a sulfadimidine model to evaluate pharmacokinetics and residues at
TRANSCRIPT
Aus dem Institut für Pharmakologie, Toxikologie und Phamazie
der Stiftung Tierärztliche Hochschule Hannover
A sulfadimidine model to evaluate pharmacokinetics and residues at various concentrations in laying hens
INAUGURAL DISSERTATION
Zur Erlagung des Grades eines
Doktors der Veterinärmedizin
(Dr. med. vet.)
durch die Stiftung Tierärztliche Hochschule Hannover
Vorgelegt von
Natthasit Tansakul
aus Lopburi/ Thailand
Hannover 2008
Wissenchaftliche Betreuung: Univ.-Prof. Dr. Manfred Kietzmann
1. Gutachter: Univ.-Prof. Dr. Manfred Kietzmann
2. Gutachter: Prof. Dr. Gerd Hamscher
Tag der mündlichen Prüfung: 11 November 2008
Dedicated to my father, with love and never ending faith &
my beloved mother
Contents
Contents
1 Introduction.................................................................................................................7
2 Literature review.........................................................................................................8
2.1 The principle of pharmacokinetics.......................................................................8
2.1.1 Definition and principle ................................................................................8
2.1.2 Pharmacokinetic parameters .........................................................................9
2.2 General considerations of sulfonamides ............................................................14
2.2.1 History and properties of sulfonamides ......................................................14
2.2.2 Pharmacokinetic of sulfonamides ...............................................................15
2.3 Veterinary drug residues ....................................................................................20
2.4 Sulfonamide residues in animals .......................................................................27
2.4.1 Sulfonamide residues in livestock ..............................................................27
2.4.2 Sulfonamides residues in poultry................................................................29
2.5 Sulfadimidime....................................................................................................35
2.5.1 Properties of sulfadimidine .........................................................................35
2.5.2 Pharmacokinetics of sulfadimidine in laying hens .....................................35
2.5.3 Residues of sulfadimidine in laying hens ...................................................38
3 Aims and objectives..................................................................................................40
4 Materials and methods ..............................................................................................41
4.1 Materials ............................................................................................................41
4.2 Methods..............................................................................................................43
4.2.1 Drug administration and sample collection ................................................43
4.2.2 Analysis (HPLC).........................................................................................45
4.2.3 Pharmacokinetic parameters and statistical analyses..................................49
5 Results.......................................................................................................................50
5.1 Method of analysis.............................................................................................50
5.1.1 Selectivity ...................................................................................................50
5.1.2 Linearity......................................................................................................52
5.1.3 Limit of detection (LOD) and limit of quantification (LOQ) .....................54
5.1.4 Recovery .....................................................................................................54
Contents
5.1.5 Precision......................................................................................................54
5.2 Plasma concentrations of sulfadimidine following various doses administration
by intravenous and oral in laying hens ....................................................................56
5.3 Pharmacokinetic characteristics of sulfadimidine in laying hens ......................58
5.3.1 Pharmacokinetic characteristics of sulfadimidine following a single
intravenous bolus administration in laying hens..................................................58
5.3.2 Pharmacokinetic characteristics of sulfadimidine following a single oral
bolus administration at different concentrations in laying hens...........................60
5.4 Sulfadimidine residue in eggs after a single oral bolus administration at various
doses in laying hens .................................................................................................65
5.4.1 Sulfadimidine residue in yolk .....................................................................65
5.4.2 Sulfadimidine residue in albumen ..............................................................66
5.4.3 Transfer rate of sulfadimidine into eggs after a single oral administration 67
5.5 Sulfadimidine residue in eggs after medication via mixed feed at various doses
for seven consecutive days in laying hens ...............................................................68
5.5.1 Sulfadimidine residue in yolk .....................................................................68
5.5.2 Sulfadimidine residue in albumen ..............................................................70
5.5.3 Sulfadimidine concentrations in plasma .....................................................73
5.6 Comparative pharmacokinetic behavior and residues of sulfadimidine between
healthy and impaired kidney hens............................................................................74
5.6.1 Comparison pharmacokinetic behaviors of sulfadimidine between healthy
and impaired kidney hens ....................................................................................74
5.6.2 Comparison sulfadimidine residue in eggs between healthy and impaired
kidney hens ..........................................................................................................77
6 Discussion .................................................................................................................80
6.1 Method of analysis.............................................................................................80
6.2 Pharmacokinetic behavior of sulfadimidine following single bolus via
intravenous injection and various doses of single bolus via oral administration in
laying hens ...............................................................................................................82
6.3 Sulfadimidine residues in egg components after a single bolus via oral and
medicated feed at different concentrations ..............................................................86
6.4 Comparison pharmacokinetic characteristics and residues of sulfadimidine
between healthy and impaired kidney laying hens following intravenously
administration ..........................................................................................................89
Contents
7 Summary ...................................................................................................................92
8 Zusammenfassung.....................................................................................................94
9 References.................................................................................................................96
10 Appendix...............................................................................................................127
10.1 Glossary of abbreviations ..............................................................................127
10.2 Tables.............................................................................................................130
11 Acknowledgement ................................................................................................139
Parts of the thesis have been published:
TANSAKUL N., F. NIEDORF and M. KIETZMANN (2007):
A sulfadimidine model to evaluate pharmacokinetics and residues at various
concentrations in laying hen.
Food Addit. Contam. 24(6), 598–604
Introduction
7
1 Introduction
Currently, more attention is being paid to food safety. Veterinary drug residue is one
of many global issues concerning food contamination. The use of veterinary drugs in
food-producing animals has the potential to generate residues in animal-derived
products and poses a health hazard to the customer. Low-level drug contamination of
animal’s feed is one causative channel of residues in animal products. Even very low
levels of antibiotic contaminations may result in relevant residues in food, as
confirmed by studies on coccidiostats in poultry feed (ROKKA et al., 2005;
KENNEDY et al., 1998) and sulfadimidine in swine (BIEHL et al., 1981;
McCAUGHEY et al., 1990a). In the present study, sulfadimidine (SDD) was used as
a model to determine the pharmacokinetic characteristics and residue risk at various
doses in laying hens.
In former research, pharmacokinetic characteristics of SDD in chicken have been
described (KIETZMANN, 1980; GEERTSMA et al., 1987; NOUWS et al., 1988;
JIAN and PUNIA, 2001). Different doses and routes of SDD residue have also been
studied in laying hens (BLOM, 1975; SEIB, 1991; ROUDAUT, 1993; ROUDAUT
and GARNIER, 2002; FURUSAWA, 2001; KAN, 2003). These studies indicated a
transfer of sulfonamides into the eggs, but mainly provided data focusing on
therapeutic doses. Therefore, information about the pharmacokinetic behavior and
residue risk following the administration of SDD at low doses are scarce. Specially,
very little data are available on residue levels in hens after low-level sulfonamides
contaminations of layer feed. In order to determine whether substance carry-over may
result in relevant residues even in untreated animals, pharmacokinetic characteristics
and residue of SDD at low to high doses in laying hens after a single bolus oral
administration and medication via feed were observed. Moreover, the effect of kidney
dysfunction on kinetic behaviour and residue as compared to healthy laying hens was
also investigated.
Literature review
8
2 Literature review 2.1 The principle of pharmacokinetics 2.1.1 Definition and principle
The term pharmacokinetics was first introduced by DOST (1953) in order to describe
the characteristic of drugs after an aplication. It is the study of the time course of drug
and xenobiotic concentration in the body. To increase understanding of a drug’s
behaviour, it is important to examine the four major processes after drug
consumption: absorption, distribution, biotransformation and excretion.
Absorption is described as the process which a compound passes from its site of
administration into the bloodstream. Absorption is influenced by many factors such as
the properties of cell membrane, drug properties and route of administration and
physio-pathological state of the animal (CURRY, 1974). An indication of the rate of
drug absorption is obtained from the peak plasma concentration (Cmax) and time
reaching the maximum concentration (Tmax). Distribution is the process whereby a drug is transported to all the tissues and organs
(LABAUNE, 1989). After entering the systemic circulation, in whatever route of
administration, drugs are conveyed throughout the body and reach their site of action.
There are four major factors responsible for the extent and rate of distribution. These
are the physicochemical properties of the drug, the concentration gradient established
between the blood and tissue, the ratio of blood flow to tissue mass, and the affinity of
the drug for tissue constituents and serum protein binding (RIVIERE et al., 1991).
Only the fraction free-form (unbound) of drug is capable of exiting the circulation to
distribute through the body and exert activity at the site of action (BOTSOGLOU and
FLETOURIS, 2001). The parameter which defines the process of distribution is the
volume of distribution (Vd).
Literature review
9
Biotransformation (Metabolism) is the principle mechanism of elimination for the
transformation of drugs or xenobiotics into metabolites by the chemical reaction
(LABAUNE, 1989). The two classes of enzymatic pathways of biotransformation are
phase I (non-synthesis) and phase II (conjugation). Phase I corresponds to
functionalisation processes including oxidation, reduction, hydrolysis, hydration and
isomerisation reactions. Phase II reactions involve conjugation of the drug or phase I
metabolite with the endogenous substrate such as glucuronic acid, sulfate, acetate and
methyl group (RIVIERE et al., 1991). Although some drugs are eliminated from the
body by uncharged, most drugs undergo metabolism where liver is the main organ of
reaction (BOTSOGLOU and FLETOURIS, 2001). In addition, the liver’s function
may change the drug’s form to being inactive and easy to excrete but some drugs may
be converted to an activating form.
Excretion is the process by which the parent drug or its metabolites are removed from
the body fluids. The kidney is the most important site of drug excretion. There are
three renal mechachnisms: glomerular filtration, carrier-mediated proximal tubular
secretion and pH-dependent passive tubular resorption in the distal nephron
(RIVIERE et al., 1991). Renal insufficiency usually significantly affects drug
excretion (BOTSOGLOU and FLETOURIS, 2001). The systemic clearance (CL) and
elimination half-life (T1/2el) are important parameters referring to the overall rate of
elimination (metabolism and excretion). Although most compounds are excreted
primarily by the renal, some drugs are partially or completely excreted through the
bile. It has been reported that there is an extensive species variation among animals in
their general ability to secrete drugs in the bile e.g. chicken are characterised as good
biliary excreters, whereas sheep and rabbit are characterised as moderate and poor
excreters (BOTSOGLOU and FLETOURIS, 2001).
2.1.2 Pharmacokinetic parameters
The drug plasma concentration-time profile can be mathematically predicted if
quantitative estimates of the rate and extent of absorption, distribution and elimination
are available (RIVIERE et al., 1991). After administration, the drug may distribute
into the entire accessible region instantly. In such a case, the body is considered as a
Literature review
10
homogeneous container. The disposition of the drug can be described as a one-
compartment open model, as generalised in figure 1.
Administration K01 K10
Figure 1: Schematic of one-compartment open model
Mathematical expression of one-compartment with first-order input, first-order output
and no lag time can be performed following this equation:
C(T) = D*K10 / V*(K01-K10) * (exp(-K10*T)-exp(-K01*T))
C(T) is the plasma drug concentration at any time (T) and D is the dose (amount) of
drug to be administered. K01 and K10 are the apparent first-order absorption and
distribution (elimination) rate constants, respectively. V is an apparent volume of
distribution based on the area under the curve and the coefficient represents the zero-
time intercept (in unit of concentration) of the back-extrapolated disposition phase
corresponding to the zero-time intercept of the absorption phase (BAGGOT, 2001).
2.1.2.1 Area under the curve
The area under the curve (AUC) is the area under a concentration of the analysis
versus time curve. Theoretically, if C(t) denotes the concentration of analysis at time
t, then AUC can be described in equation 1.1. Moreover, the AUC can be simply
obtained from the relationship between dose (D) and total clearance (CL), following
equation 1.2.
AUCab =a ∫b c(t)dt (1.1)
AUC = D/ CL (1.2)
0 1
Literature review
11
2.1.2.2 Elimination half-life
The elimination half-life time (T1/2el) indicates the rate of elimination. It is the time
required drug plasma concentration at 50 % of the dose present in the circulation. It is
related to the elimination rate constant (Kel or K10) by the equation 1.3. However,
with the relationship between the volume of distribution and the systemic (body)
clearance of the drug, the general equation for calculating of the elimination half-life
is given in equation 1.4 (BAGGOT, 2001).
T1/2el = 0.693/ Kel (1.3)
T1/2el = 0.693 . Vd(area)/ CL (1.4)
After carrying out the elimination half-life five times, 97 % of a drug could be
eliminated or only 3 % of the dose may remain in the body. However, the hybrid
parameter corresponds with both Vd and CL. Thus, the body can be affected
pathologically making the derived T1/2el an unreliable indicator of drug elimination
(RIVIERE et al., 1991). Regarding the time unit, the Tmax is the observed time after
drug administration at which peak plasma concentration occurs. Generally, the Tmax
can theoretically be calculated by equation 1.5. However, estimating Tmax in equation
1.6 was obtained by using the WinNonLin® program. According to the relationship
with Tmax, Cmax is the maximum concentration at Tmax, this being calculated by
equation 1.7.
Tmax = (ln . Ka/Kel) / Ka- Kel (1.5)
Tmax = ln(K01/K10)/ (K01-K10) (1.6)
ln Cmax = ln Yo – (Kel . Tmax) (1.7)
Yo is the theoretically calculated zero time plasma concentration.
Literature review
12
2.1.2.3 Volume of distribution
The apparent volume of distribution (Vd) indicates the apparent space (L/kg) in the
body available to contain the drug. Thus, the volume of distribution at the steady state
Vss can be calculated after intravenous administration. Vd is the apparent distribution
which relates the plasma concentration of a drug to the total amount of drug in the
body at any time after pseudodistribution equilibrium has been attained (equation
1.7).
Vd = Dose/Cp0 (1.7)
Cp0 is the drug concentration at time zero. The volume of distribution can be
calculated by equation 1.8 (BAGGOT, 2001). The volume of distribution can also be
calculated from the total clearance and half-life by using equation 1.9 (LABAUNE,
1989).
Vd(area) = Dose/ AUC . β (1.8)
Vd = T1/2el . CL / 0.693 (1.9)
2.1.2.4 Clearance
The systemic clearance (CL) of a drug represents the sum of the clearances by the
various organs that contribute to eliminating of the drug. Total body clearance is the
sum of the clearances of each organ (renal, hepatic and others) of elimination
(RIVIERE et al., 1991; BAGGOT, 2001). The systemic clearance can be generalised
by the following equation:
CL = F . Dose/ AUC (1.10)
F is the fraction of absorption which assumes complete systemic availability after
intravenous administration. From the relationship with volume of distribution, the
systemic clearance can be obtained by:
Literature review
13
CL = Vd(area) . β (1.11)
β is the apparent first-order rate constant fraction associated with elimination per unit
of time after intravenous or extra-vascular administration.
2.1.2.5 Bioavailability
The absolute bioavailability is determined by comparing the area under the curve
(AUC) after extra-vascular administration, with the AUC after intravascular
administration. The “first-pass effect” can substantially reduce the systemic
availability (absolute oral bioavailability) of lipid soluble drugs undergo extensive
biotransformation in the liver (BAGGOT, 2001). The usual method for estimating
systemic availability (F) of drugs after extra-vascular administration of the dose is the
method of corresponding areas, following the equation:
F = AUC(extra-vascular) / AUC (intravascular) (1.12)
2.1.2.6 Mean residence time
The mean residence time (MRT) represents the average time the molecules of a drug
reside in the body following the administration of a single dose (BAGGOT, 2001).
The molecules introduced into the body when the drug is administered, have the same
probability of being absorbed, distributed, metabolised or eliminated. The distribution
curve resulting from this (plasma concentration vs time) can be characterised by its
mean value (MRT) and the variance of this mean (LABAUNE, 1989). Based on the
area under the curve, MRT can be calculated by the following equation:
MRT = AUMC/ AUC (1.13)
AUMC is the area under the (first) moment curve obtained from the product of plasma
concentration and time versus time from time zero to infinity and AUC is the total
area under the curve (zero moment) (BAGGOT, 2001).
Literature review
14
2.2 General considerations of sulfonamides
2.2.1 History and properties of sulfonamides
After the discovery of the prototype of sulfonamides (Prontosil) by Gerhard Domagk
in 1935, the active portion known as sulfanilamide was found. Sulfonamides are a
group of compounds containing the structure SO2NH2 (see table 4 page 35). Over
5400 derivatives of sulfanilamide were created, but approximately 20 derivative
forms are generally available. Sulfonamides, the first synthesis chemotherapeutic,
play an important role as effective antimicrobials inhibiting both gram-positive and
gram-negative bacteria, as well as some protozoa, such as coccidials (PRESCOTT,
2000; BARRAGRY, 1994). The fundamental act of sulfonamides was described by
Woods and Fildes in 1940, who discovered para - aminobenzoic acid (PABA) as a
metabolic substrate for folic acid (WOODS, 1940). Sulfonamides interfere with
multiplication of the bacterial cell by completely competitively binding the PABA
molecule and prevent the folic acid formation required for DNA synthesis. Therefore,
sulfonamides are usually bacteriostatics.
Generally, sulfonamides are weak organic acids, relatively insoluble in water, more
soluble in alkaline than acid pH, with their variability and extent bound to plasma
protein (15%-90%) having a wide range of pKa values from 2.65 to 10.4 (SPOO and
RIVIERE, 2001). Sulfonamides are relatively nontoxic when used in normal
therapeutic doses (BARRAGRY, 1994). Nevertheless, except for the sulfapyrimidine
group (sulfamethazine, sulfadiazine and sulfamerazine; KAHN, 2005), sulfonamides
are predisposed to precipitation in renal tubules due to low water solubility of
acetylated forms (RIVIERE et al., 1991; PRESCOTT, 2000). Some other toxic effects
have been documented e.g. allergies in humans (CHOQUET-KASTYLEVSKY et al.,
2002), keratoconjunctivitis sicca in dogs (COLLINS et al., 1986), and haemorrhagic
syndrome in chickens from sulfaquinoxaline administration (DAFT et al., 1989).
Sulfadimidine has been reported to exhibit carcinogenicity in mice (LITTLEFIELD et
al., 1989 and 1990), but a recent report showed that it would not occur at a normal or
low dose (LIONEL et al., 1999).
Literature review
15
In the veterinary field, sulfonamides were proposed for treatment and, in the past, as
growth promoters for some decades. In addition, the indications of sulfonamides for
many species of animals are well established (SPOO and RIVIERE, 2001; PLUMB,
2005). It is reportedly effective against a wide variety of infectious diseases and
commonly therapeutic especially for cattle and swine. In poultry, it has been used for
treating of cocidiosis, infectious coryza, pullorum disease and flow typhoid
(GIGUÈRE et al., 2006). However, owing to theirs low cost, ease of administration
and a wide range of application, sulfonamides have been used extensively; resulting
in a rapidly rise of bacteria-resistance, cross-resistance among sulfonamides and
residues appearing in animal products (EINSTEIN, 1994; AIELLO, 1998; JEMMI
and KÖNIG, 1999; PRESCOTT, 2000; SMITH et al., 2007).
2.2.2 Pharmacokinetic of sulfonamides
2.2.2.1 Absorption
Various routes of administration such as p.o. via drinking water and mixed feed, i.v.,
i.m., i.p., intrauterine and topical are generally used for sulfonamides, indicating that
they are extensively applied and well absorbed (KAHN, 2005). Most sulfonamides
are well absorbed by the gastrointestinal tract except some long-acting compounds
such as sulfamethoxypyridazine (RIVIERE et al., 1991; EINSTEIN, 1994).
Nonetheless, the other exceptions are some so-called enteric drugs e.g.
phthalylsulfathiazole, succinylsulfathiazole, sulfaquinoxaline and sulfaguanidine
(SPOO and RIVIERE, 2001; FREY et al., 1996). However, the absorption may be
delayed in pathological animals (BOTSOGLOU and FLETOURIS, 2001), in adult
ruminants and when administered with food to monogastric animals (PRESCOTT and
BAGGOT, 1993; PLUMB, 1991).
Bioavailability shows the percentage fraction of a drug that reaches systemic
circulation following non i.v. application. In chickens, oral bioavailability of
sulfadiazine has been reported at approximately 100 % (LÖSCHER et al., 1990) and
80%, respectively (BAERT et al., 2003). In a study on the bioavailability of
sulfadimidine administered to yearling cattle in different oral dosage forms, it was
Literature review
16
shown that 81% of the dose is available systemically from an oral solution, 63% from
a rapid-release bolus and 32% from a slow-release bolus (BEVILL et. al., 1977). The
estimation of bioavailability of sulfadimidine was based on the cumulative urinary
excretion of the drug (BAGGOT, 2001). Varying times required to reach maximum
plasma concentration in broiler for other sulfonamides have been recorded; for
sulfaquinoxaline of 5.5 h (EL-SAYED et al., 1995), for sulfadiazine of 1.6 h to 2.7 h
(LÖSCHER et al., 1990; BEART et al., 2003), for sulfadimidine approximately 2 h
(KIETZMANN, 1980; JAIN and PUNIA, 2001).
