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Title: Detection of alpha2 agonists in equine urine by LCMSn'


1
Detection of alpha-2 agonists in equine urine by
LC/MSn.
M.Fidani, E. Pasello, P. De Iuliis and
M.Montana U.N.I.R.E. Lab. s.r.l., 70. Gramsci
Street, Settimo Milanese (MI) 20019, ITALY
ABSTRACT Alpha-2 agonists (romifidine, clonidine,
detomidine, medetomidine, xylazine) are used for
sedation and analgesia in horses. ELISA test is
used for screening of these drugs but, if this
technique improves the sensivity, there are often
cross-reactions between these substances. The
confirmation of these drugs was made by GC-MS
analysis, but its necessary a derivatization
step. A rapid and sensitive method has been so
developed coupling HPLC with mass spectrometry
after an SPE extraction . The reversed phase LC
column and isocratic conditions used resulted in
a seven minutes analysis time. Linear ion trap
was used for mass spectrometry. Urine samples
were spiked with the drugs and Recovery, LOD and
LOQ were calculated. The method was tested on
real samples demonstrating his great efficiency.
INTRODUCTION Alpha-2 agonists (Fig. 1) are
indicated for use as sedative and analgesic (pain
relief) for minor surgical and diagnostic
procedures in mature horses and yearlings. These
drugs can also be abused (Doping) in the race
horse to reduce athletic performance. For this
reason, it was necessary to develop a rapid and
sensitive method to confirm these drugs in equine
urine. Actually, in our laboratory the screening
of these drugs is made with ELISA test (TCC. and
Neogen) . Considering that ELISA test gives
often cross-reactions between these substances,
its our purpose to apply this method to detect
these drugs.
MATERIALS AND METHODS Reagents and
chemicals Romifidine and clonidine were obtained
from Boeringher Ingelheim (Boeringher, Bracknell,
UK), xylazine from Sigma Aldrich (St. Louis, MO,
USA) and detomidine (Domosedan) and medetomidine
(Domitor) from Orion Pharma (Espoo,
Finland). Methanol, formic acid , and water (all
HPLC grade) were purchased from J.T Baker
(Deventer, The Netherlands). Samples Preparations
Each standard (1 mg) was dissolved in 10 ml of
methanol (100 µg/mL). Then 0,5 mL of this
solution was diluted 1200 with water to yeld a
final concentration of 0,5 µg/mL. 100 µl of
this solution was added to 0,9 ml of urine to
obtain a final concentration of 50 ng/ml of the
drug. These urines were used to calculate the
respective recoveries. Drug recovery Recoveries
of the 5 alpha 2 agonists, spiked at 50 ng/ml
each, were studied using urine samples from
different horses. Comparisons were made between
each of the spiked urine exctracts and the
corresponding blank urine extracts spiked after
extraction (taken as 100 recovery). Drug
recoveries were found to range between 82 for
clonidine and 95 for xylazine (Table 1). Solid
phase extraction method One milliliter of
centrifuged urine was adjusted to pH 6,8 with
phosphate buffer (1M, 2 mL) and enzyme hydrolyzed
with ß glucuronidase (500 IU, 0,2 ml, Helix
Pomatia, St. Louis, USA) for 2 h at 55C. After
hydrolysis urine aliquots were spiked with 50 ng
of int. std. Xylazine was used like internal
std. for the confirmation of the other alpha-2
agonists and clonidine was used like int. std to
confirm the xylazine. Extraction cartridges
(Strata Screen-C, Phenomenex, , San Josè, CA,
USA) were conditioned with methanol (3 mL) and
0,1M phosphate buffer (pH 6.0, 3ml). Each sample
was passed trough a cartridge, which was then
washed with and 0,1M phosphate buffer (pH 6.0,
3ml) and methanol (3 mL). The cartridge was dried
for 5 min. with compressed air at 30 psi and
eluted with methylene chloride / isopropanol /
ammonium hydroxide (80/18/2, 2ml). Instrumentation
An LTQ linear ion-trap mass spectrometry
equipped both with APCI and ESI source (
Thermo-Electron Corporation, San Josè, CA, USA)
connected to a Surveyor Autosampler-MS Pump
(Thermo-Electron Corporation) was
used. Separation was performed at ambient
temperature, in isocratic conditions, in a
reversed-phase with polar-endcapping Synergi
Polar-RP column (150 x 2mm, 4µm Phenomenex ,
San Josè, CA, USA). The mobile phase was
composed of 55 formic acid 0,1 (solvent A) and
45 methanol (solvent B) at a flow rate of 0,25
mL/min. The injection volume was 5 µl. Compound
identities were confirmed in the positive
electrospray ionization (ESI) MS-MS mode , with
full-scan product acquisition in the m/z 60-275
range. A capillary voltage of 22 V, a capillary
temperature at 275 and a spray voltage of 8 kV
were employed. The nitrogen sheath and auxiliary
gas flow rates were set at 20 and 10 arbitrary
LTQ units, respectively. Data acquisition for
MS/MS was performed in 4 segments the 1st (0-3
min.) with 2 scan events for romifidine and
clonidine , the 2nd (3-4 min.)for xylazine, the
3rd (4-5,5 min.) for detomidine and the 4th for
medetomidine (5,5-8 min.). The collision energy
was 46 for romifidine, 48 for clonidine, 38
for xylazine, 28 for detomidine and 23 for
medetomidine
Figure 1 Molecular structure of clonidine,
romifidine, xilazyne, detomidine and medetomidine
.
