title: A rapid and sensitive method to determine tacrolimus in rat whole blood using liquid-liquid extraction with mild temperature ultrasonication and LC-MS/MS.
abstract: Tacrolimus (TAC) is an immunosuppressant widely used in organ transplantation, but its extremely low aqueous solubility causes poor intestinal absorption. There have been efforts to develop an alternative TAC formulation with an improved dissolution rate and oral bioavailability (BA), and the development of a rapid and sensitive analytical method for its in vivo pharmacokinetic study is an essential prerequisite. Thus, here, we develop a novel method to determine TAC in rat whole blood based on liquid chromatography and tandem mass spectrometry, and liquid-liquid extraction (LLE) with mild temperature ultrasonication. For rapid and efficient separation of TAC from other hydrophobic compounds, a C8 column was chosen with isocratic mobile phase elution. With the help of the high specificity and the high sensitivity of multiple reaction monitoring in positive ion mode, the present method showed good performance including specificity, linearity (r(2) ≥ 0.996 within 1-200 ng/mL), sensitivity (the lower limit of quantitation at 1 ng/mL), intra- and inter-day accuracy (88.7-104.5 %) and precision (≤10.3 %), and recovery (94.7-102.6 %). Also, the stability of TAC and ascomycin, the internal standard, in rat whole blood was confirmed before and after the sample preparation. The validated method was satisfactorily applied to a pharmacokinetic study to determine TAC in rat whole blood following oral administration of the marketed product (Prograf(®), Astellas Pharma). In the present study, LLE with mild temperature ultrasonication was successfully expanded to the determination of a drug from whole blood or plasma for the first time. Therefore, the present method can contribute to the rapid in vivo evaluation of novel TAC formulations, and will be able to contribute to the development of TAC formulations with a higher dissolution rate and a higher BA.
authors.affiliation: College of Pharmacy, Dankook University, 119 Dandae-ro, Dongnam-gu, Cheonan, 330-714, Chungnam, South Korea.
authors.forename: Jun Seo
authors.initials: JS
authors.lastname: Park
authors.affiliation: College of Pharmacy, Dankook University, 119 Dandae-ro, Dongnam-gu, Cheonan, 330-714, Chungnam, South Korea.
authors.forename: Ha Ra
authors.initials: HR
authors.lastname: Cho
authors.affiliation: College of Pharmacy, Dankook University, 119 Dandae-ro, Dongnam-gu, Cheonan, 330-714, Chungnam, South Korea.
authors.forename: Myung Joo
authors.initials: MJ
authors.lastname: Kang
authors.affiliation: College of Pharmacy, Dankook University, 119 Dandae-ro, Dongnam-gu, Cheonan, 330-714, Chungnam, South Korea. analysc@dankook.ac.kr.
authors.forename: Yong Seok
authors.initials: YS
authors.lastname: Choi
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards; D059625:Liquid-Liquid Extraction / Q000379:methods / Q000592:standards*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D016559:Tacrolimus / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards*; D013696:Temperature; D013997:Time Factors; D000069453:Ultrasonic Waves
chemical_list: D016559:Tacrolimus
keywords: LC–MS/MS; Mild temperature liquid–liquid extraction; Pharmacokinetics; Rat whole blood; Tacrolimus