International Food Research Journal 17: 885-892 (2010)

Sulfonamides determination in chicken meat products from

Malaysia

Cheong, C.K., Hajeb, P., *Jinap, S. and Ismail-Fitry, M.R.

Centre of Excellence for Food Safety Research, Faculty of Food Science and
Technology, Universiti Putra Malaysia, 43400 UPM, Serdang, Malaysia

Abstract: Sulfonamides (SAs), synthetic antibiotics, are commonly used by veterinarians in chicken for
therapeutic, prophylactic or as growth promoter and halt the growth of bacteria in animal production. Four
common SAs, Sulfadiazine (SDZ), Sulfamethazine (SMZ), Sulfamethoxazole (SMX) and Sulfaquinoxaline
(SQX), were determined in chicken breast and liver samples using reverse phase HPLC using UV detector
at 266nm. The concentration of SAs detected in samples from 11 states in Peninsular Malaysia ranged from
0.006-0.062 µg/g in breast meat samples and 0.08-0.193 µg/g in liver samples. Except for sample from Johor,
concentration of SAs in all the samples were lower than MRLs established by Malaysia (0.1 µg/g). Exposure
of sulfonamides in Malaysian consumers ranged from 0.002-0.088 µg/kg body wt. /day. The highest value of
sulfonamides exposure was found in Johor with an estimated daily intake (EDA) of Sulfamethoxazole (SMX)
in Johor.

Keywords: sulfonamides, HPLC-UV, chicken breast meat and liver, Malaysia

Introduction and possible antibiotic resistance (Shao et al., 2005).

Therefore, to ensure the safety of the food to the
Antibiotics are chemical substances that can consumers, SAs are set at 0.1 µg g-1 of food producing
inhibit the growth of, and even destroy, harmful animals for the maximum residue limit (MRL) by the
microorganisms. Sulfonamides (SAs) are synthetic European Union Regulation (1990) and Malaysian
antibiotics with a wide spectrum against most gram- Food Regulation 1985 (2006).
positive and many gram-negative organisms. They The aim of this study is to determine level of four
are regularly used by veterinarians in chickens for SAs, Sulfadiazine (SDZ), Sulfamethazine (SMZ),
therapeutic, prophylactic, or growth-promoting Sulfamethoxazole (SMX) and Sulfaquinoxaline
purposes and halt the growth of bacteria in animal (SQX,) in chicken muscle and liver samples marketed
production. They are also used to treat many kinds in Malaysia.
of infections caused by bacteria and certain other
microorganisms such as infectious diseases of Materials and Methods
digestive and respiratory tracts (Forth et al., 1987).
Sulfonamides (SAs) inhibit multiplication of bacteria Chemicals and standard solutions
by acting as competitive inhibitors of p-aminobenzoic Acetonitrile and methanol (HPLC grade), were
acid in the folic acid metabolism cycle (Hela et al., obtained from fisher scientific (Fisher Scientific
2003). UK Limited), whereas acetone, methylene chloride,
The extensive use of SAs as a result of their low acetic acid, ammonium acetate and n-hexane were
cost has resulted in the increase of many sulfonamide- purchases from Merck (Darmstadt, Germany).
resistant strains of bacteria. Use of SAs in chickens Deionized water was obtained through a Millipore-
may result in SAs residues being present in the Q50 Ultrapure water system (Sartorius). The four
marketed tissues if inadequate withdrawal times sulfonamides, sulfadiazine (SDZ), sulfamethoxazole
for the chickens have not been observed or if these (SMX), sulfaquinoxaline (SQX) and sulfamethazine
drugs have been indecently administered (Kishida, (SMZ), were obtained from Sigma (St. Louis, MO,
2007). As a consequence of their extensive usage, USA). The stock solutions (c = 1mg/ml) were
considerable attention has been paid to the potential prepared by dissolving 0.01 g of each SA standard
human health risk due to their carcinogenic potency with 10 mL of 90% acetonitrile (n-hexane saturated).

*Corresponding author.
Email: [email protected], [email protected] © All Rights Reserved
Tel: +60389468393 ; Fax: +60389423552
886 Cheong, C.K., Hajeb, P., Jinap, S. and Ismail-Fitry, M.R.