Figure 2 Left Extracted Ion Chromatograms of
m/z 230232 (Clonidine), m/z 159160
(Romifidine), m/z 90 (Xylazine), m/z 81
(Detomidine), m/z 85 (Medetomidine). Right The
relative full scan LC-MS/MS spectra.
Figure 4 Detection of romifidine in an urine
sample by LC-MS/MS. a) extracted-ion chromatogram
of m/z 159160 and full scan LC-MS/MS spectrum of
romifidine in a positive urine. b) extracted-ion
chromatogram of m/z 81 and full scan LC-MS/MS
spectrum of romifidine in a spiked urine at 50
ng/ml.
Figure 3 Detection of detomidine in an urine
sample by LC-MS/MS. a) extracted-ion chromatogram
of m/z 81 and full scan LC-MS/MS spectrum of
detomidine in a positive urine. b) extracted-ion
chromatogram of m/z 81 and full scan LC-MS/MS
spectrum of detomidine in a spiked urine at 50
ng/ml.
CONCLUSION An efficient LC-MS/MS method for
detecting alpha-2 agonists at low ng/ml level in
equine urine was developed. Equine urine was
enzyme hydrolyzed and extracted by SPE with
Strata Screen-C columns. Both APCI and ESI
ionization mode were tested and the better
results, in term of sensivity, were obtained in
positive electrospray ionization mode. The high
recovery, the isocratic condition and the short
LC/MS run time show that the method can be
applied successfully to detect alpha-2 agonist
drugs. For romifidine, clonidine and xylazine
its possible to apply this method also for a
definitive confirmation of a positive samples
cause are fulfilled the AORC criteria for mass
spectrometry. For a definitive confirmation of
detomidine and detomidine these citeria are not
fulfilled and its possible to apply this method
only to a screening analysis.
Table 1 Recovery for the alpha-2 agonists
Table 2 MS parameters for the alpha-2 agonists
REFERENCES S. Scrimshaw, S. Mayoss, J.Fox and
J.Roberts (2002) Development of a rapid lateral
flow immunoassay for the detection of the alpha-2
agonist romifidine in equine plasma. Proc. 14th
int. Conf. racing Anal. Vet. Eds D.W. Hill and
W. T. Hill. RW Publications, Newmarket, pp
421-426.. P.L Toutain and V. Lassourd (2002)
Pharmacokinetic/Pharmacodinamic assessment of
irrelevant drug concentrations in horse plasma or
urine for a selection of drugs.. Proc. 14th int.
Conf. racing Anal. Vet. Eds D.W. Hill and W. T.
Hill. RW Publications, Newmarket, pp 19-28. J.
Keledjian, N. Basgallop and S.M.R. Stanlley
(2002) A Rapid and sensitive liquid
chromatography mass spectrometry target screen of
post race urine samples for basic drugs. Proc.
14th int. Conf. racing Anal. Vet. Eds D.W. Hill
and W. T. Hill. RW Publications, Newmarket, pp
386-391.. Ramos, F., Banobre,M.C., da Conceicao
Castilho, M.and da Silveira, M.I.H. (1999)
Solid-phase extraction (SPE) for multi residue
analysis of ß2agonists in bovine urine. J. Liq.
Chrom. Rel. Technol. 22, 2307-2320. P. Sarkar,
Shelley Mackenzie and R. Leavitt (1996)
Analytical methods for the detection and
confirmation of romifidine in equine serum. Proc.
11th int. Conf. racing Anal. Vet. Eds D.E Auer
and E. Houghton. RW Publications, Newmarket,
pp141-146. England, G.C.W., Clarke, K.W. and
Gossens, L. (1991) Sedative and cardiovascular
effects of romifidine, alone and in combination
with butorphanol, in the horse. J. vet. Anaesth.
18, 25-29. Steven G. Kamerling, Winter M. T.
Cravens, Lyndon M. Goodly, Cleo a. Bagwell (1988)
Analgesic and sedative effects of detomidine in
the horse. Proc. 7th int. Conf. racing Anal. Vet.
Eds Thomas Tobin, Jerry Blake, Mike Potter and
Thomas Wood, pp 41-45.
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