title: Validated LC-MS/MS method for the determination of 3-hydroxflavone and its glucuronide in blood and bioequivalent buffers: application to pharmacokinetic, absorption, and metabolism studies.
abstract: The purpose of this study is to develop an UPLC-MS/MS method to quantify 3-hydroxyflavone (3-HF) and its metabolite, 3-hydroxyflavone-glucuronide (3-HFG) from biological samples. A Waters BEH C8 column was used with acetonitrile/0.1% formic acid in water as mobile phases. The mass analysis was performed in an API 5500 Qtrap mass spectrometer via multiple reaction monitoring (MRM) with positive scan mood. The one-step protein precipitation by acetonitrile was used to extract the analytes from blood. The results showed that the linear response range was 0.61-2500.00 nM for 3-HF and 0.31-2500.00 nM for 3-HFG. The intra-day variance is less than 16.5% and accuracy is in 77.7-90.6% for 3-HF and variance less than 15.9%, accuracy in 85.1-114.7% for 3-HFG. The inter-day variance is less than 20.2%, accuracy is in 110.6-114.2% for 3-HF and variance less than 15.6%, accuracy in 83.0-89.4% for 3-HFG. The analysis was done within 4.0 min. Only 10 μl of blood is needed due to the high sensitivity of this method. The validated method was successfully used to pharmacokinetic study in A/J mouse, transport study in the Caco-2 cell culture model, and glucuronidation study using mice liver and intestine microsomes. The applications revealed that this method can be used for 3-HF and 3-HFG analysis in blood as well as in bioequivalent buffers such HBSS and KPI.
authors: Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, The University of Houston, 1441 Moursund Street, Houston, TX 77030, USA.
authors: Beibei
authors: B
authors: Xu
authors: Guanyi
authors: G
authors: Yang
authors: Shufan
authors: S
authors: Ge
authors: Taijun
authors: T
authors: Yin
authors: Ming
authors: M
authors: Hu
authors: Song
authors: S
authors: Gao
mesh_terms: D000042:Absorption; D000818:Animals; D002021:Buffers; D018938:Caco-2 Cells; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005419:Flavonoids / Q000097:blood* / Q000493:pharmacokinetics; D020719:Glucuronides / Q000097:blood*; D006801:Humans; D008297:Male; D051379:Mice; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002021:Buffers; D005419:Flavonoids; D020719:Glucuronides; C041477:3-hydroxyflavone
keywords: 3-HF; 3-HFG; 3-Hydroxyflavone; 3-hydroxyflavone; 3-hydroxyflavone-glucuronide; AUC; Absorption; CE; CXP; DP; HBSS; Hank's balanced salt solution; I.S.; KPI; LLOQ; MPA; MPB; Metabolism; Pharmacokinetics; QC; UDPGA; UPLC; UPLC–MS/MS; area under the curve; collision cell exit potential; collision energy; declustering potential; internal standard; lower limit of quantification; mobile phase A; mobile phase B; potassium phosphate buffer; quality control; ultra-performance liquid chromatography; uridine-5′-diphosphate-β,d-glucuronic acid ester
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