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. 2022 Nov;36(11):e5457.
doi: 10.1002/bmc.5457. Epub 2022 Aug 1.

Bioanalytical assay for the quantification of the tyrosine kinase inhibitor EAI045 and its major metabolite PIA in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry

M Merve Susam  1   2 , Jing Wang  3 , Alfred H Schinkel  3 , Jos H Beijnen  1   2   3 , Rolf W Sparidans  4
Affiliations

Bioanalytical assay for the quantification of the tyrosine kinase inhibitor EAI045 and its major metabolite PIA in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry

M Merve Susam et al. Biomed Chromatogr. 2022 Nov.

Abstract

EAI045 is a tyrosine kinase inhibitor (TKI) that targets the mutant epidermal growth factor receptor (EGFR). It was developed to control resistance to available EGFR TKIs. In this study, a major metabolite of EAI045, (5-fluoro-2-hydroxyphenyl)(1-oxo-1,3-dihydro-2H-isoindol-2-yl)acetic acid (PIA), was discovered as a hydrolysis product of the parent drug. A validated assay for both analytes in mouse plasma and tissue homogenates from brain, kidney, liver, lung, spleen, and small intestine with content was set up using LC-MS/MS. Samples were prepared by protein precipitation with acetonitrile and with PLX4720 as internal standard. Separation was performed on a bridged ethylene hybrid C18 column by gradient elution with 0.1% v/v formic acid and methanol. Using positive electrospray, detection was performed in selected reaction monitoring mode. A linear calibration range of 2-2,000 ng/ml was used and validated for both analytes. Precision values ranged between 2.0 and 7.5% for EAI045 and between 2.2 and 12.1% for the metabolite, and accuracy values were between 91.1 and 107.6% for EAI045 and between 87.6 and 100.6% for the metabolite. Both analytes were sufficiently stable under the relevant analytical conditions. Finally, the assay was applied to analyze mouse plasma and tissue levels in a pharmacokinetic study in FVB/NRj wild-type female mice treated with oral EAI045.

Keywords: EAI045; EGFR-TKI; LC-MS/MS; metabolite; mouse matrices.

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Figures

FIGURE 1
FIGURE 1
Chemical structures and product spectra of the protonated ions of (a) EAI045, m/z 384.1 at 15 V, (b) PIA [phenyl‐(iso)indol‐acetic acid], m/z 302.1 at 26 V and (c) PLX4720, m/z 414.1 at 31 V with the expected fragments used for quantification.
FIGURE 2
FIGURE 2
Representative chromatograms of double blank pooled plasma samples and lower limit of quantitation spiked plasma samples showing (a) EAI045, (b) PIA and (c) PLX4720. Signals were given an artificial offset.
FIGURE 3
FIGURE 3
Bland–Altman plots for reanalysis data from seven mouse matrix samples from (a) EAI045 (n = 28) and (b) PIA (n = 27).
FIGURE 4
FIGURE 4
Chromatograms of (a) EAI045 in plasma (37.0 ng/ml, black trace) and kidney (14.9 ng/ml, red trace) and (b) PIA in plasma (133 ng/ml, black trace) and kidney (30.8 ng/ml, red trace). Signals were given an artificial offset.
FIGURE 5
FIGURE 5
Plasma concentration–time curve (a) and log plasma concentration‐time curve (b) of EAI045 and PIA in wild‐type FVB/NRj mice (n = 5) during 4 h after oral administration of 20 mg/kg EAI045.
FIGURE 6
FIGURE 6
Tissue concentration of (a) EAI045 and (b) PIA in wild‐type FVB/NRj mice (n = 5) in six tissues, 4 h after oral administration of 20 mg/kg EAI045.
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