L. Dirikolu, E. T. McFadden, K. J. Ely, H. ElkHoly, A. F. Lehner, and K. Thompson 141 CLINICAL RELEVANCE Clonidine is classified as a class 3 performance-enhancing agent by the Asso- ciation of Racing Commissioners International and thus has the potential to in- fluence the outcome of a race. In this study, the authors developed and validat- ed a sensitive gas chromatograph and mass spectrometer method to determine the pharmacokinetic parameters of clonidine in equine plasma samples after IV administration of a single dose (0.025 mg/kg) of clonidine in horses. At this dose, clonidine produced rapid and profound sedation, which could be quickly reversed with yohimbine. Clonidine was able to produce an analgesic effect but failed to provide maximal analgesia in all horses; the limited analgesic effect persisted for about 60 minutes. Clonidine in Horses: Identification, Detection, and Clinical Pharmacology* L. Dirikolu, DVM, MSVc, PhD a E. T. McFadden, BS a K. J. Ely, BS a H. ElkHoly, DVM, MS, PhD a A. F. Lehner, MS, PhD b K. Thompson, DVM c ■ INTRODUCTION In veterinary medicine, a number of the α 2 - receptor agonist medications are marketed as sedatives, hypnotics, and analgesics, with their principal use being the chemical restraint of large and small animals. In human medicine, however, members of the α 2 -receptor agonist family are used primarily as antihypertensive agents. 1–3 In recent years, the usefulness of the α 2 -adrenoceptor agonist drugs has been recog- nized in equine practice. 4 For clinical purposes, these agents produce sedation and analgesia and, thus, are useful for premedication and markedly potentiate the effects of other seda- tive or analgesic agents. 5,6 The α 2 -receptor agonists, of which clonidine is the best-known example, were first synthe- *This research was supported by grants from the Birmingham Racing Commission Breeding and Development Fund, Birmingham, Alabama, and Tuskegee University Research and Graduate Studies Development Fund, Tuskegee, Alabama. a Department of Biomedical Sciences c Department of Large Animal Clinics College of Veterinary Medicine, Nursing and Allied Health Tuskegee University Tuskegee, AL 36088 b Livestock Disease and Diagnostic Center College of Agriculture University of Kentucky Lexington, KY 40512-4125
Transcript
L. Dirikolu, E. T. McFadden, K. J. Ely, H. ElkHoly, A. F. Lehner, and K. Thompson
141
CLINICAL RELEVANCE
Clonidine is classified as a class 3 performance-enhancing agent by the Asso-
ciation of Racing Commissioners International and thus has the potential to in-
fluence the outcome of a race. In this study, the authors developed and validat-
ed a sensitive gas chromatograph and mass spectrometer method to determine
the pharmacokinetic parameters of clonidine in equine plasma samples after IV
administration of a single dose (0.025 mg/kg) of clonidine in horses. At this
dose, clonidine produced rapid and profound sedation, which could be quickly
reversed with yohimbine. Clonidine was able to produce an analgesic effect but
failed to provide maximal analgesia in all horses; the limited analgesic effect
persisted for about 60 minutes.
Clonidine in Horses: Identification, Detection, andClinical Pharmacology*
L. Dirikolu, DVM, MSVc, PhDa
E. T. McFadden, BSa
K. J. Ely, BSa
H. ElkHoly, DVM, MS, PhDa
A. F. Lehner, MS, PhDb
K. Thompson, DVMc
■ INTRODUCTIONIn veterinary medicine, a number of the α2-
receptor agonist medications are marketed assedatives, hypnotics, and analgesics, with theirprincipal use being the chemical restraint oflarge and small animals. In human medicine,
however, members of the α2-receptor agonistfamily are used primarily as antihypertensiveagents.1–3 In recent years, the usefulness of theα2-adrenoceptor agonist drugs has been recog-nized in equine practice.4 For clinical purposes,these agents produce sedation and analgesiaand, thus, are useful for premedication andmarkedly potentiate the effects of other seda-tive or analgesic agents.5,6
The α2-receptor agonists, of which clonidineis the best-known example, were first synthe-
*This research was supported by grants from theBirmingham Racing Commission Breeding andDevelopment Fund, Birmingham, Alabama, andTuskegee University Research and Graduate StudiesDevelopment Fund, Tuskegee, Alabama.