2.2.2.2 Distribution
It has been reported that sulfonamides penetrate widely into tissue and fluid
throughout the body including CNS, synovial fluid (joint), urine, bile and milk (SPOO
and RIVIERE, 2001). However, distribution of individual sulfonamides depends on
many factors such as pKa value, ionisation state, the vascularity of the absorption site,
lipophilicity, plasma protein-binding properties and species (BOTSOGLOU and
FLETOURIS, 2001). In addition, total plasma protein concentration in domestic
mammals is similar to that in humans (range of 6.0-8.5 g/dL), but it is approximately
3.8-5.2 g/dL in galliform species due to the low albumin concentration (BAGGOT,
2001). The different degree of plasma-binding protein in ruminants ranging from less
than 20% sulfanilamide (NIELSON and RASMUSSEN, 1977) to 94%
sulfadimethoxine (VAN GOGH, 1980) has been reported. In chickes, the percentage
of plasma protein-binding has been recorded as being 22 % for sulfadimidine
(NOUWS et al., 1988) and 40-60% for sulfamerazine (ATEF et al., 1978; OSHIMA
et al., 1964). Moreover, it has been reported that plasma protein-binding of the
metabolites of sulfamonomethoxine and sulfadimethoxine was higher than that of the
parents in laying hens, possibly affecting their excretion rates (VREE et al., 1987).
The volume of distribution (Vd) of SDD following single oral administration at 100
mg/kg in broilers was 0.55 L/kg (JAIN and PUNIA, 2001). The Vd of sulfadiazine in
chickens has been recorded as being 0.43 L/kg after i.v. administration of 33.34
mg/kg BW (BAERT et al., 2003) and 0.96 L/kg after i.v. injection of 100 mg/kg BW
(LÖSCHER et al., 1990).
Literature review
17
Following feeding trials to laying hens, sulfamonomethoxine and sulfadimethoxine
were reported which quickly distribute through the body, reaching a constant level in
various tissues 8 hours after the application, the highest concentration being in the
kidney and the lowest occurring in adipose tissues (FURUSAWA and MUKAI,
1995). This is supported by a latter paper which revealed that sulfadiazine,
sulfadimidine, sulfadimethoxazole, sulfaquinoxaline and sulfamonomethoxine were
well distributed though tissues of laying hens (FURUSAWA and KISHIDA, 2002).
Sulfadimidine has been found to significantly distribute into milk and eggs, remaining
in milk up to 2 days (PAULSON et al, 1992) and for weeks in eggs (SEIB, 1991) after
withdrawal of the drugs. Moreover, transfer and distribution of certain veterinary
drugs including sulfonamides in laying hens have been interestingly studied
(FURUSAWA, 2001).
2.2.2.3 Biotransformation
Sulfonamides are usually easily and extensively metabolised (KAHN, 2005). They are
primarily metabolised in the liver, but they are also biotransformed in other tissues
e.g. kidney, lung, brain, adrenal, blood, neuron, skin and gastrointestinal tract,
BOTSOGLOU and FLETOURIS, 2001). Acetylation, at the N4-position, is the major
process of most sulfonamides, the other metabolism including aromatic
hydroxylation, glucoronidation, O-dealkylation, deamination and sulfatation (VREE
et al., 1985).
Metabolisation is influenced by several factors e.g. species, age, pathological effect
and the specific sulfonamides administrated. Canine species are considered to lack the
ability to acetylate due to a high rate of deacetelation (VREE et al., 1983; TRETTIEN
et al., 1994; CAMPBELL, 1999), while swine are unable to hydroxylate this drug
(SPOO and RIVIERE, 2001; BOTSOGLOU and FLETOURIS, 2001). The
acetylation pathway of sulfadimidine is predominant in swine due to the 6-
hydroxymethylsulfadimidine metabolite in plasma, edible tissues and urine not being
able to be detected (NOUWS et al, 1986). Some different metabolism processes have
been reported e.g. sulfadimethoxine is mainly glucoronidated at the N1-position in
human whereas it is acetylated at the N4-position in pigs (VREE et al., 1985). In
general, metabolites of sulfonamides are more water-soluble and rapidly eliminated
Literature review
18
than the parent drug (NOUWS et al., 1988). However, the desaminosulfadimidine
half-life of elimination can vary from 1-9 days, while sulfadimidine and other
metabolites have a shorter half-life of 10-20 hours in pigs (MITCHELL and
PAULSON, 1986). NOUWS et al., (1991) have observed that the N4-acetyl
metabolite of sulfamethoxazole in plasma was 24.5% for cows and 100% for newborn
calves.
In poultry, approximately 6-20 % of most sulfonamides are metabolised by
acetylation (KIETZMANN, 1980, VREE and HEKSTER, 1985; NOUWS et al.,
1988). The low yield of plasma N4-acetylsulfonamide may be due to a high rate of
deacetylation in chickens (OIKAWA et. al., 1977), a high faecal excretion rate or to a
preferred metabolic pathway by hydroxylation or deamination (JAIN, 1994).
However, FURUSAWA (2000) found that the main pathway of in vitro hepatic
biotransformation of sulfamonomethoxine was hydroxylation (2, 6-diOH-SMM) in
laying hens. N4-acetyl metabolites of sulfonamides have no antimicrobial activity and
most are less water-soluble (except for sulfadiazine, sulfamerazine and
sulfadimidine).
2.2.2.4 Excretion
Kidneys play the primarily route of elimination for most sulfonamides either as parent
substance or metabolite; by glomerular filtration (uncharged), active carrier-mediated
proximal tubular secretion and passive reabsorption of nonionised drugs from the
distal part of the renal tubules (PRESCOTT, 1993 and 2000; PLUMB, 1991 and
2005). Other less significant routes of excretion are via milk, bile, faeces, tears and
sweat (SPOO and RIVERE, 2001). In the case of sulfadimidine it has been reported
that 11-37% and 24.5% of the dose was excreted uncharged into the urine in cattle
and swine, respectively (NOUWS et al., 1988; BEVILL et al., 1977; DUFFEE et al.,
1984).
It has been indicated that sulfadimidine might be extensively metabolised in turkeys
where only 8.6% (of oral dose) and about 17% (of i.v. dose) of the parent compound
was found in urine and faeces (HEATH et al., 1975). In addition, it has been reported
Literature review
19
that sulfadimethoxine was renally excreted at approximately 17.9% and 58.4% in the
form of a parent drug and its metabolites, respectively, in cattle (BOURNE et al.,
1981). In swine, approximately 48% of the dose was excreted into urine as uncharged
sulfathiazole and about 19% of the dose was excreted into urine as acetylsulfathiazole
(BOURNE et al., 1978).
The elimination half-life of sulfonamides varies according to the species and
individual drug; for example, sulfadimethoxine has an approximate half-life of 12.5 h
in cattle, 8.6 h in goats, 11.3 h in horses, 15.5 h in swine, 13.2 h in dogs and 10.2 h in
cats (GIGUÈRE et al., 2006), whereas sulfadimidine has an estimate half-life of 8-11
h in cattle, 3-8 h in goats, 10-13 h in horses, 9-16 h in swine, 4-17 h in dogs and 7-10
h in chickens (FREY and LÖSCHER, 2002). Varying withdrawal time of
sulfonamides from edible tissues of poultry have been reported; 2 days for
sulfadimethoxine from broiler tissues (NAGATA et al., 1994) and approximately 4-7
days for sulfadimidine, sulfanilamide and sulfaguanidine in fattening chicks
(VLAHOVIĆ et al., 1996).
Sulfadimidine has been documented as being generally more rapidly eliminated after
injection than compared to oral medication by mixing feed or drinking water
(BOTSOGLOU and FLETOURIS, 2001). Approval drugs treatment for coccidiosis in
chickens have been established. For example, the withdrawal time of sulfadimidine is
10 days after administration via drinking water of 0.1% for 2 days or 0.05% for 4
days, whereas it is 5 days after a dose of 0.05% for 6 days of sulfadimethoxine
(KAHN, 2005).
Literature review
20
2.3 Veterinary drug residues
At present, quality and safety of food from animal products are widely concerning
public and health agencies around the world. Since veterinary drugs have played an
important role in the field of animal husbandry and agro-industry for some decades,
increasing occurrence of residues and resistance have become interesting issues.
Residues of veterinary medicinal products, as defined by the European Union, are
"pharmacologically active substances (whether active principles, excipients or
degradation products) and their metabolites which remain in foodstuffs obtained from
animals to which the veterinary medicinal product in question has been administered",
(ANONYMOUS 1, 1990). In addition, the Center for Veterinary Medicine (CVM), an
agency under the Food and Drug organization (FDA) in the USA, has defined
residues as follows: “A residue means any compound present in edible tissues that
results from the use of a drug, and includes the drug, its metabolites, and any other
substance formed in or on food because of the drug's use”, (ANONYMOUS 2, 2004).
Recently, among positive samples, the EU reported that 51-52 % non-compliant
results of residues in animals were antibacterial (ANONYMOUS 3, 2004 and
ANONYMOUS 10, 2006). Accordingly, prudent usage of drugs should be strongly
considered (BOTSOGLOU and FLETOURIS, 2001).
2.3.1 Maximum Residue Limits (MRLs)
The European Commission sets Maximum Residue Limits (MRLs) after adoption by
the Standing Committee, following advice from the Committee for Veterinary
Medicinal Products (CVMP). MRL is the maximum concentration of a residue
following administration of a veterinary drug which is legally permitted or acceptable
in food. Determining MRL is a requirement of European legislation under a Council
Regulation (2377/90). Under this legislation, substances must be entered into one of
four annexes to the Regulation; Annex I: The data in the dossier are considered
adequate to establish a final MRL. Annex II: No MRLs required on the basis of the
data supplied. Annex III: established provisional MRLs but some clarification of
further studies must be supplied before final MRLs can be set. Annex IV: No MRLs
due to unacceptable risk to public health and prohibited for use in food producing
animals. In the EU, the MRL of sulfonamides for edible tissues is 100 µg/kg
Literature review
21
(ANONYMOUS 1, 1990). The MRLs values of sulfonamides established by some
health agencies are given in the table 1. However, MRLs values of sulfonamides have
not been set for eggs only. The withdrawal period is defined as the time taken for the
level of residues in the tissues (muscle, liver, kidney, skin/fat) or products (milk, eggs,
honey) to be depleted below the MRLs after the last dose has been given to the
animals. Regulatory requirements for withdrawal time vary among countries. The
selection of times listed in table 2 (KAHN, 2005) serves only as a general guideline.
Drug Target animal
species
Target tissue MRLs
µg/kg
Health
agency
All sulfonamides All animals
Bovine, Ovine,
Caprine
Muscle, fat, liver,
kidney
Milk
100
25
JECFA
JECFA
Sulfadimethoxine Turkey, Chicken,
Duck, Cattle
Not specified
Edible tissues
Milk
100
10
USDA
USDA
Sulfadiazine Swine, Poultry,
Fish
Muscle, liver, kidney,
milk (not egg)
100 EMEA
Sulfamerazine Trout Edible tissues 0 USDA
Sulfamethazine Cattle, Swine,
Chicken, Turkey
Swine
Edible tissues
Muscle, liver, kidney,
milk (not egg)
100
100
USDA
EMEA
Sulfaquinoxaline Cattle, Chicken,
Turkey
Poultry
Edible tissues
Muscle, liver, kidney,
milk (not egg)
100
100
USDA
EMEA
Sulfathiazole Swine Edible tissues 100 USDA
Table 1: Example of MRLs of sulfonamides produced by health agencies
Literature review
22
Drug Species Withdrawal time
(days)
Milk discard
(h)
Sulfadimidine Cattle
Swine
10a
14
96
-
Sulfabromethazine Cattle 10 96
Sulfadimethoxine Cattle 7 60
Sulfonamide combinationb Cattle 10 96
Trimethoprim/Sulfadiazine Cattle 3 7
Trimethoprim/Sulfadoxine Cattle 5 (p.o.)c -
Table 2: Drug withdrawal and milk discard times of sulfonamides (Modified from
KAHN, 2005)
a) 28 days for slow-release bolus
b) 8% sodium sulfadimidine: 8% sodium sulfapyridine: 8% sodium sulfathiazole
c) 28 days following parenteral administration
2.3.2 Possible explanation causes of veterinary drug residues in animals
Veterinary drug residues are one of the major problems for food contamination, if
natural forms of contamination (mycotoxins, microorganism or etc.) and organic
pollutant (dioxin etc.) are excluded. There are many factors influencing the build-up
of residues in animal products such as drug’s properties and their pharmacokinetic
characteristics, physicochemical or biological processes of animals and their products
(tissues, milk and eggs). Drug residues are usually accumulated in the liver or kidney
rather than other tissues (DOYLE, 2006). It has been noted that different residue
levels can be found in different tissue positions such as site and route of
administration (KORSRUD et al., 1994; KAARTINEN et al., 1999) or even between
breast and thigh of chickens (REYES-HERRERA et al., 2005).The most likely reason
for drug residues may result from human management such as improper usage
including extra-label or illegal drug applications (KENNEDY et al., 2000; FAJT,
2001; STOLKER et al., 2004; LUNESTAD and GRAVE, 2005; LI et al., 2006).
However, the most obvious reason for unacceptable residues might be due to failure
to keep to the withdrawal period including using overdose and long-acting drugs
Literature review
23
(SHEARER, 1999). Inadequate good sanitary care during animal or product
transportation (WHIPPLE et al., 1980) including the cross-contamination of animal
feeding stuffs with inadvertently applied drugs (BIEHL et al., 1981; McCAUGHEY
et al., 1990c), environmental and animal to animal transfer of drugs (McCAUGHEY
et al., 1990b; KIETZMANN et al., 1995; KENNEDY et al., 2000) may also cause
residues.
As mentioned earlier, a low-level drug contamination of animal feed is one large
causative channel of residues in animal products. Drug cross-contamination or drug
carry-over is a form of feed contamination that may result when the substance in
question is transferred (carried) from an acceptable to an unacceptable location or
feeding stuff. Drugs carry-over may come from dust and drug powder that have
electrostatic or moisture properties (HARNER et al., 1996). In addition, a recently
report showed various antibiotic agents including tylosin, tetracycline, sulfadimidine
and chloramphenicol were detected in dust on a swine farm (HAMSCHER et al.,
2003). Drug carry-over may also arise from medicated feed remaining in the mixers,
bins, conveyors, elevators and bulk trucks during or between batches of
manufacturing or delivering processes (HARNER et al., 1996). However, it seems
that approximately 5% carry-over between feeding batches is technically unavoidable
even with good manufacturing practice (BOTSOGLOU and FLETOURIS, 2001). The
potential contamination of some veterinary drugs in swine, broilers and layers feeds
have been documented (AERTS, 1990). The result of this study revealed that more
than 50% of the investigated feeds were generally contaminated with drugs at low
levels.
Studies carried out drug cross-contamination in feed have been published; low-level
contamination of sulfadimidine, ca. 1% of normal dosage, with detectable residues in
pig’s tissues (CROMWELL et al., 1980; McCAUGHEY et al., 1990a). Recently,
unintentionally contaminated polyether ionophores (coccidiostats) in animal feed,
resulting in detectable residues in eggs are more interesting in the EU
(ANONYMOUS 4, 2007). Carry-over of some anticoccidials e.g. diclazuril,
robenidine, halofuginone and nicarbazin in combination with narasin into eggs have
been studied (MORTIER et al., 2005). This demonstration showed that some drugs
presented at 5% in feed could cause residues in eggs. This value was also similar in
Literature review
24
the study of monensin carried out on feed mill (KENNEDY et al., 1998). In addition,
violative residues of nicarbazin in eggs and broiler liver have been reported following
ingestion of low-level contaminated feed (McEVOY et al., 2003). Furthermore,
finding of feed containing lasalocid at 0.1 mg/kg produced detectable residues at 6-8
µg/kg in egg when fed to hens have also been published (KENNEDY et al., 1996).
The transfer of some veterinary substances exposed at a very low-level into animal
products has been documented; McCRACKEN and KENNEDY (1997) showed a
metabolite of furazolidone (AOZ) residue in pig’s tissues after low-dose treatment
(0.5-2.3 mg/kg) for five days. Drug cross-contamination may create serious problems
among different species. The horse is a crucial example since it takes a very serious
problem of carry-over of monensin due to its LD50 at only 2-3 mg/kg (LANGTHON
et al., 1985). Thus, mortality of the horse can occur from an inadvertent low-level
contamination of monensin.
Drug low-level contamination generally may not generate a violation problem on
public health. However, extensive use of drugs may increase the risk of an adverse
effect of residues on the customer including the occurrence of antibiotic resistance.
Therefore, prudent use in the manner of preventing feed contamination is necessary.
2.3.3 Adverse effect of veterinary drug residues
Although the adverse effects of veterinary drug residues have been discussed, in this
thesis only four hazards will be considered according to DOYLE (2006). Firstly,
veterinary drug residues can cause allergic or toxic reactions in humans; penicillin is
one crucial example of this. The occurrences of penicillin-induced hypersensitivity
after milk or beef consumption have been cited (ORMEROD et al., 1987; REYES-
HERRERA et al., 2005). However, with regard to other antibiotics, macrolide and
cepharosporin residues are unlikely to be an allergenic hazard (DEWDNEY et al.,
1991). Although sulfonamides are potentially induced skin hypersensitivity reactions
(CHOQUET-KASTYLEVSKY et al., 2002), however, no allergic cases involving of
exposure to residues of sulfonamides in food have been reported (PAIGE et al., 1999).
Fortunately, exposure of antibiotics via oral channels usually sensitises to a lesser
extent than the parenteral route does (DEWDNEY et al., 1991).
Literature review
25
Secondly, chronic toxic effects occurring with prolonged exposure to a low level of
antibiotics also might generate side effects or tissue-degeneration. It has been noted
that most residues of veterinary drugs appear at a low level in animal products but
adverse effects are likely to occur due to acute rather than long-time exposure
(PAIGE et al., 1997). Although sulfadimidine reportedly has the potential to induce
thyroid teratogenic in mice (LITTLEFIELD et al., 1989 and 1990), it would not occur
if received at therapeutic level (LIONEL et al., 1999), thus implying that it might be
safe when there is low-dose exposure. Recently, however, there have been
cooperation studies within EU countries to evaluate risks of long-term or low-dose
exposure in foods (ANOYMOUS 5, 2004). So far, no report has been confirmed.
Thirdly, veterinary drug residues potentially develop antibiotic-resistant bacteria.
Since the initial widespread use of antibiotics over 60 years ago, situations have been
recognised in which an antibiotic has lost its effectiveness in controlling infection,
even when the dose is increased. These may increase the risk of treatment failures in
humans. LANZ et al. (2003) observed 581 cases of clinical Escherichia coli isolated
from different animal species in Switzerland between 1999 and 2001. They found that
the most frequent antimicrobial resistance was sulfonamides, tetracycline and
streptomycin. There are several mechanisms of bacteria resistance to sulfonamides
such as development of a form of dihydropteroate synthase, which is highly selective
for PABA and has less affinity to sulfonamides (MANDELL et al., 1996; ALLEN et
al., 1993). Moreover, an altering the recycling or utilising of dihydropteroic acid
within the cell, (CRAIG and WHITE, 1976), increasing synthesis of PABA
(GREENE, 1998) and destroying the sulfonamides (WHILCKE, 1988) have been
reported. Resistance to sulfonamides is usually persistent and irreversible.
Fortunately, this does not imply cross-resistance to other drug groups (CAMPBELL,
1999). The relationship of antibiotic-resistance between animals and humans has been
observed e.g., in the Netherlands the emergence of fluoroquinolones-resistant
Campylobacter species among poultry and humans has been reported (ENDTZ et al.,
1991). In Germany and the USA, increasing incidents of its fluoroquinolones-
resistance have also been found (HELMUTH and PROTZ 1997; SMITH et al., 1998).
Finally, there is disruption of normal human flora in the intestine. Antibiotic residues
may decrease the amount of commensal bacteria (normal flora) in humans, therefore
Literature review
26
creating opportunity for pathogenic bacteria to invade and cause illness. An increase
in E. coli resistance in faeces of mice given a low-dose of ampicillin and
chlortetracycline was observed. However, this failed to show the effect on humans
studied (CORPET, 1987). Recently, one study has reported that tetracycline-
resistance of commensal E. coli in swine is frequently linked to ampicillin- and
trimethoprim-sulfonamides- resistance (DEWULF et al., 2007). However, this was
controversial since several other reports stated that antibiotic residues in food of
animal origin would probably not disrupt the intestinal microflora in humans
(CERNIGLIA and KOTARSKI, 2005). The resistance gene of commensal flora in the
animal gut, which is a large reservoir, may be one probable route of transmission
leading to impacts on human health (DEWULF et al., 2007).
Literature review
27
2.4 Sulfonamide residues in animals
2.4.1 Sulfonamide residues in livestock
Sulfonamides have been classified in group B1 (veterinary medicines and
contaminants) of annex I of Directive 96/23/EC (1996), (ANONYMOUS 6, 1996).
