Screening, quantification, and confirmation of phenylbutazone and oxyphenbutazone in equine plasma by liquid chromatography-tandem mass spectrometry.
A sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated for screening, quantification, and confirmation of phenylbutazone and oxyphenbutazone in equine plasma. Analytes were recovered from plasma by liquid-liquid extraction followed by separation in a reversed-phase column and identification by mass spectrometry with selected reaction monitoring in negative electrospray ionization mode. Extraction recovery for both analytes was >80%. Limits of detection, quantification, and confirmation for both analytes were 0.01 microg/mL (S/N>or= 3), 0.05 microg/mL, and 0.05 microg/mL, respectively. The assay with d9-labeled phenylbutazone as internal standard (IS) was linear over a range of 0.05-20 microg/mL (r2>0.995). Intra- and interday precision in terms of coefficient of variation was less than 15%. Intra- and interday accuracy (bias%) was within 80-120%. Hemolysis of red blood cells decreased analyte signal intensity but did not affect quantification results because an isotope-labeled IS was used. Analytes were stable in plasma for 24 h at room temperature, 9 days at 4 degrees C, and 45 days at -20 degrees C and -70 degrees C. The method was successfully used in screening, quantification, and confirmation of phenylbutazone in post-competition plasma samples obtained from racehorses. The method is simple, rapid, and reliably reproducible.
You
Youwen
Y
University of Pennsylvania, School of Veterinary Medicine, Department of Clinical Studies, New Bolton Center Campus, Kennett Square, Pennsylvania 19348, USA.
Uboh
Cornelius E
CE
Soma
Lawrence R
LR
Guan
Fuyu
F
Li
Xiaoqing
X
Rudy
Jeffrey A
JA
Chen
Jinwen
J
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D004300:Doping in Sports; D006461:Hemolysis; D006736:Horses; D010113:Oxyphenbutazone / Q000097:blood*; D010653:Phenylbutazone / Q000097:blood*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D010653:Phenylbutazone; D010113:Oxyphenbutazone