DETECTION OF DICYANDIAMIDE (DCD) IN MILK POWDER

Background Information

• Jan 2013, trace level toxic Dicyandiamide (DCD) was detected in milk and milk products from New Zealand. The government has already banned sale of tainted products.

• DCD is listed as indirect food additives by FDA. Even though there is no international regulation yet, high dosage of DCD can be toxic to human being.

Dicyandiamide (DCD)Physical Properties

• IUPAC Name 2-Cyanoguanidine

• Other Names Cyanoguanidine, dicyanodiamide, N-cyanoguanidine,1-cyanoguanidine, Guanidine-1-carbonitrile, dicyandiamin, Didin, DCD, Dicy

• Molecular formula C2H4N4 • Molar mass 84.08 g/mol• Appearance White crystals • Density 1.400 g/cm3 • Melting Point 209.5 °C• Boiling Point 252 °C• Solubility in water 41.3 g/l

DCDWhat is DCD

DCD or Dicayndiamide is a synthetic compound used in pasture lands to prevent nitrification of cow’s urine and production of green house gases NO & N2O.

Is an amine derived from Guanidine

Toxic limits are not established

• DCD is developed about 30 years ago.

• Widely used in a number of industries including electronics, pharmaceuticals, and in food packaging.

• Since 2004, New Zealand farmers have used DCD to reduce their environmental footprint by slowing the rate that soil bacteria convert ammonia into nitrate and nitrous oxide.

• DCD is a nitrification inhibitor which the dairy industry uses to reduce nitrate leaching into waterways and greenhouse gas emissions.

• DCD is completely biodegradable in soil and leaves no residues in soil. It is not applied in the NZ summer months, but just before times of high drainage in autumn, winter and spring.

• DCD is also a mitigation technology that has been incorporated into New Zealand’s annual greenhouse gas inventory submitted annually to the UN’s Framework Convention on Climate Change.

DCD has used to synthesis of Melamine

Used in fertilizers, explosives, oil well drilling mud, pharmaceuticals, and dyestuffs

Used in pasture land to prevent nitrification & greenhouse gas emission

Melamine Phobia, SLASS 17th July 2009

7

China - 2008

–

Structures of high nitrogen compounds

Production and use

2-Cyanoguanidine is produced by treating cyanamide with base. It is produced in soil by decomposition of cyanamide. A variety of useful compounds are produced from 2-cyanoguanidine, guanidines and melamine. It is also used as a slow fertilizer. Formerly, it was as a fuel in some explosives. It is used in the adhesive industry as a curing agent

ITI analysis of Milk food for DICYANDIAMIDE (DCD)

Materials required for analysis

Analytical methodology

Weighing

•1g Milk powder into screw cap valve

Dissolve

•In 1ml of de-ionized water

Extracti

on

•Add 2ml of Acetonitrile (ACN) vortex for 2-3 min to and kept for separation

Analytical methodology contd..

Cleanup

•Transfer the ACN layer to MAS-QuChERS cartridge

•Repeat the above extraction process and transfer the Acetonitril layer to the Cartridge

•Combine ACN layer vortex for 30s and centrifuge 5500rpm for 5min to 8min

Supernatan

t

•Separate the supernatant into another screw cap valve

Concentrati

ng

•Evaporate to dryness with slow nitrogen stream at 400C

Analytical methodology contd..

Final make

up

•Dissolve the residue in 1ml of acetonitrile

Filtration

•Filter the final makeup through 0.22µm membrane filter

Injection

•Inject 5µL solution to HPLC/UV

LC-UV (ITI) vs LC-MS/MS

Analysis by LC-MS/MS

ITI Method

Sample Weighing & Dissolving

Extraction

Method of peer reviewed journal

Clean upSPE

Concentration SeparationBy LC

Analysis by LC-UV

Sample Weighing & Dissolving

Extraction Clean upSPE

Concentration SeparationBy LC

LC - UV

LC - MS

High Performance Liquid Chromatographic (HPLC/UV) Condition

Detector: Ultra Violet (UV)Column: Unisol Amide (HILIC)Mobile Phase:

Solvent A - 10mM Ammonium Acetate (pH=4.0) Solvent B – Acetonitrile

A: B = 15:85 (v/v)Detector: UV 220 nmInjection Volume: 5 µLColumn Temperature: 290CFlow Rate: 0.8mL/min

Method of establishment of (LOD)

• LOD was established with seven independent sample blanks fortified at lowest acceptable concentration (0.3mg/kg) of the Analyte and analyzed for DCD on individual fortified samples.

• LOD = Analyte concentration corresponding to a mean sample blank value + 3s (definition)

= ( ) + 3 s = 0.219 + 3 * 0.045 = 0.35 ppm

(s=standard deviation, for n=7)

Limit of Quantification (LOQ)

Is the lowest concentration of an analyte that can be determined with acceptable precision and accuracy under the stated conditions of the analysis.

