1. Logistics and Health Risk Assessment of Veterinary Drug and Additive
in Animal Feed after ASEAN Economic Community (AEC)
Presented by Preechaya Yodrasing
ID 51216325 ENTM/M
7. Objective
To find out a environment that suitably for measurement the
quantity of residue veterinary in terms of livestock product,
and its measuring method will perform by ultrasonic-assisted
extraction technique (UAE) - SPE - LC-MS/MS.
To determine the level of residue veterinary in terms of
livestock product, within ASEAN+3 and the current domestic
market.
To evaluate the risk for contaminated residue of veterinary in
feed of used animal.
Page 7
8. Expected Results
To understand the overview of livestock product not only
in-house and outer market but also imported product that
may conceal a residue of veterinary.
To know a dispersion trend of residue after the AEC was
formally announce and a quantity of residue in the country.
To be model of management and monitoring a residue,
that may cause a risk to consumer especially after the
AEC was formally announce.
Page 8
10. 200,000
150,000
100,000
50,000
6,000
5,000
4,000
3,000
2,000
1,000
Thailand’s total of animal feed import from ASEAN (2010-2013)
(Division of Animal Feed and Veterinary Products Control, 2013)
0
2010 2011 2012 2013
0
2010 2011 2012 2013
Volume (Tons)
Value (Million Bath)
Page 10
11. Ratio of quantity and animal feed imported value, segmentation by type
Supplementary
20.28%
Other feed
4.93%
Thailand’s total imports of animal feed from ASEAN in 2013
Raw material
66.40%
Mixed
material
9.35%
Other feed
3.97%
Raw material
17.70%
Mixed material
49.97%
Supplementary
27.39%
Countries Volume
(Ton)
Value (million
bath)
Vietnam 53.67 4.23
Malaysia 116.14 6.45
Singapore 4,719.02 548.82
Indonesia 6.54 2.45
China 8,603.36 939.38
Korea 1,439.68 78.91
Japan 1,520.96 143.27
Ratio of quantity imports
Ratio of value imports
(Division of Animal Feed and Veterinary Products Control, 2013) Page 11
12. Related Agencies of animal feed import.
1. Customs Department, Ministry of Finance.
48 locals customs facilitations
(Customs Facilitations, 2014)
Page 12
13. Related Agencies of animal feed import (Cons.)
3. Division of Animal Feed and Veterinary
Products Control, Department of Livestock
Development, Ministry of Agriculture and
Cooperatives.
Proceeding to evaluation, control and
preserve for animal feed entrepreneur
( Producer, importer and trader) follow the
Feed Quality Control Act, 1982.
2. Division of Veterinary Inspector and
Quarantine, Department of Livestock
Development, Ministry of Agriculture and
Cooperatives.
Responsibly for animal quarantine,
both of domestic and international, also
control the movement of animal carcasses
and livestock products. There are 43
outposts for in and out animal quarantine
control.
Regulations
The Act of Drug B.E. 2510 (1967)
The Act of Feed Quality Control B.E. 2525 (1982) and revised B.E. 2542 (1999)
Page 13
14. Imported process of animal feed is divided into two
phases: pre-import and date of import.
Import permit for animal feed
A license to import for animal
feed into Thailand
Import Entry
Importer
Division of Animal
Feed and Veterinary
Products Control
Phases I: pre-import
Customs Facilitation
Page 14
15. Phases II: date of import
Inspection of the permit and
other documents
Sampling for disease
inspection and residue
Seizure or Withholding
for further inspection
Customs Facilitation
Animal Quarantine
The Act of Feed
Quality Control
- DNA Ruminant
- Melamine
Issue the permit
Return the country of
origin and legal actions
Bureau of Quality Control
of Livestock Products
- Kasetsart University
- Chulalongkorn University
- Central laboratory
Inspect and
release products
pass
Consideration
Not passed
Passed
by
Page 15
16. The outpost that mainly imported
animal feed.
A=Bangkok Port
B=Laem Chabang Port
C=Ladkrabang Cargo Clearance
D=Suvarnabhumi Airport Cargo
Clearance
CD
A
B
Laos
Vietnam
Cambodia
China
Myanmar
Page 16
17. Antimicrobial agent used in feed
(Frank, 2010)
• Antimicrobials for the production of medicated feed is
confirmed by national feed manufacturers’ associations.
1
3
2
Therapy
prevention
Growth promoter
Page 17
18. Prohibition of using chemical product in
feed additive and its object Act.