The sulfonamide group is primarily approved for swine and cattle production. It has
potentially raised a residue problem in edible tissues. It has been found in animal
products for decades, as shown in the following reports. In Germany, sulfonamides
residues found during 1991-1992 ranged from 14-33,600 µg/kg (BERGNER et al.,
1993). The violation sulfonamides residues were found at 2.2-5.4% (1992-1995) in
Switzerland (ROLAND et al., 1996), 5.2-11.3% (1986-1990) in the UK (KAY and
PENNY, 1996), 0.2-1.3% (1990-1995) in Canada (NEIDERT and
SASCHENBRECKER, 1996) and 0.3-5.6% in the USA, respectively
(ANONYMOUS 7, 2003). The report of the EU in 2003 followed by the monitoring
program, SANCO, indicated that the sulfonamide group is one of the most commonly
occurring contaminating drug (ANONYMOUS 8, 2004). This conclusion agrees with
the recent reports by KENNEDY et al., (2000) and WANG et al., (2006). Fortunately,
a recent report by the EU showed a large decrease in sulfonamides detected in animal
products amounting to approximately 0.03% (106 samples out of 362,551 targeted
samples) of non-compliant results (ANONYMOUS 10, 2006). Additionally, most
sulfonamides found in animal products which included suspected samples were
sulfadiazine (47 samples), sulfadimidine (30 samples) and sulfadimethoxine (21
samples) (ANONYMOUS 10, 2006). Nonetheless, the remaining of unexpected
substances found in animal products proved that adequate residue prevention for the
consumer has not yet been achieved.
In swine production, an emergence of sulfonamides residues in pork and its products
has been worryingly observed (CROMWELL et al., 1980; ASHWORTH et al., 1986;
McCAUGHEY et al., 1990a, b, c). Sulfonamides have generated a huge problem
concerning their residues in animal products in the USA for decades (CORDEL,
1989). Recently, it has been reported that in the USA that sulfonamides contamination
still amounts to over 4% (DEY et al., 2003). Sulfadimidine is one classical example of
Literature review
28
drug residue in pork since it has been used at approximately 75% in hogs markets in
the USA (AUGSBURG, 1989) and frequently found in swine tissues (BEVILL et al.,
1986). Sulfadimidine needs nine times more for withdrawal time than sulfathiazole,
therefore deducing that it is prone to accumulate more than sulfathiazole as the data
showed, (BEVILL et al., 1986). The withdrawal time of sulfadimidine from swine
tissues have been reported starting at 7-8 days (GARWACKI et al., 1996; KORSRUD
et al., 1996), 14-15 days (WHIPPLE et al., 1980; AUGSBURG, 1989; PRIPWAI et
al., 2004) or even 30 days (PAPAPANAGIOTOU et al., 2005). Studies by
ASHWORTH et al. (1986) have shown that swine fed with sulfadimidine
concentrations greater than 8 mg/kg produced residues at a level greater than 100
µg/kg in muscle and 400 µg/kg in the liver, whereas, being fed up to 2 mg/kg could
be tolerated in withdrawal feeds before liver residues exceeded 100 µg/kg. However,
this study was contradicted those of the University of Kentucky and University of
Nebraska in 1981 (ANONYMOUS 9, 1981) which showed that a very little amount of
contamination of sulfamethazine (sulfadimidine) at 1 mg/kg in pig feed could
generate a high incidence of residues in pig’s liver. The contamination of
sulfadimidine in pig has been studied which transfers via cross-contamination
(McCAUGHEY et al. (1990c), and animal to animal (WHIPPLE et al., 1980;
McCAUGHEY et al., 1990b). The result revealed violative residues in an
unmedicated group, detected in the kidney and diaphragm, after exposure to the
contaminated housing for 6-24 h. In addition, they found the contaminating of
sulfadimidine in the faeces or urine in untreated pigs. This result was supported by
KIETZMANN et al., (1995). Furthermore, it was suggested that sulfadimidine was
preferentially retained in the mixer (McCAUGHEY et al., 1990a) and detected in
tissues of swine being found in feed at 2 g/tonne (BIEHL et al, 1981).
Sulfonamides residues in milk have also been observed. Sulfadimidine has created a
public health problem concerning its residue, this being highly detectable
approximately 40-70% in milk samples from supermarkets in the USA (PAIGE and
KENT, 1987; GRASSIE, 1988). A recent report showed that sulfadimidine was the
second most frequently drug presented in dairy products following Beta-lactams
(ANONYMOUS 7, 2003). Though, BLÜTHGEN et al., (2000) reported a carry-over
percentage of sulfadimidine from feed to milk of less than 1%. In contrast to the
studies on lactating diary cows by McEVOY et al., (1999) who indicated no violative
Literature review
29
contamination of sulfadimidine detected in milk samples after 7 days of withdrawal,
taken from the cows fed the concentrate containing 2 or 10 mg/kg. Unfortunately, the
peak level of sulfonamides in milk were found around the MRL value established by
the EU following the experiment that fed cows consist of sulfadiazine, sulfadimidine
or chlortetracycline at 250 mg/kg for 21 days, which is the normal dose used
(McEVOY et al., 1999 and 2000), In Japan, a survey of sulfadimidine,
sulfamethoxypyrimidine, sulfachloropyridazine, sulfathiazole and sulfamethoxazole
residues in milk were found below the detectable level (FURUSAWA and KISHIDA,
2005).
2.4.2 Sulfonamides residues in poultry
Poultry meat and eggs are very commonly consumed by humans. However, there may
be situations where contaminations of drug residues in poultry products occur.
Veterinary drugs and feed additives, especially anticoccidials and antibacterials e.g.
sulfonamides are drugs most commonly used on poultry farms. They can be easily
absorbed and distributed through the body of chickens, accumulated in various tissues
and transferred into their products (KAN and PETZ, 2000; WEISS et al, 2007).
Interestingly, sulfonamides were detected in 41.6% (5 out of 12 non-compliant
results) among antibacterials found in eggs (ANONYMOUS 10, 2006).
Residue of sulfadimethoxine has been observed in broiler tissues following dietary
administration at 25, 50 and 100 mg/kg (NAGATA, 1994). The residue was very
rapidly eliminated from tissues with less than 0.1 µg/g remaining within two days
after the drug withdrawal. This result is in the range of the study of TAKAHASHI
(1991), who reported that sulfadimethoxine was rapidly disappeared from plasma and
tissues (except skin) on the 3rd-5th day post dosed via drinking water. Compared to
sulfadimethoxine residue in laying hens, it has been found that it quickly distributed
through the body and reached a constant level in various tissues at 8 hours after feed
(FURUSAWA and MUKAI, 1995). However, it may appear below 0.1 µg/g in yolk
and albumen up to 7 and 3 days after the drug withdrawal, respectively (NAGATA,
1992). The comparison experiment of depletion of 3 sulfonamides; sulfadimidine,
sulfanilamide and sulfaguanidine, from muscle and liver after six days continuous
feeding in fattening chicks has been demonstrated (VLAHOVIĆ et al., 1996). The
Literature review
30
result showed that sulfaguanidine was the fastest eliminated, 4 days for all tissues,
whereas sulfanilamide and sulfadimidine needed 3 days longer to sink to below 0.1
µg/g in the liver. In turkeies, sulfadimidine given by drinking water was also
observed. It was found that concentrations fluctuated between 0.1-0.4 ppm in the
kidney, liver, and skin after 72 hours of withdrawal and as long as 14 days (HEATH
et al., 1975). Feed containing sulfadimethoxine (60 mg/kg) and sulfaquinoxaline (100
mg/kg) in turkeies was also studied by EPSTEIN and ASHWORTH (1989); presented
sulfadimethoxine and sulfaquinoxaline needed 2-3 and 4-6 days respectively for liver
and muscle tissues to decrease to the level of 0.1 µg/g.
In daily egg production incidents of residues in this product occur easily if withdrawal
time is ignored. There might be some parent drugs, including theirs metabolites
remaining in eggs, resulting from improper or extra-label used as feed additives for
laying hens older than 16 weeks (SHAIKH et al., 1999). However, cross-
contamination of the medicated feed into the feed free-drugs may be the cause of
residues in eggs (SERRATOSA et al., 2006). The distribution of 5 sulfonamides;
sulfadimidine, sulfadiazine, sulfamethoxazole, sulfamonomethoxine and
sulfaquinoxaline, following administration via medicated feed at 100 mg/kg in laying
hens has been studied (FURUSAWA and KISHIDA, 2002). The result showed that
sulfaquinoxaline was the highest residue found in samples (plasma, muscle, liver,
ovary and oviduct) whereas sulfadimidine was the lowest, after seven days of the
treatment.
Sulfonamide residues occurring in egg compositions have previously been studied.
Therefore, variations of withdrawal times of sulfonamides for eggs have been
reported; 9 to 10 days for sulfaquinoxaline (FURUSAWA et al., 1998; CAVALIERE
et al., 2003; SHAIKH et al., 2004) and for a combination of sulfaquinoxaline:
sulfadimidine: sulfamerazine (ROMAVARY and SIMON, 1992), 7 days for
sulfadimethoxine (NAGATA et al., 1992) and at least 4 days of sulfadiazine given by
drinking water (ATTA and EL-ZEINI, 2001). Additionally, KAN and PETZ (2000)
have interestingly summarised the concentrations and ratio of sulfonamide transfer
into egg white and egg yolk, given in table 3. However, up-to-date data are added in
this table.
Literature review
31
Content (mg/kg) Ratio
Drug White
(W)
Yolk
(Y) W/Y
Exposure
method Reference
Sulfadimethoxine4.6 1.7 2.71
400 mg/kg in
feed, 5 days
Furusawa
et al., 1994
0.86 0.37 2.32
100 mg/kg in
feed, 14 days
Kan and Jacobs,
1998
0.15 0.05 3
25 mg/kg in
feed, 21 days
Nagata
et al., 1989
0.25 0.1 2.5
50 mg/kg in
feed, 21 days
Nagata
et al., 1989
0.5 0.2 2.5
100 mg/kg in
feed, 21 days
Nagata
et al., 1989
0.04 0.02 2
10 mg/kg in
feed, 14 days
Nagata
et al., 1992
35 9 3.89
5000 mg/L in
water, 5 days
Roudaut, 1993
Sulfadiazine 0.14 0.015 9.33
20 mg/kg in
feed, 14 days
Kan and Jacobs,
1998
0.015 <0.008 >1.8
1.3 mg/kg in
feed , 21 days
Tomassen
et al., 1996
0.04 <0.008 >5
3.8 mg/kg in
feed, 21 days
Tomassen
et al., 1996
0.10 0.022 4.55
8.1 mg/kg in
feed, 21 days
Tomassen
et al., 1996
0.22 0.15 1.47
0.2 g/L drinking
water, 5 days
Atta et al., 2001
0.32 0.18 1.78
0.4 g/L drinking
water, 5 days
Atta et al., 2001
Table 3: Sulfonamide residues in egg yolk and albumen (modified from KAN and
PETZ, 2000)
Literature review
32
Content (mg/kg) Ratio
Drug White
(W)
Yolk
(Y) W/Y
Exposure
method Reference
Sulfanilamide 35 43 0.81
1000 mg/L in
water, 8 days
Bloom, 1975
0.15 0.14 1.07
20 mg/kg in
feed, 14 days
Kan and
Jacobs, 1998
Sulfachlorpyrazine 0.52 0.17 3.05
50 mg/kg in
feed, 14 days
Kan and
Jacobs, 1998
Sulfadimidine 56 45 1.24
1000 mg/L in
water, 8 days
Blom, 1975
97 83 1.17
2000 mg/L in
water, 8 days
Blom, 1975
20 5 4
5x 100 mg/kg
BW, 5 days
Geertsma
et al., 1987
0.03 0.01 3
20 mg/kg in
feed,14 days
Kan and
Jacobs, 1998
70 30 2.33
1000 mg/L in
water, 5 days
Krieg, 1966
51 34 1.5
1000 mg/L in
water, 5 days
Rodaut, 1993
38 23 1.65
94 mg/kg in
feed, 5 days
Sieb, 1991
Table 3(cont): Sulfonamide residues in egg yolk and albumen (modified from KAN
and PETZ, 2000)
Literature review
33
Content (mg/kg) Ratio
Drug White
(W)
Yolk
(Y) W/Y
Exposure
method Reference
Sulfaquinoxaline 50 36 1.39
400 mg/L in
water, 8 days
Blom, 1975
2.3 2.0 1.15
200 mg/L in
feed, 7 days
Furusawa
et al., 1998
0.54 0.26 2.07
20 mg/kg in
feed, 14 days
Kan and
Jacobs, 1998
0.95 1.57 0.61
60 mg/kg in
feed, 14 days
Nose et al.,
1982
3.7 1.4 2.64
100 mg/kg in
feed, 7 days
Petz, 1993
80 20 4
350 mg/L in
water, 3 days
Rana et al.,
1993
3.4 2.9 1.17
500 mg/kg in
feed, 12 days
(intermittent)
Righter et al.,
1970
8 2 4
400 mg/L in
water, 3 days
Romvary and
Simon, 1992
8.3 2 4.15
6000 mg/L in
water
Sakono et al.,
1981
Sulfapyridine <0.1 <0.005 -
50 mg/kg in
feed, 14 days
Kan and
Jacobs, 1998
Sulfisoxazole 0.05 0.02 2.5
100 mg/kg in
feed, 14 days
Kan and
Jacobs, 1998
Table 3 (cont): Sulfonamide residues in egg yolk and albumen (modified from KAN
and PETZ, 2000)
Literature review
34
Table 3(cont): Sulfonamide residues in egg yolk and albumen (modified from KAN
and PETZ, 2000)
Content (mg/kg) Ratio
Drug White
(W)
Yolk
(Y) W/Y
Exposure
method Reference
Sulfaguanidine 0.32 0.64 0.5
100 mg/kg in
feed, 14 days
Kan and
Jacobs, 1998
Sulfamerazine 0.22 0.03 7.33
100 mg/kg in
feed, 14 days
Kan and
Jacobs, 1998
23 6 3.83
2000 mg/kg in
feed, 25 days
Onodera
et al., 1970
Sulfamethoxazole 20 1.8 11.11
2000 mg/kg in
feed, 5 days
Oikawa et al.,
1977
39 2.9 13.45
4000 mg/kg in
feed, 5 days
Oikawa et al.,
1977
0.42 0.12 3.5
50 mg/kg in
feed, 14 days
Kan and
Jacobs, 1998
Sulfamono-
methoxine 6.8 1.5 4.53
400 mg/kg in
feed, 5 days
Furusawa and
Mukai, 1995
0.20 0.04 5
25 mg/kg in
feed, 21 days
Nagata et al.,
1989
0.35 0.07 5
50 mg/kg in
feed, 21 days
Nagata et al.,
1989
1.0 0.20 5
100 mg/kg in
feed, 21 days
Nagata et al.,
1989
Literature review
35
2.5 Sulfadimidine
2.5.1 Properties of sulfadimidine
Sulfadimidine (SDD), known as sulfamethazine, is one of the most widely used
sulfonamides in animals. It has reportedly been effectively used against a wide variety
of infectious diseases, a common therapeutic drug, especially for cattle, swine and
poultry. It is approved for treating coccidiosis and has been used as a growth promoter
in swine and cattle. It is not approved for use in lactating dairy cows and laying hens
older than 16 weeks (BOTSOGLOU and FLETOURIS, 2001). General properties of
sulfadimidine are given in table 4 (SUKUL and SPITELLER, 2006).
Chemical structure
Nomenclature 2-(4-Aminobenzensulfonamido)-4,6-dimethylpyrimidine
Other names Sulfamethazine, Sulfadimethylpyrimidine, Sulfamethiazine,
Sulfadine, sulfodimesin, ect.
Formula C12H14N4O2S
Molecular weight 278.33
pKa value 7.45± 0.50 (most acid)
2.79± 0.24 (most basic)
Koc value (L/kg) 1 at pH 1, 61.2 at pH 4, 48.2 at pH 7, 1 at pH 10
Table 4: General properties of SDD (modified from SUKUL and SPITELLER, 2006)
2.5.2 Pharmacokinetics of sulfadimidine in laying hens
Pharmacokinetic characteristics of SDD in different species have been interestingly
summarised by SPOO and RIVIERE, (2001) but complete pharmacokinetic values of
SDD in poultry are limited. However, in this present study the focus on SDD in laying
hens will be reviewed. Sulfadimidine has been reported as being rapidly absorbed by
the digestive tract of the hen, after it being given 2 g/L of SDD via drinking water
Literature review
36
(GOREN et al., 1984).This is in agreement with ROUDAUT and GARNIER (2002)
who studied SDD at the same route and dose. Furthermore, GOREN et al. (1984)
showed no significant difference in the SDD concentration in the plasma between
broilers and layers after intravenous or crop inoculation. Following a single oral
dosage of SDD at 100 mg/kg in laying hens, GEERTSMA et al. (1987) found that its
peak plasma concentration reached the range of between 44-73 µg/mL within 2-3
hours. Therefore, rapid absorption is confirmed. Compared to SDD treated in broilers,
following oral application at 100 mg/kg, the absorption rate constant (Ka) at 1.29 h-1
with the Tmax of 2.07 h (JAIN and PUNIA, 2001), Ka at 0.28 h-1 with T max of 1.42
(recalculated) (REDDY et al., 1988; CRAIGMILL el al., 2006) and Tmax of 2.0 h
(KIETZMANN, 1980) have been reported. Thus, all studies indicated that SDD was
also quickly absorbed in broilers.
Disposition of SDD into tissues and eggs has been presented (BLOM, 1975;
GEERTSMA et al., 1987; SHAIKH and CHU, 2000; ROUDAUT and GARNIER,
2002). BLOM (1975) has interestedly reported that plasma protein binding of SDD
was about 24-27%. This is in agreement with later results documented by NOUWS et
al. (1988), who showed the percentage degree of plasma protein-binding at 22% when
SDD concentration in plasma was greater than 50 µg/mL. However, at the low plasma
concentration (less than 50 µg/mL) the degree of plasma protein-binding was
increased to 53%.
The metabolism pathway of SDD in laying hens is similar to that of other
sulfonamides. Acetylation and hydroxylation are the major processes but their
metabolites may not be higher than 20% (VREE and HEKSTER, 1985). However,
GEERTSMA et al. (1987) and NOUWS et al. (1988) described that there may have
been an additional unknown metabolic pathway in poultry, since 57% of the dose
remained unaccounted for in the faecal excretion. In addition, NOUWS et al. (1988)
have demonstrated that N4-acetyl-sulfadimidine followed by hydroxylmethyl-
sulfadimidine and 5-hydroxy-sulfadimine in plasma were found only at about 6.6-
7.2%, 1.7-4.5% and 0.91-2.0% of the dose administered at 100 mg/kg, respectively.
However, its acetylation occurred only at 12% in broilers (KIETZMANN, 1980).
Moreover, the finding of SDD metabolites being higher than metabolites of
sulfadiazine in broilers has been documented (LÖSCHER et al., 1990).
Literature review
37
Sulfadimidine is generally more rapidly eliminated after injection than by oral
application via feed or drinking water (BOTSOGLOU and FLETOURIS, 2001).
Some previous papers reported that SDD decreased more rapidly than
sulfaquinoxaline (LÜDERS et al., 1974) and sulfadiazine (LÖSCHER et al., 1990) in
broilers. FURUSAWA and KISHIDA (2002) have studied the comparison of the
distribution among sulfadimidine, sulfadiazine, sulfamethoxazole, sulfaquinoxaline
and sulfamonomethoxine, following administration via medicated feed at 100 mg/kg
in laying hens. The results revealed SDD as the lowest concentration found in plasma
and tissues of these drugs after stop treatment. Thus, SDD was the most rapidly
excreted of all those drugs considered. Regarding the studies by NOUWS et al. (1988)
and GEERTSMA et al. (1987), the findings of the renal clearance of the metabolites
of SDD was approximately 10 times greater than SDD as a parent drug, indicating
that its metabolites were excreted faster than those of the parent drug.
However, varying data of the biological half-life values of SDD in laying hens were
recorded; NOUWS et al. (1988) and GEERTSMA et al. (1987) reported the final half-
life of 2.0 h, 2.9 h and 6.4 h post single intravenous, single oral and multiple dosing
orally, respectively, whereas the elimination half-life of SDD at 4.7 h (GOREN et al.,
1984) and as long as 16 h (BLOM, 1975) has also been reported. Interestingly,
REDDY et al. (1988) have reported that the elimination half-life was 15.5 h.
Nevertheless, it was only 8.9 h when recalculated by the WinNonlin® program
(CRAIGMILL el al., 2006). However, these results were in a similar range as those
from previous studies on broilers; KIETZMANN (1980) reported that SDD reached
peak plasma level at 87.4 µg/mL within 4-8 h with a calculated half-life of 5.1 h after
a single oral treatment of 150 mg/kg. Furthermore, JAIN and PUNIA (2001) have
showed the profile of SDD following oral treatment at 100 mg/kg as achieving its
peak level (Cmax at 83 µg/mL) of 2.1 h and the elimination half-life of 6.1 h. In
general, a wide range of SDD residues remained in various edible tissues of poultry,
the fluctuated level in muscle, skin, kidney, fat and liver being 0.01-0.47 ppm on day
ten (RIGHTER et al., 1971.). In the liver and skin of turkey SDD residues were
detected at 0.1-0.4 ppm after 14 days post treatment (HEATH, 1975). Variation of
withdrawal times of SDD has also been reported, approximately three days for
broilers (LÜDERS et al., 1974.), about 5-6 days for muscle and 7 days for the liver of
Literature review
38
fattening chickens (VLAHOVIĆ et al., 1996) and four days for tissues of hens
(NOUWS et al., 1988) post administration.
