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
Z...
Dicyandiamide (DCD)
Physical Properties
• IUPAC Name 2-Cyanoguanidine
• Other Names Cyanoguanidine, dicyanodiamide, N-cyan...
DCD
What is DCD
DCD or Dicayndiamide is a synthetic compound used in pasture
lands to prevent nitrification of cow’s urine...
• DCD is developed about 30 years ago.
• Widely used in a number of industries including electronics, pharmaceuticals, and...
DCD has used to synthesis of Melamine
Used in fertilizers, explosives, oil well drilling mud, pharmaceuticals, and
dyestuf...
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 o...
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
Extraction
• A...
Analytical methodology contd..
Cleanup
• Transfer the ACN layer to MAS-QuChERS cartridge
• Repeat the above extraction pro...
Analytical methodology contd..
Final make
up
• Dissolve the residue in 1ml of
acetonitrile
Filtration
• Filter the final m...
LC-UV (ITI) vs LC-MS/MS
Analysis by LC-MS/MS
ITI Method
Sample
Weighing &
Dissolving
Extraction
Method of peer reviewed jo...
LC - UV
LC - MS
High Performance Liquid Chromatographic
(HPLC/UV) Condition
Detector: Ultra Violet (UV)
Column: Unisol Amide (HILIC)
Mobil...
Method of establishment of (LOD)
• LOD was established with seven independent
sample blanks fortified at lowest acceptable...
Limit of Quantification (LOQ)
Is the lowest concentration of an analyte that can
be determined with acceptable precision a...
Establishment of Recovery
Known amount of DCD was spiked in three levels (0.5 mg/kg, 1.0
mg/kg and 2.0 mg/kg) to a matrix ...
Establishment of Linearity
• The linearity of standard plot was expressed in terms of
linear regression (R2)from plot of t...
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
DAD1A,Sig=220,4Ref...
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
DAD1A, Sig=220,4Ref=o...
Calibration curve
A r e a
2 0
4 0
6 0
8 0
1
2
3
D C D , D A D 1 A
R e l . R e s % ( 1 ) : 8 . 7 9 0
A r e a = 4 2 . 4 1 0 ...
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 Parameters
Method Validation
parameter
CML Result Literature Results
Linearity (R2)
0.996
0.997
Reco...
Summary of
DCD in tested milk powder samples
Brand of Milk Powder DCD
Concentration/mg/kg
(Average)
DCD
Concentration/mg/k...
Summary of
DCD in tested milk powder samples
Item No
Milk
powder
Brand
Batch NO
Date of
Manufacture
Date of
Repacking
Test...
QA/QC
• Linearity of Calibration R2>0.995
• Analyze in replicate
• Spike recovery
• Bracketed sample analysis
• Calibrated...
Managing the situation
• Directing the Research Scientists to established
the method within a one month period
• Submissio...
Capitalizing the opportunity
• To assure health & safety of People in Sri Lank the
country should have the Analytical capa...
Thank you
Press conference by dir iti 220813
Press conference by dir iti 220813
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Press conference by dir iti 220813

DCD කියන්නේ මොනවාද නවසීලන්තයෙන් ආනයනය කරනු ලබන කිරිපිටි වර්ග කිහිපයක DCD රසායනිකය අඩංගු බවට කාර්මික තාක්‍ෂණ ආයතනය (ITI) මඟින් තහවුරු කර ගැනීමත් සමඟ ඒ පිළිබදව සමාජය තුල ඇත්තේ කුතුහලය පිරුනු භියකි. මෙහි සැබෑ තත්වය කුමක්ද යන්නත් DCD, මෙලමයින්, වේ ප්‍රෝටීන් හා බොටියුලිනම් යනු මොනවාද යන්නත් පැහැදිලි කිරිම සදහා මාධ්‍ය සාකච්ඡාවක් ඊයේ (22) කාර්මික තාක්ෂණ ආයතනයේදී (ITI) පැවැත්විණ. DCD කියන්නේ මොනවාද Presentation එක බලන්න
Published on: Mar 4, 2016
Published in: Education      Technology      Business      
Source: www.slideshare.net


Transcripts - Press conference by dir iti 220813

  • 1. DETECTION OF DICYANDIAMIDE (DCD) IN MILK POWDER
  • 2. 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.
  • 3. 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
  • 4. DCD What 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
  • 5. • 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.
  • 6. 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
  • 7. Melamine Phobia, SLASS 17th July 2009 7 China - 2008 –
  • 8. Structures of high nitrogen compounds
  • 9. 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
  • 10. ITI analysis of Milk food for DICYANDIAMIDE (DCD)
  • 11. Materials required for analysis
  • 12. Analytical methodology Weighing • 1g Milk powder into screw cap valve Dissolve • In 1ml of de-ionized water Extraction • Add 2ml of Acetonitrile (ACN) vortex for 2- 3 min to and kept for separation
  • 13. 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 Supernatant • Separate the supernatant into another screw cap valve Concentrating • Evaporate to dryness with slow nitrogen stream at 400C
  • 14. 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
  • 15. LC-UV (ITI) vs LC-MS/MS Analysis by LC-MS/MS ITI Method Sample Weighing & Dissolving Extraction Method of peer reviewed journal Clean up SPE Concentration Separation By LC Analysis by LC-UV Sample Weighing & Dissolving Extraction Clean up SPE Concentration Separation By LC
  • 16. LC - UV LC - MS
  • 17. 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 nm Injection Volume: 5 µL Column Temperature: 290C Flow Rate: 0.8mL/min
  • 18. 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)
  • 19. 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
  • 20. Establishment of Recovery Known 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 Recovery Recovery (%) = (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
  • 21. 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 .
  • 22. 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 DAD1A,Sig=220,4Ref=off(SUJITHA12072013000011.D) Area:44.2157 5.409
  • 23. 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 DAD1A, Sig=220,4Ref=off (SUJITHA04072013000002.D) Area:108.365 5.407
  • 24. Calibration curve A r e a 2 0 4 0 6 0 8 0 1 2 3 D C D , D A D 1 A 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
  • 25. Anchor Full Cream – Fonterra Brand DCD detected
  • 26. Anchor Full Cream – Fonterra Brand Spiked with DCD
  • 27. Anchor Full Cream – Fonterra Brand DCD not detected
  • 28. DCD not detected
  • 29. Anchor 1+
  • 30. Anchor Full Cream
  • 31. Maliban non fat
  • 32. Highland
  • 33. Palawatta
  • 34. Summary of Validation Parameters Method Validation parameter CML Result Literature Results Linearity (R2) 0.996 0.997 Recovery Low 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
  • 35. Summary of DCD 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- 0.36 Max-0.96 Foreign Brand 2 (Diamond) 0.67 (n=2) Min- 0.65 Max-0.69 Foreign Brand 4 (Maliban N/F) 0.66 (n=4) Min- 0.61 Max-0.72 Foreign Brand ( Anchor1+) 0.68 (n=4) Min- 0.62 Max-.0.73 Local Brand 1(Highland) Not Detected (n=5) Local Brand 2(Palawaththa) Not Detected (n=5)
  • 36. Summary of DCD in tested milk powder samples Item No Milk powder Brand Batch NO Date of Manufacture Date 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* 3 Lanka Sathosa 1320 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*
  • 37. 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
  • 38. 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)
  • 39. 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
  • 40. Thank you