Working individual standard and mixed standard Collection of samples

solution of these four SAs were prepared by diluting A total of 66 samples consisted of breast meat
the stock solutions with 50% methanol in 0.01 M and liver were purchased from wholesale markets in
ammonium acetate (pH4.6). The stock standard was eleven states at Peninsular Malaysia which included
stored at 4 °C. Perlis, Kedah, Penang, Terengganu, Pahang, Kelantan,
Perak, Selangor, Melaka, Negeri Sembilan and Johor.
Instrumentation The samples were immediately frozen to -20°C,
The HPLC system consisted of a Waters 600, placed in ice-box (-20°C) during transportation and
Controller HPLC pump and Waters 486 Tunable immediately placed in the -20°C freezer upon arrival
Absorbance detector was used to analyze SA residues in the laboratory. Deep-frozen samples were kept at
in chicken breast meat and liver samples. The mobile -20ºC until analysis.
phase used was 0.01 M ammonium acetate at pH 4.6
and acetonitrile. Chromatographic separation was Sulfonamide extraction
performed with gradient elution on reverse phase The sulfonamide extraction was carried out
TSKgel ODS-80TM, C18 (5 μm, 4.6 mm x 25.0 cm) following the method described by Ismail-Fitry et
analytical column following the methods described al. (2008). Breast meat and liver samples were cut
by Ismail-Fitry et al (2008) (Table 1). The flow rate into small portion (dimension) and blended An
was 1.0 ml/min and 20-µl volume of the sample was accurately weighed 10g amount of sample was
injected. The sulfonamides were detected at 266nm place in 200ml PTE centrifuge bottle with 30ml
(Figure 1). 90% acetonitrile (n-hexane saturated) is added.
Other apparatus used were a rotary evaporator The sample is homogenized for 1 minute with a
(N-1001S-W, EYELA, 1L, Tokyo, Japan), , equipped homogenizer (IKA Labortechnik, Staufen, Germany)
with an aspirator (A-1000S, Tokyo, Japan) and a and centrifuged at 3500 rpm for 10 min (Clements
digital water bath, , (SB-1000, Tokyo, Japan), a 2000, benchtop, Sydney, Australia). The supernatant
nitrogen-evaporating unit (Pierce, Reacti-Therm was transferred into 250 ml round bottom flask,
Heating Module, Rockford, IL, USA), bowl whereas the residue (sample) was extracted with
cutter/mixer (ADE SL-18, Hamburg, Germany), 20 ml acetone after sonication (Ultrasonik 104X,
Homogenizer Ultra Turrax basic (IKA Labortechnik, Neytech, Bloomfield, CT, USA) for 5 minutes. Then
Staufen, Germany), benchtop centrifuge (Clements the mixture was centrifugated at 3500 rpm for 10
2000, Sydney, Australia) and Eppendorf microfuge minutes; the supernatant was poured into the pear-
(EBA 12, Hettich Zentrifugen, Germany). shaped flask and evaporated at 50 °C until near to
dryness. Then, 5ml methylene chloride was added,
mixed using vortex and transferred into test tube; this
step was repeated 3 times. The methylene chloride
was then dried under nitrogen at 50 °C. The solution
Table 1. Gradient of mobile phases used in HPLC was reconstituted with 1 ml 50 % methanol in acetate
determination of SA residues in chicken breast meat buffer and mixed using vortex (Stuart, Oregon, USA).
and liver samples.
Then 2ml n-hexane was added and the mixture was
mixed again using vortex. The mixture was then
filtered and 20-µl of filtrate was injected into the
Time (min) A (%) B (%)
HPLC system.

0 95.0 5.0 Linearity, Recovery, Limit of Detection (LOD) and

18 63.0 37.0 Limit of Quantification (LOQ)
The linearity, R2, of SDZ, SMZ, SMX and SQX
23 63.0 37.0 were obtained from the calibration graphs, which
25 95.0 5.0 composed of seven points by plotting peak areas of
SA standards having concentrations from 0.025 to 1.0
30 95.0 5.0 μg g-1. The correlations of determination (R2) for all
35 95.0 5.0 SAs were more than 0.99. The LOD were determined
by measuring the peak height of the blank chicken
A: 0.01M ammonium acetate pH 4.6 samples. LOQ were measured based on a signal-
B: Acetonitrile to-noise ratio (S/N) 10:1. In order to determine the
recovery, 10 g of blended samples were spiked with

International Food Research Journal 17: 885-892

 Sulfonamides determination in chicken meat products from Malaysia 887

Figure 1. HPLC chromatogram of A: SAs standards (0.1µg g-1 SAs); B: SAs in chicken breast meat sample.