aDepartment of Biomedical SciencescDepartment of Large Animal ClinicsCollege of Veterinary Medicine, Nursing and Allied Health
Tuskegee UniversityTuskegee, AL 36088
bLivestock Disease and Diagnostic CenterCollege of AgricultureUniversity of KentuckyLexington, KY 40512-4125
In a recent study, it was suggested that vari-ous α2-receptor agonists were being dissolved invitamin B12 solutions containing alcohol andinjected IV into horses before races.17 This isadministered in small doses (about 0.02 mg/kg)shortly before post time to reduce the intensityof racing-related pulmonary hypertension and,by extension, the associated exercise-inducedpulmonary hemorrhage (EIPH). The rationalefor administering α2-receptor agonists is thatmost horses experience pulmonary hyperten-sion during running, leading to EIPH, a con-siderable problem in the horseracing industry.EIPH acutely interferes with the racing per-formance of horses by compromising the ex-change of oxygen and carbon dioxide in thealveolar capillaries, and repeated bouts of EIPHresult in chronic and cumulative damage to thelung.18 Known as an antihypertensive in humanmedicine, clonidine could reduce pulmonaryarterial blood pressure in racing horses and,thus, potentially reduce the incidence or severi-ty of EIPH. On the other hand, as an α2-ago-nist agent, clonidine may also have the abilityto tranquilize or sedate horses and may alsohave some bronchodilator activity.
Medications capable of improving the racingperformance of horses are classified by the As-
sized in the early 1960s and found to producevasoconstriction when applied topically, appar-ently mediated through α2-receptors. Duringclinical testing of clonidine as a nasal decon-gestant, it caused hypotension, sedation, andbradycardia in humans, which led to its intro-duction as an antihypertensive in human med-icine.7 For a number of years, clonidine hasbeen marketed under several brand names forhuman use as a centrally acting α2-receptor ag-onist to decrease blood pressure.8 Evidencefrom clinical studies and animal experimentssuggests that the effect of clonidine is largelyrelated to its action at postsynaptic α1-recep-tors in the central nervous system to producean overall decrease in sympathetic outflow.9
Other centrally mediated effects of clonidineinclude sedation10,11 and analgesia,12,13 and, assuch, clonidine has the potential to producesedative or tranquilizing effects and bron-chodilation in horses. Because of the markedantihypertensive effect of clonidine, only smalldoses (0.075 to 1.5 mg/day) are used in anti-hypertensive therapy in humans.14,15 Conse-quently, numerous difficulties have been en-countered in making an exact determination of the resulting extremely low plasma drugconcentration.16
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Figure 1. Chemical structure (A) and calculated isotopic profile (B) of underivatized clonidine.
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sociation of Racing Commissioners Interna-tional (ARCI) based on their performance-en-hancing potential. Clonidine is currently clas-sified as an ARCI class 3 agent.19 As such,clonidine is considered to have the potential toinfluence the outcome of a race, and its ad-ministration to a horse shortly before post timewould clearly contravene the rules of racing inmost jurisdictions. As such, useful screeningand confirmation methods for clonidine inequine serum, plasma, or urine are required.
Clonidine is a basic lipid-soluble drug with ahigh volume of distribution, and plasma con-centration after administration is very low inhumans and various animal species.20 Despitethe numerous analytic methods described inthe literature, the problem of quantitativelymeasuring clonidine in low concentrations inbiologic matrices remains difficult. Further-more, at this time, no validated and peer-reviewed analytic method exists for the quanti-tative determination of clonidine in thebiologic fluids of horses. We have previously
described the detection, quantification, andpharmacokinetics of the related α2-receptor ag-onist guanabenz in performance horses.21 It isessential for the welfare and integrity of theracing industry that accurate and specific ana-lytic tests exist for the quantitation of this drugin biologic fluids of racing horses to control in-advertent or intentional misuse.
The objective of this study was to developand validate a peer-reviewed and peer-replicat-ed sensitive and specific gas chromatogra-phy–mass spectrometry (GC/MS) quantitativemethod for clonidine in horses. In addition,we wanted to determine the duration of thepharmacologic effects of clonidine in perform-ance horses to establish scientifically defensiblewithdrawal times and/or a threshold level forclonidine.
■ MATERIALS AND METHODSHorses
Four mature Thoroughbred mares (7 to 8years old) were used for this study. The animals
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Figure 2. Selected ion chromatogram of TBDMS derivative of clonidine extracted from plasma with standard peakat 13.7 minutes. The overlap of ions m/z 252, 254, 93, and 286 is displayed.
Figure 3. Electron impact mass spectrum of TBDMS derivative of clonidine (molecular weight, 343; A) and un-derivatized clonidine (B).
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were maintained on grass hay and feed, whichwas 50:50 mixture of sweet feed and an alfalfa-based protein pellet (14% protein). Horseswere fed twice a day. The animals were vacci-nated annually and dewormed quarterly. Aroutine clinical examination was performedbefore each experiment to assure that the ani-mals were healthy and sound. During experi-mentation, horses were provided water and hayad libitum. Animals used in these experimentswere managed according to the rules and regu-lations of the Tuskegee University InstitutionalAnimal Care Use Committee, which also ap-proved the experimental protocol.
