Detection of alpha-2 agonists in equine urine by
n
LC/MS .
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 it’s 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, it’s 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 1:200 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 β glucuronidase (500 IU, 0,2 ml,
Helix Pomatia, St. Louis, USA) for 2 h at 55°C.
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 230+232 (Clonidine), m/z
159+160 (Romifidine), m/z 90 (Xylazine), m/z 81 (Detomidine), m/z 85
(Medetomidine). Right : The relative full scan LC-MS/MS spectra.
RESULTS AND DISCUSSION
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.
Drugs
Recovery (%)
Romifidine
86
Clonidine
82
Xylazine
95
Detomidine
87
Medetomidine
91
Table 1 : Recovery for the alpha-2 agonists
Method sensivity
The method was proved both in APCI and ESI ionization mode.
The best results, in term of better LOD were obtained in positive electrospray ionization mode.
The limits of detection (LOD), calculated on the basis of a minimal accepted value of the
signal-to-noise ratio of 3 were: 2 ng/ml for clonidine and romifidine, 1 ng/ml for xylazine, 0,5
ng/ml for detomidine and medetomidine.
The limits of quantification (LOQ) calculated on the basis of a minimal accepted value of the
signal-to-noise ratio of 10 were: 5 ng/ml for clonidine and romifidine, 3 ng/ml for xylazine and
2 ng/ml for detomidine and medetomidine.
Detection and confirmation of alpha 2 agonists
The separation and MS/MS full scan spectra of the alpha-2 agonists is illustrated in Fig. 2.
Fig 3 shows the extracted ion-chromatograms of m/z 159+160 and product ion-spectra
(MS/MS) of the precursor ions m/z 258/260 (MH+) of romifidine in a spiked urine and in a
positive sample.
Fig 4. shows the extracted ion-chromatograms of m/z 81 and product ion-spectra (MS/MS) of
precursor ion m/z 187 (MH+) of detomidine in a spiked urine and in a positive sample.
Both the retention time and the product-ion spectrum of romifidine and detomidine in the real
sample matched well with the standard in the spiked urine.
Drugs
Precursor
Ion (m/z)
Product
Ions (m/z)
Collision
Energy (%)
Isolation
Width(m/z)
Romifidine
259
159/160,
178/179,
241/243.
46
4
Clonidine
231
213/215
162/164
48
4
Xylazine
221
90,147,164
38
3
Detomidine
187
81
28
3
Medetomidine
201
95
23
3
Table 2 : MS parameters for the alpha-2 agonists
Figure 4 Detection of romifidine in an urine sample by LC-MS/MS.
a) extracted-ion chromatogram of m/z 159+160 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.
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 it’s 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
it’s possible to apply this method only to a screening analysis.
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. R&W 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. R&W 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. R&W 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 ß2–agonists 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. R&W 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.