Consist of
1. Avoparcin
2. Beta-agonist
3. Chloramphenicol 4. Carbadox
5. Olaquindox 6. Nitrofurans gr.
7. Nitroimidazole gr. 8. Diethylstilbestrol
9. Dioxin 10. Melamine Ammeline
Ammelide Cyanuric acid
Page 18
19. The contaminated in situation that residues drug
and other additives in animal feed of AEC.
In Malaysia, according from the study of Veterinarian services in 2012 found that, the
amount of chicken fed up to 50% are resistant to antibiotics, Ampicillins, Sulfonamides
and Tetracycline. And for an imported products, were found to be resistant to both
Ampicillin, Streptomycin and other antibiotics in proportions ranging from 50 to 87% The
most obviously pathogens in food such as. Listeria, Salmonella and E. coli. That is
detrimental to control food quality. From the workhouses industry and manufacturing to
consumers in the long term. (TheFishSite, 2014)
In China, they have been found to have used beta-agonist for a while, for example in
2006, there are 336 people were taken to hospital after ate pork and its entrails
(pathogens to clenbuterol) and in 2009. Police officers arrested 9 suspects who selling
pork meat those pathogens to beta-agonist. That caused more than 70 customers in
Guangdong got sick. And in 2011, Chinese media have presented a farmer, in the
province of Henan, was mixed clenbuterol into pig feed. (ASTV, 2011)
Page 19
20. The contaminated in situation that residues drug
and other additives in animal feed of Thailand.
Residue monitoring results in 2010 - 2012
**Contaminated assessment of residues drug in beef*** (2013)
From 314 beef samples, found that there are had 128 samples contaminated
which are 40 percentage of a sampling (range of amount is 174.81 – 39,092.74 ppb)
Department of Livestock Development, 2013
Page 20
21. The contaminated in situation that residues drug
and other additives in animal feed of Thailand.
Year Type of
sample
Sampling
places
Sample
Quantity
Detected
Quantity
Type of residues
2011 Poultry feed Poultry farms 304 - -
Pig feed Pig farms 1,297 15 Tetracyclines,
Sulphonamides
2012 Poultry feed Poultry farms 315 - -
Pig feed Pig farms 1,340 17 Chlortetracycline,
Oxytetracycline,
Tylosin A,
Sulfamethazine
(SMZ)
Department of Livestock Development, 2013
Page 21
23. Literature Review
Pleadin et al. (2010) were determine the level of clenbuterol residues in muscle tissue of
pigs after repeat administration in a growth-promoting dose. An anabolic dose of
clenbuterol (20 μg/kg body mass per day) was administered orally to experimental group
(n= 12) for 28 days. Determination of residual clenbuterol was by enzyme-linked
immunosorbent assay (ELISA) as a screening method and liquid chromatography tandem
mass spectrometry (LC-MS/MS) as a confirmation method. Result that the highest
clenbuterol content in the muscle of treated animals was recorded on day 0 of
treatment cessation (4.40± 0.37 ng/g) and significantly (p<0.05) exceeded the maximum
residue limit (MRL) of 0.1 ng/g. On day 3 of withdrawal, it was 0.49±0.22 ng/g and on
day 7 0.10±0.02 ng/g (at MRL); on day 14 of treatment discontinuation, clenbuterol content
was below the limit of detection (<0.1 ng/g) in all samples.
Page 23
24. Literature Review (cons.)
Wijayanti et al. (2011) were investigate the effect of doxycycline (a tetracycline derivative)
administered at disease-prevention dose given daily in the feed and drinking water on the
residue level in the broiler-chicken meat. Doxycycline at concentration of 100 ppm was
mixed in the drinking water (1 g of doxycycline in 10 L of drinking water) and feed (1 g of
doxycycline in 10 kg of feed). Analysis of doxycycline level was performed using high
performance liquid chromatography with extraction method referring to the standard of
Association of Official Analytical Chemistry. The result showed that the highest residue
level was found at the first week in the group given doxycycline in drinking water
(0.96±0.15 μg/g),while the highest was found at the third week in the group given
doxycycline in feed (0.15±0.02 μg/g). The level of doxycycline residue exceeding the
level permitted by the Indonesian National Standard (0.1 μg/g). It can be concluded that
doxycycline is not recommended to be given daily both through water or feed to
commercial chickens as a disease-prevention agent.