2.5.3 Residues of sulfadimidine in laying hens
Sulfadimidine residues in tissues and eggs of chickens have been studied. RIGHTER
(1971) has studied the comparison of tissues residues of SDD between calves and
chickens after application of SDD at 0.4% in mixed feed and 0.1% in drinking water.
The results showed that the residue was lower than 0.1 ppm after 8 days for calves but
longer than 10 days for chickens. Moreover, the report stated that residue in hepatic,
renal and skin tissues of chickens given medicated feed was more slowly eliminated
than residue in tissues in those given drinking water. Sulfadimidine residue in eggs
following drug administration via drinking water was also documented elsewhere
(KRIEG et al., 1966; BLOM, 1975; ROUDUAT, 1993 and 2000).
KRIEG et al. (1966) and BLOM (1975) have observed SDD residue in eggs after
administration via drinking water at 1 g/L. The result revealed that SDD concentration
in albumen was 70 and 56 µg/mL, respectively, which was higher than that found in
plasma, 50 and 47 µg/mL, respectively. GEERTSMA and colleagues (1987) have
studied residue of SDD and its metabolites in eggs following a single and multiple
oral applications (gelatine capsule form) at 100 mg/kg. Similar ranges of maximum
concentration residues in whole egg were found at 20.1 and 37 µg/g for both
experiments, respectively. They also found the residue in eggs after treatment up to 8
days. This value is similarly reported by PENSABENE et al. (1998), who observed an
occurrence of SDD in eggs fed by a single dose capsule of 75 mg/kg. In addition, they
demonstrated that SDD deposition in the whole eggs quickly declined from 13.9-15.5
ppm, during the first two days, reaching 0.6 ppm on the third day after treatment. This
range of time was also supported by later studies; KAN and PETZ (2000), SHAIKH
and CHU (2000). SDD residue in eggs and tissues, given at 100 mg/kg by feed
medication was also studied (SIEB, 1991). This observation detected SDD in albumen
greater than 100 ppb even 15 days post administration and presented the residue
preferential accumulated in the kidney and liver. This is in agreement with
FURUSAWA and KISCHIDA (2002), who found a greater amount of SDD in the
liver than in other tissues (muscle, plasma, and ovary), a comparison study of the
Literature review
39
distribution of sulfadimidine, sulfadiazine, sulfamethoxazole, sulfamonomethoxine
and sulfaquinoxaline, administered via medicated feed at 100 mg/kg in laying hens.
Moreover, the ratio of sulfadimidine in the tissues concerning egg formation to that in
the plasma was found to be significantly greater than those drugs. Thus, it may be
deduced that SDD is prone to transfer into egg components more easily than those
drugs. In addition, SDD may excrete into egg ca. 1% of the dose after a single
application (GEERTSMA et al, 1987; ROUDUAT et al., 2001).
Distribution of total 14C residues of SDD in eggs and tissues, after single (121.4 mg of
[14C] SDD) or multiple (274.6 mg/day of [14C] SDD) oral doses has been studied
(SHAIKH and CHU, 2000). They reported a higher concentration of SDD in the
albumen than in the yolk on the first two days. Thereafter, it was the opposite case,
this remaining detectable for up to 9 days in yolk. No metabolites of SDD were
detectable in either albumen or yolk, indicating that the parent drug was the main
marker residue in eggs. This result was attributed to support the study of SHAIHK et
al., (1999) and GEERTSMA (1987), who concluded that known metabolites of SDD
residues in eggs could be ignored. ROUDAUT and GARNIER (2002) observed drug
residues of sulfadimethoxine (0.5 g/L) and sulfadimidine (1.0 and 2.0 g/L) in eggs
post administration via drinking water for 5 days. The results revealed that 0.9-1.4%
of the application dose was deposited in the eggs and sulfadimidine was more easily
transferred to the eggs than sulfadimethoxine (1.4% versus 1.0%). Moreover,
approximately 3 times the amount of residues than that in yolk was found in albumen.
Thus, the albumen was recommended as the component for monitoring the drug
residues in eggs. This is in contrast to a recent study which suggested that yolk is an
appropriate matrix for monitoring sulfaquinoxaline in eggs (SHAIKH et al., 2004). In
general, the depletion of SDD and its metabolites in eggs may take at least 7-8 days
following the reports below; after cessation of medication via drinking water (BLOM,
1975; ROUDAUT and GARNIER, 2002), or by oral administration in gelatine
capsule form (NOUWS et al., 1986; GEERTSMA et al., 1987; PENSABENE et al,
1998; SHAIKH and CHU, 2000). However, it has also been documented that its
residue was detected for up to 15 days (SIEB, 1991).
Aims and objectives
40
3 Aims and objectives
The present studies were undertaken to elucidate on pharmacokinetic characteristics
of the veterinary drug and its residues in egg components following application at
various doses. Sulfadimidine and laying hens were used as models. The specific aims
were as follows:
- To develop the analytic procedure of sulfadimidine in the incurred residue samples.
- To evaluate the pharmacokinetic profiles at various doses.
- To observe the drug residues in plasma and eggs components from high to low-level
via oral and medicated feed administration.
- To investigate cross-contamination (drug carry-over) of the drug, in particular,
exposure at low levels.
- To compare pharmacokinetic behaviours and residue levels in egg components
between healthy and hens with impaired kidney function.
Materials and methods
41
4 Materials and methods
4.1 Materials
4.1.1 Chemical agent
Acetic acid 85% Applichem, Darmstadt Germany
Acetonitrile (HPLC far UV) Labscan, Dublin Ireland
Cyclohexane (extra pure) Merck, Darmstadt Germany
Ethyl acetate (HPLC grade) Labscan, Dublin Ireland
Formalin Merck, Darmstadt Germany
Hydrochloric acid 37% Merck, Darmstadt Germany
Methanol (HPLC grade) Labscan, Dublin Ireland
n-hexane (HPCL grade) Labscan, Dublin Ireland
Phosphate hydrogen monohydrate Merck, Darmstadt Germany
Phosphoric acid 85% Merck, Darmstadt Germany
Potassium dichromate Merck, Darmstadt Germany
Sodium chloride (analytical grade) Merck, Darmstadt Germany
Sulfadimidine standard (≥ 99%) Sigma-Aldrich, Steinheim Germany
Sulfadoxine standard (analytical grade) Sigma-Aldrich, Steinheim Germany
Water (HPLC grade) Karl Hecht KG, WD 3V, Germany
4.1.2 Instrument and material
Agitator (micro tube, eppendorf) Heildolph® REAK top, Germany
Agitator horizontal (Tube) IKA® Vibrax VXR basic, Brazil
Analytical column LiChropher®100 RP-18, end capped,
125×4, 5 µm, Darmstadt Germany
Auto sampler Gilson, model 231, USA
Centrifuge tube 2 mL Eppendorf® safe-lock, Germany
Chromatographic software 32-Karat™ Software, version 3.0.,USA
Column oven Spark Holland B.V., The Netherlands
Materials and methods
42
Cooling centrifuge Centrifuge 5403, Eppendorf, Hamburg
Germany
Detector UV detector, Varian® 2050, USA
Evaporator Labor technik Barkey, Bielefeld
Germany
Glass pasture pipettes 150 mm Brand, Wertheim Germany
Guard cartridge LiChorCart® 100 RP-18e (5µm),
Darmstadt Germany
Heparin sodium Euro OTC, Kamen Germany
HPLC vial WICOM, Darmstadt Germany
Nitrogen gas Westfaland, Germany
pH meter WTW pH320, Weilheim Germany
Pipett tips 20 µl, 100 µl, 2 mL Greiner, Frickenhausen Germany
Polypropylene test tube 10 mL Sarstedt, Nümbrecht Germany
Solvent delivery system Beckman®, Programmable solvent
module 116, USA
Sonicator Bandelin sonorex®, Berlin Germany
4.1.3 Composition of buffer solution
Mobile phase 0.05 mol/L phosphate buffer pH 3
13.8 g NaH2PO4 · H2O
2000 mL Water (HPLC grade)
pH 3 adjust with Phosphoric acid
4.1.4 Animals
Six healthy laying hens each group (Lohmann Brown), 22-26 weeks of ages (1.95 ±
0.15 kg), were housed at controlled temperature (20 ± 2°C) and light (14 h/day) in
accordance with the animal welfare acts (33-42502-04/917, LAVES). Water and a
commercial standard laying hen mash (deuka Deutsche Tiernahrung, Germany) were
supplied ad libitum.
Materials and methods
43
4.2 Methods 4.2.1 Drug administration and sample collection
4.2.1.1 Sulfadimidine experiment I
Groups of six hens each were treated with SDD via single intravenous injection (i.v.),
a single oral bolus administration (p.o.) and medicated feed. Animals were fasted
(feed) overnight before treatment. For a group of single i.v. administration, SDD was
injected as a single bolus via the vena axillaris at 100 mg/kg body weight (BW). For a
single oral bolus application (5 groups), each group of hens was treated at 1, 3, 10, 30
and 100 mg/kg BW. For the three groups of medicated feed, SDD was mixed into
feed resulting in a dose of 3, 10 and 30 mg/kg BW and fed for seven consecutive
days.
Blood samples of 1.5 mL were taken from the brachial vein at predetermined
intervals, as shown in table 5 and 6. All blood was heparinised to prevent blood
clotting. This was centrifuged at 4100 g for 10 min to separate the plasma, which was
placed into a 2 mL centrifuge vial for drug extraction. Eggs were collected for 7 days,
starting at 20 h after the treatment. Egg yolk and egg albumen were separated, and
placed into 10 mL polypropylene test tubes. All samples were stored at –20°C
pending analysis.
Table 5: Experiment method of single bolus administration of SDD
Route of
administration
Dose
(mg/kg BW)
Time determined
for plasma sample
Egg sample
Intravenous 100
Oral 1
Oral 3
Oral 10
Oral 30
0.25 (only for i.v.),
0.5, 1, 2, 4, 6, 8,
12, 24, 32, 48, 60,
72 h.
Collected daily
after dosed when
hens had laid eggs.
Oral 100
Materials and methods
44
Route of
administration
Dose
(mg/kg BW)
Feed duration
(day)
Plasma sample Egg sample
Mixed feed 3 7
Mixed feed 10 7
Mixed feed 30 7
Collected daily Collected daily
after dosed if
eggs were laid.
Table 6: Experiment method of medicated feed of SDD
4.2.1.2 Sulfadimidine experiment II
Hens were divided into two groups, one for healthy hens (n=4) and another for
impaired kidney hens (n=6). Potassium dichromate (K2Cr2O7), a renal toxic
substance, was prepared as a solution in water at 200 mg/mL (EICHLER, 1965). To
induce kidney damage hens were subcutaneously injected with K2Cr2O7 at 15 mg/kg
BW and allowed a stage of kidney damage for 7 days. Thereafter, an i.v.
administration of SDD at 100 mg/kg BW was applied for both groups. Blood samples
were collected following experiment I (see 4.2.1.1). Eggs were collected for 7 days,
starting at 6 h after the treatment. Egg yolk and egg albumen were separated, and
placed into 10 mL polypropylene test tubes. All samples were stored at –20°C prior to
analysis. At the end of the experiment, birds were sacrificed and their renal tissues
were examined histologically to confirm pathological lesions. The protocol for
experiment II is presented in table 7.
Hen Route of
administration
Dose
(mg/kg BW.)
Time determined
for plasma sample
Egg sample
Healthy i.v. 100
Impaired
kidney
i.v. 100
0.5, 1, 2, 4, 6, 8,
12, 24, 32, 48, 60,
72 h.
Collected daily
after dosed
when hens
have laid eggs.
Table 7: Protocol for experiment II of SDD
Materials and methods
45
4.2.2 Analysis (HPLC)
4.2.2.1 Preparing standard solution
Sulfadimidine (SDD), standard solutions were prepared in distilled water at a
concentration of 0.001 mg/mL to 1 mg/mL. Sulfadoxine (SDX), as an internal
standard, was dissolved in methanol at concentrations of 50 µg/mL.
4.2.2.2 Extraction process
Plasma: A 10 µL aliquot of SDX solution (50µg/mL) was added to 490 µL of
plasma. Then, 100 µL of 0.1 N HCl and 1 mL of ethyl acetate were added. Thereafter,
the sample was mixed for 1 min using a vortex mixer and centrifuged at 23000 g for 5
min. 600 µL supernatant was taken into a centrifuge cup and evaporated under a
gentle N2 steam at 40°C. The residue was mixed for 1 min with 700 µL eluent and
centrifuged at 23000 g for 5 min. A 500 µL aliquot of the clearly upper phase was
collected into a HPLC vial for injection (100 µL) into the HPLC system.
Yolk: A 1 g accurately weighed sample of yolk was put into a 10 mL test tube. The
yolk sample was mixed with 2.9 mL distilled water and internal standard (10 µL of
100µg/mL) of SDX. A 500 µL of 0.1 N HCl and 2 ml of ethyl acetate were added
before mixing the sample for 2 min with a vortex mixer. The amount of excessive
NaCl (approximately 1.2 g) along with 100 µL of 1 N HCl and 500 µL of n-Hexane
were added into the colloid. Afterwards it was mixed for 2 min and centrifuged at
4100 g for 5 min: 1.5 mL supernatant was put into 10 ml test tube. The remainder was
re-extracted with 1.5 mL ethyl acetate, then mixed for 2 min and centrifuged at 4100
g for 5 min to collect the 1.5 mL upper phase. Thereafter, combined with the previous
one, the extract was evaporated to dryness with a N2-evaporator. The residue was
reconstituted with 700 µL of eluent. After mixing for 2 min, the solution was
transferred into a centrifuge cup and centrifuged at 23100 g for 5 min. 500 µL of the
supernatant was filled in the HPLC vial for analysis.
Albumen: A 1 g portion of albumen sample was weighed into test tube, and then 0.5
mL distilled water and10 µL of SDX (100 µg/mL) were added. The mixed sample
Materials and methods
46
was added with 0.5 mL of 0.1 N HCl, 0.9 mL of acetonitrile and 0.1 mL of n-Hexane,
respectively. After mixing for 2 min, excessive NaCl (approximately 1.2 g) and 2 mL
of ethyl acetate were added. The sample was mixed for 2 min with a vortex mixer and
centrifuged at 4100 for 5 min. 2 mL of the upper phase was evaporated under N2
steam at 40°C. The remnant was dissolved with 700 µL mobile phase, mixed for 1
min and centrifuged at 4100 g for 5 min, and then 500 µL of the clearly phase was put
into the HPLC vial for the HPLC injection at 100 µL.
4.2.2.3 HPLC conditions
A mobile phase consisting of 0.05 mol/L phosphate buffer (pH 3.0) and acetonitrile at
85:15, v/v and 89:11, v/v was used for plasma and egg analysis, respectively. A flow
rate of 1.5 mL/min and a wavelength of 268 nm were applied and the temperature of
the column oven was set to 40°C throughout.
The validation method is involves the checking of data or programs for correctness,
compliance with standards and conformance with the requirement specification. To fit
the intended purpose, the typical validation characteristics considered are selectivity,
linearity, limit of detection, limit of quantification, recovery, accuracy, and precision.
Selectivity: The ability of the bioanalytical method to measure and differentiate the
analyte in presence of components that may be expected to present regarded as
“selectivity”. The selectivity of the analytical method must be demonstrated by
providing data to show the absence of interference peaks with regard to degradation
products, synthetic impurities and the matrices.
Linearity: Linearity is defined of an analytical procedure as the ability (within a
given range) to obtain test results of variable data which are directly proportional to
the concentration in the sample. At least five different concentration levels should be
used. The linearity is expressed by the linear regression coefficient (R2). The
calibration curve is the relationship between experimental response value and known
concentration of the analyte. At least duplication of five different concentrations was
used for the curve. Table 8 provide the concentrations used for linear calibration curve
of sulfadimidine in plasma, yolk and albumen.
Materials and methods
47
Matrix Concentration
Plasma 25, 100, 500, 1000, 2000, 5000 (ng/mL)
Yolk 50, 200, 1000, 5000, 10000, 20000 (ng/g)
Albumen 50, 200, 1000, 5000, 10000, 20000 (ng/g)
Table 8: Matrix and concentration used for linearity curve of SDD
Limit of detection: The limit of detection (LOD) is the lowest concentration of an
analyte that the bioanalytical procedure can reliably differentiate from background
noise. However, the LOD may not necessarily quantities as an exact value. The LOD
was calculated from the confidence interval of the calibration curve according to DIN
32645.
Limit of quantification: The limit of quantification (LOQ) is the lowest
concentration of an analyte in sample that can be determined quantitatively with
suitable precision (less than 20% of SD) and accuracy (80-120%). The LOQ was
determined by preparing standard solutions at the estimated LOQ level (based on the
preliminary tests which provide precision and accuracy values).
Recovery: Recovery is the extraction efficiency of an analytical process, reported as
the yield of an analyst obtained after complete sample extraction and processing steps
of the method in comparison to the theoretical maximum defined by complete transfer
from the natural matrix into the solution actually measured. The recovery of the
analyte need not be 100%, but the extent of recovery of an analyte and or internal
standard should be consistent, precise and reproducible.
Recovery = [Cfrt – C unf / Cadd ] . 100
Cfrt = concentration determined in fortified sample
Cunf = concentration determined in unfortified sample
Cadd = concentration of fortification
Materials and methods
48
Accuracy: Accuracy of an analytical procedure expresses the closeness of agreement
between the expected and the measured mean value. Accuracy is determined by
replicate analysis of samples containing known amounts of the analyte. The
measurements of 5 samples per concentration were calculated. Table 9 is given
different spiking concentrations in plasma, yolk and albumen. The mean value should
be within 15% of the actual value except at the LOQ, where it should not deviate by
more than 20%.
Matrix Sample amount Concentration
Plasma 5 0.025, 0.10, 0.50 (µg/mL)
Yolk 5 0.10, 1.0 (µg/g)
Albumen 5 0.10, 1.0 (µg/g)
Table 9: Spiked SDD at different concentrations in plasma, yolk and albumen
Precision: Precision presents the reproducibility of repeated measurements at the
same homogeneous sample under the prescribed condition. Precision is usually
expressed in term of the relative standard deviation (RSD), which is useful in case of
the comparison of samples with different concentrations.
The precision may be called “intra-day” if operated within short interval of time or
within batch and “inter-day” when operated in different day or between batches. The
measurements of 6 replicate determinations per concentration per day which at least 3
different days were operated. The intra- and inter-day precision determined at each
concentration level should not exceed 15% except for the LOQ level, where it should
not exceed 20%. Table 10 provided matrix, sample amounts and concentration for
precision test of SDD. Intra-day precision, sw and inter-day precision, sb were
calculated as:
∑∑ ⋅
=Tagesserie
TagesserieTagesseriew f
sfs
)( 2
Tage
gesamtTagesserie
b fXX
s ∑ −=
2)(
Materials and methods
49
Matrix Sample amount/ day Concentration
Plasma 6 0.025, 0.050, 0.075, 0.10 (µg/mL)
Yolk 6 0.050, 0.125 (µg/g)
Albumen 6 0.050, 0.125 (µg/g)
Table 10: Matrix, sample amounts and concentrations for precision test of SDD
4.2.3 Pharmacokinetic parameters and statistical analyses
Pharmacokinetic parameters were calculated using a program WinNonLin®version
4.1, Pharsight® USA. A first-order one-compartment model was used throughout the
calculation. The area under the curve (AUC), half-life of absorption (K01_HL),
elimination half-life (K10_HL), total body clearance (CL), time of maximum plasma
concentration (Tmax) and maximum plasma concentration (Cmax) were calculated.
In the experiment II, comparison between health and impaired kidney hens, statistical
data were performed using a software package, Prism 4 for Windows version 4.01
(GraphPad Software, Inc, San Diego USA). The data were evaluated using Mann-
Whitney test (two-tailed). The significance value was presented at p ≤ 0.05.
Results
50
5 Results
5.1 Method of analysis
5.1.1 Selectivity
To determine the selectivity of the analysis of SDD in the matrices; plasma, albumen
and yolk, the control and fortified samples were examined. Figure 2, 3 and 4
illustrates the typical chromatograms, which showed no interference and clearly
differentiate between SDD spiked into the matrices at the LOQ level (black arrow)
and the internal standard (I.S.; SDX).
