Rapid, sensitive and selective HPLC-MS/MS method for the quantification of topically applied besifloxacin in rabbit plasma and ocular tissues: Application to a pharmacokinetic study.
Besifloxacin is a fourth-generation broad-spectrum fluoroquinolone registered for the topical treatment of bacterial conjunctivitis. In this study, a rapid, sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for quantification of besifloxacin in rabbit plasma and ocular tissues using nateglinide as the internal standard (IS). The analyte and IS were separated on a Sepax GP-Phenyl column by isocratic elution with methanol-acetonitrile-5 mM ammonium formate-formic acid (29:55:16:0.1, v/v/v/v) as the mobile phase at a flow rate of 1.2 mL/min, and the total run time was 3.0 min. An electrospray ionization (ESI) source was applied and operated in the positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification, and the monitored transitions were 394.2→377.1 for besifloxacin and m/z 318.3→166.1 for the IS. The calibration curve was linear over the range of 0.103-206 ng/mL for plasma and 2.06-2060 ng/mL for tears, aqueous humor, conjunctiva and cornea with correlation coefficient (r) greater than 0.99. The lower limit of quantification (LLOQ) for besifloxacin was 0.103 ng/mL for plasma and 2.06 ng/mL for other ocular tissues with good accuracy and precision. Intra- and inter-batch precision were both lower than 15% and accuracy ranged from 85% to 115% at all QC levels. The method was successfully applied to the pharmacokinetic study of besifloxacin in rabbit plasma and ocular tissues after single and multiple topical administrations.
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, PR China.
Xiao-Fei
XF
Gu
Changzhou Yabang Pharmacy Research Institute Company Ltd., Changzhou 213163, PR China.
Bai-Yang
BY
Mao
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR China.
Min
M
Xia
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR China.
Yang
Y
Yang
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR China.
Jia-Li
JL
Zhang
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR China.
Da-Song
DS
Yang
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR China.
Wei-Xin
WX
Wu
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, PR China; Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing 210009, PR China.
Ying-Xiang
YX
Du
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR China; Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing 210009, PR China. Electronic address: ddw888@vip.sina.com.
Bin
B
Di
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR China; Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing 210009, PR China. Electronic address: sumengxiang@cpu.edu.cn.
Meng-Xiang
MX
Su
D060433:Administration, Ophthalmic; D000287:Administration, Topical; D000818:Animals; D001082:Aqueous Humor / Q000187:drug effects / Q000378:metabolism; D001381:Azepines / Q000008:administration & dosage* / Q000097:blood* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards; D003228:Conjunctiva / Q000187:drug effects / Q000378:metabolism*; D003315:Cornea / Q000187:drug effects / Q000378:metabolism*; D024841:Fluoroquinolones / Q000008:administration & dosage* / Q000097:blood* / Q000378:metabolism; D008297:Male; D011817:Rabbits; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards*; D013997:Time Factors
D001381:Azepines; D024841:Fluoroquinolones; C522124:besifloxacin
Besifloxacin; Fluoroquinolone; LC–MS/MS; Ocular pharmacokinetics; Rabbit; Topical