Validated HPLC-UV method for quantification of paxalisib, a pan PI3K and mTOR inhibitor in mouse plasma: Application to a pharmacokinetic study in mice.

Paxalisib is a pan-PI3K and mTOR inhibitor, currently entering into Phase II clinical trials as a potential drug to treat glioblastoma patients. We report the development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of paxalisib in mouse plasma as per the US Food and Drug Administration regulatory guidelines. From the mouse plasma, paxalisib and the internal standard (IS; filgotinib) were extracted using ethyl acetate as an extraction solvent. The chromatographic separation of paxalisib and the IS was accomplished on a Symmetry C18 (250 × 4.6 mm, 5.0 µm) column maintained at 40°C using 10 mm ammonium formate and acetonitrile in gradient conditions at a 0.8 ml/min flow-rate. The injection volume was 20 µl. The elution was monitored using a photo-diode array detector set at λmax 280 nm. Paxalisib and the IS eluted at 6.5 and 5.9 min, respectively with a total run time of 10 min. The calibration curve was linear over the range of 111-4,989 ng/ml. Inter- and intraday precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated and the results met the acceptance criteria. The validated HPLC method was extended to assess the pharmacokinetic parameters of paxalisib in mice.

Validated LC-MS/MS method for the determination of copanlisib in mouse dried blood spots.

Copanlisib is a dual PI3K-δ inhibitor, used in follicular lymphoma treatment. In this research, we report a validated LC-MS/MS method for quantifying copanlisib from a mouse dried blood spot (DBS). We validated the method in line with the guidelines of the US Food and Drug Administration. The liquid-liquid extraction technique was used to extract copanlisib from the DBS discs. We used an Atlantis dC18 column and isocratic mobile phase for the chromatographic separation of copanlisib and the internal standard (idelalisib). The flow was 0.90 ml/min. Under the optimized chromatographic conditions, the retentions of copanlisib and the internal standard were 0.98 and 0.93 min, respectively. Each injection total run time was 2.50 min. The MS/MS ion transitions monitored were m/z 481.31 â†’ 128.00 and 416.10 â†’ 176.10 for copanlisib and the internal standard (IS) idelalisib, respectively. We have used a broad calibration range (1.01-4,797 ng/ml) with a determination coefficient (r2 ) of 0.997. All of the evaluated parameters met the acceptance criteria. Hematocrit did not influence the DBS copanlisib concentrations. We have used the validated method to derive the intravenous pharmacokinetic parameters by quantifying copanlisib in mouse plasma.

Validated liquid chromatography-tandem mass spectrometry method for simultaneous quantitation of bendamustine and copanlisib in mouse plasma: Application to a pharmacokinetic study in mice.

In this study, we report the development and validation of an LC-tandem mass spectrometry method for the simultaneous quantitation of bendamustine and copanlisib in mouse plasma as per the US FDA regulatory guidelines. The sample processing involves extraction of bendamustine and copanlisib along with internal standard (IS; warfarin) from 50 µL mouse plasma using a liquid-liquid extraction method. The chromatographic separation of bendamustine, copanlisib and the IS was achieved on an Atlantis dC18 column using an isocratic mobile phase (5 mM ammonium acetate:methanol, 20:80 v/v). Bendamustine, copanlisib and the IS eluted at 0.88, 1.39 and 0.74 min, respectively, with a total run time of 2.5 min. The calibration curve ranged from 3.99-2996 and 4.33-3248 ng/mL for bendamustine and copanlisib, respectively. Inter- and intra-day precision and accuracy, stability in processed samples and upon storage, dilution integrity and incurred sample reanalysis were investigated for both the analytes. The intra- and inter-day precisions were in the ranges of 2.01%-5.05% and 2.74%-6.13% and 1.98%-7.64 and 8.62%-9.04% for bendamustine and copanlisib, respectively. Stability studies showed that both analytes were stable on bench top for 6 h, in auto-sampler for 24 and at -80°C for 30 days. The validated method was successfully applied to a pharmacokinetic study in mice.

Pharmacokinetics of Darolutamide in Mouse - Assessment of the Disposition of the Diastereomers, Key Active Metabolite and Interconversion Phenomenon: Implications to Cancer Patients.

