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Controlling Therapeutic Substances - A European Harmonised Approach:
Determination of the Detection Time for eltenac following IV Administration to Horses.
M.Montana*, F.Baia*, L.Bartolino*, S.Carli†, P.De Iuliis*, L.Ricci‡, E. Pasello*.
*U.N.I.R.E. LAB, via Gramsci 70, 20019 Settimo Milanese, Milan, Italy
† Department of Sciences and Technologies for Food Safety,via Celoria 10, 20133 Milan,Italy
‡U.N.I.R.E., via Cristoro Colombo 283/A 00147 Rome, Italy
ABSTRACT
In Europe, a major initiative is underway to develop a fair and effective approach for controlling substances commonly used for treating racehorses during training. The following factors were considered in the development of this approach: 1) ensuring fair competition; 2) protecting the welfare of
racehorses and 3) protecting the breed.
The Rules of Racing are not intended to discourage the proper veterinary treatment of racehorses if such treatment does not threaten any of these three important factors. However, the use of medication to treat illness and injury during normal racehorse training carries an inherent risk of incurring
a positive drug test on race-day if insufficient time has elapsed for elimination of the drug from the horse. Recognising this, the EHSLC co-ordinated a series of administration studies in France, Germany, Great Britain, Hong Kong, Ireland and Italy, with the aim of producing information for
horseracing veterinarians and to encourage good veterinary practice while ensuring a harmonised approach to drug screening.
Pharmacokinetic studies and risk analysis/risk management procedures have been applied in order to establish screening method limitations for therapeutic substances. Administration studies were then performed at manufacturer’s recommended therapeutic dose levels and dosing regimens for
a number of proprietary formulations commonly used in equine veterinary practice. The detection times for the substances in urine have been determined using analytical methods with limits of quantification established by the pharmacokinetic studies and risk analysis procedures.
Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used in the management of injury. They are also the most common group of drugs reported as prohibited substances. And were therefore the first group of substances chosen by the EHSLC for publication of detection time guidelines,
along with the diuretic furosemide.
The poster reports the studies to determine the detection time for eltenac. Eltenac (Telzenac, Schering-Plough Animal Health, 0.5 mg/Kg) was administered intravenously to six horses in resting condition. All urine samples were collected every day for 15 days post administration. Eltenac was
isolated from urine by solid phase extraction and the extracts analysed by GC-MS. The analytical method used b-naphthoic acid as internal standard and was validated in terms of precision and accuracy. The detection time was determined as the time at which the urinary concentration of eltenac
in all the horses in the study was less than the selected screening method limit derived from the pharmacokinetic/pharmacodynamic studies and risk analysis/risk management exercises.
INTRODUCTION
In Europe, a major initiative is underway to develop a fair and effective approach for controlling substances commonly used for treating racehorses during training. The following factors were
considered in the development of this approach :1) ensuring fair competition; 2) protecting the welfare of racehorses and 3) protecting the breed.
The Rules of Racing are not intended to discourage the proper veterinary treatment of racehorses if such treatment does not threaten any of these three important factors. However, the use of
medication to treat illness and injury during normal racehorse training carries an inherent risk of incurring a positive drug test on race-day if insufficient time has elapsed for elimination of the drug
from the horse. Recognising this, the EHSLC co-ordinated a series of administration studies in France, Germany, Great Britain, Hong Kong, Ireland and Italy, with the aim of producing information
for horseracing veterinarians and to encourage good veterinary practice while ensuring a harmonised approach to drug screening.
Pharmacokinetic studies and risk analysis/risk management procedures have been applied in order to establish screening method limitations for therapeutic substances. Administration studies
were then performed at manufacturer’s recommended therapeutic dose levels and dosing regimens for a number of proprietary formulations commonly used in equine veterinary practice. The
detection times for the substances in urine have been determined using analytical methods with limits of quantification established by the pharmacokinetic studies and risk analysis procedures.
Non-steroidal anti-inflammatory drugs (NSAIDs) are amongst the most commonly prescribed agents for use in horse. Muscoloskeletal pain and inflammation are the most common indications for
chronic administration of NSAIDs to horses and they are also the most common group of drugs reported as prohibited substances. And were therefore the first group of substances chosen by the
EHSLC for publication of detection time guidelines, along with the diuretic furosemide.
