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
5
Cvetnićb, Željko Cvetnićc
6
a
7
cesta 143, HR-10000 Zagreb, Croatia
8
b
Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia
9
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
10 11
ABSTRACT
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The distribution of sulfamonomethoxine (SMM) and trimethoprim (TMP) in egg yolk and
13
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
15
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
20
and 2 (µg kg-1): 6521 and 7329 in yolk, 1370 and 1539 in white. TMP residues were measured
21
above LOD (0.3 µg kg-1) in yolk for both doses on day 37 post-treatment.
22 23
Key words: Sulfamonomethoxine; Trimethoprim; Elimination; Laying hens; Egg; Yolk;
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White; LC-MS/MS
25 26
∗
Corresponding author. Tel.: +385 1 612 3601, fax: +385 1 612 3636
1
27 28
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
60
so on. However, dispersible powder and suspensions of SMM/TMP are prohibited for use in
61
laying hens. On the other hand, there may be situations where these compounds can be
62
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.,
73
2012) or combinations of trimethoprim and sulfonamides (Atta & El-zeini, 2001; Nagata,
74
Saeki, Iida, Kataoka, & Shikano, 1991) have been studied in the eggs of laying hens. In
75
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
85 86
2.1. Animal treatment and sampling
87 88
A total of 50 laying hens (ISA Brown) were obtained from a breeding station and were
89
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
4
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
103
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
114 115
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).
5
125
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.
149
6
150
2.4. Instrumentation
151 152
The following equipment was used in sample preparation: blender model 7011HS
153
(Waring Commercial, Connecticut, USA), dispersing system Polytron model T-2000
154
(Kinematica, Inc., Switzerland), vortex model Minishaker MS2 (IKA® -WERKE GMBH &
155
CO.KG, Staufen, Germany), ultrasonic bath Iskra (ISKRA PIO, Slovenia), vacuum manifold
156
Supelco (Supelco Inc, Bellefonte, PA), centrifuge Rotanta 460R (Hettich Zentrifugen,
157
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
189
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
191
trueness as recovery (%).
192 193
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
8
199
concentrations in egg white and yolk were analyzed using the t-test. The differences were
200
considered significant when p ≤ 0.05.
201 202
3. Results and discussion
203 204
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
206
assumption that the chemotherapeutic SMM/TMP combination may be occasionally and
207
accidentally or intentionally used for laying hens via drinking water and this may cause
208
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;
210
Roudaut & Garnier, 2002; Vandenberge et al., 2012).
211
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
218
concentrations.
219
No residues were detected in the egg yolk and white of the non-treated control group
220
of hens. Therefore, no contamination or other interfering residues were present. The
221
deposition and depletion of SMM in egg yolk and white during and after administration of
222
SMM/TMP combination via drinking water within 7 days (day 1 to 7) using two
9
223
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.
225
During administration of the drug, residue concentrations of SMM in yolk were in the
226
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
228
for dose 2. The maximum measured concentration of SMM in the yolk and white of group 2
229
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
231
withholding for both groups 1 and 2 (µg kg-1): 5920 and 9453 in yolk; 4831 and 6050 in
232
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.
235
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.
244
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.
250
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
252
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.
361 362
Atta, A.H, & El-zeini, S. A. (2001). Depletion of trimethoprim and sulphadiazine from eggs
363
of laying hens receiving trimethoprim/sulphadiazine combination. Food Control, 12, 269–
364
274.
365 366
Blom, L. (1975). Plasma half-lives and the excretion into egg albumen and yolk of three
367
sulphonamides and pyrimethamine after medication of laying hens. Acta pharmacologica et
368
toxicologica, 37, 79–93.
369
15
370
Bourne, D. W. A. (1992). Mathematical Modeling of Pharmaceutical Data: With Examples
371
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
374
Dermatology, 10, 205-215.
375 376
Donoghue, D. J., Hairston, H., & Podhorniak, L.V. (1997). Modeling drug residue uptake by
377
eggs: evidence of a consistent daily pattern of contaminant transfer into developing
378
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
384
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
389
pharmacologically active substances and their classification regarding maximum residue
390
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,
393
summary
394
EMEA/MRL/026/95.
report
(2)
committee
for
veterinary
medicinal
products,
EMEA.
16
395 396
Furusawa, N., & Mukai, T. (1995). Disappearance pattern of sulfamonomethoxine from eggs.
397
Jappan Poultry Science, 32, 34–41.
398 399
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
417
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
420
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