BACKGROUND: Darolutamide is recently approved for the treatment of non-metastatic castrate resistance prostate cancer. Hitherto, no stereoselective pharmacokinetic data have been published pertaining to darolutamide and its diastereomers in animals or humans. The key aims of the experiment were to examine darolutamide, S,S-darolutamide and S,R-darolutamide with respect to (a) assessment of in vitro metabolic stability and protein binding and (b) characterization of in vivo oral and intravenous pharmacokinetics in mice. METHODS: In vitro (liver microsomes stability and protein binding) and in vivo experiments (oral/intravenous dosing to mice) were carried out using darolutamide, S,S-darolutamide and S,Rdarolutamide. Besides, tissue levels of darolutamide, S,S-darolutamide and S,R-darolutamide were measured following oral and intravenous dosing. Appropriate plasma/tissue samples served to determine the pharmacokinetics of various analytes in mice. Liquid chromatography in tandem with mass spectrometry procedures enabled the delineation of the plasma pharmacokinetics, in vitro and tissue uptake data of the various analytes. RESULTS: Chiral inversion was absent in the metabolic stability study. However, darolutamide showed profound stereoselectivity (S,S-darolutamide greater than S,R-darolutamide) after either intravenous or oral dosing. S,R-darolutamide but not S,S-darolutamide showed conversion to its antipode post oral and intravenous dosing to mice. Regardless of oral or intravenous dosing, active keto darolutamide formation was evident after administration of darolutamide, S,S-darolutamide or S,R- darolutamide. Tissue data supported the observations in plasma; however, tissue exposure of darolutamide, S,Sdarolutamide and S,R-darolutamide was much lower as compared to plasma. CONCLUSION: In lieu of the human pharmacokinetic data, although the administration of diastereomeric darolutamide was justified, it is proposed to delineate the clinical pharmacokinetics of S,Rdarolutamide and S,S-darolutamide relative to darolutamide in future clinical pharmacology studies.

Enantioselective in vitro ADME, absolute oral bioavailability, and pharmacokinetics of (-)-lumefantrine and (+)-lumefantrine in mice.

Lumefantrine (LFN) is a chiral antimalarial drug. Enantioselective in vitro attributes and absolute oral pharmacokinetics for (-)-LFN and (+)-LFN have been characterized in mice. No stereoselectivity was seen with either of the enantiomers when compared with rac-LFN in the executed in vitro studies (solubility, metabolic stability, protein binding, permeability and blood partitioning). Post intravenous or oral administration of rac-LFN, the AUC0-∞ and MRT of (+)-LFN was higher over (-)-LFN, which is reflected in higher clearance value for (-)-LFN. Following (-)-LFN intravenous administration to mice, the key PK parameters were comparable to (-)-LFN from rac-LFN; however, post intravenous administration of (+)-LFN alone to mice, the AUC0-∞ was 1.3-fold higher than (+)-LFN from rac-LFN. Similarly, post oral administration of (-)-LFN to mice, both AUC0-∞ and Cmax were 1.3-fold higher than (-)-LFN from rac-LFN. On other hand, (+)-LFN showed 1.4-fold higher AUC0-∞ and 1.7-fold higher Cmax post oral administration over (+)-LFN from rac-LFN.

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

Different options on performing incurred sample reanalysis (ISR) on dried blood spot (DBS) cards were investigated using drugs belonging to various therapeutic areas: (a) darolutamide (to treat prostate cancer) and (b) filgotinib (to treat rheumatoid arthritis). The proposed novel methodology included the generation of half-DBS and quarter-DBS discs after initial blood collection using the full-DBS discs. Accordingly, blood collection via DBS was performed in male BALB/c mice following intravenous and oral dosing of darolutamide; in male Sprague Dawley rats following intravenous and oral dosing of filgotinib. The ISR data generated from the full-DBS disc, half-DBS disc and quarter-DBS disc were compared for the assessment of the proposed methodology. Quantification of darolutamide and filgotinib was accomplished using liquid chromatography-electrospray ionization/tandem mass spectrometry methods. Darolutamide and filgotinib ISR samples, which were collected and prepared using full-, half- and quarter-DBS discs, met the acceptance criteria for ISR analysis. In conclusion, this is the first report showing a viable tool for the performance of ISR on DBS cards. The use of quarter- or half-DBS discs would aid in not only ISR but also in long-term storage experiments of analytes because it would avoid the need for additional blood sampling in patients.

