Article’s

Midostaurin (PKC412) is an orally bioavailable multi-kinase inhibitor approved for the treatment of FLT3-mutated Acute Myeloid Leukemia (AML) and advanced systemic mastocytosis. Due to its high lipophilicity ($\log P \sim 4.6$) and narrow nanogram-level therapeutic window, establishing a sensitive and robust analytical method is essential. This study demonstrates the development, optimization, and validation of a Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method for Midostaurin compliant with ICH Q2(R2) guidelines. Chromatographic separation was achieved on a Hypersil GOLD C18 column ($100\text{ mm} \times 2.1\text{ mm}$, $1.8\,\mu\text{m}$) using an isocratic mobile phase of 0.1% v/v formic acid in water and acetonitrile (25:75 v/v) at a flow rate of $0.30\text{ mL/min}$. Mass spectrometric detection was performed in ESI+ mode utilizing Multiple Reaction Monitoring (MRM) targeting $m/z\ 571.20 \to 398.20$ for Midostaurin and $m/z\ 576.25 \to 310.10$ for Midostaurin-$d_5$ (Internal Standard). Midostaurin eluted at $2.15\text{ min}$ with a total run time of $3.50\text{ min}$. High linearity was obtained over $0.50\text{ ng/mL}$ to $100.00\text{ ng/mL}$ ($r^2 = 0.9994$). Limits of Detection and Quantitation were $0.15\text{ ng/mL}$ and $0.50\text{ ng/mL}$, respectively. Forced degradation testing per ICH Q1A(R2) demonstrated the stability-indicating nature of the method.