• LOQ =Analyte concentration corresponding to a mean sample blank Value + 6s (definition)

= ( ) + 6 s = 0.219 + 6*.045 = 0.5ppm

Establishment of RecoveryKnown amount of DCD was spiked in three levels (0.5 mg/kg, 1.0 mg/kg and 2.0 mg/kg) to a matrix of milk powder with predetermined and verified DCD concentration (0.44 mg/kg).Then DCD of spiked samples were extracted as per the above method given in section 3 and quantified for DCD. Recovery percent was calculated as per the formula given below

Calculation of RecoveryRecovery (%) = (C1 – C2) /C3 * 100

Where C1 is the concentration determined in the fortified sample C2 = the concentration determined in unfortified sample

C3 = concentration of fortification

Establishment of Linearity

• The linearity of standard plot was expressed in terms of linear regression (R2)from plot

of the integrated peak area verses concentration of the DCD standard (mg/kg).

• The curve equation y = mx + c calculated with linear regression method, which was used to determine sample DCD concentrations. This equation was obtained over a range of concentration, in accordance with the levels of DCD found in milk powder. R2 value of the equation (y= mx+ c) of the curve was 0.997 shows the good linearity of the analytical method .

Chromatogram of DCD Standard 0.5ppm

min4 4.25 4.5 4.75 5 5.25 5.5 5.75 6 6.25

mAU

0

5

10

15

20

25

30

35

DAD1 A, Sig=220,4 Ref=off (SUJITHA\12072013000011.D)

5.4

09

Chromatogram of DCD 2.0 standard

min4 4.25 4.5 4.75 5 5.25 5.5 5.75 6 6.25

mAU

0

5

10

15

20

25

30

35

DAD1 A, Sig=220,4 Ref=off (SUJITHA\04072013000002.D)

Area

: 108

.365

5.4

07

Calibration curve

A m o u n t [n g / u l ]0 1

A r e a

0

2 0

4 0

6 0

8 0

1

2

3

4

D C D , D A D 1 A

C o r r e l a t i o n : 0 . 9 9 8 5 4

R e l . R e s % ( 1 ) : 8 . 7 9 0

A r e a = 4 2 . 4 1 0 5 9 6 3 * A m t + 2 . 5 4 6 6 8 1 8

Anchor Full Cream – Fonterra Brand DCD detected

Anchor Full Cream – Fonterra Brand Spiked with DCD

Anchor Full Cream – Fonterra Brand DCD not detected

DCD not detected

Anchor 1+

Anchor Full Cream

Maliban non fat

Highland

Palawatta

Summary of Validation ParametersMethod Validation

parameterCML Result Literature Results

Linearity (R2) 0.9960.997

RecoveryLow Level Mid Level High Level

118%101%83%

80%-90%

Limit of Detection (LOD) 0.35 mg/kg

Limit of Quantification (LOQ) 0.5 mg/kg 0.5 mg/kg

Summary ofDCD in tested milk powder samples

Brand of Milk Powder DCD Concentration/mg/kg

(Average)

DCD Concentration/mg/kg

Foreign Brand 1 (Fonterra) 0.64 (n=10) Min- 0.36Max-0.96

Foreign Brand 2 (Diamond) 0.67 (n=2) Min- 0.65Max-0.69

Foreign Brand 4 (Maliban N/F) 0.66 (n=4) Min- 0.61Max-0.72

Foreign Brand ( Anchor1+) 0.68 (n=4) Min- 0.62Max-.0.73

Local Brand 1(Highland) Not Detected (n=5)Local Brand 2(Palawaththa) Not Detected (n=5)

Summary ofDCD in tested milk powder samples

Item NoMilk

powder Brand

Batch NODate of

ManufactureDate of

Repacking Test value

mg/kg

1 Anchor 0605 H 1843 2013/03/16 2013/07/03 ND*

2 Lakspray 06 2013/01/29 2013/05/02 ND*

3Lanka

Sathosa1320 0FX1 2013/03/08 2013/07/19 ND*

4 Milgro SL60 B 653 2012/10/29 2013/04/02 ND*

5 Ratthi 0594 I 0663 2012/11/17 2013/03/07 ND*

6 Maliban 12354 A1 2012/09/10 2012/12/20 ND*

7 Nespray 305703768F 2012/12/07 2013/02/26 ND*

8 Lakspray 06 2012/09/30 2013/03/06 ND*

QA/QC

• Linearity of Calibration R2>0.995• Analyze in replicate• Spike recovery • Bracketed sample analysis• Calibrated Analytical Balance traceable to SI

units• Calibrated micropipette• De-ionized water EC<18MΩ

Managing the situation

• Directing the Research Scientists to established the method within a one month period

• Submission of the confidential report to Ministry• Expedite the procurement fastest way for

consumables required for the analysis • Trying hard to manage the client services• Staff commitment to work extra hours on the

national issue (Day & Night including week ends)

Capitalizing the opportunity• To assure health & safety of People in Sri Lank the

country should have the Analytical capability in compliance with the developed countries.

• ITI evolved on long path to achieve the most Competent Human resources on Testing & calibration in line with ISO/IEC 17025:2005

• What missing is the high tech equipment which needs to meet present level of quantification ppb, ppt

• ITI proved its position on this issue to all stakeholders• So its time for all of you to capitalize the opportunity

to make ITI as a reference laboratory in the region

Thank you