Accepted Manuscript Distribution of sulfamonomethoxine and trimethoprim in egg yolk and white Nina Bilandžić, Đurđica Božić, Božica Solomun Kolanović, Ivana Varenina, Luka Cvetnić, Željko Cvetnić PII: DOI: Reference:

S0308-8146(15)00087-4 http://dx.doi.org/10.1016/j.foodchem.2015.01.076 FOCH 17027

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

19 August 2014 10 December 2014 15 January 2015

Please cite this article as: Bilandžić, N., Božić, Đ., Kolanović, B.S., Varenina, I., Cvetnić, L., Cvetnić, Ž., Distribution of sulfamonomethoxine and trimethoprim in egg yolk and white, Food Chemistry (2015), doi: http:// dx.doi.org/10.1016/j.foodchem.2015.01.076

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1

Distribution of sulfamonomethoxine and trimethoprim in egg yolk and

2

white

3 4

Nina Bilandžića,∗∗, Đurđica Božića, Božica Solomun Kolanovića, Ivana Vareninaa, Luka

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Cvetnićb, Željko Cvetnićc

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a

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cesta 143, HR-10000 Zagreb, Croatia

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b

Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia

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c

General Department, Croatian Veterinary Institute, Savska cesta 143, HR-10000 Zagreb, Croatia

Department of Veterinary Public Health, Laboratory for Residue Control, Croatian Veterinary Institute, Savska

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ABSTRACT

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The distribution of sulfamonomethoxine (SMM) and trimethoprim (TMP) in egg yolk and

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white was measured during and after administration of a SMM/TMP combination in laying

14

hens in doses of 8 g l-1 and 12 g l-1 in drinking water for 7 days. The SMM concentration

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reached maximal levels on day 2 of the post-treatment period for both doses (µg kg-1): 5920

16

and 9453 in yolk; 4831 and 6050 in white, in doses 1 and 2, respectively. Significant

17

differences in the SMM and TMP concentrations between yolk and white in post treatment

18

period were found. SMM dropped below the LOD (1.9 µg kg-1) in yolk after day 16 and 19

19

for doses 1 and 2. TMP reached maximal levels on day 3 after drug administration for doses 1

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and 2 (µg kg-1): 6521 and 7329 in yolk, 1370 and 1539 in white. TMP residues were measured

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above LOD (0.3 µg kg-1) in yolk for both doses on day 37 post-treatment.

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Key words: Sulfamonomethoxine; Trimethoprim; Elimination; Laying hens; Egg; Yolk;

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White; LC-MS/MS

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Corresponding author. Tel.: +385 1 612 3601, fax: +385 1 612 3636

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E-mail address: [email protected] (N. Bilandžić).

1. Introduction

29 30

Sulfonamides are a group of synthetic antibiotics and chemotherapeutics used for

31

prophylactic and therapeutic purposes in veterinary medicine. They are known to inhibit both

32

Gram-positive and Gram-negative bacteria, some Chlamydia, Nocardia and Actinomyces

33

species, and some protozoa including coccidia and Toxoplasma species. As structural analogs

34

and competitive antagonists of para-aminobenzoic acid, sulfonamides inhibit dihydropteroate

35

synthetase, the enzyme that catalyzes the synthesis of dihydrofolic acid (folic acid). They are

36

also widely used in animal feed as growth promoters, and to prevent and treat a series of

37

diseases, such as infectious diseases of the digestive and respiratory tracts (Campbell, 1999).

38

Sulfonamides are well adsorbed after oral dosing and biotransformed in the liver by the

39

glucuronization or acetylation procedures, and then rapidly excreted mainly via the kidneys,

40

and to a limited extent, in bile and faeces. Potential undesirable effects in humans are urinary

41

tract disorders, porphyria and hypersensitivity reactions. Due to damage to the homeopathic

42

system, they may cause thrombocytopenia, anaemia, leukopaenia, and various changes to the

43

skin (Šeol, Matanović, & Terzić, 2010). Trimethoprim (TMP) is a synthetic antibacterial

44

diaminopyrimidine. It is often administered together with sulfonamides, and their activity

45

becomes synergistic, resulting in an increased bactericidal effect and additional antibacterial

46

activity against different bacteria (Hela, Brandtner, Widek, & Schuh, 2003).

47

The presence of sulfonamide residues in food is a potential human health risk due to

48

toxicological and carcinogenic potency and possible antibiotic resistance (EMEA, 1995).