For the pharmacokinetics study, clonidinewas administered as a single IV dose at 0.025mg/kg in 10% ethanol solution (prepared inisotonic saline solution), and blood sampleswere collected as described below. Clinicalpharmacology of clonidine experiments fol-lowed a rigorous standard protocol to reducevariability from extraneous effects. Horses wereplaced in individual stalls in the early morningand allowed to acclimate to the stall for 7 hoursbefore each experiment. For the determinationof the sedative actions of clonidine, horses weredosed again with 0.025 mg/kg of clonidine asa single IV dose; the potency and duration ofthe sedative effects of clonidine in these horseswere determined as described below. In addi-tion, after a minimum 1-week washout inter-val following the completion of the sedationstudies, the potency and duration of the anal-gesic effects of clonidine in these horses fol-lowing a single IV dose at 0.025 mg/kg weredetermined as described below.
Pharmacodynamic ParametersHead Drop
Sedation was assessed by measuring the de-gree of head drop following administration ofclonidine. A pretreatment floor-to-chin heightwas determined at 30, 15, and 0 minutes be-
fore IV injection of clonidine to establish abaseline value in each horse. The degree ofhead drop was then measured at 5, 10, 15, 20,25, and 30 minutes after injection and every15 minutes thereafter until head drop meas-urements returned to baseline values. All relat-ed clinical signs associated with the sedative ef-fects of clonidine in horses, such as ataxia anddrooping of the eyelids and lower lip, were alsoreported. In a separate experiment to evaluatereversal of the effects of clonidine, the sameprotocol described above was followed, exceptthat two horses received IV injections of 0.12mg/kg yohimbine (an α2-receptor antagonist)20 minutes after injection of clonidine; theyohimbine powder was first dissolved in 2.5 mlof dimethyl sulfoxide (DMSO).
AnalgesiaA heat projection lamp was used to deter-
mine thermal antinociception, which has beenused as a measure of analgesia in anotherstudy.21 Briefly, focused radiant light or heat
TABLE 1. Interpretation of FragmentsObserved in Clonidine Mass SpectrumShown in Figure 3A
was used as a noxious stimulus and was direct-ed at the pastern of the horse from a constantdistance to elicit the classic flexion–withdrawalreflex. Hoof withdrawal reflex latency (HWRL)was defined as the time between initiation oflamp illumination and withdrawal of the hoof.The reflex times were adjusted by varying theintensity of the heat output with a rheostat sothat the HWRL for control measurements was3 to 4 seconds, with the actual HWRL record-ed on an electronic timer built into the lamp.The duration of light exposure to the pasternswas limited to 10 seconds to prevent skin dam-age. A secondary unfocused light beam (shamlight) was used to confound the horse, reduc-ing the possibility that the flexion–withdrawalreflex was to visual rather than thermal percep-tion of the focused light beam.
HWRL was measured at 30 and 15 minutesand immediately before injection of clonidine,and these times (–30, –15, and 0 minutes)were used to establish a mean baseline value forHWRL in each horse. The HWRL then wasmeasured at 5 and 15 minutes after injectionof clonidine and every 15 minutes thereafteruntil HWRL returned to control values. TheHWRL was expressed as a percentage of the
baseline values (100%), with 9 seconds beingmaximum analgesic effect (300%).
Pharmacokinetic ParametersSample Collection
Horses were given a single IV dose of cloni-dine (0.025 mg/kg in 10% ethanol solutionprepared in isotonic saline solution), and plas-ma concentrations were determined in a phar-macokinetics study. The skin over the left jugu-lar vein was washed with povidone–iodinescrub (Poviderm, Burns Veterinary Supplies,Westbury, NY) and rinsed with ethanol. An IVcatheter (Abbocath-T, 14-gauge × 5.5-inch,Abbott Animal Health, North Chicago, IL)was inserted into the left jugular vein and su-tured in place. Clonidine was administeredwith great care as a single IV dose into the rightjugular vein. Blood samples were collectedfrom the left jugular vein for analyses at 0, 5,10, 20, 30, and 45 minutes and 1, 2, 4, 6, 8,10, 24, 48, 72, and 96 hours into heparinizedVacutainer plasma tubes (Becton Dickinson,Franklin Lakes, NJ) and then centrifuged at4˚C at 2,000 ×g for 15 minutes; the plasmawas stored and refrigerated in 5-ml aliquotsuntil assayed.