Eltenac is an acetic acid derivative in the class of NSAIDs that appears to be effective and relatively safe when used in horses (Prugner et al 1991). Eltenac is an efficacious NSAID in horses when
used at recommended doses:0.5-1.0 mg /kg i.v. once daily for 3 days ( Hamm et al 1997 ) and 0.5 mg/kg i.v. once daily for 5 days (Dyke et al 1998). In addition, the effects of eltenac are long
lasting with a dosing interval of once every 24 h recommended for most conditions ( Dyke et al 1998)
The aim of the present study focuses on the determination of Detection time for eltenac. Detection time for urine is the interval between the time of the last administration and the time at which
urine concentrations of all investigated horses (e.g. n= 6) are observed below an appropriate RLOD (Recommended Limit of Detection) established by the EHSLC using the routine screening
analytical method.
The RLOD is based upon the value of the IUC (Irrilevant Urine Concentration) calculated from the pharmacokinetic studies of Toutain et al. 2002 and the application of suitable risk analysis/risk
management procedure.
Cl
O
S
OH
NH
O
Cl
C
OH
eltenac
b-naphthoic acid
Figure 1:Molecular structure of eltenac and b-naphtoic acid.
MATERIALS AND METHODS
Drugs administration and sampling protocol
220
283
373
Six standardbred males and geldings in resting condition, weighing 320- 480 kg and aged 3-10 years were used for the experiment. Eltenac (Telzenac, Schering-Plough Animal Health, 0.5 mg/Kg) was
administered intravenously once daily for 5 days. Urine samples were collected from the horses during the days of administration (5 days) and 10 days after, over a total period of 15 days.
Urine samples were transferred to the pre-analytical lab within 2 hours from collection and centrifuged at 2000 rpm for 15 min. Every sample of urine collected was divided into aliquots and stored
refrigerated at –20°C until analysed.
Reagents and materials
Eltenac was obtained from Schering-Plough Animal Health and the internal standard (IS) ß naphthoic acid was from Sigma-Aldrich (USA).
Organic Solvents were purchased from J. T. Baker (The Netherlands). N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) were obtained from Supelco / Sigma-Aldrich (Italy)
Strata-X cartridges were obtained from Phenomenex (USA).
All the other chemicals used were analytical grade.
Sample preparation
220
373
283
Three quality control urine samples (1 mL volume) were prepared from pooled blank equine urine (previously centrifuged) and fortified with eltenac to a final concentration of 10, 50,100 ng/ mL in triple.
Aliquots of 1 mL of each post administration urine sample and quality control samples were spiked with ß naphthoic acid (IS) to a final concentration of 500 ng/mL. Samples were added with 2 mL of NaOH
(0.2 N) and subjected to basic hydrolysis at 55°C for 1 hour.
Solid phase extraction method
Automated extraction was performed on the Gilson Aspec XL4 solid phase extraction unit. Samples were applied onto Strata -X cartridges, conditioned with methanol (3 mL) and water (3 mL) with a flow rate
of 1 mL/min.The columns were washed with water (3 mL) and hexane
(1 mL) and dried with air for 2 min.
The analyte was elute with trichloromethane:isopropanol:acetonitril (70:20:10 v/v, 4 mL).
The eluate was evaporated to dryness in a vacuum concentrator and the residue treated with MSTFA (50 μL).
Gas chromatography/mass spectrometry
Figure 2: Product ion chromatogram and mass spectrum of an eltenac spiked
urine and a post administration urine.
The TMS derivative samples were analysed with a full scan EI GC/MS method using a Varian Gas–chromatograph (USA) equipped with a Varian 8400 Autosampler (USA) interfaced to a ITD mass
spectrometer Saturn 2000 Varian (USA). A CP-Sil 8 CB column (10 m X 0.53 mm ID X 0.25 μm, VARIAN) with a methyl-deactivated precolumn ( 60 cm X 0.1 mm ID X 0.34 μm, VARIAN) was used with
Helium as a carrier gas. The initial column temperature, 80°C was maintened for 0.5 min , the oven was then programmed to reach 280°C in increments of 20°C/min and then maintened at 280°C for 1.5 min.
The injector and transfer line temperatures were 280°C and 250°C respectively. Aliquots of 0.5 μL of the derivatised urine isolates were injected in the splitless mode. Positive ion EI mass spectra were
recorded at 70 eV.
kCounts
RESULTS AND DISCUSSION
400
8
The analytical screening method allowed the identification of the drug in post administration urine samples at a
concentration of eltenac equal to or higher than the concentration corresponding to the RLOD as shown in Figure 2.
Analytical proportional response of the drug for the 3 levels (low 10 ng/mL, medium 50 ng/mL, high 100 ng/mL) and
repeatibility of IS is shown in Figure 3.