49

TMP can cause changes in the bone marrow and can have significant effects on some organ

50

weights in humans and animals (Li, Sun, Zhang, & Pang, 2013). Improper administration or

51

misuse of these antibiotics can cause elevated residues in meat, milk, eggs and fish. To

2

52

safeguard human health, the European Union (EU) set maximum residue limits (MRLs) as a

53

sum of all sulfonamides at the level of 100 µg kg-1 in meat, liver, kidney, fat and milk but no

54

limit is assigned for eggs (European Commission, 2010). Today there are more than 16

55

commonly used sulfonamide compounds on a global scale in veterinary medicine, including

56

SMM and antibacterial diaminopyrimidines TMP (Weiss et al., 2007). SMM in combination

57

with TMP is used for the disease prevention and treatment of domestic poultry, such as

58

fattening chickens, fattening turkeys, layers and breeders infected with coccidiosis,

59

colisepticaemia, salmonellosis, pasteurellosis, staphylococcus infection, infectious rhinitis and

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so on. However, dispersible powder and suspensions of SMM/TMP are prohibited for use in

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laying hens. On the other hand, there may be situations where these compounds can be

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accidentally or intentionally used, such as in medicated feed or water intended for broilers or

63

water for layers (Atta & El-Zeini, 2001). However, the most common cause of residues in

64

eggs is of a management nature and is mainly related to failures to meet withdrawal times

65

(Donoghue & Myers, 2000; JECFA, 2004). After drug administration to laying hens, residues

66

will appear in egg white or yolk or both, and the distribution between egg yolk and white is of

67

great importance with regard to drug residues. Deposition of the drug is determined by the

68

physiochemical properties of the drug and the physiology of the chicken and egg formation

69

(Alaboudi, Basha, & Musallam, 2013).

70

However, there is limited data available regarding the deposition of SMM and TMP

71

residues in eggs. Depletion of residues of some sulfonamides (Furusawa, Tsuzukida, &

72

Yamaguchi, 1998; Romvary & Simon, 1992; Roudaut & Garnier, 2002; Vandenberge et al.,

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2012) or combinations of trimethoprim and sulfonamides (Atta & El-zeini, 2001; Nagata,

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Saeki, Iida, Kataoka, & Shikano, 1991) have been studied in the eggs of laying hens. In

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Croatia, the chemotherapeutic SMM/TMP is available as Trisulfon powder (Krka, Slovenia).

76

Administration in domestic poultry (fattening chickens, fattening turkeys, layers and breeders) 3

77

is most commonly performed at a concentration of 8 g l-1 via drinking water during 3 to 5

78

days. For preventive treatment, half the treatment dose is used during 5 to 7 days.

79

The aim of the present study was to investigate SMM and TMP residue transfer in egg

80

yolk and egg white during and after drug administration via drinking water in two doses.

81

Quantification was performed using a sensitive method of liquid chromatography–tandem

82

mass spectrometry (LC-MS/MS).

83 84

2. Materials and methods

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2.1. Animal treatment and sampling

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A total of 50 laying hens (ISA Brown) were obtained from a breeding station and were

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set up at approximately 21 weeks of age. The hens were fed on balanced ration, free from

90

therapeutic agents and fresh clean drinking water was available ad libitum. Chickens were

91

randomly divided into three groups in separate cages, two treatment groups (each n=10) and

92

one control group (n=10). In cages, hens had free access to water and feed. Birds consumed

93

an average of 225 ml water per day. Prior to drug administration animals were weighed. The

94

average weight of the animals was 1.65 kg. The two therapeutic groups were administered a

95

SMM/TMP combination using the chemotherapeutic Trisulfon powder (active dose 40 mg g-1

96

sodium SMM and 20 mg g-1 TMP; Krka, Slovenia) in two concentrations of combined drug,

97

i.e. 8 and 12 g l-1 via drinking water within 7 days (day 1 to 7). Group 1 (dose 1) received

98

concentrations via water (8 g l-1): SMM 320 mg l-1 and TPM 160 mg l-1. Hens of group 1

99

received medicated water with 8 g l-1, which corresponded to concentrations for SMM

100

43.6 mg kg-1 b.w. day-1 and for TMP 21.8 mg kg-1 b.w. day-1. Group 2 (dose 2) received

101

concentrations via water (12 g l-1): SMM 480 mg l-1 and TPM 240 mg l-1. Therefore, hens in

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102

group 2 received daily average doses of 65.5 mg kg-1 b.w. day-1 for SMM and 32.7 mg kg-1

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b.w. day-1 for TMP.

104

Eggs collected prior to administration in the treatment groups and eggs of the control

105

group were used as the blank control. Eggs were collected for 7 days during treatment, while

106

in the post-treatment period, they were collected on the first four days and subsequently every

107

third day over a total of 37 days (days 1, 2, 3, 4, 7, 10, 13, 16,…37). Eggs were labelled with

108

the date and stored at 4°C until delivery to the laboratory. For the determination of SMM and

109

TMP in egg white and yolk homogenate, the bulk of five eggs were separated for white and

110

yolk and homogenized accordingly. Therefore, four samples of egg white and yolk were

111

prepared per collection day. Samples were stored and frozen at -20°C until analysis.