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Figure 4. The chemical structure and isotopic profile of TBDMS derivative of clonidine (molecular weight, 343).(A) Clonidine [2-(2,6-dichloroanilino)-1,3-diazacyclopentene(2)] derivatized at the N-1 (left) or N-3 (right) po-sitions; note the essentially identical chemical structure by symmetry. (B) Isotopic profile of either structure.
Analytic Detection of ClonidineStandard solutions of clonidine (Sigma
Chemical, St. Louis) and isoxsuprine (internalstandard; Sigma Chemical) were prepared inN,N-dimethylformamide (DMF; Sigma Chem-ical) and stored in a refrigerator. These stan-dards were allowed to come to room tempera-ture (23˚C) before use. With each analytic run,serial dilutions were made from the stock cloni-dine standard and added to blank plasma sam-ples. Extraction standards were prepared by theaddition of a known volume of a clonidine so-lution to blank plasma samples at a range of 1to 250 ng/ml (0, 1, 2.5, 5, 10, 25, 50, 100, and250 ng/ml). A known volume of an isoxsuprinestandard (10 µl of 10 µg/ml in DMF) wasadded to each sample and standard and blankplasma samples as an internal standard.
The plasma standards and blanks (1 ml/sam-ple) were placed in culture tubes. The plasmasamples were alkalinized with 0.75 µl of am-
Extraction of ClonidineThe analytic procedure was adapted from
that described by Arrendale et al.22 Clean 16 ×150 mm Pyrex screw-cap culture tubes withpolytetrafluoroethylene (PTFE) cap liners weresilanized with trimethylchlorosilane (TMCS;Pierce Chemical, Rockford, IL) before use byrinsing them first with methanol, followed bya 5% solution of TMCS in hexane (Fisher Sci-entific), and twice more with methanol (FisherScientific). The silanized tubes were allowed toair-dry at room temperature.
The analytic method used was as follows:Plasma samples (1 ml) were pipetted into 15-ml screw-cap tubes. Then, 10 µl of 10 µg/mlinternal standard was added to each tube, fol-lowed by 75 µl of ammonium hydroxide (con-centrated) and dichloromethane:isopropanol
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L. Dirikolu, E. T. McFadden, K. J. Ely, H. ElkHoly, A. F. Lehner, and K. ThompsonR
Figure 5. Typical calibration curve for clonidine extracted from equine plasma samples, indicating coefficient of de-termination (r 2) of 0.999603. “Response” on the y axis refers to Clonidine Area × (Internal Standard Concentra-tion/Internal Standard Area).
(Sigma Chemical):ethylacetate (Fisher Scien-tific) (6:1:3). The tubes were shaken on a re-ciprocating shaker for 10 minutes and cen-trifuged at 300 ×g for 5 minutes. The aqueouslayer was then aspirated to waste. Thedichloromethane:isopropanol:ethylacetate ex-tracts were decanted into silanized test tubes,DMF (15 µl) was added as a “keeper solvent,”and all extracts were evaporated under a streamof nitrogen at 40˚C in a waterbath.
For derivatization, each dried sample was dis-solved in 50 µl of N-methyl-N-(tert-butyl-dimethyl-silyl)trifluoroacetamide + 1% tert-butyldimethylchlorosilane (MTBSTFA + 1%TBDMCS; Pierce Chemicals, Rockford, IL),vortex mixed briefly, incubated at 75˚C for 2hours to produce the tert-butyl-dimethylsilyl(TBDMS) derivatives of both clonidine and in-ternal standard, and then transferred to anautosampler vial equipped with a 200 µl spring-loaded insert. In addition, alternative derivati-zations using N,O-bis-trimethylsilyltrifluo-roacetomide plus 1% TMCS (BSTFA + 1%TMCS; Pierce Chemicals), Barb-Prep (AlltechAssociates, Deerfield, IL), and N-methyl-N-tri-methlysilyltrifluoroacetamide (MSTFA) + 1%
TMCS (MSTFA + 1% TMCS; Pierce Chemi-cals) were evaluated for the detection and quan-titation of clonidine.3,22,23 Each solution wastransferred to a micro insert in an automaticliquid sampler vial (as described above), fromwhich 1 µl was injected for chromatography.