Figure 2: Typical chromatogram obtained from fortified plasma at 0.025 µg/mL (A)
and control plasma (B)
Results
51
Figure 3: Typical chromatogram obtained from fortified albumen at 0.05 µg/g (C) and
control albumen (D)
Figure 4: Typical chromatogram obtained from fortified yolk at 0.05 µg/g (E) and
control yolk (F)
Results
52
5.1.2 Linearity The linear calibration curve of SDD in plasma, within the range of 25- 5000 ng/mL,
was obtained by plotting the peak area against the amount. For yolk and albumen, the
linearity was determined within the range of 50- 20000 ng/g. The calculation of linear
regression showed R2= 0.9990 for plasma (Fig. 5), R2= 0.9994 for yolk (Fig. 6) and
R2= 0.9991 for albumen (Fig. 7).
y = 0.0014x + 0.0628R2 = 0.999
0
2
4
6
8
0 1000 2000 3000 4000 5000 6000Concentration (ng/mL)
Res
pons
e
Figure 5: The linear calibration curve of SDD in plasma
Results
53
y = 0.0008x + 0.0204R2 = 0.9994
0
5
10
15
20
0 5000 10000 15000 20000 25000Concentration (ng/g)
Res
pons
e
Figure 6: The linear calibration curve of SDD in yolk
y = 0.0009x - 0.0401R2 = 0.9991
0
5
10
15
20
0 5000 10000 15000 20000 25000Concentration (ng/g)
Res
pons
e
Figure 7: The linear calibration curve of SDD in albumen
Results
54
5.1.3 Limit of detection (LOD) and limit of quantification (LOQ) The method provided the LOD of SDD in plasma at 0.01 µg/mL and 0.02 µg/g for
both yolk and albumen. According to the results, the LOQ concentration of SDD at
0.025 µg/mL for plasma and 0.05 µg/g for both yolk and albumen were found,
respectively.
5.1.4 Recovery
Collated average recoveries of SDD from different spiking concentrations in plasma,
yolk and albumen are presented in table 11. The values of recoveries greater than 80
% were satisfied with relative standard deviation (RSD) between 2.22% and 6.66%
except at the LOQ level of plasma (10.20 %).
Matrix Spiked concentration
(µg/mL or µg/g) n
Recovery
(%)
Relative standard
deviation, RSD. (%)
0.025 5 94.57 10.20
0.1 5 86.81 2.22 Plasma
0.5 5 84.70 3.29
0.1 5 97.06 6.66 Yolk
1.0 5 88.74 3.54
0.1 5 90.05 5.09 Albumen
1.0 5 90.14 6.15
Table 11: Average recoveries of SDD at different spiking concentrations of plasma
(µg/mL), yolk (µg/g) and albumen (µg/g)
5.1.5 Precision
The precision of SDD in plasma, yolk and albumen are given in table 12. An
overview, the intra-day and inter-day repeatability are less than 15 % except at the
LOQ level (0.025 µg/mL) of plasma for intra-day (16.95%).
Results
55
Table 12: Precision data of SDD (intra-day and inter-day) by different spiking
concentrations of plasma (µg/mL), yolk (µg/g) and albumen (µg/g)
Matrix Concentration
(µg/mL or µg/g)
Intra-day
(%)
Inter-day
(%)
0.025 16.95 8.37
0.050 6.80 4.47
0.075 5.18 2.02 Plasma
0.100 4.46 1.77
0.050 6.82 7.60 Yolk
0.125 8.07 4.20
0.050 9.58 3.35 Albumen
0.125 5.42 1.66
Results
56
5.2 Plasma concentrations of sulfadimidine following various doses
administered intravenously and orally in laying hens
Average plasma concentrations (Mean ± SD) of SDD in laying hen at predetermined
interval times following a single intravenous bolus and various doses via oral
treatment are given in table 13. Following i.v. administration at 100 mg/kg BW, the
maximum concentration of SDD was 129.40 ± 19.69 µg/mL rapidly declining to 0.06
± 0.06 µg/mL at 48 h after injection. Thereafter it was undetectable.
For the groups treated orally, the maximum plasma level of SDD reached 72.35 ±
15.49 µg/mL after 4 h of oral application at 100 mg/kg BW, whereas the maximum
concentration of 23.33 ± 1.36 µg/mL was detected 2 h after oral treatment with 30
mg/kg BW. For all low doses of oral administration at 1, 3 and 10 mg/kg BW, the
maximum plasma levels achieved 0.5 h after application were 0.53 ± 0.23, 2.09 ± 1.06
and 5.29 ± 2.49 µg/mL, respectively (see table 13). Semi-logarithmic plots of plasma
concentration-time curve of SDD (Mean ± SD) following i.v. and p.o. treatment are
given in figures 8 and 9, respectively. As shown in the plots, SDD was rapidly
depleted from the plasma, which showed concentrations lower than 0.025 µg/mL
within 48 h after i.v. injection and within 6-32 h after p.o. administration.
Dose (mg/kg BW.) 100 (i.v.) 100 (p.o.) 30 (p.o.) 10 (p.o.) 3 (p.o.) 1 (p.o.)
Time (h)
0.25 129.40 ± 19.69 - - - - -
0.5 121.68 ± 16.25 30.78 ± 15.82 18.60 ±11.14 5.29 ± 2.49 2.09 ±1.06 0.53 ±0.23
1 113.16 ± 15.03 40.96 ± 8.79 20.92 ± 8.93 5.08 ± 1.71 1.65 ± 0.35 0.51 ± 0.10
2 101.18 ± 11.11 68.27 ± 15.91 23.33 ± 1.36 4.41 ± 1.15 0.78 ± 0.18 0.21 ± 0.02
4 83.93 ± 16.34 72.35 ± 15.49 18.32 ± 4.25 1.64 ± 0.50 0.21 ± 0.07 0.06 ± 0.02
6 64.36 ± 13.45 69.08 ± 23.77 10.76 ± 2.06 0.56 ± 0.27 0.07 ± 0.01 <0.025
8 52.89 ± 12.40 65.53 ± 19.55 5.54 ± 1.33 0.33 ± 0.13 <0.025 ND
12 29.98 ± 9.92 51.00 ± 18.70 1.27 ± 0.55 0.03 ± 0.04 ND ND
24 4.37 ± 2.01 23.08 ± 10.72 <0.025 ND ND ND
32 0.03 ± 0.09 2.67 ± 2.10 ND ND ND ND
48 0.06 ± 0.06 <0.025 ND ND ND ND
Table 13: Average SDD plasma concentration (µg/mL, Mean ± SD), (n=6)
Drug administered via i.v. and p.o. route at different doses (1, 3, 10, 30 and 100 mg/kg BW)
ND = not detected, < 0.025 = below the LOQ
57
Results
58
5.3 Pharmacokinetic characteristics of sulfadimidine in laying hens 5.3.1 Pharmacokinetic characteristics of sulfadimidine following a single
intravenous bolus administration in laying hens
Pharmacokinetics values following plasma concentrations in laying hens after a single
intravenous bolus injection at 100 mg/kg BW were calculated by first-order one
compartment model. Figure 8 depicts a semi-logarithmic plot of average plasma
concentration of six hens versus time. As presented in the plot, SDD was rapidly
depleted from the plasma, which lowered to less than 0.025 µg/mL within 48 h. Table
14 presents the calculation of the pharmacokinetics parameters of SDD after i.v.
application. The calculated maximum plasma concentration (Cmax) of 132.20 ± 16.95
µg/mL was found 15 minutes after the dose and could be detected over LOQ (0.025
µg/mL) up to 48 hours. The elimination half-life (K01_HL) of 5.87 ± 1.14 h and the
total body clearance (CL) of 94.15 ± 27.84 mL/h/kg were found.
0.010
0.100
1.000
10.000
100.000
1000.000
0 6 12 18 24 30 36 42 48
Time (h)
Plas
ma
conc
entra
tion
( µ
g/m
L)
Figure 8: Plasma concentration of sulfadimidine (Mean ± SD) versus time after a
single intravenous bolus injection in laying hens (n=6)
Results
59
Hen (number) Parameter Unit 1 2 3 4 5 6 Mean SD
AUC µg*h/mL 1285.54 674.95 1212.25 997.63 1254.40 1307.43 1122.03 245.81
K10_HL h 6.78 4.44 6.14 4.82 5.65 7.41 5.87 1.14
CL mL/h/kg 77.79 148.16 82.49 100.24 79.72 76.49 94.15 27.85
Cmax µg/mL 131.38 105.41 136.94 143.37 153.90 122.23 132.20 16.95
Vss mL/kg 761.15 948.70 730.22 679.77 649.77 818.14 767.58 105.44
MRT h 9.78 6.40 8.85 6.96 8.15 10.70 8.47 1.64
Table 14: Pharmacokinetic values (Mean ± SD) of sulfadimidine after intravenous
injection of 100 mg/kg BW in laying hens
AUC = area under the curve, K01_HL = the absorption half-life,
K10_HL = the elimination half-life, CL = the systemic clearance,
CL_F = the total body clearance (for extra vascular models),
Tmax = the time of peak concentration, Cmax = the maximum concentration,
Vss = the volume of distribution at steady state, K01 = the apparent first-order
absorption rate constant, K10 = the apparent first-order elimination rate constant
Results
60
5.3.2 Pharmacokinetic characteristics of sulfadimidine following a single oral
bolus administration at different concentrations in laying hens
Based on plasma concentrations following a single oral bolus administration of
various doses (1, 3, 10, 30 and 100 mg/kg BW.), pharmacokinetic profiles were
calculated which are presented in tables 15-18. However, an oral application group of
1 mg/kg BW was not calculated due to inadequate quality data for performing kenetic
values. Figure 9 shows semi-logarithmic plots of average plasma concentration versus
time with six hens in each group. SDD was very quickly eliminated from the plasma
within 6-32 h. The calculated pharmacokinetic parameter showed the maximum
plasma concentrations as being 2.42 ± 1.15, 6.20 ± 1.79, 25.32 ± 5.78 and 76.50 ±
19.85 µg/mL following oral application of 3, 10, 30 and 100 mg/kg BW, respectively.
However, a wide range of the AUC values between 4.18 ± 1.22 to 1379.30 ± 428.13
h*µg/mL was observed. SDD had rapid absorption behaviour as presented in the
results of the rate of absorption of half-life (K01_HL) are 0.21 ± 0.26 to 1.20 ± 0.44 h
after various doses administered orally. Moreover, the biological half-life (K10_HL)
of SDD range was between 0.89 ± 0.16 h to 9.10 ± 1.58 h.
0.001
0.010
0.100
1.000
10.000
100.000
1000.000
0 6 12 18 24 30 36 42 48
Time (h)
Plas
ma
conc
entra
tion
( µ
g/m
L)
p.o. 100 mg/kg
p.o. 30 mg/kg
p.o. 10 mg/kg
p.o. 3 mg/kg
p.o. 1 mg/kg
Figure 9: Plasma concentration of sulfadimidine (Mean ± SD) versus time after a
single oral bolus administration of different doses in laying hens
Results
61
Hen (number) Parameter Unit 1 2 3 4 5 6 Mean SD
AUC µg*h/mL 1533.92 1400.57 1210.57 1734.85 1777.25 618.43 1379.30 428.13
K01_HL h 0.90 0.70 1.28 1.42 1.93 10.99 1.20 0.44
K10_HL h 10.49 9.67 10.80 8.63 8.53 6.48 9.10 1.58
CL_F mL/h/kg 65.19 71.40 82.59 57.64 56.27 161.70 82.47 40.00
Tmax h 3.48 2.87 4.46 4.42 5.35 3.16 3.96 0.94
Cmax µg/mL 80.55 81.74 58.39 97.68 93.49 47.15 76.50 40.00
K01 1/h 0.77 0.99 0.54 0.49 0.36 0.70 0.64 0.22
K10 1/h 0.77 0.07 0.06 0.08 0.08 0.11 0.08 0.02
Table 15: Pharmacokinetic properties of SDD after a single oral bolus administration
at 100 mg/kg BW
Hen (number) Parameter Unit 1 2 3 4 5 6 Mean SD
AUC µg*h/mL 160.65 122.04 163.32 175.77 175.25 112.20 151.54 27.53
K01_HL h 0.89 0.47 0.18 0.07 0.62 1.82 0.67 0.63
K10_HL h 2.37 2.48 3.56 3.39 3.00 1.86 2.78 0.65
CL_F mL/h/kg 186.75 245.82 183.69 170.68 171.18 267.38 204.25 41.63
Tmax h 1.98 1.40 0.80 0.42 1.77 2.66 1.51 0.81
Cmax µg/mL 26.34 23.03 27.16 33.00 26.86 15.54 25.32 5.78
K01 1/h 0.80 1.46 3.93 9.44 1.12 0.38 2.86 3.46
K10 1/h 0.29 0.28 0.19 0.20 0.23 0.37 0.26 0.07
Table 16: Pharmacokinetic properties of SDD after a single oral bolus administration
at 30 mg/kg BW
Results
62
Hen (number) Parameter Unit 1 2 3 4 5 6 Mean SD
AUC µg*h/mL 17.30 25.49 17.10 17.82 19.80 13.20 18.45 4.06
K01_HL h 0.96 0.03 0.03 0.76 0.58 0.03 0.40 0.42
K10_HL h 0.97 2.42 1.49 0.79 0.60 2.27 1.42 0.78
CL_F mL/h/kg 577.96 392.31 584.83 561.17 504.99 757.66 563.15 119.24
Tmax h 1.39 0.20 0.17 1.10 0.85 0.21 0.65 0.53
Cmax µg/mL 4.58 6.89 7.35 5.96 8.60 3.78 6.20 1.79
K01 1/h 0.72 21.55 24.47 0.91 1.20 19.81 1.44 11.60
K10 1/h 0.72 0.29 0.46 0.91 1.16 0.31 0.64 0.35
Table 17: Pharmacokinetic properties of SDD after a single oral bolus administration
at 10 mg/kg BW
Hen (number) Parameter Unit 1 2 3 4 5 6 Mean SD
AUC µg*h/mL 3.50 4.68 5.16 5.26 4.38 2.07 4.18 1.22
K01_HL h 0.28 0.03 0.05 0.05 0.15 0.70 0.21 0.26
K10_HL h 0.94 1.15 0.91 0.76 0.87 0.70 0.89 0.16
CL_F mL/h/kg 858.03 640.84 581.65 567.58 684.27 1451.47 797.31 337.09
Tmax h 0.70 0.17 0.23 0.22 0.46 1.01 0.46 0.33
Cmax µg/mL 1.54 2.55 3.30 3.93 2.42 0.75 2.42 1.15
K01 1/h 2.48 21.69 13.52 12.89 4.53 0.99 9.35 8.03
K10 1/h 0.74 0.60 0.76 0.91 0.80 0.99 0.80 0.14
Table 18: Pharmacokinetic properties of SDD after a single oral bolus administration
at 3 mg/kg BW
To observe the relation of dose (mg/kg BW) and some pharmacokinetic parameters,
the Cmax, K10_HL, Tmax and CL_F values were plotted. Figures 10-13 elucidate that
after a single oral bolus administration of SDD at different doses, the Cmax, K10_HL
and Tmax increased while the CL_F decreased with increasing doses.
Results
63
0
20
40
60
80
100
120
0 20 40 60 80 100 120dose [mg/kg BW]
Cm
ax [ µ
g/m
L]
Figure 10: Cmax values versus dose after a single oral bolus administration of SDD at
different doses (Mean ± SD, n = 6)
0
2
4
6
8
10
12
0 20 40 60 80 100 120 dose [mg/kg BW]
K10
_HL
[h]
Figure 11: K10_HL values versus dose after a single oral bolus administration of SDD
at different doses (Mean ± SD, n = 6)
Results
64
0
1
2
3
4
5
6
0 20 40 60 80 100 120 dose [mg/kg BW]
Tm
ax [h
]
Figure 12: Tmax values versus dose after a single oral bolus administration of SDD at
different doses (Mean ± SD, n = 6)
0
200
400
600
800
1000
1200
0 20 40 60 80 100 120 dose [mg/kg BW]
CL
[ml/h
/kg]
Figure 13: CL values versus dose after a single oral bolus administration of SDD at
different doses (Mean ± SD, n = 6)
Results
65
5.4 Sulfadimidine residue in eggs after a single oral bolus
administration at various doses in laying hens
5.4.1 Sulfadimidine residue in yolk
Following various single oral dose treatments of SDD at 1, 3, 10, 30 and 100 mg/kg
BW, the residues in yolk are summarised in table 19. The residue of 14.32 ± 6.47
µg/g was found in yolk one day after application at 100 mg/kg BW. For sub-
therapeutic doses at 3, 10 and 30 mg/kg BW, the residues on the first day after the
dose treatment were 0.11 ± 0.05, 0.42 ± 0.08 and 3.08 ± 1.52 µg/g, respectively.
However, no drug residue in yolk above LOQ (0.05 µg/g) was detected after oral
treatment of 1 mg/kg BW.
The residue was detectable above LOQ up to 8 days after administration at 100 mg/kg
BW. In addition, it could be found up to 7, 3 and 1 day for the dose of 30, 10 and 3
mg/kg BW, respectively.
Day after Dose (mg/kg BW)
treatment 100 30 10 3 1
1 14.32 ± 6.47 3.08 ± 1.52 0.42 ± 0.08 0.11 ± 0.05 ND
2 6.62 ± 2.72 0.51 ± 0.01 0.07 ± 0.02 <0.05 ND
3 3.67 ± 1.17 0.44 ± 0.12 0.06 ± 0.02 ND ND
4 2.74 ± 0.69 0.32 ± 0.05 0.04 ± 0.01 ND ND
5 1.98 ± 0.56 0.20 ± 0.04 <0.05 ND ND
6 1.21 ± 0.59 0.13 ± 0.02 ND ND ND
7 0.43 ± 0.32 0.07 ± 0.03 ND ND ND
8 0.12 ± 0.14 <0.05 ND ND ND
9 <0.05 <0.05 ND ND ND
10 ND ND ND ND ND
Table 19: Sulfadimidine residue in yolk (µg/g, Mean ± SD) after a single oral
administration of different doses; ND = not detected, < 0.05 = below the LOQ
Results
66
5.4.2 Sulfadimidine residue in albumen
Table 20 shows SDD residue in albumen following a single oral bolus administration
at different concentrations in laying hens. Within the normal dose of 100 mg/kg BW
the residue of SDD was found at a high amount in albumen at 49.34 ± 7.03 µg/g, one
day after of the dose, and detectable above the LOQ for up to 6 days. In the case of
the low administered doses, the residue levels of 0.28 ± 0.12, 0.98 ± 0.24 and 6.25 ±
1.85 µg/g were detected on day one following oral application with 3, 10 and 30
mg/kg BW, respectively. Interestingly, the detection of SDD residue at 0.07 ± 0.02
µg/g in albumen was observed after a dose of 1 mg/kg BW.
Day after Dose (mg/kg BW)
treatment 100 30 10 3 1
1 49.34 ± 7.03 6.25 ± 1.85 0.98 ± 0.24 0.28 ± 0.12 0.07 ± 0.02
2 6.10 ± 0.43 0.16 ± 0.04 ND ND ND
3 0.83 ± 0.16 0.15 ± 0.14 ND ND ND
4 0.38 ± 0.12 ND ND ND ND
5 0.20 ± 0.17 ND ND ND ND
6 0.12 ± 0.03 ND ND ND ND
7 <0.05 ND ND ND ND
8 ND ND ND ND ND
Table 20: Sulfadimidine residue in albumen (µg/g, Mean ± SD) after a single oral
administration of different doses; ND = not detected, < 0.05 = below the LOQ
Results
67
5.4.3 Transfer rate of sulfadimidine into eggs after a single oral administration
In the presented residues the calculation of the drug transfer rate (TR) into the eggs
was equated. The sum of residues (Rtotal) in the albumen and yolk were calculated
using the measured concentration in the albumen and yolk (Calbumen, Cyolk) and the
mass of the albumen and yolk (malbumen, myolk) at day i as:
[1] iyolkiyolkialbumenialbumenitotal mcmcR ,,,,, ⋅+⋅=
For the mass of yolk and albumen, an average weight of six representative eggs was
used (13.84 g and 36.28 g, respectively). The transfer rate was calculated from the
sum of residues of all eggs measured up to 10 days after administration as:
[2] 100[%]
10
1,
⋅=∑=
=
dose
RTR
t
iitotal
Interestingly, the calculation reveals that the transfer rates of SDD into the eggs after
single oral doses are dose-dependent (table 21). Decreasing doses are related with a
diminishing transfer rate.
Dose Drug Residue Transfer-ratec
intakea in whole eggb
(mg kg-1 BW) (mg) (µg) (%)100 195 2497.16 1.2830 61.5 303.60 0.4910 19.1 43.17 0.233 5.8 11.68 0.201 1.8 2.54 0.14
Table 21: Transfer rate of SDD into eggs after single oral administration
a calculated from dose × average BW, (data not shown)
b calculated according to equation [1]
c calculated according to equation [2]
Results
68
5.5 Sulfadimidine residue in eggs after medication via mixed feed at
various doses for seven consecutive days in laying hens
5.5.1 Sulfadimidine residue in yolk
In table 22 SDD residue levels in yolk are presented after application via mixed feed.
The average residue levels following medicated feed administration of 10 and 30
mg/kg BW for 7 days were 0.14 ± 0.06 and 1.43 ± 0.57 µg/g, respectively. However,
no residue level was found in the group medicated with 3 mg/kg BW. Figure 14 and
15 elucidate residue levels of each hen per day. The residues in yolk following
medication with 10 and 30 mg/kg BW gradually rose after the first day of application.
However, as compared to the albumen (see table 23), all SDD residues were slowly
accumulated in the yolk.