112 113

2.2. Solvents, reagents and standards

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All reagents were of analytical, HPLC or LC-MS grade. LC grade dichloromethane

116

without ethanol as a stabilizer, LC-MS grade methanol and aromatic sulfonic acid (SO3H)

117

solid phase extraction (SPE) cartridges (500 mg per 3 ml) were purchased from J.T. Baker

118

(Deventer, Netherlands). Acetone (LC grade) was supplied by Merck (Darmstadt, Germany),

119

sodium sulphate (anhydrous) by Carlo Erba (Milan, Italy), ammonium formate (97%), acetic

120

acid min. 99.8% and formic acid (ACS reagent ≥ 96%) by Sigma Aldrich Chemie GmbH

121

(Steinheim am Albuch, Germany) and sodium chloride and 25% ammonia solution by

122

Kemika (Zagreb, Croatia). Nitrogen 5.0 and 5.5 were purchased from SOL spa (Monza,

123

Italy). Ultra pure water (18 MΩxcm) was obtained using the system NIRO VV UV UF 20

124

(Nirosta d.o.o. Water Technologies, Osijek, Croatia).

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SMM and TMP were obtained from Sigma-Aldrich (St. Louis, MO, USA). Internal

126

standards sulfamethazine phenyl 13C6 (SMZ-13C6) was purchased from the Cambridge

127

Isotope Laboratory (Andover, MA, USA).

128

Mixed stock solution for SMM and TMP was prepared at concentration of 10 µg ml-1

129

in methanol and further diluted ten-fold in methanol for the preparation of working solutions.

130

The internal standard stock solution was prepared at a concentration of 100 µg ml-1. The

131

working mixed solution of internal standards was at 1 µg ml-1. Working solutions were used

132

for standard curve and matrix fortification. For calibration curves, standards were prepared in

133

the mobile phases A and B (1:1).

134 135

2.3. Sample preparation

136 137

Homogenized yolk and white egg samples were weighed to 10 g and spiked with the

138

internal standard. For extraction, 50 ml of the acetone/dichlormethane mixture (1:1, v/v) was

139

added and mixed with the dispersing system for 1 minute. To remove water from the mixture,

140

10 g sodium chloride and 10 g sodium sulphate were added. After each addition, samples

141

were shaken for 30 seconds. After centrifugation (3000 x g, 5 minutes), 5 ml of supernatant

142

was taken and 250 µl of acetic acid was added, followed by vortex mixing. SPE columns were

143

previously activated with two times 6 ml of an acetone/dichlormethane / acetic acid mixture

144

(47.5:47.5:5). Samples were added to the column and columns were washed by adding 5 ml

145

water and 5 ml of methanol. For elution, 5 ml of methanol with 2.5% of ammonia was added.

146

Samples were evaporated with nitrogen at 50 ± 5°C. Residues were dissolved in 100 µl of

147

methanol and 100 µl of mobile phase B and vortexed. Samples were filtered through 0.45 µm

148

regenerated cellulose membrane filters prior to injection in the LC-MS/MS.

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2.4. Instrumentation

151 152

The following equipment was used in sample preparation: blender model 7011HS

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(Waring Commercial, Connecticut, USA), dispersing system Polytron model T-2000

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(Kinematica, Inc., Switzerland), vortex model Minishaker MS2 (IKA® -WERKE GMBH &

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CO.KG, Staufen, Germany), ultrasonic bath Iskra (ISKRA PIO, Slovenia), vacuum manifold

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Supelco (Supelco Inc, Bellefonte, PA), centrifuge Rotanta 460R (Hettich Zentrifugen,

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Tuttlingen, Germany) and nitrogen evaporation system N-EVAP model 112 (Orgamonation

158

Associates Inc., Berlin, MA, USA).

159

Analysis of SMM and TMP was carried out by liquid chromatography with a tandem

160

mass spectrometry Agilent LC-MS/MS system consisting of HPLC 1200 and Triple Quad

161

LC/MS 6410 mass spectrometer (Palo Alto, CA, USA).

162 163

2.5. Chromatographic and MS parameters

164 165

Chromatographic separation was achieved by gradient elution on a X Terra RP18 Waters

166

4.6 x 150 mm, 3.5 µm (Waters, Milford, MA, USA) column at 40°C. The mobile phase

167

consisted of phase A, 5 mM ammonium formate acidified at pH 3.5 with formic acid and

168

phase B, 5 mM ammonium formate prepared in methanol. The gradient condition started with

169

0% B, then was ramped linearly to 100% from 10–12.10 min, further to 16 min back to 0%,

170

and further column stabilization for 3 min. The flow rate was 0.6 ml min-1 and injection

171

volume was 15 µl.

