AB SCIEX Quantitation and Identification of Dicyandiamide in Milk and ...

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LC-MS/MS Analysis of Emerging Food Contaminants Quantitation and Identification of Dicyandiamide in Milk and other Protein-Rich Foods Fanny Fu1 and André Schreiber2 1 AB SCIEX Taipei (Taiwan), 2AB SCIEX Concord, Ontario (Canada)

Introduction Recent issues with adulteration of food using nitrogen rich compounds to make the protein content of food appear higher than the actual value highlighted the need for both food manufacturers and regulatory agencies to utilize fast and accurate analytical techniques to proactively ensure product safety. In 2007, melamine and cyanuric acid in wheat gluten added to pet food caused renal failure and sickened and killed large numbers of cats and dogs. In 2008, Chinese authorities discovered the adulteration of milk and infant formula with melamine by several Chinese producers. There were hundreds of thousands of victims and six confirmed deaths in China, as 1-4 well as product recalls in many countries. In response to the melamine contamination a large number of analytical methods were developed for the detection of melamine and its analogues, including several published by the United States Food and Drug Administration (FDA) that also targeted 4-8 cyanuric acid. However, the Kjeldahl method, the traditional standard technique for measuring protein content by indirectly measuring the nitrogen content in food, remains the most widespread methodology. As long as protein content in food is not determined directly, economic adulteration with nitrogen rich compounds will continue to be a serious concern. Analytical methods to detect potential adulterants (non-protein nitrogen sources), including amidinourea, ammelide, ammeline, biuret, cyanuric acid, cyromazine, dicyandiamide, melamine, triuret, and urea (Figure 1) have been developed and validated 4, 5 to test milk products and bulk protein. Recently, traces of dicyandiamide were found in milk produced in New Zealand. Milk producers and government agencies moved quickly to reassure there was no risk to health. Here we present a fast, easy, and sensitive LC-MS/MS method for the detection of dicyandiamide and other nitrogen rich compounds in milk and other protein-rich foods with limits of quantitation down to low μg/kg.

Experimental Sample Preparation Simple liquid extraction of food samples was performed using 4 the following procedure : • Add 10 mL of acetonitrile containing 2% formic acid to 1 g of a homogenized sample. • Mix thoroughly and sonicate for 10 minutes. • Centrifuge for 10 minutes. • Transfer an aliquot of 50 μL of the extract into and autosampler vial and dilute with 950 μL acetonitrile resulting in a total dilution factor of 200. Further dilution of the extract might be necessary if the sample is heavily contaminated. LC The target compounds were separated using a normal phase gradient on a Hydrophilic Interaction Chromatography (HILIC) column. LC separation was achieved using the Eksigent ekspert™ ultraLC 100 system with a Phenomenex LUNA HILIC 3u (100 x 2 mm) column with a mobile phase of acetonitrile and water containing 0.1% formic acid and 10 mM ammonium formate at a flow rate of 0.2 mL/min (Table 1). A sample volume of 10 μL was injected.

p1

NH2

N

H2N

OH

OH

N

N

N

HO

NH2

NH

N

N

N

OH

O

N

H2N

O

O

N

N

OH

NH2

N

NH

H2N

N

NH2

O

O

O

O

NH

N

N

NH

NH2

O

CN H2N

NH

H2N

NH2

H2N

NH

NH2

H2N

NH

NH

NH2

H2N

NH

NH2

Figure 1. Potential adulterants (non-protein nitrogen sources), including melamine, cyanuric acid, ammelide, ammeline, cyromazine, dicyandiamide, urea, biuret, triuret, amidinourea, (top left to bottom right)

Table 1. LC gradient used for the separation of dicyandiamide and other potential adulterants Mobile phase A (%): water with 0.1% formic acid and 10 mM ammonium formate

Mobile phase B: 95% actetonitrile with 0.1% formic acid and 10 mM ammonium formate

0.0

0

100

2.0

0

100

2.1

50

50

4.3

50

50

4.4

0

100

10.0

0

100

Time (min)

MS/MS ®

The AB SCIEX QTRAP 5500 was used with the Turbo V™ source and an Electrospray Ionization (ESI) probe. The mass spectrometer was operated in Multiple Reaction Monitoring (MRM) mode using fast switching between negative and positive polarity. Two selective MRM transitions were monitored for each analyte using the ratio of quantifier and qualifier ion for 13 15 identification (Table 2). C3 N3-melamine was used as an internal standard.

Table 2. MRM transitions used for the detection of dicyanamide and other potential adulterants Compound

Polarity

Q1 (amu)

Q3 (amu)

Dicyandiamide 1

positive

85

68

Dicyandiamide 2

positive

85

43

Melamine 1

positive

127

85

Melamine 2

positive

127

68

Cyanuric acid 1

negative

128

42

Cyanuric acid 2

negative

128

85

Ammelide 1

positive

129

86

Ammelide 2

positive

129

70

Ammeline 1

positive

128

86

Ammeline 2

positive

128

69

positive

133

89

13

C314N3Melamine

Results and Discussion First, the limit of detection (LOD) and reproducibility were evaluated using injections of dicyandiamide standards and spiked matrix samples.

LC-MS/MS data was processed using the MultiQuant™ software version 2.1.

p2

The MRM ratios calculated across the dynamic range for identification were found well in between the expected 25% 9 tolerance of the standard ratio of 0.392. The MRM ratios were automatically calculated and reported using the ‘Multicomponent’ query in the MultiQuant™ software.