Matrix Yolk (µg/g)
Dose (mg/kg.BW) 30 10 3
Day 1 0.26 ± 0.14 0.04 ± 0.01 ND
Day 2 1.23 ± 0.52 0.10 ± 0.03 ND
Day 3 1.50 ± 0.70 0.13 ± 0.03 ND
Day 4 2.04 ± 0.60 0.15 ± 0.03 ND
Day 5 1.80 ± 0.52 0.16 ± 0.02 ND
Day 6 1.55 ± 0.45 0.20 ± 0.02 ND
Day 7 1.64 ± 0.55 0.20 ± 0.02 ND
Mean ± SD 1.43 ± 0.57 0.14 ± 0.06 ND
Table 22: SDD residue level in the yolk (µg/g, Mean ± SD) following medication via
feed for seven consecutive days in laying hens; ND = not detected
Results
69
0
1
2
3
0 1 2 3 4 5 6 7
Time (day)
Con
cent
ratio
n ( µ
g/g)
Hen 1Hen 2Hen 3Hen 4Hen 5Hen 6
Figure 14: SDD residue level in the yolk, each hen following medicated via feed for 7
days of 30 mg/kg BW
0
0.1
0.2
0.3
0 1 2 3 4 5 6 7
Time (day)
Con
cent
ratio
n ( µ
g/g)
Hen 1Hen 2Hen 3Hen 4Hen 5Hen 6
Figure 15: SDD residue level in the yolk each hen following medicated via feed of 10
mg/kg BW for seven days
Results
70
5.5.2 Sulfadimidine residue in albumen
Table 23 presents SDD residue concentrations in albumen following medication via
feed. Figure 16 to 18 give the residue levels for each hen per day. High concentrations
of SDD residue in the albumen of 2.50 ± 1.10 µg/g were found after the first day of
application at 30 mg/kg BW via medicated feed. For the group administration of 3 and
10 mg/kg BW, the average residue levels of day one were 0.13 ± 0.03 and 0.42 ± 0.07
µg/g, respectively. Interestingly, the residue levels in the albumen were estimated as
being equal to those found in the plasma (see table 24), which confirm a rapid
accumulation of SDD residue in the albumen.
Matrix Albumen (µg/g)
Dose (mg/kg BW) 30 10 3
Day 1 2.50 ± 1.10 0.42 ± 0.07 0.13 ± 0.03
Day 2 5.40 ± 2.58 0.62 ± 0.15 0.15 ± 0.02
Day 3 4.86 ± 1.44 0.61 ± 0.15 0.15 ± 0.01
Day 4 4.99 ± 1.93 0.64 ± 0.05 0.14 ± 0.01
Day 5 3.77 ± 1.25 0.45 ± 0.06 0.13 ± 0.02
Day 6 2.84 ± 1.25 0.40 ± 0.12 0.14 ± 0.02
Day 7 2.97 ± 0.90 0.43 ± 0.14 0.13 ± 0.01
Mean ± SD 3.90 ± 1.18 0.51 ± 0.10 0.14 ± 0.01
Table 23: SDD residue level in the albumen (µg/g, Mean ± SD) following medication
via feed for seven consecutive days in laying hens
Results
71
0
2
4
6
8
10
0 1 2 3 4 5 6 7Time (day)
Con
cent
ratio
n ( µ
g/g)
Hen 1Hen 2Hen 3Hen 4Hen 5Hen 6
Figure 16: SDD residue levels in the albumen of each hen following medication via
feed for seven days of 30 mg/kg BW
0.0
0.2
0.4
0.6
0.8
1.0
0 1 2 3 4 5 6 7Time (day)
Con
cent
ratio
n ( µ
g/g)
Hen 1Hen 2Hen 3Hen 4Hen 5Hen 6
Figure 17: SDD residue levels in the albumen of each hen following medication via
feed for seven days of 10 mg/kg BW
Results
72
0.0
0.1
0.2
0.3
0 1 2 3 4 5 6 7
Time (day)
Con
cent
ratio
n ( µ
g/g)
Hen 1Hen 2Hen 3Hen 4Hen 5Hen 6
Figure 18: SDD residue levels in the albumen of each hen following medication via
feed for seven days of 3 mg/kg BW
Results
73
5.5.3 Sulfadimidine concentrations in plasma
The residue concentrations of SDD in the plasma following seven consecutive days of
medication via feed are given in table 24. The average residue concentrations of 0.09
± 0.01, 0.45 ± 0.09 and 3.38 ± 1.24 µg/g were found after medication via feed with 3,
10 and 30 mg/kg BW, respectively. All residue levels appeared approximately equal
in the albumen (see table 23).
Matrix Plasma (µg/mL)
Dose (mg/kg BW) 30 10 3
Day 1 4.87 ± 2.05 0.58 ± 0.17 0.11 ± 0.03
Day 2 3.33 ± 1.97 0.52 ± 0.11 0.09 ± 0.03
Day 3 4.70 ± 1.88 0.47 ± 0.07 0.09 ± 0.02
Day 4 3.99 ± 1.52 0.44 ± 0.05 0.09 ± 0.03
Day 5 2.36 ± 2.41 0.43 ± 0.07 0.10 ± 0.03
Day 6 2.91 ± 1.08 0.31 ± 0.10 0.07 ± 0.01
Day 7 1.48 ± 0.95 0.37 ± 0.12 0.09 ± 0.02
Mean ± SD 3.38 ± 1.24 0.45 ± 0.09 0.09 ± 0.01
Table 24: SDD residue in the plasma (µg/mL, Mean ± SD) following medication via
feed for seven consecutive days in laying hens
Results
74
5.6 Comparative pharmacokinetic behavior and residues of
sulfadimidine between hen with healthy and impaired kidneys
5.6.1 Comparison of pharmacokinetic behaviours of sulfadimidine between hen
with healthy and impaired kidneys
Kidney damage was induce in those hens with impaired kidneys by subcutaneously
injection of K2Cr2O7 at 15 mg/kg BW and the stage of kidney damage allowed for
seven days. Thereafter, SDD was injected via i.v. at 100 mg/kg BW in the same day
for both healthy and impaired kidney groups. Most birds in the group of impaired
kidney hens showed depression, lethargy and increased water intake while feed intake
was decreased. At the end of the experiment, the birds were sacrificed. Kidneys gross
lesion in the renal dysfunction group showed a swollen kidney. According to the
histopathological confirmation by the Institute of Pathology (TiHo, Hannover), all
hens in the impaired kidney group were found to have multifocal fibrosis, interstitial
nephritis, tubular epithelial cell degeneration and necrosis, depicting of the induced
kidney damage.
Figure 19 depicts the comparison of the plasma concentration in semi-logarithmic
plots among normal kidney hens and kidney-damaged hens. The graphs show some
hens in the impaired kidney group having longer elimination than the normal hens.
Based on those plasma concentrations, the kinetics parameters were calculated and
presented in tables 25 and 26. Similar values of certain pharmacokinetic parameters
between normal kidney hens and impaired kidney hens were Cmax of 131.15 ± 13.88
and 124.51 ± 30.12 µg/mL, K01_HL of 8.23 ± 1.32 and 12.37 ± 3.86 h, CL of 66.52 ±
16.60 and 53.16 ± 24.35 mL/h/kg, respectively. Statistical significance of healthy
hens as compared to the impaired kidney hens was determined using a Mann-
Whitney, two-tailed test and applied for all pharmacokinetic values. The results
revealed p > 0.05 for all pharmacokinetic parameters. Therefore, the kinetic
behaviours of SDD were not statistically significantly different in healthy hens as
compared to kidney damaged hens.
Results
75
0.001
0.01
0.1
1
10
100
1000
0 10 20 30 40 50 60 70
Time (h)
Con
cent
ratio
n ( µ
g/m
L)
Healthy hens
Impaired kidney hens
Figure 19: Plasma concentration of sulfadimidine (Mean ± SD) versus times after a
single i.v. bolus administration at 100 mg/kg BW in laying hens
Hen (number)
Parameter Unit 1 2 3 4 Mean SD
AUC µg*h/mL 1846.40 1124.80 1863.07 1442.26 1569.14 354.41
K10_HL h 8.83 6.71 8.62 7.77 8.23 1.32
Cmax µg/mL 130.12 116.12 149.71 128.62 131.15 13.88
CL_F mL/h/kg 54.19 88.90 53.67 69.33 66.52 16.60
MRT h 14.18 9.68 12.44 11.21 11.88 1.91
Vss mL/kg 768.51 861.12 667.90 777.44 768.75 79.11
Table 25: Pharmacokinetic parameters of healthy hens
Results
76
Hen (number)
Parameter Unit 1 2 3 4 5 6 Mean SD
AUC µg*h/mL 1455 1116 1716 3884 2090 3467 2288 1128
K10_HL h 13.24 7.30 9.34 16.58 10.98 16.77 12.37 3.86
Cmax µg/mL 76.13 105.95 127.35 162.37 131.93 143.31 124.51 30.12
CL_F mL/h/kg 68.72 89.55 58.26 25.74 47.83 28.83 53.16 24.35
MRT h 19.11 10.53 13.47 23.92 15.84 24.19 17.84 5.58
Vss mL/kg 1313.44 943.79 785.23 615.87 757.96 697.77 852.34 250.68
Table 26: Pharmacokinetic parameters of impaired kidney hens
Results
77
5.6.2 Comparison of sulfadimidine residue in eggs between healthy and impaired
kidney hens
The residue levels of SDD in yolk following a single i.v. bolus treatment at 100 mg/kg
BW for normal kidney hens are given in table 27. Unfortunately, the hens had laid
only a few eggs. The residue in the yolk of two hens was 44.03 and 35.16 µg/g after
the first day of an application. The concentration of 0.53 µg/g was found in one hen
on day nine. The table 28 presents SDD levels in the yolk of each impaired kidney
hen after i.v. dosed with 100 mg/kg BW. Lower residue levels were found on the first
day after application compared to those of normal group. However, SDD residue
could be detected up to the 8th day after injection.
Hen (Number)
Day 1 2 3 4 Mean SD
1 44.03 - - 35.16 35.59 6.27
2 13.73 - - - - -
3 10.19 - 8.08 7.71 8.66 1.33
4 - - 5.67 - - -
5 4.53 - 4.64 3.27 4.15 0.76
6 3.13 - 2.5 - 2.81 0.31
7 1.58 - 0.68 - 1.13 0.45
8 0.12 - - 1.63 0.87 0.75
9 - - - 0.53 - -
Table 27: SDD residue levels in yolk (µg/g) after i.v. administration
at 100 mg/kg BW in healthy hens (n=4)
Results
78
Hen (Number)
Day 1 2 3 4 5 6 Mean SD
1 14.82 15.88 21.80 1.5 - 6.23 12.05 8.10
2 7.03 - - - - - - -
3 - 4.66 7.01 5.84 - - 5.84 1.17
4 3.86 4.59 5.51 12.98 - - 6.73 4.21
5 3.41 3.37 4.62 6.94 - - 4.58 1.67
6 1.16 1.16 2.90 3.49 - - 2.18 1.20
7 - 0.57 - 1.49 - - 1.03 0.65
8 - - 0.90 0.13 - - 0.51 0.54
Table 28: SDD residue levels in yolk (µg/g) after i.v. administration
at 100 mg/kg BW in impaired kidney hens (n=6)
Tables 29 and 30 indicate residue levels of SDD in the albumen following a single i.v
bolus treatment at 100 mg/kg BW for normal kidney and impaired kidney hens,
respectively. The residue in the albumen of two normal hens was 18.78 and 44.96
µg/g on the first day after application. The concentration of 0.09 µg/g was found on
day eight in one hen. For the impaired kidney group, the residue levels in the albumen
on the first day after the dose were 3.24 to 25.77 µg/g. Very rapid depletion of the
residue in the albumen on the second day after dose was observed for both groups.
Results
79
Hen (Number)
Day 1 2 3 4 Mean SD
1 18.78 - - 44.96 31.87 18.51
2 4.01 - - - - -
3 1.01 - 0.71 0.66 0.19 0.19
4 - - 0.66 - - -
5 0.19 - 0.18 0.14 0.17 0.026
6 0.20 - 0.09 - 0.055 0.050
7 0.04 - - - - -
8 - - - 0.09 - -
Table 29: SDD residue levels in albumen (µg/g) after i.v. administration
at 100 mg/kg BW in healthy hens (n=4)
Hen (Number)
Day 1 2 3 4 5 6 Mean SD
1 25.77 9.12 18.94 3.24 - 10.16 13.45 8.88
2 7.03 - - - - - - -
3 - 0.77 0.35 5.47 - - 0.66 0.10
4 0.22 0.23 0.23 0.76 - - 0.36 0.26
5 0.09 0.14 0.14 - - - 0.12 0.03
6 - - 0.10 0.11 - - 0.105 0.007
7 - - - 0.07 - - - -
Table 30: SDD residue levels in albumen (µg/g) after i.v. administration
at 100 mg/kg BW in impaired kidney hens (n=6)
Discussion
80
6 Discussion Unintentionally cross-contamination of veterinary drugs into feeding stuffs may
generate a violative residue problem in animal products. Therefore, the studies in this
thesis were conducted to investigate the potential residue deposition, in particular that
derived from low levels of contaminated feed. The pharmacokinetic characteristics
and residue depletion were studied, using sulfadimidine (SDD) application by
different doses and routes in laying hens as a model. The drug was applied as a single
i.v. injection (100 mg/kg BW), as a single oral bolus administration (1, 3, 10, 30 and
100 mg/kg BW) and medicated in mixed feed for 7 consecutive days (3, 10 and 30
mg/kg BW). Plasma and egg components (egg yolk and egg albumen) were samples
of interest. A comparison between healthy and impaired kidneys from those animals
was also observed.
The following discussion is divided into four major parts. The first part covers on the
methods of analysis, the second part dealing with drug plasma concentrations at
various doses via i.v. and p.o application. Based on plasma concentration versus time,
the pharmacokinetic parameters were calculated. In the third part, following an oral
and medicated feed application at different doses, drug residues were analysed in the
matrices of interest. A focus on drug concentrations as to whether residue in eggs at
low levels can be created residue in eggs was investigated. Finally, the data profiles
comparing of pharmacokinetic properties and the residues between the normal and
kidney dysfunction hens were also evaluated.
6.1 Method of analysis
In the present study, high performance liquid chromatography with spectroscopic UV
detection (HPLC-UV) was employed. The HPLC is one of the most widely used
techniques for determining sulfonamide residues in plasma, milk, meat and poultry
(AGARWAL, 1992; WANG et al., 2006). In this experiment, the UV wavelength was
carried out at 268 nm, which was in the range (265-275 nm) of those in previous
research (PETZ, 1983; HORII et al., 1990; PENSABENE et al., 1998; SHAIKH et al.,
1999). The retention behaviour of sulfonamides was dependent on its ionisation and
Discussion
81
polarity of mobile phase. Therefore, the pH of mobile phase plays an important role in
the chromatographic separations (WANG et al., 2006). For this, the optimum pH of
the buffer of 3.0 consisting of 0.05 mol/L phosphate hydrogen monohydrate was used
throughout. Traditionally, extraction and sample clean-up process involving the
determination were laborious, time-consuming, using a large amount of both volume
of samples and hazardous organic solvent, in particular for biological matrices such as
egg components which are rich in protein and lipid (AGARWAL, 1992; AERTS et
al., 1995). Therefore, a new approach was developed which had only a few steps,
using less volume of organics solvent and needing a volume of samples of only 0.5
mL of plasma and 1 g of egg components for an extraction.
To meet the criteria of validation, this present method provided good specificity,
linearity, limit of detection, limit of quantification, accuracy, precision and recovery.
The calculation of linear regression for plasma (0.025-5 µg/mL), yolk and albumen
(0.05-20 µg/g) showed R2 = 0.9990, 0.9994 and 0.9991, respectively. The limit of
detection (LOD) was 0.01 µg/mL and 0.025 µg/g for plasma and egg components,
respectively. However, these values were within the range for sulfonamides presented
in previous papers (FURUSAWA, 1999b; FURUSAWA, 2003b; KISHIDA et al.,
2004). The methods found the limit of quantification (LOQ) at 0.025 µg/mL for
plasma and 0.05 µg/g for both yolk and albumen. It has been recorded that the LOQ
value of sulfonamides in the plasma and eggs ranges from 0.02-0.10 µg/mL or µg/g
(FURUSAWA, 2002; KISHIDA et al., 2004; KISHIDA, 2007).
Several previous studies showed a variation in recovery values of approximately 60–
99% (TSAI et al., 1995; SHAIKH et al., 1999; FURUSAWA, 2002; ROUDAUT and
GARNIER, 2002). In this study, there was a high yield recovery of 84-97% with RSD
values less than 10% for all samples except for plasma spiked at 0.025 µg/mL
(10.20%), indicating that the methods were extreamly accurate. Moreover, these
methods provided intra- and interday precision values less than 10% except at the
LOQ of plasma (16.9%).
Discussion
82
6.2 Pharmacokinetic behaviour of sulfadimidine following single
bolus via intravenous injection and various doses of single bolus via
oral administration in laying hens
Due to different methods, dosage, route of administration, drug forms and duration of
medication the comparison of kinetic profiles of the drug are prevented (ROUDAUT
and GARNIAR, 2002). Little information on sulfonamides kinetic profiles of different
concentrations in laying hens is available. Therefore, the present experiment was
studied.
The pharmacokinetic values in this experiment were best described mathematically by
a first-order one compartment model. As showed in the results, the maximum plasma
concentration (Cmax) of SDD following an i.v. application at 100 mg/kg reached
132.20 ± 16.95 µg/mL, whereas it was detected at 76.50 ± 19.85 µg/mL after 4 h of
p.o. application. The results were of a similar level to those in former studies on
chickens; the Cmax value of SDD after i.v. injection has been recorded at 130 µg/mL
(NOUWS et al., 1988) or after p.o. application at 98.63 µg/mL and 82.98 µg/mL,
respectively, (REDDY et al., 1988; JAIN and PUNIA, 2001). However, the smaller
Cmax value at 59 µg/mL following p.o. treatment has also been reported (GEERTSMA
et al., 1987). In agreement with other studies, the time need to reach a peak was 2-4 h
(NOUWS et al., 1988; JAIN and PUNIA, 2001). The results confirmed that SDD was
rapidly absorbed. Consequently, SDD concentrations were found to be at an equal
level after 4-6 h for both i.v. and p.o administration. Thereafter, drug level following
i.v. injection showed more rapid depletion than p.o. application.
Following i.v. and p.o. administration, the calculated AUC showed similar levels at
1122.03 ± 245.81 and 1379.30 ± 428.13 µg*h/mL, respectively. It is important to note
that the slightly higher value in the case of oral rather than i.v. application may vary
due to the physiological aspect of each hen since different hens were used in these
two experiments. Additionally, the large amount reabsorbed within the cloaca in most
avian species (NOUWS et al., 1988 and LIERZ, 2003) or the occurrence of an
entrohepatic recircular pattern (EHC) may account for the higher AUC value after
Discussion
83
oral rather than for i.v. application. The findings, nonetheless, revealed that SDD has
very good absorption properties following oral treatment.
The present experiment provided the volume distribution of SDD following i.v.
injection at 0.77 ± 0.10 L/kg. According to NIELSON and RASMUSSEN (1975), the
observed volume lesser than 1 L/kg indicated that the concentration of the drug in
tissues was similar or less than in the plasma. Thus, the apparent volume of
distribution in this study was less than 1, indicating that the extent of SDD
concentration distributed in the plasma or blood was more than in the chicken body.
As compared to the same route and dose of application, a lower amount of the Vss of
SDD following i.v. injection has been reported by BLOM, 1975 (0.61 L/kg) and
REDDY et al., 1988 (0.43 L/kg). Regarding this present result, it is assumed that
intra-vessel administration of SDD penetrates better in chickens than some other
species since lower Vss values (0.24-0.40 L/kg) were reported in cattle and sheep
(BEVILL et al., 1977; WITCAMP et al., 1992; ELSHEIKH et al., 1991). However,
similar levels of Vss (0.50-0.62 L/kg) have also been reported in pigs and dogs
(RIFFAT et al., 1982; DUFFEE et al., 1984; YUAN et al., 1997).
BOTSOGLOU and FLETOURIS (2001) reported that SDD is more quickly
eliminated after injection than oral application via feed or drinking water. This is in
accordance with the results established in this present study where the elimination
half-life (T1/2el) after i.v. injection (5.87 ± 1.14 h) was shorter than p.o. administration
(9.10 ± 1.58 h). Thus, that the level of SDD decreases more quickly after i.v. injection
than p.o. application can be confirmed. Correspondingly, these values correlated well
with CL values, since the finding CL value in oral treatment was 82.47 ± 40 mL/h/kg,
relatively lower than that in i.v. application (94.15 ± 27.85 mL/h/kg). In contrast to
other reports, this presented CL value of SDD in hens was lower than that recorded at
around 200 mL/h/kg (GOREN et al., 1984) but of a slightly larger volume than that
reported at around 60 mL/h/kg (NOUWS et al., 1988).
The current findings showed that SDD, after oral administration in chickens, has a
relatively longer T1/2el than those published to date (2.9-6.4 h) in hens (NOUWS et al.,
1988; GEERTSMA et al., 1987; JAIN and PUNIA, 2001) and in broilers
(KIETZMANN, 1980). Nevertheless, a wide range of the T1/2el of 1.4-16 h following
Discussion
84
i.v. injection has also been reported (LÖSCHER et al., 1990; GOREN et al., 1984;
REDDY et al., 1988; BLOM, 1975). Thus, different experimental conditions such as
pharmacokinetic model, animal physiologys, species, breed, age, drug formulation
and dosage may be an explanation for these varying data (EL-SAYED et al., 1995;
BOTSOGLOU and FLETOURIS, 2001; FURUSAWA, 2001; ROUDAUT and
GARNIER, 2002). Furthermore, the absorption rate of the drug following oral
treatment depends on the concentration and duration time remaining in the crop which
consequents affects on the T1/2el value (KIETZMANN, 1980).