Gas Chromatography–Mass SpectrometryAnalysis
Samples were analyzed using Perkin ElmerAutoSystem XL Gas Chromatography andTurboMass Mass Spectrometer (Perkin Elmer,Norwalk, CT) set in positive ion mode. Thegas chromatography conditions used for iden-tification of the corresponding clonidine peakwere as follows: column, HP-5ms [(5%phenyl)-95% methylpolysiloxane)] 30 m,0.50-µm film thickness, 0.25-mm internal di-ameter (Agilent Technologies, Wilmington,DE) (helium flow rate: 1.5 ml/min); injector,250˚C; transfer line, 250˚C; the oven was tem-perature programmed from 70˚C (2 minutesat initial temperature) with a 20˚C/min rate ofincrease to 280˚C (holding temperature, heldfor 10 minutes). The mass spectrometer wasset to acquire from mass:charge ratio (m/z)
TABLE 2. Accuracies and Within-Run and Between-Run Precisions of GC/MS AssayUsed to Quantify Clonidine in Horse Plasma Samples
Theoretic Measured Concentration Accuracy (%; Coefficient ofConcentration (ng/ml) (ng/ml; mean ± SD; n = 6) mean ± SD) Variation (%)
Within-run
2.5 2.40 ± 0.146 96.13 ± 5.87 6.11
25 25.13 ± 2.25 103.2 ± 5.66 5.48
200 203.48 ± 6.04 101.8 ± 2.99 2.93
Mean 100.38 4.84
Between-run
2.5 2.53 ± 0.167 101.3 ± 6.80 6.71
25 23.83 ± 1.947 95.33 ± 7.92 8.3
200 204.5 ± 6.09 102.3 ± 3.28 3.2
Mean 99.64 6.07
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50 to 650 at 1 scan/sec (scan/s). In quantita-tive experiments, selected ion monitoring(SIM) was performed for ions m/z 93, 252,254, and 286 for clonidine and m/z 178 forinternal standard. The m/z 178 of TBDMS–isoxsuprine derivative is the major fragmenta-tion product and was monitored as the internalstandard ion. For TBDMS–clonidine deriva-tive, quantitation was based on the most abun-dant ion, m/z 252. Mass Spec Calculator Pro(Version 4.03, 1998, Quadtech Associates,Fairfield, CA) was used to assist in the inter-pretation of full-scan spectra where necessary.For chromatographic and mass spectrometricidentification of clonidine and isoxsuprine,Chromatographic and Mass Spectrometric Crite-ria for Identification Guidelines (available fromthe Association of Official Racing Chemists[AORC], Lexington, KY) were used.24
A standard curve was constructed by plot-ting standard clonidine concentration versusthe ratio of clonidine:internal standard peakareas. Standard curves were generated with Sig-ma Plot for Windows (Aspire Software Inter-national, Leesburg, VA). The areas of peakscorresponding to clonidine and internal stan-dard were recorded, and the internal standardvalues were used to normalize the clonidineareas. Integrated peak values were entered into
QuattroPro for Windows (Borland SoftwareCorporation, Scotts Valley, CA) for statisticalanalysis of standards and for interpretation ofunknown amounts of clonidine. An estimateof clonidine concentration in unknown sam-ples was obtained by comparing unknown:in-ternal standard area ratio obtained from theunknown sample and interpolated on the stan-dard curve.
Validation of the AssayThe GC/MS method for the quantitation of
clonidine was validated by applying criteria inthe most recent Standard Operating Procedureavailable to us from Dr. Rick Sams.a The quan-titative method of clonidine was validated byexamining the measurement of consistency ofresults (within-run and between-run), correla-tion (coefficient of determination of the stan-dard curve), and extraction efficiency of the as-say. The within-run precision was calculatedfrom similar responses from six repeats of threecontrol samples (2.5, 25, and 200 ng/ml) inone run. The between-run precision was deter-mined by comparing the calculated response
L. Dirikolu, E. T. McFadden, K. J. Ely, H. ElkHoly, A. F. Lehner, and K. Thompson
TABLE 3. Extraction Efficiencies and Measured and Expanded Uncertainties of GC/MSAssay Used to Quantify Clonidine in Horse Plasma Samplesa
Theoretic Extraction Efficiency (%; Measured ExpandedConcentration (ng/ml) mean ± SEM; n = 6) Uncertainty (%) Uncertainty (%)
2.5 96 ± 9.6 3.92 9.79
25 80 ± 6.0 2.45 6.12
100 75.5 ± 6.1 2.49 6.22
Mean 83.83 2.95 7.38aUncertainty was determined using the method of A2LA. (Uboh C: Personal communication, Equine Toxicology and
Research Laboratory, University of Pennsylvania, School of Veterinary Medicine, Kennet Square, PA, 2002)
Mean ± expanded uncertainty = 95% confidence range.
aSams R: Personal communication, Professor andDirector, OSU Analytical Toxicology Laboratory,College of Veterinary Medicine, Ohio State Univer-sity, 2002.
(in ng/ml backfit of the standard curve) of thelow (2.5 ng/ml), middle (25 ng/ml), and high(200 ng/ml) control samples over three consec-utive daily runs (total of six runs). The assayaccuracy for within-run and between-run wasestablished by determining the ratio of calcu-lated response to expected response for low(2.5 ng/ml), middle (25 ng/ml), and high (200ng/ml) control samples over six runs.