172

The triplequad mass spectrometer consisted of an ESI ion source and was operated in

173

positive ion mode. The ion source was heated to 350°C with a gas flow of 11 l min-1 and

174

capillary voltage at 4000 V. Fragmentor voltage (F) and collision energy (CE) were optimized

7

175

by injecting 2 µl of the 10 µg ml-1 standard solution. Ultimately, data were acquired according

176

to the multiple reaction monitoring approach (MRM), by selecting the most intense ion

177

transition from the precursor to product ions. Analytes were quantified both by the isotope

178

dilution and the matrix-matched approach, calculating the response factors for the scanned

179

product ions. A summary of the optimized MS/MS conditions for Multiple Reaction

180

Monitoring (MRM) analysis is provided in Table 1.

181 182

2.6. Method validation parameters

183 184

Limit of detection (LOD), limit of quantification (LOQ), precision and trueness were

185

determined according to the criteria laid down by Commission Decision 2002/657/EC

186

(European Commission, 2002). Limit of detection LOD and limit of quantification LOQ were

187

obtained by adding three and ten times the standard deviation of the 20 blank samples to the

188

mean blank value. Method precision and trueness was determined by fortifying egg samples

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with SMM and TMP at 5, 10 and 15 µg kg-1 and 2.5, 5 and 7.5 µg kg-1 in six replicates for

190

each level. The method precision was calculated as the coefficient of variation % (CV) and

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trueness as recovery (%).

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2.7. Statistical analysis

194 195

Statistical analysis was performed using the SPSS Statistics 17.0 software package.

196

Concentrations were expressed as mean ± standard deviation. Half-life of elimination for

197

SMM and TMP in yolk and white after the end of treatment was calculated using a linear

198

regression approach (Bourne, 1992). The differences between the TMP and SMM

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concentrations in egg white and yolk were analyzed using the t-test. The differences were

200

considered significant when p ≤ 0.05.

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3. Results and discussion

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In the poultry industry, the widespread use of veterinary drugs, such as antimicrobial

205

compounds, may lead to the presence of residues in eggs. This study is based on the

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assumption that the chemotherapeutic SMM/TMP combination may be occasionally and

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accidentally or intentionally used for laying hens via drinking water and this may cause

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substantial residues in eggs over a long period after administration. There are few studies

209

regarding the determination of sulfonamides and TMP in eggs (Atta & El-Zeini, 2001;

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Roudaut & Garnier, 2002; Vandenberge et al., 2012).

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The performance method parameters limit of detection (LOD), limit of

212

quantification (LOQ), precision and recovery are reported in Table 2. As SMM and TMP are

213

prohibited in eggs, mixed egg yolk and white was spiked at the lowest concentrations possible

214

to detect. For TMP, LOD and LOQ were calculated at lower levels (0.3 and 1.2 µg kg-1) than

215

for SMM (1.9 and 7.4 µg kg-1). Satisfactory values for precision (< 8.5%) and recovery (94.2-

216

106.6%) were achieved for both analytes. Obtained validation parameters for the method used

217

were shown to meet the criteria for detecting residues of SMM and TMP at very low

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concentrations.

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No residues were detected in the egg yolk and white of the non-treated control group

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of hens. Therefore, no contamination or other interfering residues were present. The

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deposition and depletion of SMM in egg yolk and white during and after administration of

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SMM/TMP combination via drinking water within 7 days (day 1 to 7) using two

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concentrations of 8 g l-1 (dose 1) and 12 g l-1 (dose 2) are presented in Fig. 1 and 2 for SMM

224

and Fig. 3 and 4 for TMP.

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During administration of the drug, residue concentrations of SMM in yolk were in the

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range of 257 to 5358 µg kg-1 for dose 1 (Fig. 1) and 358 to 8101 µg kg-1 for dose 2 (Fig. 2),

227

and in egg white were in the range of 778 to 4737 µg kg-1 for dose 1 and 1025 to 6018 µg kg-1

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for dose 2. The maximum measured concentration of SMM in the yolk and white of group 2

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was 1.6 and 0.8 times higher, respectively, than the values determined in group 1. The SMM

230

concentration was increased gradually and reached maximal levels on the second day of drug

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withholding for both groups 1 and 2 (µg kg-1): 5920 and 9453 in yolk; 4831 and 6050 in

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white, respectively. Thereafter, SMM concentrations gradually decreased during the 37 days

233

of the post-treatment period. A chromatogram of an SMM in egg yolk (group 1) day after

234

treatment is presented in Fig. 5.

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Using a linear regression approach, the half-life of elimination after medication was

236

determined. After the end of treatment, SMM concentrations decreased more rapidly in group

237

1 than 2 and the half-life of elimination was 4.58 and 5.37 days in yolk. In the post-treatment

238

period, SMM residues were retained longer in white than in yolk, with half-life elimination of

239

8.04 and 9.94 days in white for doses 1 and 2, respectively. The average ratio of the SMM

240

concentration between yolk and white within the first 4 days after the end of treatment was

241

1:0.82. However, after day 7 of the post-treatment period, SMM residues decreased much

242

slower in egg white, with an average ratio in white and yolk of 3.75–25.4:1 following dose 1,

243

and 2.72–34.4:1 following dose 2.

