B: 50:50 ACN: 0.1% formic acid / 10 mM ammonium formate in H2O
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