Validated LC-MS/MS Method for Simultaneous Quantitation of SAFit-1 and SAFit-2 in Mice Plasma: Application to a Pharmacokinetic Study.

SAFit-1 and SAFit-2 are selective FKBP51 (FK506-binding protein 51) ligands. In this paper, we present the development and validation data of an LC-MS/MS method for the simultaneous quantitation of SAFit-1 and SAFit-2 in mice plasma as per FDA regulatory guideline. SAFit-1 and SAFit-2 along with internal standard were extracted from mice plasma using liquid-liquid extraction method. Chromatographic resolution of SAFit-1, SAFit-2 and the internal standard (warfarin) was achieved on an X-Terra phenyl column using 0.2% formic acid:acetonitrile (20:80, v/v) as an eluent, which was delivered at a flow-rate of 0.9 mL/min. The MS/MS ion transitions monitored were m/z 748.4→420.4, 803.7→384.3 and 309.2 →163.2 for SAFit-1, SAFit-2 and the internal standard, respectively. The linearity range was 2.45-2446 ng/mL for both SAFit-1 and SAFit-2. The intra- and inter-day accuracy and intra- and inter-day precision were in the range of 0.90-1.07 and 2.38-10.8%, respectively for SAFit-1; 0.97-1.15 and 0.23-12.5%, respectively for SAFit-2. Both SAFit-1 and SAFit-2 were found to be stable in stability studies (up to three freeze-thaw cycles and for long-term at -80°C for 30 days) and processed (bench-top for 3 h and in in-injector for 16 h) samples. The application of the validated method was shown in a pharmacokinetic study in mice.

Enantioselective LC-ESI-MS/MS method for quantitation of (-)-lumefantrine and (+)-lumefantrine in mice plasma and application to a pharmacokinetic study.

We developed and validated a simple, sensitive, selective, and reliable LC-MS/MS-ESI method for the direct quantitation of lumefantrine (LFN) enantiomers [(-)-LFN and (+)-LFN] in mice plasma as per regulatory guideline. LFN enantiomers and carbamazepine (internal standard) were extracted from mice plasma using Strata X SPE (solid-phase extraction) cartridges. Good resolution between enantiomers was achieved on a Chiralpak IA-3 column using an isocratic mobile phase (0.1% of diethyl amine in methanol), which was delivered at a flow rate of 0.8 mL/min. Detection and quantitation were performed using multiple reaction monitoring mode following the transitions m/z 530.27 → 512.30 and 237.00 → 194.00 for LFN enantiomers and the internal standard, respectively, in the positive-ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 2.39-895 ng/mL for each enantiomer. The intra- and inter-day precisions were in the range of 1.03-6.14 and 6.36-8.70 and 2.03-4.88 and 5.82-11.5 for (-)-LFN and (+)-LFN, respectively. Both (-)-LFN and (+)-LFN were found to be stable under different stability conditions. The method was successfully used to delineate stereoselective pharmacokinetics of LFN enantiomers in mice after an oral administration of rac-LFN (20 mg/kg). The pharmacokinetic results indicated that the disposition of LFN enantiomers was stereoselective in mice.

Preparation and optimization of nilotinib self-micro-emulsifying drug delivery systems to enhance oral bioavailability.

Objective: The objective of the current research was to prepare self-micro-emulsifying drug delivery systems (SMEDDS) for BCS class II drug, nilotinib to enhance its oral bioavailability.Methodology: Different types of excipients like oil, surfactant, and co-surfactant were evaluated for drug solubility. Among the screened excipients, Capryol 90, Transcutol HP, and Tween 80 were selected as oil, co-surfactant, and surfactant, respectively, to construct a ternary phase diagram to identify a homogenous mixture. Characterization performed for the prepared SMEDDS for its particle size/droplet size, emulsification time, phase separation, droplet morphology, in vitro drug release, and oral bioavailability.Results: Prepared SMEDDS showed the highest of 87% drug release in in vitro drug release experiment. SMEDDS drug release was superior over suspension formulation, which could be attributed to oil/surfactant ratios and particle size of the SMEDDS. The acquired pharmacokinetic parameters indicate that twofold increase in systemic exposure of SMEDDS compared with nilotinib suspension formulation. A similar twofold increase in relative oral bioavailability was also observed when compared SMEDDS formulation with suspension formulation. Delayed Tmax (time to reach peak plasma concentrations) was observed with SMEDDS over suspension formulation, which was evident by slow rate of absorption of nilotinib from SMEDDS.Conclusion: This research demonstrated that SMEDDS could be an effective approach to improve solubility and oral bioavailability for the BCS class II poorly soluble nilotinib.