Standard curve correlability was measuredby the mean coefficient of determination (r2)for six consecutive daily runs. The extractionefficiency was determined by comparing theresponse (in area) of low (2.5 ng/ml), middle(25 ng/ml), high (100 ng/ml), and internalstandard (100 ng/ml) standards spiked toblank plasma eluent before evaporation to theequivalent extracted standards. The lower lim-it of detection (LOD) was calculated from sixconsecutive runs. The concentration calculatedfrom the mean of the responses at zero con-centration (y-intercepts) was determined. TheLOD was defined as the concentration calcu-lated from the mean response at zero concen-
tration plus two times the stan-dard deviations (the upper 95%confidence limit for zero).25 Inaddition to this determination ofthe LOD, an alternate calculationwas performed utilizing the ana-lyte’s peak height compared tothe baseline noise in the m/z 252fragmentation chromatogram. Bythis method, the LOD was de-fined as the lowest concentrationof analyte producing a peakgreater than or equal to threetimes the baseline noise of the ionchromatogram. The lower limitof quantitation (LOQ) was de-fined as the concentration calcu-lated from the mean of the zeroresponses plus five times the stan-dard deviation.25
■ RESULTSWe developed a sensitive quantitative
GC/MS method for clonidine employing a liq-uid extraction procedure and derivatizationwith MTBSTFA + 1% TBDMCS to detectand quantify clonidine in plasma samples fromhorses. Figure 1 shows the chemical structureand expected isotopic profile of underivatizedclonidine. The selected ion chromatogram ofTBDMS derivative of clonidine extracted fromplasma spiked with 250 ng/ml of clonidinestandard is shown in Figure 2. Figure 3 andTable 1 present the full-scan mass spectrum ofderivatized clonidine peak as shown in Figure2, as well as the underivatized clonidine stan-dard (AORC library entry) and the interpreta-tion of the derivatized clonidine spectrum. Thechemical structure of the TBDMS derivative ofclonidine along with its predicted isotopic pro-file is shown in Figure 4. For quantitation, theintense ion at m/z 178 was chosen for SIM-MSof TBDMS isoxsuprine derivative (internal
Figure 6. Head drop following IV injection of clonidine at 0.025 mg/kg.
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standard, data not shown) andthe intense ion at m/z 252 waschosen for SIM-MS of TBDMSclonidine derivative. The result-ant standard curve was linearfrom 1 ng/ml to 250 ng/ml, withthe r 2 value of the assay (n = 6)being 0.997 ± 0.005 (SD). Figure5 presents a typical standardcurve for clonidine, with theLOD for this method being 0.3ng/ml and the LOQ being 0.6ng/ml. In addition, an alternatecalculation for LOD was per-formed utilizing the analyte’speak height compared with thebaseline noise in the m/z 252fragmentation chromatogram. Bythis method, the LOD was de-fined as the lowest concentrationof analyte producing a peak greater than orequal to three times the baseline noise of theion chromatogram and was 0.0154 ng on col-umn (1 ng/65 µl, data not shown).
For within-run, the coefficient of variation(CV) for 2.5 ng/ml (n = 6) was 6.11%, for 25ng/ml (n = 6) was 5.48%, and for 200 ng/ml(n = 6) was 2.93%, with the mean CV of4.84% (Table 2) less than the 15% CV valueacceptable for the assay validation. In addition,the within-run accuracies for this method (n =6 for each value) were 96.13% ± 5.87 (SD),103.2% ± 5.66 (SD), and 101.8% ± 2.99 (SD)for 2.5, 25, and 200 ng/ml standard solutions,respectively (Table 2); the accuracy require-ments for validation of the assay are between85% and 115%.
For between-run, the CV for 2.5 ng/ml (n =6) was 6.71%, for 25 ng/ml (n = 6) was 8.30%,and for 200 ng/ml was 3.20%, with the meanCV of 6.07% (Table 2). In addition, the be-tween-run accuracies for this method (n = 6 foreach value) were 101.3% ± 6.80 (SD), 95.33%
± 7.92 (SD), and 102.3% ± 3.28 (SD) for 2.5,25, and 200 ng/ml standard solutions, respec-tively (Table 2). The extraction efficiency wasdetermined in three different concentrations(n = 6 for each value): low (2.5 ng/ml) = 96%± 9.6 (SD), middle (25 ng/ml) = 80% ± 6.0(SD), and high (100 ng/ml) = 75.5% ± 6.1(SD; Table 3). The measurement uncertaintyand expanded uncertainty were determined inthree different concentrations (n = 6 for eachvalue): low (2.5 ng/ml) = 3.92% and 9.79%,middle (25 ng/ml) = 2.45% and 6.12%, andhigh (100 ng/ml) = 2.49% and 6.22%, respec-tively (Table 3).