0.1 µg g-1 of standards SAs to the samples followed was employed to determine the variation in SA
by homogenization for 1 min. concentrations among samples and locations.
A p < 0.05 was considered to indicate statistical
Exposure assessment significance.
The estimated daily intake (EDI) values for
different Sulfonamides (µg/kg body wt. /day) from Results and Discussion
chicken consumption in eleven states of Peninsular
Malaysia were calculated using the averages chicken The limit of detection (LOD) of SDZ, SMZ, SMX,
consumption reported by Ministry of Health Malaysia and SQX in the samples was 0.008, 0.007, 0.008, and
in each state (2006). The average Sulfonamide values 0.005μg g-1, respectively. Meanwhile, the limit of
in samples (µg/g) from each state were multiplied quantification (LOQ) was 0.023, 0.022, 0.025, 0.021
by mean chicken consumption (g/day) and by the μg g-1 for SDZ, SMZ, SMX and SQX, respectively,
average weight of an individual (50 kg) (Hajeb et al., which was lower than the MRL specified by Malaysia
2009). and EU countries of 0.1 μg g-1. The recoveries range
from 82.0 to 98.9 % and RSDs from 0.7 to 7.6 %;
Statistical analysis these values are still within the criteria of the Codex
The statistical analysis has been performed using for residue analysis (recovery of 70-110 % and RSD
using SPSS (Version 11.5, SPSS Inc., Chicago, IL, of <20 %).
USA). A two-way analysis of variance (ANOVA) Samples of chicken breast meat and liver samples

International Food Research Journal 17: 885-892

888 Cheong, C.K., Hajeb, P., Jinap, S. and Ismail-Fitry, M.R.

Table 2. Sulfonamides (SAs) concentrations in chicken breast meat and liver samples from different states of
Peninsular Malaysia.

Sulfonamides (mean ± S.D) (µg/g)

Location
Samples Sulfadiazine Sulfamethazine Sulfamethoxazole Sulfaquinoxaline
(SDZ) (SMZ) (SMX) (SQX)
 
Kedah Breast meat 0.013±0.003 0.020± 0.009 - -
liver 0.019±0.003 0.013± 0.003 - -

Perlis Breast meat - 0.039± 0.002 0.008± 0.003 0.005± 0.003

liver 0.027±0.009 0.014± 0,734 0.015± 0.001 -

Penang Breast meat 0.016±0.011 - - -

liver 0.021±0.001 0.014± 0.005 0.013± 0.009 -

Terengganu Breast meat 0.011±0.005 0.012± 3,631 - -

liver 0.007±0.001 0.007± 0.001 0.008± 0.004 -

Pahang Breast meat 0.049±0.013 - - -

liver 0.012±0.005 0.007± 0.005 0.014± 0.002 -

Kelantan Breast meat - 0.011± 0.004 - -

liver - 0.019± 0.002 0.037± 0.007 -

Perak Breast meat 0.030±0.003 0.029± 0.002 - -

liver - - 0.009± 0.004 -

Johor Breast meat 0.008±0.003 - - -

liver 0.010±0.003 0.008± 0.002 0.152± 0.072 -

Malacca Breast meat 0.012±0.004 0.007± 0.003 - -

liver - 0.004± 0.019 0.015± 0.001 -

Selangor Breast meat - 0.009± 0.002 - -

liver 0.010±0.003 0.009± 0.004 0.006± 0.004 -

N.Sembilan Breast meat 0.009±0.001 0.010± 0.002  - -

liver - 0.012± 0.004 - -

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 Sulfonamides determination in chicken meat products from Malaysia 889

Table 3. Estimated daily intake of Sulfonamides (µg/kg body wt. /day) from chicken consumption in different
states of Peninsular Malaysia.