Other sulfonamides profiles studied in poultry have also been reported. Sulfadiazine
applications via i.v. in broilers have been recorded; the T1/2el of 2.7 h with an apparent
volume of distribution at 0.96 L/kg (LÖSCHER et al., 1990) and another recent
report, the T1/2el at 3.2 h with the Vss value of 0.43 L/kg (BAERT et al., 2003). As
compared to this present data, SDD showed a longer time of elimination since the
T1/2el (5.9 h) was higher than those previously reported. This is also supported by an
article of REDDY et al. (1988) who presented the T1/2el of SDD at 3.9 h, whereas T1/2el
of suldiazine was 2.3 h. On the contrary, from those kinetic profiles, it could be
deduced that SDD applied in hens is faster excreted than sulfaquinoxaline treated in
broilers since sulfaquinoxaline treatment in broilers has been reported a T1/2el of 12.6
h, a Vss value of 0.44 L/kg and a CL value of 17 mL/h/kg (EL-SAYED et al., 1995).
When comparing the same dose (100 mg/kg) following oral administration both
results of this present study and of REDDY et al. (1988) documented that the T1/2el of
SDD was 9.1 h and 15.6 h, respectively. This was much higher than the T1/2el reported
at 6 h for sulfadiazine (LÖSCHER et al., 1990) and at 4.5 h (REDDY et al., 1988).
Thus, the finding of this experiment are inconsistent with those previously reported
which presented SDD as being eliminated faster than sulfadiazine in broilers
following an oral dose (LÖSCHER et al., 1990). Furthermore, from this it can be
deduced that SDD has a longer elimination time than sulfadiazine, not only after i.v.
injection but also after oral application. In addition, the T1/2el of SDD following oral
application at 100 mg/kg as established in this experiment was comparatively higher
than that of sulfadoxine, 6 h, (JAIN and PUNIA, 2000) but lower in comparison to
sulfamethoxypyridazine, 13.2 h, (BAJWA and SINGH, 1977; CRAIGMILL et., al
2006) and sulfadimethoxine, 11.9 h, (BAJWA and SINGH, 1977) or 15.1 h (REDDY
Discussion
85
et al., 1988). Nevertheless, in this present case no flip-flop phenomenon was shown
since the entire absorption rate constant values (K01) were higher than the elimination
rate constant values (K10), following an overnight fast before treatment.
According to SPOO and RIVIERE (2006), sulfonamides are classified as short-acting
when the drug level in blood remains above 50 µg/mL for less than 12 h, and as
intermediate-acting if drug levels are obtained between 12-24 h. Thus, SDD in this
present study may be considered as short-acting in the case of administration via i.v.,
and intermediate-acting following p.o. application at 100 mg/kg.
In the experiments of sub-therapeutic concentrations administered via oral route, the
kinetic profiles of the dose of 3, 10, 30 mg/kg were calculated except for the group
application at 1 mg/kg due to its insufficiency data. A comparison of pharmacokinetic
parameters of SDD at different concentrations following oral administration showed
dose-dependent behaviours of Cmax, Tmax and T1/2 el. The SDD characteristic has more
rapidly absorption even at a low dose of administration, proved by a decrease of the
absorption rate half-life (K01_HL) to 0.2 h of 3 mg/kg compared to 1.2 h of 100
mg/kg. For this finding, the Tmax values were reached more quickly from 3.96 h to
0.46 h after reducing the drug concentrations. Moreover, the plot in figure 10
indicating high correlation between Cmax and different doses demonstrates a dose-
dependent trend. Furthermore, decreasing doses were consorted with larger volumes
of total body clearances. Correspondingly, the elimination half-life was shorter at a
low dose (0.9 h of 3 mg/kg) rather than at a high concentration (9.1 h of 100 mg/kg).
The rate-limited absorption may explain the deviations from dose-independent total
clearance, which is expected when using first-order kinetics. However, the almost
equal clearance value following i.v. and p.o. application with the same dose (100
mg/kg) suggested that the excretion is rate-limited, with both processes resulting in an
over-proportional decrease of the AUC after decreasing doses of SDD. In addition,
the extent and the rate of metabolism which have an influence on the elimination of
sulfonamides (MENGELERS et al., 2001) and capacity-metabolism or extensive
reabsorption from the cloaca (NOUWS et al., 1988 and LIERZ, 2003) may also
partially support the present profiles.
Discussion
86
SDD was mainly excreted by the renal. It has been reported that its major metabolite
form is N4-actyl in laying hens (NOUWS et al., 1988; KIETZMANN, 1980), but this
form does not occur over 20% (VREE, 1985). The underlying physiological
mechanisms involved in this complex pattern of SDD absorption and excretion in
chickens remain to be clarified. The limited data gathered in this study, however, do
not allow me to make further interpretations or create more complex pharmacokinetic
models. Nevertheless, as first-order kinetics may not be readily assumed for SDD in
chickens, pharmacokinetic characteristics formerly established on therapeutic levels
could not be extrapolated on sub-therapeutic levels.
6.3 Sulfadimidine residues in egg components after a single bolus via
oral and medicated feed at different concentrations
This study focused on residues incurred in eggs from the treatment of SDD in hens,
specifically exposed to low levels. The experiment was divided into two parts; firstly,
six laying hens in each group were orally treated with a single bolus at 1, 3, 10, 30 or
100 mg/kg, and secondly, they received medicated feed at 3, 10 or 30 mg/kg for seven
successive days. The results of the first experiment demonstrated that the residues of
SDD could be quickly detected as early as the first day in both yolk and albumen,
except for yolk at the lowest dose (1 mg/kg BW). The results obviously showed
higher level residue concentration, in particular when received at the highest dose in
the albumen (49.34 µg/g) than yolk (14.32 µg/g). It should be noted that only on the
first two days post oral dose did the residue level remain around 2-4 times higher in
the albumen than in the yolk. Thereafter, the residue level in the albumen rapidly
declined, most obviously found in the group administered the highest dose (100
mg/kg BW). The residue level in the albumen of this group quickly fell from 49.34
µg/g to 6.10 µg/g on the following day, and remaining at a low concentration (< 1
µg/g) for 5 days before being undetectable. Compared to yolk, the residue was
detected at 14.32 µg/g on the first day, then dropping to 6.62 µg/g on the following
day. Thereafter, the residue level in yolk decreased slightly and could be detected
below 1 µg/g on day 7 after dose. These findings indicate that SDD is prone to
accumulate during the initial phase in the albumen, the reverse being true for the yolk.
This behaviour may occur during the drug exchange within egg components,
Discussion
87
especially during shell formation (KAN and PETZ, 2000; ROUDAUT and
GARNIER, 2002). The rapid depletion pattern of SDD in the albumen was supported
by the previous research (GEERTSMA et al., 1987; PENSABENE et al., 1998;
SHAIKH and CHU, 2000; FURUSAWA, 2001; ROUDAUT and GARNIER, 2002).
In the present study, SDD residue in yolk was detectable over a longer period after a
single oral administration of the therapeutic dose (up to 8th day, compared to 6th day in
albumen, whereas it was eliminated from the plasma after only 32 h). Consequently,
the SDD level in yolk exceeded that in the albumen after about 2 days, which is in
agreement with previous reports (GEERTSMA et al., 1987; SHAIKH and CHU
2000). Moreover, slower depletion behaviour of residue in yolk than in albumen could
be seen even in hens exposed to the drug at sub-therapeutic doses (30 and 10 mg/kg
BW). Therefore, the yolk should be used in general as an appropriate matrix for
monitoring SDD residue in eggs, as previously suggested for sulfaquinoxaline
(SHAIKH et al, 2004). Nevertheless, we found no residues in yolk after receiving the
drug at 1 mg/kg. This may suggest that using of the albumen as an alternative matrix
is the best choice in case of egg contamination of SDD at very low levels, i.e. 1-3 %
of the recommended dose.
In the second experiment the SDD residue rapidly appeared in egg components within
the first day after starting the feed medication treatment. This tallied with the previous
study by GEERTSMA et al. (1987), the present results showing that residue levels of
the SDD in albumen reflected the concentration in the plasma rather than in the yolk.
A gradual increase then a levelling off of the SDD levels in those matrices 2-3 days
after feeding start should be noted. KAN and PETZ (2000) described that residue
concentration of drugs in plasma and albumen generally becomes constant 2-3 days
after the start treatment, whereas 8-10 days are needed in case of the yolk.
Nevertheless, the present findings may partly support these results due to the constant
level of SDD in yolk being achieved in only 2 days with medicated feed with sub-
therapeutic concentration. An appropriate withdrawal time of 8 days for SDD is
suggested for laying hens following treatment of the recommended dose. This
duration is in agreement with the results of GEERTSMA et al. (1987). This contrast
with previous research which reported that the withdrawal time should be longer than
2 weeks since the residue in eggs was detected up to 15-20 days (SIEB, 1991;
Discussion
88
ROUDAUT and GARNIER, 2002) or longer than 10 days for chickens (RIGHTER et
al., 1971). However, my findings showed prolonged withdrawal time of SDD rather
than of other sulfonamides e.g. sulfdimethoxine (7 days; NAGATA et al., 1992;
FURUSAWA et al., 1994) or sulfadiazine (4 days; ATTA and EL-ZEINI, 2001),
nonetheless shorter than sulfaquinoxaline (9-10 days; FURUSAWA et al., 1998;
CAVALIERE et al., 2003; SHAIKH et al., 2004).
Unfortunately, this experiment did not determine the SDD metabolites in eggs.
However, it has been documented that the parent drug was the major substance
residue found in both albumen and yolk since trace level metabolites were detectable
(GEERTSMA et al., 1987; SHAIKH et al., 1999) or not found (SHAIKH and CHU,
2000). Furthermore, it has been shown that SDD rather than other sulfonamides was
found preferentially in the tissues involving egg formation e.g. oviduct (FURUSAWA
and KISCHIDA, 2002). This indicated that the drug might be easily accumulated in
eggs where drug deposition in eggs may occur during the plumping or calcification
phase (DONOHUE and HAIRSTON, 2000; ROUDAUT and GARNIER, 2002).
Following oral treatment, the transfer rates of SDD into eggs were calculated. This
was approximately 1 % for the normal dose. The transfer rates of SDD in the present
study commensurated with those reported by GEERTSMA et al., (1987);
FURUSAWA (2001) and ROUDAUT and GARNIER (2002). Comparison transfer
rates between SDD and certain antibiotics into eggs have been noted (FURUSAWA,
2001). Furusawa reported that SDD had the highest transference (1.54 %) of all
studied agents, i.e. sulfadimethoxine (0.65 %), sulfamonomethoxine (0.77 %),
amprolium (0.21 %) and tylosin (0.005 %). Nonetheless, as stated in the present
results, the transfer rate of SDD application at the low concentrations was less than
0.5 %. Thus, this information is reflected in the reduced risk incurred in the residue
level in eggs when hens are exposed to drug at low levels.
Drugs such as sulfonamides, which have adequate water solubility properties, were
mostly had residues at a higher level in the albumen than in the yolk (FURUSAWA,
2001). Similar observations were also reported in studies on some antibiotics, e.g.
oxolinic acid (ROUDAUT, 1989), enrofloxacin (GORLAR et al., 1997) and
olaquidox (KEUKENS et al., 1996). Recently, KAN (2003) studied the relation of
Discussion
89
physicochemical properties such as pKa value, lipid solubility or protein binding in
order to predict the distribution pattern of drugs into and between egg components.
However, none of those properties could clearly explain the drug penetration
behaviours. Thus, the patterns of drug deposited in eggs remain to be elucidated.
Cross-contamination awareness of veterinary drugs in feed and transference to animal
products has been described elsewhere. However, little information on drug cross-
contamination on layer feed and eggs is available (KENNEDY et al., 2000;
McEVOY, 2002). Furthermore, studies carried out on low levels in contaminated feed
inducing residue problems in animal products, specifically in eggs, are scarce. To our
knowledge, the above cited studies failed to observe residues occurring in eggs after
receiving various concentrations of drugs via oral or medicated feed. This is the first
study of its kind showing measurable residues of SDD in egg components even at the
intake at a very low concentration of 1% of the therapeutic dose. Moreover, the
pharmacokinetics findings of dose-dependence are a noticeable feature of interest
namely that residue occurring in eggs should be determined at the relevant
concentrations and should not be predicted from known high-level kinetics. These
data may be of benefit concerning risk assessment, reducing and controlling residues
occurring in animal products.
6.4 Comparison of pharmacokinetic characteristics and residues of
sulfadimidine between healthy and impaired kidney laying hens
following intravenous administration
Renal excretion is the principle process for eliminating of drugs that are
predominantly ionised at physiological pH and for compounds with limited solubility
in lipid (drug metabolites) (BAGGOT, 2001). There are many causes of kidney
disease in birds e.g. viral infection, bacterial infection, toxins, obstruction (gout) and
vitamin A deficiency. Avian renal tubular cells in particular are susceptible to injuries,
renal dysfunction and are frequently encountered (SCHMIDT, 2006). However,
pathognomonic signs are rare when renal disorder occurs (LIERZ, 2003). Unlike
other species, chickens have two complex types of nephorns, reptilian (cortical) and
mammalian (meddullary)-type (MORILD et al., 1985). Most birds excrete urate as a
Discussion
90
waste product, instead of toxic urea. This process requires a lesser amount of water. In
birds this is probably a method for conserving water by resorption within the coaca
(LIERZ, 2003). The manifestation of renal damage in birds is different from
mammals in that it is usually associated with deposition of gout crystals within the
lumen and cytoplasm of tubular cells due to the regular production of urate as an end-
product of nitrogen metabolism in avians. The deposition of urate is in turn harmful to
the tubular cells, causing deterioration of tubular lesions. However, avian renal
tubular cells and kidney function may be reversible from the impaired stage. The bird
is one species that has a relatively good chance of recovery as long as the disability
caused by injury is removed in time.
Generally, SDD is mainly excreted by the kidney (PRESCOTT, 2000). In this
experiment, similar values of the pharmacokinetic parameters between hens with
normal kidney functions and those with impaired kidney functions were found: Cmax
of 131.15 ± 13.88 and 124.51 ± 30.12 µg/mL, K01_HL of 8.23 ± 1.32 and 12.37 ±
3.86 h, CL of 66.52 ± 16.60 and 53.16 ± 24.35 µg/mL, respectively. However,
relatively slow elimination of SDD in kidney damaged hens than in healthy hens
could be observed. As stated in the results, the SDD level was detected over the LOQ
value for up to 60 h for the group of impaired kidney hens, whereas it was
undetectable at 48 h for healthy hens after drug application (see Fig. 21). A noticeable
feature of this experiment following Mann-Whitney (two-tailed) calculation,
nonetheless, showed no statistically significant (p > 0.05) of the SDD kinetic
behaviours between healthy and kidney damaged hens.
SDD, as a parent drug, has been observed in some species (humans, ruminants and
swine) mainly being excreted via glomerular filtration, whereas its metabolites were
predominantly eliminated by tubular secretion (VREE and HEKSTER, 1985;
NOUWS et al., 1986). It has been reported that the use of potassium dichromate as a
renal damage agent mainly induces tubular lesion in animals e.g. rabbits and dogs
(EICHLER, 1965). According to the results in this present study, the histo-pathology
of impaired kidney hens was also mainly tubular lesion. Thus, it might be deemed that
the SDD application in hens is preferably excreted by glomerular filtration rather than
by tubular secretion when similar SDD excretion behaviour in the healthy group as in
the impaired kidney hens is presented in the results. Additionally, NOUWS et al.
Discussion
91
(1988) reported that an additional metabolic pathway exists for sulfonamides in
chickens. Unfortunately, this present study did not consider SDD metabolites.
However, limited rates of absorption and excretion of SDD were found in experiment
I of this study. Thus, the capacity-limited absorption, metabolism and excretion of
SDD in hens may also be involved. Therefore, these reasons may partly explain the
insignificant characteristics occurring between both groups of hens in this present
experiment.
For SDD residue in eggs, unfortunately few eggs were laid. However, this correlated
with experiment I of this study which found that SDD residue highly accumulated in
both albumen and yolk on the first day of the dose. Additionally, it should be noted
that the high concentration of SDD residue was detected in the albumen (31.87 µg/g)
and yolk (35.59 µg/g) on the first day after treatment in the healthy hens group,
whereas it was found at a lower level in both albumen (13.45 µg/g) and yolk (12.05
µg/g) for the kidney dysfunction hens group. This results from the pathological effect
maybe also occurring in the tissues involving egg formation. It is noteworthy that the
maximum SDD residue in the yolk of hen number four in the impaired kidney group
was found later on the third day after drug application. Individual kinetic profiles of
this hen may partly account for its CL value being low, whereas AUC, T1/2el and Cmax
were greater than other hens. However, a similar withdrawal time of SDD to the
experiment I could be documented due to the SDD residue detectable up to 8th-9th day
post dose for both groups. Furthermore, no obviously different level of SDD residue
among those hens could be seen after a few days of treatment. Correspondingly, no
statistically significant difference (p > 0.05) in the SDD kinetic behaviours between
healthy and kidney damaged hens was found.
Summary
92
7 Summary
The studies presented in this thesis were undertaken to elucidate the potential residue
risk derived from low levels of contaminated feed. Sulfadimidine (SDD) was used as
a model to determine the residue risk at various doses and routes of application in
laying hens. To achieve the aims of the studies, groups of six hens each aged between
22-26 weeks old were used for two experiments. In experiment I, six groups of hens
received either a single intravenous (i.v.) bolus of 100 mg/kg BW or per oral (p.o.) at
1, 3, 10, 30 and 100 mg/kg BW. Three additional groups of hens were fed a feed
consisting of SDD with a resulting dose of 3, 10 and 30 mg/kg BW for seven
consecutive days. Experiment II dealt with the comparison of pharmacokinetic
behaviours and residue of SDD between healthy and impaired kidney hens after i.v.
injection at 100 mg/kg. Drug levels were determined with HPLC-UV for the matrices
of interest (plasma, yolk and albumen).
The methods of analysis provided good specificity, linearity, limit of detection, limit
of quantification, accuracy, precision and recovery. The calculations of linear
regression for all the matrices were R2 > 0.9990. The limit of detection (LOD) was
0.01 µg/g and 0.025 µg/mL and the limit of quantification (LOQ) was 0.025 µg/mL
and 0.05 µg/g for plasma and egg components, respectively. The intra- and interday
precision values were less than 10% except at the LOQ of the plasma (16.9%). The
pharmacokinetic parameters were calculated assuming first-order kinetics, except for
one group (1 mg/kg BW.) due to insufficient data quality. The results revealed that
SDD was rapidly absorbed after treatment at the therapeutic dose. Furthermore, at
sub-therapeutic doses, our data demonstrated that SDD was absorbed even more
rapidly with a decrease in the calculated absorption rate half-life value (K 01_HL) to
0.2 h at 3 mg/kg BW as compared to 1.2 h at 100 mg/kg BW. Correspondingly, the
Tmax value decreased from 4 h to 0.5 h. The elimination of SDD was quickly depleted
from the plasma. Within 48 h after a single bolus treatment via i.v. and 6-32 h after
p.o. administration at various doses, the concentrations were lower than the LOQ.
Following single oral doses, the Cmax, Tmax and K10_HL increased while the CL
decreased with increasing doses. Therefore, these characteristics might be deemed
that dose-dependent. Regarding the SDD residue in egg components, our data
Summary
93
revealed that, at the initial phase after a single oral administration and during
medication via feed the SDD concentration in the albumen was 2 to 4 times higher
than in the yolk. However, the SDD residue in yolk could be detected longer over
LOQ, up to the 8th day as compared to the 6th day in the albumen, whereas it was
eliminated from the plasma after only 32 h. For other lower concentrations, SDD
levels were detected approximately 2 days longer in the yolk than in the albumen. The
ingestion of sub-therapeutic doses of SDD at 1% to 3% of the recommended dose led
to measurable residue in eggs. The transfer rates of SDD deposited into egg
components were dose-dependent, which might be deemed to reduce the risk of
residue occurring in eggs. Results of the comparison between the kinetic profiles and
residues in eggs of SDD obtained from healthy and impaired kidney hens showed no
statistically significant difference in the kinetic behaviours. This may link to rate-
limited absorption and elimination. Thus, moderate kidney damaged in laying hen
may have no affect on kinetic behaviours of SDD.
In conclusion, our study demonstrated that low levels intake of SDD by ingesting of
contaminated feed may generate a residue problem in eggs. The data showed that an
application at only 1% of the therapeutic dose resulted in a measurable concentration
in eggs. These results suggest that residue risks should be examined at relevant
concentrations and may not be estimated from known high-level kinetics. Thus,
similar protocol approaches should be applied to other drug substances in order to
avoid a residue risk for the consumer.