Page 24
25. Summary of analytical methods for monitoring of Beta-agonist
Compounds Target
species for
feed use
Sample treatment Analytical
technique
Method performance Ref.
Extraction
procedure
Clean up Detection
system
Recovery
(%)
Limit
(ng/g)
Salbutamol
Ractopamine
Clenbuterol
Pig vortex SPE (C18
cartridges)
LC-MS 83-110 LOD:10
LOQ: 50
Zhang,
2009
7 Beta-agonist Not
specified
sonication SPE (polymeric
cartridge)
LC-MS/MS 82-110 LOD: 0.87
LOQ: 0.5-
0.92
Juan,
2010
Cimaterol
Salbutamol
Terbutaline
Ractopamine
Not
specified
shaking+
centrifuge
SPE (MCX
cartridge)
UPLC 90.1-101.4 LOD: 5-6.5
LOQ: 16.7-
21.7
Qu, 2011
12 Beta-agonist Not
specified
mechanical
shaking
SPE (mix-mode
cartridge)
UPLC-Q/TOF
MS
89.2-103.3 LOD: 1-5
LOQ: 5-50
Xiu-Juan,
2013
20 prohibited
veterinary drugs
Not
specified
vortex+
sonication
PSA sorbent LC-MS/MS 56.7-103 LOD: 0.42-
5.74 LOQ
5.70-9.81
Zhang,
2013
Page 25
26. Summary of analytical methods for monitoring of antibiotics
Compounds Target
species for
feed use
Sample treatment Analytical
technique
Method performance Ref.
Extraction
procedure
Clean up Detection
system
Recovery
(%)
Limit
(ng/g)
Oxytetracycline
Tetracycline
Chloramphenicol
Not
specified
Subcritical water
extraction
None LC-UV 82.1-90.0 Not
reported
Wang,
2008
Multi-class Cattle
Lamb
Pig
shaking+
sonication
PSA sorbent LC-MS/MS 51-116 LOQ:
3.8-65.0
Boscher,
2010
Multi-class Pig
Poultry
vortex+mechani
cal shaking
LLE, Extraction
solvent: hexane
LC-MS/MS 89.2-
103.3
Not
reported
Cronly,
2010
16 Sulfonamides Pig shaking+
centrifuge
SPE (alumina) LC-MS/MS 80-120 LOQ:
0.5-20
Liu, 2011
Chlortetracycline
Oxytetracycline
Tetracycline
Doxycycline
Pig
Poultry
vortex+
ultrasonic bath
SPE (Oasis
HLB)
LC-MS/MS 80.9-
119.5
LOD:
3-20
LOQ:
10-50
Guo, 2012
Page 26
27. Methodology
Frameworks of study
Select sampling point of animal feed
Beta-agonist Antibiotics
Ultrasonic-assisted extraction (UAE)
Solid-Phase Extraction (SPE)
LC-MS/MS
Health risk assessment
Tetracyclines
Tetracycline
Oxytetracycline
Chlortetracycline
Doxycycline
Sulfonamides
Sulfadiazine
Sulfamethazine
Sulfamethoxazole
Sulfaquinoxaline
Clenbuterol
Salbutamol
Ractopamine
Page 27
29. Analytical method
Sample preparation
-Blended and a portion of 2 g powdery feed was weighed to continue.
Page 29
30. Beta-agonist analysis
Extraction
(UAE)
Clean-up
SPE (SCX)
LC-MS/MS
- Sample 2 g
- Add Milli-Q 20 mL and 1 M HCl 2 mL
- Shaker 350 mot/min, 40 min
- Ultrasonic 15 min
- Centrifuge 4,000 rpm, 20 min
- Filtration membrane filter, GF/B
- Precondition: MeOH 3 mL and
Milli-Q 3 mL
- Wash: Milli-Q 2 mL and 2% Formic
acid in water 2 mL
- Elute: 5% Ammonia in MeOH 5 mL
- Dry nitrogen gas 40°C
Reconstitution: 0.1% Formic acid
in water:ACN (90:10) 1 mL
Page 30
31. Antibiotics analysis
Extraction
(UAE) - Shaker 400 mot/min, 1 hr.