Estimated daily intake of Sulfonamides (µg/kg body wt./day)

Location Chicken
consumption
(g/day) Sulfadiazine Sulfamethazine Sulfamethoxazole Sulfaquinoxaline
(SDZ) (SMZ) (SMX) (SQX)
 
Kedah 24.77 0.008 0.008 - -

Perlis 24.77 0.013 0.013 0.006 0.002

Penang 24.77 0.009 0.007 0.006 -

Terengganu 31.17 0.006 0.006 0.005 -

Pahang 31.17 0.019 0.004 0.009 -

Kelantan 31.17 - 0.009 0.023 -

Perak 32.77 0.020 0.019 0.006 -

Johor 28.81 0.005 0.005 0.088 -

Malacca 28.81 0.007 0.003 0.009 -

Selangor 32.77 0.007 0.006 0.004 -

N.Sembilan 28.81 0.005 0.006 - -

purchased from the eleven states in Peninsular which is a suspected carcinogen has been identified
Malaysia were analyzed for the four SAs. Table and determined in meat, fish, milk and cheese (Clark
2 shows the average concentration and standard et al., 2005, Gehring et al., 2006, Pena et al., 2004
deviation of each SAs compound in breast meat and and Wen et al., 2005), and has been rendered as the
liver samples from the states. The SAs concentrations major cause in approximately 95% of all violations
detected in the marketed samples were considered involving sulfonamides in tissues (Zhenga et al.,
acceptable if they did not reach maximum residues 2007). SMZ is more stable towards heat compare to
limits (MRLs) of 0.1 µg/g adopted by Malaysia other SAs which it need longer time to be destroied
(Malaysian Food Regulation, 2006). and detoxified (Rose et al. (1995). Sulfadiazine
Residue of SAs detected in breast meat samples (SDZ) was detected in breast meat samples from each
ranged from 0.006-0.062 µg/kg. In comparing state except for Perlis, Kelantan and Selangor. While
between states, breast meat samples from Pahang Sulfamethoxazole (SMX) and Sulfaquinoxaline
(0.049±0.013) showed the highest concentration (SQX) were only detected in samples from Perlis.
of total SAs followed by Perlis (0.039± 0.002) Concentration of SAs detected in chicken liver
and Perak (0.029± 0.002). Sulfamethazine (SMZ) samples was from 0.008-0.193 µg/kg. The highest
was detected in samples from each state except for levels of SAs detected were for SMZ follow by SMX,
Penang, Pahang and Johor. Sulfamethazine (SMZ) SDZ and SQX. In comparing between states, Johor