Zusammenfassung
94
8 Zusammenfassung In der vorliegenden Studie wurde das potentielle Riskiko einer Rückstandsbildung
durch eine Arzneistoffkontamination des Futters mit niedrigen Dosen untersucht.
Dazu wurde in einem Modellversuch Sulfadimidin an 22 bis 26 Wochen alte
Legehennen verabreicht und die Rückstände im Ei nach verschiedenen Dosierungen
gemessen. Darüberhinaus wurde geprüft, ob die Induktion eines Nierenschadens und
damit einhergehende verminderte Sulfadimidinelimination zu höheren Rückständen
im Ei führen kann. Zunächst wurden pharmakokinetische Grunddaten an einer
Gruppe von 6 Hühnern ermittelt, die mit 100 mg/kg KW Sulfadimidin i.v. behandelt
wurden. Danach wurde die Rückstandsbildung im Ei nach einmaliger oraler
Sulfadimidin-Applikation (5 Gruppen von jeweils 6 Hühnern in Dosierungen von 1,
3, 10, 30 and 100 mg/kg KW) sowie nach einwöchiger Dauermedikation mit dem
Futter (3 Gruppen von jeweils 6 Hühnern in Dosierungen von 3, 10 and 30 mg/kg
KW) untersucht. Zur Induktion eines Nierenschadens wurde Kaliumdichromat
benutzt und der Effekt durch klinische und pathohistologische Parameter abgeschätzt.
Bei allen Experimenten erfolgte die Analytik interessierender Matrices (Plasma,
Eigelb und Eiweiss) mittels einer HPLC-UV-Methode, welche hinsichtlich Spezifität,
Linearität, Nachweis- und Quantifizierungsgrenze, Richtigkeit, Präzision und
Wiederfindung validiert wurde. Dabei erwies sich die Methode als linear (R²>0.999 in
allen untersuchten Matrices). Die Nachweisgrenze wurde bei 0,01 µg/ml (Plasma)
bzw. 0,025 µg/g (Eigelb und Eiweiss) und die Quantifizierungsgrenze bei 0,025
µg/ml (Plasma) und 0,05 µg/g (Eigelb und Eiweiss) etabliert. Bei allen Matrices
zeigte sich bei allen untersuchten Konzentrationen eine Tages- und
Zwischentagespräzision mit Abweichungen von <10% vom Mittelwert (Ausnahme:
Plasma an der Quantifizierungsgrenze: 16,9%). Für die Berechnung
pharmakokinetischer Parameter wurde eine Kinetik erster Ordnung im 1-
Kompartiment-Modell angenommen. Dabei zeigte sich bei steigenden Dosierungen
eine Zunahme der Tmax, Cmax und der Halbwertszeit (K01) (nach 3 mg/kg KW: Tmax:
0,4 h, K01: 0,2 h; nach 100 mg/kg KW: Tmax: 4 h, K01: 1,2 h) sowie eine Abnahme
der Clearence. Nach i.v. Gabe war im Plasma nach 48 h und nach oraler Gabe nach 6-
32 h Sulfadimidin nicht mehr quantifizierbar. Messbare Sulfadimidinkonzentrationen
Zusammenfassung
95
wurden im Ei nach oraler Gabe von lediglich 1% der empfohlenen Dosis erreicht.
Diese wiesen sowohl nach einmaliger oraler Gabe als auch nach oraler Behandlung
über eine Woche im Eiweiss eine 2- bis 4-fach höhere Konzentration als im Eigelb
auf. Allerdings war Sulfadimidin nach Beendigung der Behandlung im Eigelb länger
nachweisbar (8 Tage) als im Eiweiss (6 Tage). Vergleichbar zu anderen
pharmakokinetischen Daten wies auch die Transferrate von Sulfadimidin ins Ei eine
Dosisabhängigkeit auf, wobei steigende Dosen mit einem höheren relativen Transfer
der Substanz ins Ei einhergingen, wodurch sich das Risiko einer Rückstandsbildung
im Ei bei subtherapeutischen Dosen zu verringern scheint. Beim Vergleich der
Kinetik von Sulfadimidin in gesunden und nierengeschädigten Hühnern zeigte sich
kein statistisch abzusicherner Unterschied. Dies könnte auf eine Sättigungskinetik
entweder der Absorption oder der Elimination hindeuten, wobei moderate
Nierenschäden zu keiner Veräbnderung des kinetischen Verhaltens von Sulfadimidin
führen würden.
Zusammenfassend zeigt die vorliegende Studie, dass die Aufnahme von
kontaminiertem Futter auch bei geringen Arzneistoffgehalten zu Rückständen in
Hühnereiern führen kann. Dabei wurden messbare Rückstände bereits nach
einmaliger Aufnahme eines Futters gemessen, welches lediglich 1% der
therapeutischen Dosis enthielt. Die deutliche Dosisabhängigkeit der Kinetik von
Sulfadimidin legt zudem nahe, dass das Rückstandsrisiko durch kontaminiertes Futter
auch von anderen Arzneimitteln bei relevanten Konzentrationen bestimmt, und nicht
von höheren Konzentrationen extrapoliert werden sollte.
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Appendix
127
10 Appendix
10.1 Glossary of abbreviations
β the apparent first-order rate constant fraction
µL microliter
µg microgram
AUC area under the curve
AUMC the area under the (first) moment curve
BW body weight
C(T) plasma drug concentration at any time
C° degree celsius
Calbumen the measured concentration in albumen
CL the systemic clearance
CL_F the total body clearance (extra-vascular models)
Cmax the peak plasma concentration
concn concentration
Cp0 drug concentration at time zero
Cyolk the measured concentration in yolk
D dose
dL deciliter
F the fraction of absorption
g gram
h hour
HCl hydrochloric acid
HPLC High pressure liquid chromatography
i.m. intramuscular
i.p. intraperitoneal
I.S. internal standard
i.v. intravenous
K01 the apparent first-order absorption rate constant
K01_HL the absorption half-life
Appendix
128
K10 the apparent first-order elimination rate constant
K10_HL the elimination half-life
K2Cr2O7 Potassium dichromate
Ka the rate constant of absorption
Kel the rate constant of elimination
Koc the organic carbon adsorption coefficient
kg kilogram
ln natural logarithm
L liter
LOD limit of detection
LOQ limit of quantification
malbumen mass of albumen
mg milligram
min minute
mL milliliter
Mol mole
MRL maximum residue limit MRT the mean residence time
myolk mass of yolk
N normal
n number
N2 nitrogen
NaCl sodium chloride
NaH2PO4 · H2O Phosphate hydrogen monohydrate
ND not detected
ng nanogram
nm nanometer
p.o. per os
R2 correlation coefficient
RSD relative standard deviation
Rtotal the sum of residues in albumen and yolk
SD standard deviation
SDD sulfadimidine
SDX sulfadoxine
Appendix
129
T time
T1/2el the elimination half-life
Tmax the maximum concentration
TR drug transfer rate into the eggs
UV Ultraviolet
V an apparent volume of distribution
v/v volume/volume
Vd the volume of distribution
Vss the volume of distribution at steady state
Appendix
130
10.2 Tables Hen (Number)
Time (h) 1 2 3 4 5 6 Mean SD
0.25 124.29 101.68 137.71 141.14 156.46 115.11 129.40 19.69
0.5 126.63 91.97 126.19 129.17 139.36 116.78 121.68 16.25
1 114.12 86.25 116.80 122.90 129.86 108.99 113.16 15.03
2 109.03 89.47 111.77 117.01 119.87 101.92 108.18 11.11
4 93.40 57.19 87.48 71.99 101.55 91.97 83.93 16.34
6 76.00 40.93 70.81 55.99 73.92 68.49 64.36 13.45
8 55.06 29.23 59.84 50.93 58.40 63.91 52.89 12.40
12 37.54 10.97 32.12 28.76 32.80 37.68 29.98 9.92
24 5.91 0.67 6.10 3.86 4.37 5.28 4.37 2.01
32 0.46 0.22 0.39 0.25 0.25 0.26 0.30 0.09
48 0.08 0.13 0.13 ND ND ND 0.06 0.06
Table 1: SDD plasma concentration (µg/mL) after i.v. administration at 100
mg/kg BW
Appendix
131
Hen (Number)
Time (h) 1 2 3 4 5 6 Mean SD
0.5 38.94 48.99 5.57 40.84 30.35 20.01 30.78 15.82
1 45.20 53.04 31.84 44.70 41.18 29.78 40.96 8.79
2 78.04 76.94 52.42 86.75 69.57 45.90 68.27 15.49
4 76.39 80.06 63.89 84.78 84.17 44.81 72.35 15.49
6 70.43 66.63 51.26 96.40 94.21 35.58 69.08 23.77
8 62.06 66.75 52.83 86.58 87.72 37.22 65.53 19.55
12 63.68 46.39 34.94 67.67 68.95 24.38 51.00 18.70
24 23.75 23.56 30.98 26.37 31.49 2.34 23.08 10.72
32 1.83 1.64 2.77 3.64 6.15 ND 2.67 2.10
Table 2: SDD plasma concentration (µg/mL) after p.o. administration at 100
mg/kg BW
Hen (Number)
Time (h) 1 2 3 4 5 6 Mean SD
0.5 20.00 16.78 26.37 33.37 14.34 0.75 18.60 11.14
1 20.14 21.70 24.35 28.43 27.08 3.84 20.92 8.93
2 21.68 21.56 23.90 24.77 24.25 23.82 23.33 1.36
4 25.83 14.16 17.35 17.47 20.18 14.95 18.32 4.25
6 10.38 8.13 10.92 11.66 14.13 9.36 10.76 2.06
8 5.52 4.28 5.70 6.23 7.60 3.94 5.54 1.33
12 1.95 0.66 1.19 1.38 1.80 0.63 1.27 0.55
24 ND ND ND ND ND ND - -
Table 3: SDD plasma concentration (µg/mL) after p.o. administration at 30
mg/kg BW
Appendix
132
Hen (Number)
Time (h) 1 2 3 4 5 6 Mean SD
0.5 1.78 6.45 7.09 4.25 8.52 3.67 5.29 2.49
1 5.41 4.92 3.95 6.31 7.35 2.54 5.08 1.71
2 4.38 5.06 3.57 4.82 5.95 2.68 4.41 1.15
4 1.69 2.49 1.44 1.30 - 1.29 1.64 0.50
6 0.46 0.98 0.44 0.30 0.81 0.39 0.56 0.27
8 0.12 0.40 0.51 0.36 0.27 0.31 0.33 0.13
12 - 0.06 ND ND 0.11 0.03 0.03 0.04
24 0.08 0.04 ND ND 0.03 0.06 0.03 0.03
Table 4: SDD plasma concentration (µg/mL) after p.o. administration at 10
mg/kg BW
Hen (Number)
Time (h) 1 2 3 4 5 6 Mean SD
0.5 1.47 2.17 2.88 3.29 2.41 0.32 2.09 1.06
1 1.46 1.56 1.84 2.06 1.89 1.11 1.65 0.35
2 0.81 0.87 0.97 0.81 0.82 0.43 0.78 0.18
4 0.19 0.29 0.22 0.24 0.22 0.09 0.21 0.07
6 0.07 0.07 0.06 0.09 0.06 0.06 0.07 0.01
8 <0.025 0.03 ND <0.025 <0.025 <0.025 <0.025 -
Table 5: SDD plasma concentration (µg/mL) after p.o. administration at 3
mg/kg BW
Appendix
133
Hen (Number)
Time (h) 1 2 3 4 5 6 Mean SD
0.5 0.78 0.75 0.55 0.13 0.52 0.47 0.53 0.23
1 0.47 0.44 0.43 0.56 0.69 0.50 0.51 0.10
2 0.23 0.20 0.18 0.25 0.20 0.23 0.21 0.02
4 0.05 0.04 0.06 0.10 0.05 0.06 0.06 0.02
6 0.03 <0.025 0.03 0.04 <0.025 0.03 0.02 0.01
8 ND ND ND ND ND ND - -
Table 6: SDD plasma concentration (µg/mL) after p.o. administration at 1
mg/kg BW
Hen (Number)
Day 1 2 3 4 5 6 Mean SD
1 15.40 19.68 10.51 20.75 5.28 - 14.32 6.47
2 6.86 7.27 6.17 10.19 2.60 - 6.62 2.72
3 3.90 4.69 3.45 4.55 1.78 - 3.68 1.17
4 3.18 3.01 2.83 3.16 1.54 - 2.75 0.69
5 2.33 2.15 1.66 2.58 1.18 - 1.98 0.56
6 1.94 1.18 0.59 1.67 0.70 - 1.21 0.59
7 0.88 0.24 0.06 0.16 0.36 - 0.43 0.32
8 0.28 0.02 - - 0.06 - 0.12 0.14
9 <0.05 <0.05 <0.05 <0.05 <0.05 - <0.05 -
Table 7: SDD residue concentration (µg/g) in yolk following p.o.
administration at 100 mg/kg BW
Appendix
134
Hen (Number)
Day 1 2 3 4 5 6 Mean SD
1 1.81 1.93 3.46 5.53 2.67 - 3.08 1.52
2 0.46 - 0.63 0.41 0.56 - 0.51 0.10
3 0.44 0.36 0.45 0.63 0.31 - 0.44 0.12
4 0.38 0.34 0.26 0.31 - - 0.32 0.05
5 0.25 0.17 0.18 - - - 0.20 0.04
6 0.16 0.14 0.10 0.13 0.13 - 0.13 0.02
7 0.09 0.04 0.08 - - - 0.07 0.03
8 <0.05 <0.05 <0.05 <0.05 <0.05 - <0.05 -
Table 8: SDD residue concentration (µg/g) in yolk following p.o.
administration at 30 mg/kg BW
Hen (Numer)
Day 1 2 3 4 5 6 Mean SD
1 0.44 0.33 0.39 0.48 0.54 0.34 0.42 0.08
2 0.07 0.08 0.06 0.06 0.10 0.04 0.07 0.02
3 0.05 0.08 0.06 0.04 0.09 - 0.06 0.02
4 0.03 0.05 0.04 0.03 0.04 - 0.04 0.01
5 <0.05 <0.05 <0.05 <0.05 <0.05 - <0.05 -
Table 9: SDD residue concentration (µg/g) in yolk following p.o.
administration at 10 mg/kg BW
Appendix
135
Hen (Number)
Day 1 2 3 4 5 6 Mean SD
1 0.08 0.16 0.07 0.14 0.15 0.06 0.11 0.05
2 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 -
Table 10: SDD residue concentration (µg/g) in yolk following p.o.
administration at 3 mg/kg BW
Hen (Number)
Day 1 2 3 4 5 6 Mean SD
1 53.36 57.61 39.05 50.05 46.64 - 49.34 7.04
2 6.33 6.69 5.56 5.97 5.93 - 6.10 0.43
3 0.85 0.78 0.62 0.81 1.07 - 0.83 0.16
4 0.45 0.49 0.33 0.42 0.20 - 0.38 0.12
5 0.28 0.26 0.14 0.18 0.12 - 0.20 0.07
6 0.16 0.12 0.09 0.14 0.08 - 0.12 0.03
7 <0.05 <0.05 <0.05 <0.05 <0.05 - <0.05 -
Table 11: SDD residue concentration (µg/g) in albumen following p.o.
administration at 100 mg/kg BW
Hen (Number)
Day 1 2 3 4 5 6 Mean SD
1 6.34 3.97 8.30 7.77 4.85 - 6.25 1.85
2 0.13 - 0.17 0.22 0.13 - 0.16 0.04
3 0.08 0.41 0.07 0.12 0.07 - 0.15 0.15
4 <0.05 <0.05 <0.05 <0.05 <0.05 - <0.05 -
Table 12: SDD residue concentration (µg/g) in albumen following p.o.
administration at 30 mg/kg BW
Appendix
136
Hen (Number)
Day 1 2 3 4 5 6 Mean SD
1 1.02 1.23 0.73 0.96 1.24 0.68 0.98 0.24
2 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 -
Table 13: SDD residue concentration (µg/g) in albumen following p.o.
administration at 10 mg/kg BW
Hen (Number)
Day 1 2 3 4 5 6 Mean SD
1 0.02 0.42 0.22 0.36 0.35 0.12 0.28 0.12
2 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 -
Table 14: SDD residue concentration (µg/g) in albumen following p.o.
administration at 3 mg/kg BW
Hen (Number)
Day 1 2 3 4 5 6 Mean SD
1 0.09 0.07 0.04 0.08 0.10 0.08 0.08 0.02
2 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 -
Table 15: SDD residue concentration (µg/g) in albumen following p.o.
administration at 1 mg/kg BW
Appendix
137
Hen (Number)
Time (h) 1 2 3 4 Mean SD
0.5 137.65 112.57 153.44 137.20 135.21 16.88
1 121.93 104.11 134.75 112.96 118.44 13.08
2 100.41 92.08 116.82 96.87 101.55 10.74
4 95.16 77.34 104.42 85.20 90.53 11.79
6 80.61 59.16 88.93 72.81 75.37 12.66
8 70.44 50.79 87.35 66.49 68.77 15.02
12 70.51 41.55 74.15 54.11 60.08 15.12
24 28.71 6.18 11.14 14.83 15.2 2 9.67
32 5.86 0.05 0.43 1.10 1.86 2.70
48 ND ND ND ND ND -
Table 16: SDD plasma concentration (µg/mL) after i.v. administration at
100 mg/kg BW in healthy hen, experiment II, (n=4)
Appendix
138
Impaired kidney hen (Number)
Time (h) 1 2 3 4 5 6 Mean SD
0.5 59.52 98.16 118.45 161.01 124.53 140.63 117.05 35.25
1 64.51 101.53 121.95 152.42 125.13 134.21 116.62 30.44
2 67.04 76.71 106.22 145.59 114.88 119.73 105.03 29.00
4 71.19 80.11 96.99 137.71 101.36 124.17 101.92 25.38
6 71.52 61.96 78.16 126.74 87.33 115.93 90.27 25.68
8 58.56 53.52 74.31 118.30 84.93 107.66 82.88 26.07
12 50.72 33.75 61.99 105.14 73.92 104.88 71.73 28.98
24 7.72 4.23 16.88 62.65 24.68 49.08 27.54 23.47
32 0.30 0.09 2.69 36.80 8.41 27.57 12.64 15.70
48 ND ND ND 0.34 ND 0.19 0.26 0.11
60 ND ND ND 0.11 ND 0.09 0.10 0.01
Table 17: SDD plasma concentration (µg/mL) after i.v. administration at 100 mg/kg
BW in kidney damaged hens, experiment II, (n=6)
Acknowledgement
139
11 Acknowledgement
I wish to express deep gratitude to my supervisor, Univ. Prof. Dr. Manfred
Kietzmann, who provided me with a great opportunity to undertake this study. I am
also highly indebted to him for his excellent guidance, supervision and advice not
only on academic aspects but also on a more personal level.
I would like to extend my grateful thanks to Dr. Frank Niedorf for his valuable
technical support, kind advice on various aspects and good corporation. Grateful
appreciation is also extended to Dr. Wolfgang Baümer and Dr. Michael Braun, for
their kind willingness to help me every time I asked for it. I will not forget with their
help with improving my German laguage skills, idiomatic and slang words and
inviting me to join them at parties, talking about culture and telling about humorous
stories.
My special thanks go to Mr. H.-H. Bohr and Ms.Victoria Garder for their technical
assistance, preparing some solutions and handling the chickens.
I express sincere thanks to Prof. Dr. Dr. Wulf Winkenwerder and his family for their
warm hospitality, helping all Thai veterinary students in Germany.
My deeply heartfelt thanks go to Assoc. Dr. Chartchai Noonpugdee, who passed away
before I finished doctoral degree. His suggestions and inspiration, helping me during
my studies at the TiHo will always remain in my memory.
My thanks go to Thai government under Prime Minister Dr. Thaksin Shinnawatra
(2003) for their full scholarship support of my Doctoral study
I would like to thank the staff of Foreign Student Affair of the TiHo, Hannover for
looking after overseas students and taking us on interesting cultural tours.
Acknowledgement
140
I also wish to thank for all doctoral students, trainees and laboratory technicians of the
Institute of Pharmacology, Toxicology and Pharmacy, TiHo Hannover, who helped
me improve my German language skills and gave me a fun time.
My immense thanks go to Asist. Prof. Dr. Ukadej Boonprakob and Asist. Prof. Dr.
Chaiyan Kasorndorkbua for proof-reading the first English version.
I am thankful to the administrators and colleagues of the Faculty of Veterinary
Medicine and Kasetsart University for their supported and devoted time during my
study period.
An exciting, funny and enjoyable life in Germany would not have occurred without
my Thai friends (P´ Wan, P´ Chu, Tom, Knot, Pae, Benz among others) who shared
experiences, happiness and activities such as Thai cooking, short trips, parties, and
sports activities.
Special thanks go to Dr. Pornchai Sanyathitiseree (P´ Rug), who helped me from the
very first day I came to Germany until the day he returned to Thailand.
Last but not least, I am heartily grateful to my wife, for her help, patience and
encouragement during the tenure of this study.
Nevertheless, any weaknesses in this dissertation are entirely my own responsibility.
Lastly, the encouragement and support of my beloved family, mother, older sisters
and brother are gratefully acknowledged.