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Significantly higher concentrations of SMM were measured in white than those in

245

yolk in group 1 in the post-treatment period at days 7, 10, 13 and 16. However, for group 2

246

there were statistically significant differences in the SMM concentrations among white and

247

yolk during the post-treatment period 1-19 days, with the exception of 4 day. There were

10

248

significant reductions in the concentrations of SMM in post treatment period (day): group 1:

249

yolk 4-16, white 10-25; group 2: yolk 3-19, white 10-28.

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The results differ from previously reported results, and SMM levels determined were

251

higher in yolk than in white. Previously, it was concluded that sulfonamides are initially

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deposited following treatment at higher concentrations in egg white compared to yolk

253

(Roudaut & Garnier, 2002). This may be explained by the fact that relative concentrations of

254

a drug in egg yolk and white depend on its relative lipid/water solubility (Blom, 1975).

255

Sulfonamides are more hydrophilic and weakly acidic substances and accordingly, they are

256

transferred in higher concentrations to egg white than yolk (Furusawa, Mukai, & Ohori, 1996;

257

Furusawa, 2003). However, due to more than one determining factor influencing the

258

distribution in egg white and yolk, it can be concluded that it is difficult to determine a

259

general trend for the deposition of sulfonamides in egg white and yolk, or even in both

260

(Alaboudi et al., 2013; Hafez, 1991). It can be concluded that a difference in factors such as

261

pH values or lipid solubility could not sufficiently explain the distribution between egg white

262

and yolk (Alaboudi et al., 2013). Also, drug distribution among egg compartments is also

263

determined by drug dose and dosing (Donoghue, Hairston & Podhorniak, 1997). The results

264

obtained in the present study suggest that the high concentrations of the drugs administered

265

were a major factor affecting the distribution between white and yolk compared to yolk

266

following treatment.

267

In the present study, SMM concentrations gradually decreased and dropped below 100

268

µg kg-1 between days 11 and 13 in yolk and between days 19 and 22 in white, after drugs

269

administration of doses 1 and 2, respectively. SMM concentrations were measured below

270

LOQ of 7.4 µg kg-1 after day 16 in yolk and day 25 in white after doses 1 and 2, respectively.

271

Also, SMM levels dropped below LOD (1.9 µg kg-1) after day 16 and 19 in yolk following

11

272

dose 1 and 2 and after day 37 in white following dose 1. However, SMM was detected above

273

the LOD in white at day 37 after administration of dose 2.

274

In a previous study, SMM was administered to laying hens for 5 days at the dietary

275

level of 400 mg kg-1 in feed. The biological half-life of SMM in whole egg was estimated to

276

be 0.88 days (Furusawa & Mukai, 1995). Previously, it was stated that feed may be an

277

important factor for the reduction of the rate of drug absorption and may influence the

278

deposition of drugs in eggs (Nielsen & Gyrd-Hansen, 1994). Drug administration via drinking

279

water may be affected by ambient temperature, as elevated temperatures greatly influence

280

water intake. However, this study was conducted in the autumn period when the temperature

281

was relatively stable and below 10°C and therefore the ambient temperature was not a factor

282

affecting the amount of water intake in chickens.

283

In a previous study, sulfadimidine (SDM) and sulfadimethoxine (SDT) levels were

284

determined in eggs following oral administration through drinking water for five days (0.5 g l-

285

1

286

compounds in yolk declined below the limit of quantification (0.005 µg g-1) within 14–15

287

days after discontinuation of treatment. In this study, it was determined that a longer period

288

was required for the SMM and TMP concentrations to fall below the LOQ values, which

289

could be explained by the high concentrations of this drug used in drinking water for chickens

290

compared to the much lower concentrations tested in previous studies. This conclusion is also

291

confirmed by the results obtained in a study of depletion of sulfadiazine and TPM in eggs of

292

laying hens after administration of 0.2 and 0.4 g l-1 of a drug in drinking water for five

293

successive days (Atta & El-Zeini, 2001). Sulfadiazine was detected up to days 4 and 6 in egg

294

yolk and the maximum concentration of sulfadiazine was 0.15 and 0.18 µg g-1 in yolk after

295

the usage of small and large dose, respectively.

for SDT, 1 and 2 g l-1 for SDM) (Roudaut & Garnier, 2002). Residue levels of these two

12

296

In this study, on the first two days during administration of TMP in doses 1 (Fig. 3)

297

and 2 (Fig. 4), TMP was not detected in yolk but was detected in white. During

298

administration, TMP levels gradually increased until day 7 of treatment (µg kg-1): group 1:

299

from 458 to 4549 in yolk, from 9.80 to 1274 in white; group 2: from 889 to 5311 in yolk,

300

from 25.5 to 1330 in white. The LC-MS/MS chromatogram of the TMP in egg yolk (group 1)

301

day after treatment is shown in Fig. 6. TMP reached maximal levels on the third day after

302

drug administration for both treatment doses (µg kg-1): 6521 and 7329 in yolk and 1370 and

303

1539 in white. Results show that TMP residues were measured in higher concentrations in

304

egg yolk than in white with an approximate ratio of 4.8:1 in the first four days of the post-

305

treatment period. During the post-treatment period, TMP levels gradually decreased. As

306

expected, the half-life of elimination in yolk and white of group 2 was longer than in group 1.