- Ultrasonic 15 min
- Centrifuge 4,500 rpm, 20 min
- Filtration by membrane filter, GF/B volume 10 mL
And add Milli-Q 40 mL
Clean-up
SPE (HLB)
LC-MS/MS
- Sample 2 g
- Add MeOH:0.2M citric acid, pH3 (1:1, v/v) 30 mL
- Precondition: MeOH 2 mL and 0.04M
citric acid, pH3 2 mL
- Wash: 0.04M citric acid, pH3 2 mL and
0.1M Sodium acetate 2 mL
- Elute: MeOH 6 mL
- Dry nitrogen gas 40°C
Reconstitution: MeOH +
10mM Formic acid 0.5 mL
Page 31
32. Health Risk Assessment
y1 y2 X%
Calculated in a percentage of amount of the residue in animal products, referred from a research.
The results obtained from this analytical
x 100 = x%
y2
y1
b
a
x%
100%
x a = b
b
Page 32
33. Veterinary Drug Animal Species Tissue
Maximum Residue Limit (훍g/kg )
Ministry of Public
Health
Department of
Livestock
Development
EU CODEX Japan
Clenbutarol Not specified Edible offal - Not detected - - -
Cattle Muscle - Not detected 0.1 0.2 0.2
Liver - Not detected 0.5 0.6 0.6
Kidney - Not detected 0.5 0.6 0.6
Ractopamine
hydrochloride
Cattle and Pig Muscle - - 10 10 10
Fat - - 10 10 10
Liver - - 40 40 40
Kidney - - 90 90 90
Tetracycline/
Oxytetracycline/
Chlortetracycline
Not specified Muscle 200 100 100 200 200
Liver 600 300 300 600 600
Kidney 1,200 600 600 1,200 1,200
Egg 400 200 200 400 400
Milk (Cattle,
100 - 100 100 100
Sheep)
Page 33
MRL index
34. Veterinary Drug Animal Species Tissue
Maximum Residue Limit (훍g/kg)
Ministry of Public
Health
Department of
Livestock
Development
EU CODEX Japan
Doxycycline Pig and Poultry Muscle - 50 100 - 50
Fat/Skin - 50 300 - 50
Liver - 50 300 - 50
Kidney - 50 600 - 50
Other Poultry Muscle - 100 100 - 100
Fat/Skin - 300 300 - 300
Liver - 300 300 - 300
Kidney - 600 600 - 600
Cattle Muscle - - 100 - 100
Liver - - 300 - 300
Kidney - - 600 - 600
Sulfonamide Not specified Muscle 100 100 100 100 100
Fat 100 - 100 100 100
Liver 100 100 100 100 100
Kidney 100 100 100 100 100
Egg - 100 - - 10
Cattle, Goat and
Sheep
Milk (μg/l) 25 - 100 25 10
Page 34
MRL index (cons.)
35. References
• ASEAN Economic Community. (2013). ASEAN Economic Community History. Retrieved October 28, 2013, from Thai-AEC.com
Website: http://www.thai-aec.com/ประวัติความเป็นมาประชา
• ASTV. (2009). บริษัทจีนฉาวอีก! เรียกคืนอาหารกล่องหลังเน้อืเปื้อนสารเคมี. Retrieved March 17, 2014, from Website:
http://www.manager.co.th/China/ViewNews.aspx?NewsID=9520000045726
• ASTV. (2011). จีนเก็บคืนผลิตภัณฑ์เนื้อหมู เหตุอื้อฉาวปนเปื้อน “สารหมูผอม”. . Retrieved March 17, 2014, from Website:
http://www.manager.co.th/China/ViewNews.aspx?NewsID=9540000034654
• Boscher, A., Guignard, C., Pellet, T., Hoffmann, L., and Bohn, T. (2010). Development of a multi-class method for the
quantification of veterinary drug residues in feedingstuffs by liquid chromatographt-tandem mass spectrometry. Journal of
Chromatography A, 1217(41), 6394-6404.
• Cronly, M., Behan, P., Foley, B., Malone, E., Earley, S., Gallagher, S., Gallagher, M., Shearan, P., and Regan, L. (2010).
Development and validation of a rapid multi-class method for the confirmation of fourteen prohibited medicinal additives in pig and
poultry compound feed by liquid chromatography-tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis,
53(4), 929-938.
