15
Figure 5 shows example calibration lines for melamine (positive polarity) and cyanuric acid (negative polarity). All calibration lines had r-values of greater than 0.998.
Note that the spiked matrix contained traces (< 10µg/kg) of cyanuric acid and the calibration line does not go through zero.
Figure 2. LC-MS/MS chromatogram of 2g/kg dicyanamide spiked into milk with a concentration of 0.01ng/mL in the final extract after 200x dilution
Figure 3 shows calibration lines for dicyandiamide spiked into milk, extracted using the described procedure with a total dilution factor of 200x. Extensive dilution is recommended to accurately quantify the target analyte in matrix samples to minimise possible ion suppression effects which cannot be compensated using an internal standard.
Coefficients of regression were determined to be greater than 0.997 for both transitions.
Figure 5. Calibration lines for melamine and cyanuric acid spiked into milk and analysed after 200x dilution
Milk samples were analysed using the developed method and tested positive for dicyandiamide. The ‘Multicomponent’ query was used to automatically calculate ratio of quantifier and qualifier ion for identification (Figure 6).
Figure 3. Calibration lines for dicyanamide spiked into milk and analysed after 200x dilution
The MRM ratios calculated across the dynamic range for identification were found well in between the expected 25% tolerance [9] of the standard ratio of 0.392. The MRM ratios were automatically calculated and reported using the ‘Multicomponent’ query in the MultiQuant™ software.
In a second step the method was extended to also detect other known potential adulterants. An example chromatogram is shown in Figure 4.
Dicyandiamide (retention time, RT=2.0 min), melamine (RT=4.6 min), ammeline (RT=4.7 min), ammelide (RT=4.8 min) were detected in positive polarity and cyanuric acid (RT=2.1 min) in negative polarity. The fast polarity switching of the QTRAP® dicyandiamide and cyanuric acid in a single run.
5500 system was used to detect
Figure 6. Milk samples tested positive for dicyandiamide, the ‘Multicomponent’ query was used to automatically calculate MRM ratios for compound identification
Summary
The method and data presented here showcase the fast, easy, and accurate solutions for the analysis of dicyandiamide and other nitrogen rich compounds in milk and other protein rich foods by LC-MS/MS. The AB Sciex QTRAP®
5500 systems provide excellent sensitivity and
selectivity for this analysis, with minimal sample preparation allowing maximised throughput for the analysis of many samples in a short time period.
Dicyandiamide was quantified in milk samples. Automatic MRM ratio calculation in MultiQuant™ software was used for compound identification.
References
1. C.A. Brown et al.: ‘Outbreaks of Renal Failure Associated with Melamine and Cyanuric Acid in Dogs and Cats in 2004 and 2007’ J. Vet. Diagn. Invest. 19 (2007) 525-531
2. H. Xin and R. Stone: ‘Tainted Milk Scandal. Chinese Probe Unmasks High-Tech Adulteration with Melamine’ Science 322 (2008) 1310-1311
3. Y.C. Tyan et al.: ‘Melamine Contamination’ Bioanal. Chem. 395 (2009) 729-735
4. S. MacMahon et al.: ‘A Liquid Chromatography–Tandem Mass Spectrometry Method for the Detection of Economically Motivated Adulteration in Protein-containing Foods’ J. Chromatogr. A 1220 (2012) 101-107
5. S. Turnipseed: ‘Determination of Melamine and Cyanuric Acid Residues in Infant Formula using LC- MS/MS’ FDA LIB 4421 (2008) 1-18
6. M. Smoker and A.J. Krynitsky: ‘Melamine and Cyanuric Acid Residues in Foods’ FDA LIB 4422 (2008) 1-28
7. T. Sakuma et al.: ‘A New, Fast and Sensitive LC-MS/MS Method for the Accurate Quantitation and Identification of Melamine and Cyanuric Acid in Pet Food Samples’ Application Note AB SCIEX (2010) # 1283110-01
8. E. Braekevelt et al.: ‘Determination of Melamine, Ammeline, Ammelide and Cyanuric Acid in Infant Formula Purchased in Canada by Liquid Chromatography-Mass Spectrometry’ Food Additives & Contaminants Part A Chem. Anal. Control
Expo. Risk Assess. 28 (2011) 698-704
Figure 4. Quantitation of five potential adulterants (non-protein nitrogen sources) in a single run using fast polarity switching with the AB Sciex QTRAP®
5500 system
9. Document N° SANCO/12495/2011 ‘Method Validation and Quality Control Procedure for Pesticide Residues Analysis in Food and Feed’ (2011)
INTERNATIONAL LABMATE - APRIL 2013
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