307

The half-life of elimination was 7.26 and 8.39 days in yolk and 4.90 and 6.78 days in white

308

after administration of doses 1 and 2, respectively. Higher concentrations of TMP in yolk than

309

in white may be explained by the lipid soluble organic base properties of TMP (Atta & El-

310

Zeini, 2001).

311

TMP dropped below 100 µg kg-1 between days 13 and 16 and between days 16 and 19

312

in yolk in the post-treatment period for doses 1 and 2, respectively. In white, TMP was

313

measured below 100 µg kg-1 between days 16 and 19, and between days 19 and 22 after the

314

administration of doses 1 and 2, respectively. The concentrations of TMP in group 1 were

315

measured below the LOD of 0.3 µg kg-1 in white at day 31 while in white TMP levels were

316

measured above the LOD on the final 37th day of measurement. However, TMP residues were

317

measured below LOD (0.3 µg kg-1) in white, but were at concentration of 1.98 µg kg-1 in yolk

318

of group 2 on day 37 of the post-treatment period.

319

Significantly higher concentrations of TMP were determined in yolk than those in

320

white in both experimental groups between 1 and 22 days of the post-treatment period, with

13

321

the exception of day 10 for dose 2. Significant reductions in the TMP concentrations were

322

found during the post treatment period between 3 and 4 day for both groups. Furthermore,

323

significantly lower levels were measured between days of measurement after drugs

324

administration (day): group 1: yolk 7-22, white 13-25; group 2: yolk 13-22, white 10-28.

325

The majority of previous studies suggest that the residence time of TMP in eggs is

326

from 9 to 11 days (Atta & El-Zeini, 2001; Nagata et al., 1991). TMP administration in laying

327

hens at concentrations of 4, 16 and 56 mg kg-1 in feed for 19 days showed that TMP levels

328

drop below the detection limit of 0.02 mg kg-1 in egg yolk and albumen at 4, 9 and 11 day

329

after the last dose (Nagata et al., 1991). Furthermore, administration of TMP at a dose of 0.2

330

and 0.4 g l-1 in water for five days resulted in a drop of TMP values to below the limit of

331

detection of 0.02 µg g-1 on day 5 and 7 after the end of treatment (Atta & El-Zeini, 2001).

332 333

4. Conclusions

334 335

In conclusion, SMM and TMP oral medication with doses 1 and 2 during 7 days

336

resulted in the deposition of SMM and TMP residues in both egg yolk and white. The

337

maximal concentrations of SMM in yolk and white were determined on day 2 of the post-

338

treatment period for both groups. There were significant differences in the SMM and TMP

339

concentrations between yolk and white in post treatment period. SMM residues were retained

340

longer in the white than in the yolk, and the half-life of elimination was 1.5–2 times higher in

341

white than in yolk. SMM concentrations in yolk dropped below the LOD (1.9 µg kg-1) after

342

day 16 and 19 after administration of doses 1 and 2. However, SMM was measured above

343

LOD only in white for group treated with dose 2. The maximal concentration of TMP in yolk

344

and white were measured on day 3 in the post-treatment period for both doses. TMP gradually

345

decreased and the half-life of elimination was higher in yolk than in white after administration

14

346

of doses 1 and 2. The concentrations of TMP in group 1 dropped below the LOD (0.3 µg kg-1)

347

value in yolk and white on day 37, the last day of measurement. However, in group 2, TMP

348

residues were still above LOQ (1.2 µg kg-1) in yolk on day 37 after the end of administration.

349

It could be concluded that the application of the two concentrations of SMM and TMP

350

causes prolonged retention of residues for more than 30 days after medication. The study

351

showed a longer period required for SMM and TMP concentration to fall below the LOQ

352

values, which could be explained by the high dosage of these drugs used in drinking water for

353

chickens, compared to much lower concentrations tested in previous studies.

354 355 356

References

357 358

Alaboudi, A., Basha, E. A., & Musallam, I. (2013). Chlortetracycline and sulfanilamide

359

residues in table eggs: Prevalence, distribution between yolk and white and effect of

360

refrigeration and heat treatment. Food Control, 33, 281–286.

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Atta, A.H, & El-zeini, S. A. (2001). Depletion of trimethoprim and sulphadiazine from eggs

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of laying hens receiving trimethoprim/sulphadiazine combination. Food Control, 12, 269–

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274.

