![]() ![]() For the first time, we report pharmacokinetics of E2 in guinea pigs after two different doses administered via subcutaneous route.Yang Chen †, Kaijing Zhao †, Fei Liu, Qiushi Xie, Zeyu Zhong, Mingxing Miao, Xiaodong Liu * and Li Liu * The high precision and accuracy with minimal sample volume, no derivatization and short assay run time of 3 mins presents a highly amenable assay for low-level quantitation of E2 in a high-throughput capability. These data suggest that the lower dose of 10 µg/kg E2 administered subcutaneously can achieve physiologically relevant concentration as observed in women lasting up to 12 h in guinea pigs.Ĭonclusion:We developed and validated a novel and sensitive LC-MS/MS assay using OVX guinea pig plasma to successfully obtain the PK profile of E2 in guinea pigs. Similarly, the half-life (t1/2) was 2x higher and plasma clearance was 2x lower after 40 µg/kg dose compared to the 10 µg/kg dose. Like the Cmax, the area under the curve (AUC0-ꚙ) was also 8x higher after 40 µg/kg dose compared to the 10 µg/kg dose suggesting a more than dose proportional increase in plasma concentration after the higher dose. E2 reached an average Cmax of ~540 pg/mL after 10 µg/kg dose whereas the average Cmax was ~4000 pg/mL (8x higher) after 40 µg/kg dose. In PK profiling study, E2 showed rapid absorption following subcutaneous dosing with high Cmax and AUC and elimination half-life of ~3 h. ![]() Dilution integrity assay passed at 10x level with no injection carryover. Stability studies met the acceptance criteria for bench-top, auto sampler, long term storage and freeze-thaw experiments. Absolute mean recovery was ~90% with negligible matrix effect. Intra- and inter-batch precision were <15% and accuracy (☘5-115%) for 6 replicates at 5 different QC concentrations on 3 consecutive days. The method was highly specific with no interference in blank matrix. The assay was linear from 3.90-1000.00 pg/mL with no chemical derivatization. Results:The developed method was validated and found to be robust and sensitive. The plasma concentration data were used to calculate PK parameters of E2 in both the treatment groups by non-compartmental analysis using Phoenix WinNonlin v.6.4. The method was applied to determine the pharmacokinetic (PK) profile of E2 in OVX guinea pigs (weighing 500-700 g) treated with 10 or 40 µg/kg E2, injected subcutaneously and blood samples (~600 µL) collected at 0, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h after dosing. Method was validated as per 2013 FDA Guidance for Bioanalytical Method Validation. Mass spec (AB Sciex 6500 Q-Trap) was operated in negative ion ESI mode with MRM transitions at m/z 271.0/143.0, 145.0, 183.0 for E2 and 275.0/147.0 for E2-d4, respectively. E2/E2-d4 were resolved on a Kinetex C18 Phenyl-Hexyl column with 0.05% Ammonium hydroxide in water (mobile phase A) and 0.05% ammonium hydroxide in methanol (mobile phase B) in 3.0 min. Liquid chromatography was performed on Agilent 1290 Ultra-High Performance Liquid Chromatograph. Extraction of E2 from 100 µL of OVX plasma was achieved by liquid-liquid extraction using 90:10 tert-butyl methyl ether: hexane solvent mixture. ![]() A working stock solution at 100.00 ng/mL was prepared in 60% methanol. Methods:E2 and E2-d4 (deuterated E2 used as an internal standard) were dissolved in methanol at 1.00 mg/mL. Plasma (with no measurable endogenous hormone levels) from ovariectomized (OVX) guinea pigs was used for preparation of calibrators and quality control specimens. Here, we describe a robust liquid-chromatography tandem mass spectrometry (LC-MS/MS) assay that was developed and validated in-house. To better understand the underlying effects of E2 on drug-induced arrhythmia a prerequisite is to be able to measure the circulating levels in vivo accurately. Because the duration of QT interval is a risk factor for drug-induced arrhythmia, an understanding of how female sex hormones including E2 affect the QT interval is important to understand drug-induced arrhythmia susceptibility in women. In females, QT interval is longer during the follicular phase when the circulating level of estradiol (E2) is high than during the luteal phase when the progesterone-to-estradiol ratio is increased. Purpose:Evidence from clinical and basic science studies suggest that sex differences in susceptibility to some arrhythmias arise directly from fundamental differences in cardiac tissue. ![]()
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