International Food Research Journal 17: 885-892

890 Cheong, C.K., Hajeb, P., Jinap, S. and Ismail-Fitry, M.R.

showed the highest SAs residues detected follow findings did not detect any evidence of misuse or
by Kelantan and Malacca. SAs residue detected in abuse of the investigated drugs. Only two sample
liver sample from Johor was 0.152± 72.727 which out of 66 (chicken liver samples from Johor) was
exceed MRLs of 0.1 µg/g. This concentration may in violation of the regulation due to the presence
illustrate inadequate withdrawal period before the of Sulfamethoxazole (SMX). Residue control
chicken was being slaughtered. SMZ was detected in Malaysia is primarily focused on consumers’
in liver samples from each state except for Perak. protection and it should be based on risk analysis,
SMX was not detected in liver samples from Kedah therefore it might be useful to prepare guidelines for
and Selangor. While SDZ were detected in samples the uniform enforcement of residue control all over
from all states except for Kelantan, Perak, Malacca the country.
and N. Sembilan. None of the liver samples showed
SQX residue. The level of SAs residues in chicken
liver was signifantly (p<0.05) higher compared to Acknowledgments
breast meat samples, except for SQX which wasn not
detected in liver samples. Liver play a major role in The authors wish to acknowledge the Ministry of
body metabolism and has a number of functions in Health, Malaysia for financial support.
the body including glycogen storage, plasma protein
synthesis and drug detoxification. Therefore, the References
levels of SAs compound in liver sample are usually
Botsoglou, N. A. and Fletouris, D. J. 2001. Drug Residues
higher than other parts of chicken. in Foods: Pharmacology, Food Safety and Analysis. p.
In comparison to those reported in other 575-577. New York: Marcel Dekker Inc.
countries, sulfonamides detected in chicken samples
in Malaysia is considered low. For instance in USA, Capleton, A. C., Courage, C., Rumsbya, P., Holmesa,
the contamination rates of sulfonamides was reported P., Stutt, E., Boxall, A. B. A. and Levy, L. S. 2006.
to over 4% (Dey et al., 2003), while in Italy it was Prioritising veterinary medicines according to their
lower (less than 1% violation). Samples of poultry potential indirect human exposure and toxicity profile.
meat surveyed in Italy showed contamination of Toxicology Letters 163: 213-223.
sulfadiazine at 0.64- 21.0 µg/kg and sulfaquinoxaline
Clark, S.B., Turnipseed, S.B., Madson, M.R., Hurlbut,
at 0.98- 116.0 µg/kg (Weiss et al., 2007). Positive
J.A., Kuck L.R. and Sofos, 2005. Confirmation of
samples detected for sulfonamides in their study sulfamethazine, sulfathiazole, and sulfadimethoxine
were always in the liver. Study on the occurrence of residues in condensed milk and soft-cheese products
veterinary drug residues, including sulfonamides, in by liquid chromatography/tandem mass spectrometry,
poultry products in Nigeria showed contamination of Journal of the Association of Official Analytical
1% in eggs and 33.1% in broilers, 23.6% in slaughter Chemists International 88: 736–743.
and 4.8% in local chickens (Kabir et al., 2004).
However, there was no report on sulfonamides Dey, B. P., Thaler, A. and Gwozdz, F. (2003). Analysis
occurrence in Malaysia or other Asian countries for of microbiological screen test data for antimicrobial
more realistic comparison. residues in food animals. Journal of Environmental
Science and Health Part B 38(3): 391–404.
Table 3 shows exposure of sulfonamides from
chicken consumption in Malaysian consumers from European Union Regulation. 1990. Establishment of
the eleven states. It ranged from 0.002-0.088 (µg/kg Maximum Residue Levels of Veterinary Medical
body wt. /day). The highest value of sulfonamides Products in foodstuffs of animal origin. Council
exposure was found in Johor with an estimated daily Regulation (EEC) No. 2377/90.
intake (EDA) of Sulfamethoxazole (SMX) in Johor.
However, there is no Allowed Daily Intake (ADI) Fuh M. R. S. and Chu, S. Y. 2003. Quantitative determination
recommended by regulatory agencies for exposure of sulfonamide in meat by solid-phase extraction and
to sulfonamides. Furthermore, there is no previous capillary electrophoresis. Analytica Chimica Acta
study on sulfonamides exposure from other countries 499: 215-221.
to be compared with.
Furusawa, N. and Hanabusa, R. 2002. Cooking effects on
sulfonamide residues in chicken thigh muscle. Food
Conclusions Research International 35: 37-42.

The residue of sulfonamides detected in chicken Gehring, T.A. Griffin, B., Williams, R., Geiseker, C.,
liver was higher than breast meat samples. Study Rushing L.G. and Siitonen, P.H. 2006. Multiresidue