• Customs Facilitations. (2014). Customs Office map. Retrieved July 14, 2014, from Customs Facilitations Website:
http://www.customs.go.th/wps/wcm/connect/Library+cus501th/InternetTH/About+Us/customs_area/2B75CC5BC8A4956AC217A77
595536FB9
• Division of Animal Feed and Veterinary Products Control. (2013). Comparison data between quantity and imported values of
animal feed in 2012-2013. Retrieved March 18, 2014, from Division of Animal Feed and Veterinary Products Control Website:
http://afvc.dld.go.th/forum.php?mod=viewthread&tid=161&extra=Array
• Division of Animal Feed and Veterinary Products Control. (2014). History of Division of Animal Feed and Veterinary Products
Control. Retrieved March 18, 2014, from Division of Animal Feed and Veterinary Products Control Website:
http://afvc.dld.go.th/forum.php?mod=viewthread&tid=74&extra=page%3D1
• Division of Veterinary Inspector and Quarantine. (2014). History of Division of Veterinary Inspector and Quarantine. Retrieved
December 2, 2014, from Division of Veterinary Inspector and Quarantine Website:
http://aqi.dld.go.th/th/index.php?option=com_content&view=article&id=174&Itemid=57
• Frank, A. (2010). Evaluation of the EU legislative framework in the field of medicated feed. Retrieved February 20, 2014, from
Food Chain Evaluation Consortium (FCEC) Website:
http://ec.europa.eu/food/food/animalnutrition/labelling/medicated_feed_report_20100224.pdf Page 35
36. • Guo, L., Chen, Y., Zhang, L., Yang, W., and He, P. (2012). Development and validation of a liquid chromatographic tandem mass
spectrometric method for determination of chlortetracycline, oxytetracycline, tetracycline, and doxycycline in animal feeds. Journal of
AOAC International, 95(4), 1010-1015.
• Juan, C., Igualada, C., Moragues, F., Leon, N., and Manes, J. (2010). Development and validation of a liquid chromatography tandem
mass spectrometry method for the analysis of beta-agonists in animal feed and drinking water. Journal of Chromatography A, 1217(39),
6061-6068.
• Liu, R., He, P., Li, Z., and Li, R. (2011). Simultaneous determination of 16 sulfonamides in animal feeds by UHPLC-MS-MS. Journal of
Chromatographic Science, 49(8), 640-646.
• Pleadin, J., Vulic, A., Persi, N., and Vahcic, N. (2010). Clenbuterol residues in pig muscle after repeat administration in a growth-promoting
dose. Journal of Meat Science, 86(3), 733-737.
• Qu, C. H., Li, X. L., Zhang, L., Xi, C. X., Wang, G. M., Li, N. B., and Luo, H. Q. (2011). Simultaneous determination of cimaterol,
salbutamol, terbutaline and ractopamine in feed by SPE coupled to UPLC. Journal of Chromatographia, 73(3-4), 243-249.
• Thai Feed Mill Association. (2014). Animal population and Feed Consumption. Retrieved February 20, 2014, from Thai Feed Mill
Association Website: http://www.thaifeedmill.com/tabid/56/Default.aspx
• TheFishSite. (2014). Malaysian Consumers urge ban of antibiotics in animal feed. Retrieved March 16, 2014, from National Bureau of
Agricultural Commodity and Food Standards Website: http://www.acfs.go.th/read_news.php?id=11863&ntype=07
• Wang, L., Yang, H., Zhang, C., Mo, Y., and Lu, X. (2008). Determination of oxytetracycline, tetracycline and chloramphenicol antibiotics
in animal feeds using subcritical water extraction and high performance liquid chromatography. Journal of Analytica Chimica Acta,
619(1), 54-58.
• Wijayanti, A. D., Wihandoyo., and Rosetyadewi, A. W. (2011). The comparison of Doxycycline residue in meat of broiler chickens
administered in feed and water. Journal of Animal Science and Technology, 34(3), 175-178.
• Xiu-Juan, W., Feng, Z., Fei, D., Wei-Qing, L., Qing-Yu, C., Xiao-Gang, C., and Cheng-Bao, X. (2013). Simultaneous determination of 12
beta-agonists in feeds by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Journal of
Chromatography A, 1278, 82-88.
• Zhang, G. J., Fang, B. H., Liu, Y. H., Wang, X. F., Xu, L. X., and Zhang, Y. P. (2013). Development of a multi-residue method for fast
screening and confirmation of 20 prohibited veterinary drugs in feedstuffs by liquid chromatography tandem mass spectrometry. Journal
of Chromatography B, 936, 10-17.
• Zhang, L. Y., Chang, B. Y., Dong, T., He, P. L., Yang, W. J., and Wang, Z. Y. (2009). Simultaneous determination of salbutamol,
ractopamine, and clenbuterol in animal feeds by SPE and LC-MS. Journal of Chromatographic Science, 47(4), 324-328.
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