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. Mitochondrial diseases affect cell structures that produce energy, causing serious health problems. Apomorphine, a medicine used to treat Parkinson’s disease, has shown protective effects in laboratory studies of cells from patients with mitochondrial disease. However, the body removes it quickly, and its effects on dopamine receptors may limit its use. D55 is a modified form of apomorphine being studied as a possible treatment. We developed and tested a laboratory method to measure D55 and apomorphine in very small blood plasma samples from mice. Plasma is the liquid part of blood. The method required only 10 microliters of plasma and provided reliable measurements. We then gave healthy male mice a single injection of either compound under the skin. D55 persisted about three times longer than apomorphine during the final stage of clearance from plasma. Overall exposure to D55 was also higher. These findings provide a way to measure D55 and help to guide further research. This study did not test whether D55 improves mitochondrial diseases, nor does it determine its safety. Further studies are needed to assess its benefits and risks.