International Food Research Journal 17: 885-892

 Sulfonamides determination in chicken meat products from Malaysia 891

determination of sulfonamides in edible catfish, Pena, A, Serrano, C., Reu, C., Baeta, L., Calderon V.,
shrimp and salmon tissues by high-performance liquid Silveira I.,  Sousa, J. C. and Peixe, L. 2004. Antibiotic
chromatography with postcolumn derivatization and residues in edible tissues and antibiotic resistance of
fluorescence detection, Journal of Chromatography B: faecal Escherichia coli in pigs from Portugal. Food
Analytical Technologies in Biomedical Life Sciences Additives and Contaminants 21: 749–755.
840: 132–138.
Posyniak, A., Zmudzki, J. and Mitrowska, K. 2005.
Greenberger P. A. 2006. Drug allergy. Journal of Dispersive solid-phase extraction for the determination
Allergy Clinical Immunology 117: S488-S470. of sulfonamides in chicken muscle by liquid
chromatography. Journal of Chromatography A 1087:
Hajeb, P. Jinap, S. Ismail, A., Fatimah, A. B., Jamilah B. and 259-264.
Abdul Rahim, M. 2009. Assessment of mercury level
in commonly consumed marine fishes in Malaysia. Rose, M. D., Farrington, W. H. H. and Shearer, G. 1995.
Food Control 20: 79–84. The effect of cooking on veterinary drug residues in
food: 3. sulphamethazine (sulphadimidine). Food
Hela W., Brandtner M., Widek R. and Schuh R. 2003. Additives and Contaminants 12: 739–750.
Determination of sulfonamides in animal tissues
using cation exchange reversed phase sorbent for Shao, B., Dong, D., Wu, Y., Hu, J., Meng, J., Tu, X.
sample cleanup and HPLC-DAD for detection. Food and Xu, S. 2005. Simultaneous determination of 17
Chemistry 83: 601-608. sulfonamide residues in porcine meat, kidney and liver
by solid-phase extraction and liquid chromatography–
Ito Y., Oka H., Matsumoto, H., Miyazaki, Y. and Nagase, tandem mass spectrometry. Analytica Chimica Acta
H. 2000. Application of ion-exchange cartridge clean- 546: 174-181.
up in food analysis V. Simultaneous determination of
sulphonamide antibacterials in animal liver and kidney Stoev, G. and Michailova, A. 2000. Quantitative
using high-performance liquid chromatography with determination of sulfonamide residues in foods
ultraviolet and mass spectrometric detection. Journal of animal origin by high-performance liquid
of Chromatography A 898: 95-102. chromatography with fluorescene detection. Journal
of Chromatography A 871: 37-42.
Kabir, J., Umoh, V. J., Audu-okoh, E., Umoh, J. U. and
Kwaga, J. K. P. 2004. Veterinary drug use in poultry Walsh, C., Duffy, G., Sheridan, J. J., Fanning, S., Blair, I.
farms and determination of antimicrobial drug S. and Mcdowell, D. A. 2005. Thermal resistance of
residues in commercial eggs and slaughtered chicken antibiotic-resistant and Antibiotic-sensitive salmonella
in Kaduna State, Nigeria. Food Control 15: 99–105. spp. on chicken meat. Journal of Food Safety 25: 288-
302.
Kao, Y. A., Chang, M. H., Cheng, C. C. and Chou, S. S.
2001. Multiresidue determination of veterinary drugs Weiss, C., Conte, A., Milandri, C., Scortichini, G.,
in chicken and swine muscles by high performance Semprini, P., Usberti, R. and Migliorati, G. 2007.
liquid chromatography. Journal of Food and Drug Veterinary drugs residue monitoring in Italian poultry:
Analysis 9: 84-95. Current strategies and possible developments. Food
Control 18: 1068–1076.
Lan, C. C., Hwang, B. S. and Tu, M. F. 2001. Effect of
microwave and roast treatment on the degradation Wen, Y., Zhang, M., Zhao Q. and Feng, Y.Q. 2005Monitoring
of sulfamethazine residue in tilapia meat. Journal of of five sulfonamides antibacterial residues in milk
Food and Drug Analysis 9: 102-106. by in-tube solid-phase microextraction coupled to
high-performance liquid chromatography, Journal
Malaysian Food Regulation. 2006. Food Act 1983 (Act Agricultural and Food Chemistry 53: 8468–8473.
281) and Regulation, International Law Book Services.
p. 10-29.
Zhang, S., Wang, Z., Nesterenko, I.S., Eremin, S.A. and
Malaysian Food Regulation 1985. 2006. Food Act 1983 Shen, J. 2007. Fluorescence polarisation immunoassay
(Act 281) & Regulations. p. 247-248. Kuala Lumpur, based on a monoclonal antibody for the detection
Malaysia: International Law Book Services. of sulphamethazine in chicken muscle International
Journal of Food Science and Technology 42: 36–44.
Papapanagiotou, E. P., Fletouris, D. J., and Psomas, E. I.
2005. Effect of various heat treatments and cold storage
on sulphamethazine residues stability in incurred piglet
muscle and cow milk samples. Analytica Chimica
Acta 529: 305-309.

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