In addition, alternative derivatizations usingBSTFA + 1% TMCS, Barb-Prep, and MSTFA+ 1% TMCS were evaluated for the detectionand quantitation of clonidine using theGC/MS and extraction method indicated inMaterials and Methods. Clonidine was not de-tected following its derivatization with Barb-Prep for 2 hours. Derivatization of clonidinewith BSTFA + 1% TMCS for 2 hours generat-
L. Dirikolu, E. T. McFadden, K. J. Ely, H. ElkHoly, A. F. Lehner, and K. Thompson
Figure 7. Reversal of sedative action of clonidine (0.025 mg/kg) follow-ing IV injection of yohimbine (0.12 mg/kg) in DMSO.
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Sedative Action of ClonidineWas Rapidly Reversed with Yohimbine
ed both mono- and bis-trimethylsilyl (TMS)derivatives of clonidine (data not shown), and,therefore, this derivatization agent was not fur-ther evaluated for the detection and quantita-tion of clonidine. Derivatization of clonidinewith MSTFA + 1% TMCS for 2 hours gener-ated bis-TMS derivative of clonidine (data notshown). The highly specific ion m/z 338 of thebis-TMS derivative of clonidine (data notshown) was a much smaller percentage of thetotal mass spectral intensity than was that of them/z 252 ion of the TBDMS derivative of cloni-dine. Correspondingly, areas acquired duringSIM acquisition of the bis-TMS derivative wereonly about one-tenth those of the TBDMS de-rivative, and, therefore, the TBDMS derivativewas chosen over the TMS derivative for quanti-tative purposes.
In clinical pharmacology studies, horses didnot have any locomotor activity for 30 to 45minutes after IV administration of clonidine at0.025 mg/kg. Clonidine produced rapid, pro-found sedation as evidenced by relaxation of
the lower lip, drooping of eyelids,and extreme head drop (Figure 6).As suggested by the rapidity of thehead drop, the horses were clini-cally sedated within minutes ofclonidine administration. Themaximum sedative effect of cloni-dine was observed within 15 to 20minutes after IV injection. Headheight remained approximately50% of the pretreatment value forabout 45 minutes after clonidineinjection. Although horses re-mained standing and were able towalk 45 minutes after IV cloni-dine administration, the headdrop persisted for 2.5 to 3 hoursafter injection (Figure 6). Figure 7illustrates the rapid reversal of thesedative action of clonidine fol-
lowing yohimbine injection in two horses given0.025 mg/kg of clonidine. In this experiment, 2to 3 minutes after IV injection of yohimbine(0.12 mg/kg), head height was approximately50% of pretreatment value; by 5 to 10 minutesafter injection, head height was within normallimits and the horses were clinically alert.
Figure 8 illustrates the rapid onset of analge-sia (within 5 minutes) following IV injectionof 0.025 mg/kg clonidine. IV administrationof 0.025 mg/kg of clonidine failed to providemaximal analgesia (300% of control value) inall horses, although the limited analgesic effectpersisted for about 60 minutes. Clonidine in-duced its maximum analgesic activity within30 minutes after administration in all horsesincluded in this study, and analgesia returnedto control values by 75 to 90 minutes afteradministration.
The mean plasma concentration (±SD) ofclonidine following 0.025 mg/kg single IV in-jection of clonidine is shown in Figure 9; asshown, the plasma concentration of clonidine
Figure 8. Hoof withdrawal reflex latency as a measurement of analgesiafollowing IV injection of 0.025 mg/kg clonidine.
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Onset of Analgesia Was Rapidafter IV Administration of Clonidine
153
was relatively closely distributedamong the four horses included inthis study. The plasma concentra-tion of clonidine rapidly declinedfollowing IV administration; at30 minutes after administration,the mean plasma concentration ofclonidine was 1.4 ± 0.45 ng/ml.The plasma concentration ofclonidine 40 minutes after ad-ministration was above the loweststandard curve concentration (1ng/ml) only in one of the horses;the plasma concentration ofclonidine in this horse was around1.1 ng/ml. Clonidine was still de-tectable up to 2 hours after a sin-gle IV injection (0.025 mg/kg),although the plasma concentra-tions were lower than 1 ng/ml af-ter 40 minutes postadministration.
■ DISCUSSION AND CONCLUSIONClonidine has been marketed for human use
for a number of years as a centrally acting α2-receptor agonist to decrease blood pressure.8
Other centrally mediated effects of clonidineinclude sedation10,11 and analgesia,12,13 and, assuch, clonidine has the potential to producesedative or tranquilizing effects as well as anal-gesia in horses.
