Validated LC-MS/MS Method for Simultaneous Quantitation of Enasidenib and its Active Metabolite, AGI-16903 in Small Volume Mice Plasma: Application to a Pharmacokinetic Study.

Enasidenib is a selective mutant isocitrate dehydrogenase 2 inhibitor approved for the treatment of relapsed and refractory acute myeloid leukemia patients. A sensitive and rapid method has been developed and validated as per regulatory guideline for the simultaneous quantitation of enasidenib and its active metabolite, AGI-16903 in mice plasma using an LC-MS/MS. Enasidenib and AGI-16903 along with internal standard were extracted from mice plasma using simple protein precipitation method. Chromatographic resolution of enasidenib, AGI-16903 and the internal standard (close analogue of AGI-16903) was achieved on a Chromolith RP-18e column using 0.2% formic acid:acetonitrile (15:85, v/v) as an eluent, which was delivered at a flow-rate of 1.2 mL/min. The MS/MS ion transitions monitored were m/z 474.1→267.2, 402.1→188.1 and 421.0→146.1 for enasidenib, AGI-16903 and the internal standard, respectively. The linearity range was 1.01-3023 ng/mL for both enasidenib and AGI-16903. The within-run and between-run accuracy and within-run and between-run precision were in the range of - 2.29 to 2.72 (as one value is in negative side). and 4.65-9.82%, respectively for enasidenib; 0.19-10.3 and 3.22-9.22%, respectively for AGI-16903. Both enasidenib and AGI-16903 were found to be stable in stability (up to three freeze-thaw cycles and for long-term at -80°C for 30 days) and processed (bench-top for 6 h and in in-injector for 24 h) samples. Application of the validated method was shown in a pharmacokinetic study in mice.

Validation of an LC-ESI-MS/MS method for the determination of apalutamide, a novel non-steroidal anti-androgen in mice plasma and its application to a pharmacokinetic study in mice.

A sensitive, specific, selective and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of apalutamide in mice plasma using apalutamide-d3 as an internal standard (I.S.). Sample preparation was accomplished through a simple protein precipitation process. Chromatography of apalutamide and the I.S. was achieved on an Atlantis dC18 column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (20:80, v/v) delivered at a flow rate of 0.8 mL/min. LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique in positive ion mode and the transitions of m/z 478 → 450 and m/z 481 → 453 were used to measure the derivative of apalutamide and the I.S, respectively. The total chromatographic run time was 2.5 min and the elution of apalutamide and I.S. occurred at 1.10 and 1.09 min, respectively. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. Linearity was established in the concentration range of 1.02-2030 ng/mL (r > 0.995) for apalutamide. The intra- and inter-day accuracy and precision for apalutamide in mice plasma were in the range of 2.11-8.44 and 2.51-6.09%, respectively. Apalutamide was found to be stable under various stability conditions. This novel method has been applied to a pharmacokinetic study in mice.