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Blom, L. (1975). Plasma half-lives and the excretion into egg albumen and yolk of three

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sulphonamides and pyrimethamine after medication of laying hens. Acta pharmacologica et

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toxicologica, 37, 79–93.

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Bourne, D. W. A. (1992). Mathematical Modeling of Pharmaceutical Data: With Examples

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from Pharmacokinetics. Technomic Pub. A G. http://www.boomer.org/c/p3/c05/c0508.html

372 373

Campbell, K. L. (1999). Sulphonamides: updates on use in veterinary medicine. Veterinary

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Dermatology, 10, 205-215.

375 376

Donoghue, D. J., Hairston, H., & Podhorniak, L.V. (1997). Modeling drug residue uptake by

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eggs: evidence of a consistent daily pattern of contaminant transfer into developing

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preovulatory yolks. Food Protection, 60, 1251–1255.

379 380

Donoghue, D. J., & Myers, K. (2000). Imaging residue transfer in to egg yolk. Journal of

381

Agricultural and Food Chemistry, 48, 6428–6430.

382 383

European Commission. (2002). Commission Decision 2002/657/EC of 14 august 2002 on

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implementing council directive 96/23/EC concerning the performance of analytical methods

385

and the interpretation of results 2002/657/EC. Official Journal of European Union, L 221, 8-

386

36.

387 388

European Commission. (2010). Council Regulation 37/2010/EU of 22 December 2009 on

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pharmacologically active substances and their classification regarding maximum residue

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limits in foodstuffs of animal origin. Official Journal of European Union, L15, 1-72.

391 392

EMEA (The European Agency for the Evaluation of Medical Products) (1995). Sulfonamides,

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summary

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EMEA/MRL/026/95.

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veterinary

medicinal

products,

EMEA.

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395 396

Furusawa, N., & Mukai, T. (1995). Disappearance pattern of sulfamonomethoxine from eggs.

397

Jappan Poultry Science, 32, 34–41.

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Furusawa, N., Mukai, T., & Ohori, H. (1996). Depletion of sulphamonomethoxine and

400

sulphadimethoxine from various tissues of laying hens. British Poultry Science, 37, 435–442.

401 402

Furusawa, N., Tsuzukida, Y., & Yamaguchi, H. (1998). Decreasing profile of residual

403

sulphaquinoxaline in eggs. British Poultry Science, 39 (2), 241–244.

404 405

Furusawa, N. (2003). Rapid high-performance liquid chromatographic determining technique

406

of sulfamonomethoxine, sulfadimethoxine, and sulfaquinoxaline in eggs without use of

407

organic solvents. Analytica Chimica Acta, 481, 255-259.

408 409

Hafez, H. M. (1991). Factors influencing drug residues in poultry products: a review. Arch

410

Gefluegelk, 55, 193-195.

411 412

Hela, W. , Brandtner, M., Widek, R., & Schuh, R. (2003). Determination of sulfonamides in

413

animal tissues using cation exchange reversed phase phase sorbent for sample cleanup and HPLC-

414

DAD for detection. Food Chemistry, 83, 601-608.

415 416

JECFA (Joint FAO/WHO Expert Committee on Food Additives). (2004). Toxicological

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evaluation of certain veterinary drug residues in food, 62nd meeting of the JECFA.

418

Monograph prepared by the JECFA. WHO Technical paper series No. 925 Geneva: WHO.

419 17

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Li, H., Sun, H., Zhang, J., & Pang, K. (2013). Highly sensitive and simultaneous

421

determination of sixteen sulphonamide antibiotics, four acetyled metabolites and

422

trimethoprim in meat by rapid resolution liquid chromatography-tandem mass spectrometry.

423

Food Control, 31, 359-365.

424 425

Nagata, T., Saeki, M., Iida, T., Kataoka, M., & Shikano, S. (1991). High performance liquid

426

chromatographic determination of trimethoprim residues in egg yolk and albumen in a

427

feeding experiment. British Veterinary Journal, 147, 346–351.

428 429

Nielsen, P., & Gyrd-Hansen, N. (1994). Oral bioavailability of sulphadiazine and

430

trimethoprim in fed and fasted pigs. Research in Veterinary Science, 56, 48–52.

431 432

Romvary, A., & Simon, F. (1992). Sulphonamide residues in eggs. Acta Veterinaria

433

Hungarica, 40, 99–106.

434 435

Roudaut, B., & Garnier, M. (2002). Sulphonamide residues in eggs following drug

436

administration via the drinking water. Food Additives & Contaminants, 19, 373-378.

437 438

Šeol, B., Matanović, K., & Terzić, S. (2010). Antimicrobial therapy in veterinary medicine.

439

Herak-Perković, V. (Ed). Medicinska naklada, Zagreb, Croatia.

