Development and validation of a novel method for simultaneous quantification of enzalutamide, darolutamide and their active metabolites in mice dried blood spots using LC-MS/MS: Application to pharmacokinetic study in mice

Abstract

<jats:p>&lt;p&gt;A simple, sensitive and rapid assay method has been developed and validated for the estimation of enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), darolutamide and ORM-15341 (active metabolite of darolutamide) on mice dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electro spray ionization in the positive-ion mode. The method utilizes liquid extraction of enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 from 3 mm punched disks from DBS cards (spiked or study samples). The extracted sample was chromatographed using an isocratic mobile phase (0.2 % formic acid : acetonitrile; 30:70, v/v) on an Atlantis dC18 column. The total run time was 2.5 min. The MS/MS ion transitions monitored were m/z 465 → m/z 209, m/z 451 → m/z 195, m/z 399 → m/z 178, m/z 397 → m/z 194 and m/z 481 → m/z 453 for enzalutamide, N-desmethyl­enzalutamide, darolutamide, ORM-15341 and the IS (apalutamide-d&lt;sub&gt;3&lt;/sub&gt;), respectively. Method validation was performed as per regulatory guideline. The assay had a good linearity over the range of 0.93-2000 ng/mL. The intra- and inter-batch accuracy and precision (%RE &amp;amp; RSD) across quality controls met the acceptance criteria for all the analytes. Stability studies showed that all the analytes were stable on DBS cards for one month. This novel method has been applied to analyze the DBS samples of enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 obtained from a pharmacokinetic study in mice.&lt;/p&gt;</jats:p>

Novel methodology to perform incurred sample reanalysis on dried blood spot cards: Experimental data using darolutamide and filgotinib

“…One‐half was used for half‐DBS ISR analysis and the other half was divided further into two halves to get quarter‐DBS disc and subsequently used for ISR analysis (Figure 1). All the DBS discs (full, half and quarter) were processed in similar lines as mentioned in the following section for the quantitation of respective drugs [see Saini, Sulochana, Zainuddin, & Mullangi (2018) for darolutamide and Dixit, Kiran, Gabani, & Mullangi (2020) for filgotinib]. ISR of all DBS discs (full, half and quarter) was done on day 50.…”

Review of DBS methods as a quantitative tool for anticancer drugs

“…Not all of the published methods involving DBS have reported the assessment of the influence of hematocrit on the assay performance. However, many reports published recently have incorporated the testing of the influence of hematocrit on quantitative data gathered on the analytes in question (Antunes, Raymundo, de Oliveira, et al, ; Antunes, Raymundo, Wagner, et al, ; Boons et al, ; Hahn et al, ; Hawwa et al, ; Irie et al, ; Jager, Rosing, Schellens, & Beijnen, ; Kralj et al, ; Nijenhuis et al, ; Renata et al, ; Saini, Sulochana, Kiran, et al, ; Saini, Sulochana, Zainuddin, & Mullangi, ; Singhal, Shah, Shah, Sharma, & Shrivastav, ; Xie et al, ; Xu et al, ). The DBS assay may be significantly impacted by hematocrit values since hematocrit is a sole determinant that has an effect on the viscosity of the blood; the viscosity of blood plays a role in the flux and diffusive properties of the analytes that are present in blood through DBS sampling system.…”

RP-HPLC-UV Method for Simultaneous Quantification of Second Generation Non-Steroidal Antiandrogens Along with their Active Metabolites in Mice Plasma: Application to a Pharmacokinetic Study

“…1c) and ORM-15341 (▶Fig. 1d) [28] and apalutamide (▶Fig. 1b) [29] in mice blood shown the validated methods application in a mice pharmacokinetic study.…”