From the ratio of the peak areas for the ion m/z 220 (eltenac) to m/z 229 (IS), precision and accuracy were evaluated using
3 aliquots for every level. Results are shown in Table 1.
Monitoring the RLOD through the screening procedure greatly simplifies the analytical process as opposed to the use of
absolute quantification and eliminates any consideration of the RLOD as a “Threshold”. According with this principle, just to
avoid the absolute quantification, the analytical approach is based on the comparison between the response of the drug
isolated from aliquots of the biological fluid under study fortified with the drug at the proposed RLOD concentration. The
results are compared and expressed not in terms of absolute concentration but rather as “lower” or higher concentration
than the proposed RLOD.
All the data referring to post administration urine samples of the six horses investigated are summarized in Figure 4. A
comparison with the response of the spiked samples underlined that the concentration of eltenac was below the RLOD at
day 8 after the last administration in all investigated horses.
7
eltenac
100 ng/mL
5
200
4
3
2
b naphthoic acid (IS)
300
6
500 ng/mL
500 ng/mL
100
50 ng/mL
500 ng/mL
1
10 ng/mL
0
0
10.75
11.00
11.25
11.50
6.6
6.7
6.8
6.9
minutes
minute
s
Figure 3: Overlaid product ion chromatogram plot of eltenac and IS spiked urines for the 3 levels (10,50,100 ng/mL).
Table 1: Validation data (Precision and Accuracy)
THE DATA :eltenac in Post Administration Urine
Samples
0,2
Quality Control
eltenac
concentration
Mean
(A eltenac/A IS)
SD
Precision *
CV%
Accuracy**
%
Low Level
10 ng/mL
0.013
0.001
10.6
86.5
Medium Level
50 ng/mL
0.108
0.013
12.2
105.9
High Level
100 ng/mL
0.200
0.017
8.3
96.9
A Eltenac / A IS
*Expressed as coefficient of variation ( C.V.%); n=3
**Expressed as: [(mean calculated spiked concentration) / (nominal spiked concentration)] x 100
0,1
CONCLUSIONS
Day 5
Day 6
Day 7
Day 8
Day 9
Sp
ike
R
LO
D
H
or
se
6
H
or
se
5
H
or
se
4
H
or
se
3
H
or
se
2
H
or
se
1
0
Days after last
administration
Detection Time
The results from this study supported a Detection Time of 8 days in urine for a level considered by the EHSLC as an appropriate RLOD for eltenac.
In the experiments, the drug was administered only to healthy horses under controlled, scientific conditions. During the experiment, these horses were not exercised under
conditions that might be expected in routine training.
The use of this drug therapeutically in unhealthy animals may result in longer detection times due to a number of factors, including variation in urine pH, altered
biotransformation and/or excretory processes.
The effects of training /exercise programmes, different diet and stable management may cause variation in drug elimination. The detection times are valid only in respect of
the particular formulation (Telzenac, Schering-Plough Animal Health) dose or dosage regimen employed (0.5 mg/Kg intravenously once daily for 5 days).
The ‘Detection Time’ given here is not synonymous with ‘Withdrawal Times’. To arrive at a withdrawal time an adequate safety margin must be added to the stated
detection time. This safety margin must be chosen by the treating veterinarian using his/her professional judgement and discretion, to allow for biological, pharmaceutical
and pharmacological variation, so as to minimise the possibility that a positive finding will occur on the day of racing.
Figure 4:Eltenac in post administration urine samples in all investigated horses.
REFERENCES
Dyke, T.M., Sams, R.A., Thomson, K.G., Ashcraft, S.M. (1998) Pharmacokinetic of multiple-dose administration of Eltenac in horses. Am. J. vet Res. 59,1447-1450.
Hamm, D., Turchi, P., Johnson, J.C., Lockwood, P.W., Thompson, K.C., Katz, T. (1997) Determination of an effective dose of Eltenac and its comparison with that of flunixin meglumine in horses after experimentally induced carpitis. Am. J. vet Res. 58,298-302.
Prugner, W., Huber, R., Luhmann, R. (1991) Eltenac, a new anti-inflammatory and analgesic drug for horses: clinical aspects. J. of vet Pharmacol. Therap. 14,193-199.
Toutain, P.L., Lassourd, V. (2002) Pharmacokinetic/ Pharmacodynamic approach to assess irrelevant plasma or urine concentrations in horse plasma in post-competition samples for drug control in horses. Equine vet. J. 34,242-249
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