440 441

Vandenberge, V., Delezie, E., Huyghebaert, G., Delahaut, P., De Backer, P., Daeseleire, D. et

442

al. (2012). Residues of sulfadiazine and doxycycline in egg matrices due to cross-

443

contamination in the feed of laying hens and the possible correlation with physicochemical,

18

444

pharmacokinetic and physiological parameters. Food Additives & Contaminants A, 29, 908-

445

917.

446 447

Weiss, C., A. Conte, C. Milandri, G. Scortichini, P. Semprini, R. Usberti, et al. (2007).

448

Veterinary drugs residue monitoring in Italian poultry: Current strategies and possible

449

developments. Food Control, 18, 1068-1076.

450

19

451 7000

6000

Concentration (µg kg-1)

5000

4000 SMM in egg yolk SMM in egg white 3000

2000

1000

0 -7 -6 -5 -4 -3 -2 -1

452

1

2

3

4

7 10 13 16 19 22 25 28 31 34 37

days

453 454

Fig. 1. Depletion of SMM in egg yolk and white during and after administration of combined

455

therapeutic SMM/TMP in a dose of 8 g l-1 (group 1) via drinking water for the 7 days.

456 457 458 459 460

20

461 18000

16000

14000

Concentration (µg kg-1)

12000

10000 SMM in egg white SMM in egg yolk

8000

6000

4000

2000

0 -7 -6 -5 -4 -3 -2 -1

1

2

3

4

7 10 13 16 19 22 25 28 31 34 37

days

462 463 464 465

Fig. 2. Depletion of SMM in egg yolk and white during and after administration of combined

466

therapeutic SMM/TMP in a dose of 12 g l-1 (group 2) via drinking water for the 7 days.

467 468

21

469 8000

7000

6000

Concentration (µg kg-1)

5000

TMP in egg yolk TMP in egg white

4000

3000

2000

1000

0 -7 -6 -5 -4 -3 -2 -1

1

2

3

4 7 10 13 16 19 22 25 28 31 34 37 days

470 471 472

Fig. 3. Depletion of TMP in egg yolk and white during and after administration of combined

473

therapeutic SMM/TMP in a dose of 8 g l-1 (group 1) via drinking water for the 7 days.

474 475

22

476 8000

7000

6000

Concentration (µg kg-1)

5000 TMP in egg yolk TMP in egg white

4000

3000

2000

1000

0 -7 -6 -5 -4 -3 -2 -1

1

2

3

4 7 10 13 16 19 22 25 28 31 34 37 days

477 478 479

Fig. 4. Depletion of TMP in egg yolk and white during and after administration of combined

480

therapeutic SMM/TMP in a dose of 12 g l-1 (group 2) via drinking water for the 7 days.

481 482

23

483

484 485 486

Fig. 5. Chromatogram of the SMM in egg yolk (group 1) day after treatment showing first

487

and second transition, and spectra of the precursor and product ions.

488 489

24

490 491

492 493 494

Fig. 6. Chromatogram of the TMP in egg yolk (group 1) day after treatment showing first and

495

second transition, and spectra of the precursor and product ions.

496 497 498 499 500 501 502

25

503 504

Table 1

505

MS/MS conditions for MRM analysis Compound

RT (min)

Precursor ion

TMP

11.3

291

SMZ 13C6

11.9

285

SMM

13.0

281

Product ion 123 230 186 124 92 108

Fragmentor (V) 85 120 135

Collision energy (V) 29 25 12 19 30 24

506 507 508 509 510 511

Table 2

512

Validation parameters for SMM and TMP quantification in eggs.

Analyte

SMM

TMP

LOD (µg kg-1)

1.9

0.3

LOQ (µg kg-1)

7.4

1.2

Spiking level (µg kg-1)

Estimated concentration n=6 (µg kg-1)

Standard deviation

Precision CV (%)

Recovery (%)

5

4.7

0.3

7.1

94.2

10

10.6

0.5

5.1

106.6

15

14.7

1.2

7.9

98.3

2.5

2.5

0.1

3.0

101.4

5

4.8

0.4

8.1

96.1

7.5

7.6

0.6

7.9

101.5

513 514

26

515 516

Highlights

517 518

► Laying hens received sulfamonomethoxine (SMM) and trimethoprim (TMP) in two doses.

519

► The distribution of SMM and TMP in egg yolk and white was measured.

520

► SMM and TMP residues was measurable for 30 days after drug administration.

521

►SMM is detectable for a longer period in egg white (> 30 days) than in yolk.

522

► TMP is measured in period of 37 day in egg yolk but not in white.

523 524 525 526 527

27

Distribution of sulfamonomethoxine and trimethoprim in egg yolk and white.

The distribution of sulfamonomethoxine (SMM) and trimethoprim (TMP) in egg yolk and white was measured during and after administration of a SMM/TMP co...
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