In a recent study, it was suggested that variousα2-receptor agonists were being dissolved in vi-tamin B12 solutions containing alcohol and in-jected IV into horses before races.17 The ration-ale for administering α2-receptor agonists is thatmost horses experience pulmonary hyperten-sion during running, leading to EIPH, a con-siderable problem in the horseracing industry.Known as an antihypertensive in human medi-cine, clonidine could reduce pulmonary arterialblood pressure in racing horses and, thus, po-tentially reduce the incidence or severity of
EIPH. On the other hand, as an α2-agonistagent, clonidine may also have the ability totranquilize or sedate horses and may also havesome bronchodilator activity. Clonidine is cur-rently classified as an ARCI class 3 agent. Assuch, clonidine is considered to have the poten-tial to influence the outcome of a race, and itsadministration to a horse shortly before posttime would clearly contravene the rules of rac-ing in most jurisdictions.
In this study, it was shown that IV injectionof clonidine at 0.025 mg/kg produced rapid,profound sedation as evidenced by relaxation ofthe lower lip, drooping of eyelids, and extremehead drop. As suggested by the rapidity of thehead drop, the horses were clinically sedatedwithin minutes of clonidine administration.The maximum sedative effect of clonidine wasobserved within 15 to 20 minutes after IV in-jection and persisted for 2.5 to 3 hours after in-jection. Administration of yohimbine quicklyreversed the sedative action of clonidine, whichis consistent with the suggestion that the major
L. Dirikolu, E. T. McFadden, K. J. Ely, H. ElkHoly, A. F. Lehner, and K. Thompson
Figure 9. Mean plasma concentration (±SD) of clonidine from fourhorses following IV injection of 0.025 mg/kg clonidine.
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pharmacologic responses to clonidine are medi-ated through α2-receptors.
Clonidine was also able to produce analgesiceffects following IV administration but failedto provide maximal analgesia (300% of controlvalue) in all horses; however, the limited anal-gesic effect persisted for about 60 minutes.Clonidine induced its maximum analgesic ac-tivity within 30 minutes after administrationin all horses included in this study, and analge-sia returned to control values by 75 to 90 min-utes after administration.
In conclusion, clinical pharmacology studiessuggest that an IV dose of clonidine at 0.025mg/kg induces sedation as quickly as that pro-duced by other α2 agonists and that sedationand analgesia were generally intense and con-
siderably long lasting. These experiments sug-gest considerable clinical potential for cloni-dine as a sedative and a relatively long-lastinganalgesic in equine medicine.
The pharmacokinetic parameters of cloni-dine have been explained by using two- andthree-compartmental open body models with afirst-order elimination process in humans andvarious animal species. Clonidine is a basiclipid-soluble drug with a high volume of dis-tribution, and the plasma concentration is verylow following its administration both in hu-mans and various animal species.20 Therefore,the evaluation of the pharmacokinetic behav-ior of this drug has been hampered by the lackof sensitive and specific analytic methods capa-ble of measuring concentrations in thepicogram to nanogram range. In this study, wedeveloped a relatively sensitive analytic method
for the detection and quantification of cloni-dine, with the LOD being 0.3 ng/ml and LOQbeing 0.6 ng/ml. Even though this analyticmethod is relatively sensitive, we were not ableto quantify clonidine in plasma samples ofhorses 40 minutes after a single dose at 0.025mg/kg IV. Clonidine was still detectable up to2 hours after a single IV injection at 0.025mg/kg, although the plasma concentrations atall time points after 40 minutes postadminis-tration were lower than our lowest standardconcentration, which was 1 ng/ml.
This study suggests that clonidine is a high-ly lipid-soluble drug that is rapidly distributedfrom vascular spaces into tissue spaces, makingthe analytic detection of this drug very difficultin plasma samples of horses. For safety reasons,
higher doses of clonidine were not investigatedin this study to determine the pharmacokinet-ic parameters of clonidine in performancehorses. Further studies are required to deter-mine the exact pharmacokinetic parameters ofclonidine in horses. Because we were not ableto quantify clonidine in plasma samples ofhorses 40 minutes after IV administration, itwas difficult to establish the relationship be-tween dose and plasma concentration and be-tween plasma concentration and pharmacody-namics. In addition, since the primary goals ofthis study were to determine the pharmacoki-netic parameters and clinical pharmacology ofclonidine, we did not attempt to quantifyclonidine in urine samples. It is possible thatdetection of clonidine in urine samples mightbe a more useful screening method than usingplasma, and urine samples might be useful to
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International, 2004. Available at www.arci.com/
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dine in the rat and cat. J Pharmacol Biopharm 7(5):
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