Figure 2 shows a chromatogram of dicyandiamide spiked into milk at 2 μg/kg with a Signal-to-Noise (S/N) of 54 and 13 for the quantifier and qualifier ion, respectively.

XIC of +MRM (2 pairs): 85.000/68.000 Da ID: Dicyandiamide 1 from Sample 3 (DC 0.01 ppb Matrix) of 20130201-DC.wiff (Turbo Spray), Smoothed,...

Max. 1.1e4 cps.

2.06

1.10e4 1.05e4

In a second step the method was extended to also detect other known potential adulterants. An example chromatogram is shown in Figure 4.

2 μg/kg dicyandiamide in milk (0.01 ng/mL in final extract)

1.00e4 9500.00 9000.00 8500.00 8000.00

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 5500 system was used to detect dicyandiamide and cyanuric acid in a single run.

7500.00 7000.00

Intensity, cps

6500.00 6000.00 5500.00 5000.00 4500.00 4000.00 3500.00 3000.00 2500.00 2000.00

1.60

1500.00 1000.00 XIC of +MRM (9 pairs): 85.000/68.000 Da ID: Dicyandiamide ...

500.00 0.00 0.0

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4 2.6 Time, min

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

4.6

4.8

Max. 1.9e4 cps.

XIC of +MRM (9 pairs): 128.000/86.000 Da ID: Ammeline 1 fr...

2.05

1.9e4 0.2

2000

dicyandiamide

Intensity, cps

Intensity, cps

1.5e4

Figure 2. LC-MS/MS chromatogram of 2 μg/kg dicyanamide spiked into milk with a concentration of 0.01 ng/mL in the final extract after 200x dilution

Max. 2972.1 cps.

4.69 2500

5.0

1.0e4

ammeline

1500 1000

5000.0 500 0.0

0.8

1.0

1.2

1.4

1.6

1.8

2.0 2.2 2.4 2.6 Time, min XIC of +MRM (9 pairs): 127.000/85.000 Da ID: Melamine 1 fro...

2.8

3.0

3.2

3.4

0 2.5 3.0 3.5 4.0 4.5 5.0 Time, min XIC of +MRM (9 pairs): 129.000/86.000 Da ID: Ammelide 1 fr...

3.6

Max. 2.2e4 cps.

4.66

2.2e4

5.5

6.0

6.5 Max. 5073.4 cps.

4.77

5000

2.0e4

0.0

Intensity, cps

ammelide

3000 2000 1000

0 4.5 5.0 5.5 6.0 6.5 3.0 3.5 4.0 4.5 Time, min XIC of -MRM (6 pairs): 128.000/42.000 Da ID: Cyanuric acid 2 from Sample 12 (5mix 1.0ppb (200ppb) neg) of 20130130-M.wiff (Turbo Spray), Smo... 3.0

3.5

4.0

5.0 Time, min

5.5

6.0

6.5

7.0

Max. 1.3e4 cps.

2.05

1.3e4 1.2e4 1.0e4

cyanuric acid

8000.0 6000.0 4000.0

1.44

2000.0 0.0 0.0

Coefficients of regression were determined to be greater than 0.997 for both transitions.

melamine

1.0e4

5000.0

Intensity, cps

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 minimize possible ion suppression effects which cannot be compensated using an internal standard.

Intensity, cps

4000 1.5e4

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2 2.4 Time, min

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

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

6.5e5

6.0e5

5.5e5

dicyandiamide (1 to 40 μg/kg)

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.

5.0e5

4.5e5

4.0e5

Note that the spiked matrix contained traces (< 10 μg/kg) of cyanuric acid and the calibration line does not go through zero.

Area

3.5e5

3.0e5

2.5e5

2.0e5

1.5e5

1.0e5

5.0e4

0.0e0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09 0.10 0.11 Concentration (ng/mL)

0.12

0.13

0.14

0.15

0.16

0.17

0.18

0.19

Figure 3. Calibration lines for dicyanamide spiked into milk and analyzed after 200x dilution

p3

Summary

melamine

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 maximized throughput for the analysis of many samples in a short time period.

cyanuric acid

Figure 5. Calibration lines for melamine and cyanuric acid spiked into milk and analyzed after 200x dilution

Dicyandiamide was quantified in milk samples. Automatic MRM ratio calculation in MultiQuant™ software was used for compound identification.

References 1

Milk samples were analyzed 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).

2

3

4

5

6

7

Figure 6. Milk samples tested positive for dicyandiamide, the ‘Multicomponent’ query was used to automatically calculate MRM ratios for compound identification

8

9

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 H. Xin and R. Stone: ‘Tainted Milk Scandal. Chinese Probe Unmasks High-Tech Adulteration with Melamine’ Science 322 (2008) 1310-1311 Y.C. Tyan et al.: ‘Melamine Contamination’ Bioanal. Chem. 395 (2009) 729-735 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 S. Turnipseed: ‘Determination of Melamine and Cyanuric Acid Residues in Infant Formula using LC-MS/MS’ FDA LIB 4421 (2008) 1-18 M. Smoker and A.J. Krynitsky: ‘Melamine and Cyanuric Acid Residues in Foods’ FDA LIB 4422 (2008) 1-28 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 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 Document N° SANCO/12495/2011 ‘Method Validation and Quality Control Procedure for Pesticide Residues Analysis in Food and Feed’ (2011)

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