Quantification of clenbuterol in equine plasma, urine and tissue by liquid chromatography coupled on-line with quadrupole time-of-flight mass spectrometry.
Clenbuterol (CBL) is a potent beta(2)-adrenoceptor agonist used for the management of respiratory disorders in the horse. The detection and quantification of CBL can pose a problem due to its potency, the relatively low dose administered to the horse, its slow clearance and low plasma concentrations. Thus, a sensitive method for the quantification and confirmation of CBL in racehorses is required to study its distribution and elimination. A sensitive and fast method was developed for quantification and confirmation of the presence of CBL in equine plasma, urine and tissue samples. The method involved liquid-liquid extraction (LLE), separation by liquid chromatography (LC) on a short cyano column, and pseudo multiple reaction monitoring (pseudo-MRM) by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS). At very low concentrations (picograms of CBL/mL), LLE produced better extraction efficiency and calibration curves than solid-phase extraction (SPE). The operating parameters for electrospray QTOF and yield of the product ion in MRM were optimized to enhance sensitivity for the detection and quantification of CBL. The quantification range of the method was 0.013-10 ng of CBL/mL plasma, 0.05-20 ng/0.1 mL of urine, and 0.025-10 ng/g tissue. The detection limit of the method was 13 pg/mL of plasma, 50 pg/0.1 mL of urine, and 25 pg/g of tissue. The method was successfully applied to the analysis of CBL in plasma, urine and various tissue samples, and in pharmacokinetic (PK) studies of CBL in the horse. CBL was quantified for 96 h in plasma and 288 h in urine post-administration of CLB (1.6 micro g/kg, 2 x daily x 7 days). This method is useful for the detection and quantification of very low concentrations of CBL in urine, plasma and tissue samples.
University of Pennsylvania School of Veterinary Medicine, Department of Clinical Studies, New Bolton Center Campus, Kennett Square, PA 19348, USA.
Fuyu
F
Guan
Cornelius E
CE
Uboh
Lawrence R
LR
Soma
Yi
Y
Luo
Runbo
R
Li
Eric K
EK
Birks
Donna
D
Teleis
Jeffrey A
JA
Rudy
Deborah S
DS
Tsang
D000318:Adrenergic beta-Agonists / Q000032:analysis* / Q000493:pharmacokinetics; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002976:Clenbuterol / Q000032:analysis* / Q000493:pharmacokinetics; D005260:Female; D005554:Forensic Medicine / Q000379:methods; D006736:Horses; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D015813:Substance Abuse Detection / Q000379:methods / Q000662:veterinary*; D014018:Tissue Distribution
D000318:Adrenergic beta-Agonists; D002976:Clenbuterol