Quantification of D55, an apomorphine derivative, in mouse plasma by LC-MS/MS: method development, validation, and improved pharmacokinetics compared with apomorphine

Apomorphine exhibits anti-cell death activity in mitochondrial disease models, but its short elimination half-life and dopaminergic activity limit its therapeutic application. D55 is a modified apomorphine derivative with enhanced anti-cell death activity and no dopaminergic effects.

We established and validated a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of D55 and apomorphine in mouse plasma and characterized their pharmacokinetic profiles. Validation assessed selectivity, linearity, precision and accuracy, injection carryover, extraction recovery, matrix effects, dilution integrity, and stability. Male C57BL/6J mice (n = 5 per group) received a single subcutaneous dose of either compound (0.5 mg/kg), and plasma concentration-time data were analyzed by noncompartmental analysis.

The assay required 10 μL of plasma. For D55, intra- and inter-day precision were ≤10.3%, and accuracy ranged from 86.9% to 108.6%. For apomorphine, precision was ≤6.8%, and accuracy ranged from 99.6% to 104.0%. D55 exhibited a half-life 3.13-fold longer than that of apomorphine (78.4 vs. 24.9 min) and higher systemic exposure (AUC₀-∞), with geometric mean ratios of 2.60 on a mass-concentration basis (ng·min/mL) and 2.36 on a molar-concentration basis (nM·min).

These findings support further development of D55 as a therapeutic candidate for mitochondrial diseases.