title: Development and validation of a highly sensitive LC-MS/MS method for the determination of acacetin in human plasma and its application to a protein binding study.
abstract: A highly sensitive bioanalytical method for the quantification of acacetin in human plasma was developed and comprehensively validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A minimal volume of human plasma sample (20 μL) was prepared by simple deproteinization with 80 μL of acetonitrile. Chromatographic separation was performed using Kinetex C18 column with an isocratic mobile phase consisting of water and acetonitrile (20:80, v/v) containing 0.1 % formic acid at a flow rate of 0.3 mL/min over a total run time of 2.0 min. Mass spectrometric detection was performed using multiple reaction-monitoring modes at the mass/charge transitions m/z 285.22 → 242.17 for acacetin and m/z 277.59 → 175.04 for chlorpropamide (internal standard). The calibration curve was linear over the range of 0.1-500 ng/mL with a lower limit of quantitation of 0.1 ng/mL. The coefficients of variation for both intra- and inter-day validation were less than 11.9 %, and the intra- and inter-day accuracy ranged from 96.8 to 108 %. Mean recovery of acacetin in human plasma was within the range of 91.5-95.6 %. This validated LC-MS/MS method was successfully applied to a human plasma protein binding study that indicated extensive and concentration-independent protein binding of acacetin in human plasma.
authors.affiliation: New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, 701-310, Republic of Korea.
authors.forename: Sang-Bum
authors.initials: SB
authors.lastname: Kim
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 151-742, Republic of Korea.
authors.forename: Taehun
authors.initials: T
authors.lastname: Lee
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 151-742, Republic of Korea.
authors.forename: Hun Seok
authors.initials: HS
authors.lastname: Lee
authors.affiliation: Institute of Nanoscience and Technology, Hanyang University, Seoul, 133-791, Republic of Korea.
authors.forename: Chung Kil
authors.initials: CK
authors.lastname: Song
authors.affiliation: College of Pharmacy, Kangwon National University, Chuncheon, 200-701, Republic of Korea.
authors.forename: Hyun-Jong
authors.initials: HJ
authors.lastname: Cho
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 151-742, Republic of Korea.
authors.forename: Dae-Duk
authors.initials: DD
authors.lastname: Kim
authors.affiliation: College of Pharmacy, Gachon University, 191 Hambakmoei-ro, Yeonsu-gu, Incheon, 406-799, Republic of Korea. hjmaeng@gachon.ac.kr.
authors.forename: Han-Joo
authors.initials: HJ
authors.lastname: Maeng
authors.affiliation: College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, 1666 Youngsan-ro, Muan-gun, Jeonnam, 534-729, Republic of Korea. isyoon@mokpo.ac.kr.
authors.forename: In-Soo
authors.initials: IS
authors.lastname: Yoon
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D047309:Flavones / Q000097:blood* / Q000493:pharmacokinetics; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D011485:Protein Binding; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D047309:Flavones; C023717:acacetin
keywords: Acacetin; Human plasma; LC–MS/MS; Protein binding