Validated LC-MS/MS method for quantitation of a selective JAK1 inhibitor, filgotinib in rat plasma, and its application to a pharmacokinetic study in rats.

Filgotinib is a selective JAK1 (Janus kinase) inhibitor, filed in Japan for the treatment of rheumatoid arthritis. In this paper, we report a validated liquid chromatography coupled with tandem mass spectrometry for the quantification of filgotinib in rat plasma using tofacitinib as an internal standard (IS) as per the Food and Drug Administration regulatory guidelines. Filgotinib and the IS were extracted from rat plasma using ethyl acetate as an extraction solvent and chromatographed using an isocratic mobile phase (0.2% formic acid:acetonitrile; 20:80, v/v) at a flow rate of 0.9 mL/min on a Gemini C18 column. Filgotinib and the IS were eluted at ~1.31 and 0.89 min, respectively. The MS/MS ion transitions monitored were m/z 426.3 → 291.3 and m/z 313.2 → 149.2 for filgotinib and the IS, respectively. The calibration range was 0.78-1924 ng/mL. No matrix effect and carryover were observed. Intra- and inter-day accuracies and precisions were within the acceptance range. Filgotinib was stable for three freeze-thaw cycles: on bench-top up to 6 h, in an autosampler up to 21 h, and at -80°C for 1 month. This novel method has been applied to a pharmacokinetic study in rats.

Incurred sample reanalysis of cefuroxime in rabbit ocular tissues-A case study.

In this paper, we present the incurred sample reanalysis (ISR) data for cefuroxime in various ocular tissues of rabbits. Based on the cefuroxime concentration vs. time profile in various ocular tissues, three chosen time points enabled ISR assessment. Cefuroxime was quantitated in the ocular tissues using a published liquid chromatography-electrospray ionization/tandem mass spectrometry method operated under the multiple reaction-monitoring mode in positive ion mode. Regardless of the ocular tissue, the linearity range was 12.7-2760 ng/ml with a correlation coefficient (r2 ) of ≥0.996. All of the ISR samples representing various ocular tissues met the acceptance criteria. To the best of our knowledge, this is the first report showing the ISR of ocular tissues in any species.

Validated DBS method for filgotinib quantitation in rat dried blood spots and its application to a pharmacokinetic study in rats.

Filgotinib is a selective JAK1 (Janus kinase) inhibitor, showed efficacy in patients suffering from moderate-to-severe rheumatoid arthritis. In this paper, we present the data on the development and validation of a sensitive, selective and high-throughput LC-MS/MS (liquid chromatography with tandem mass spectrometry) method for the quantitation of filgotinib from rat dried blood spot (DBS) cards. To the DBS disc cards, 0.2% formic acid enriched with internal standard (IS) was added and sonicated. Thereafter the extraction of filgotinib and the IS (tofacitinib) was accomplished using ethyl acetate as an extraction solvent. The resolution of filgotinib and the IS was achieved on a Gemini C18 column with an isocratic mobile phase, which is a mixture of 0.2% formic acid:acetonitrile (20:80, v/v) at a flow-rate of 0.9 mL/min. The total run time was 2.90 min and the retention time of filgotinib and the IS was ~1.31 and 0.89 min, respectively. Filgotinib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transition of 426.3→291.3 and m/z 313.2→149.2, respectively, for quantitation. The calibration range was 1.37-1937 ng/mL. No matrix effect and carry over were observed. All the validation parameters met the acceptance criteria. The validated method has been applied to a pharmacokinetic study in rats. A good correlation between DBS and plasma concentrations for filgotinib was observed.

Uptake and pharmacokinetics of cefuroxime in rabbits after intravitreal, intracameral, and topical dosing: relevance to human ocular injection of cefuroxime.

Cefuroxime is one of the widely used antibiotics. The objective of this study was to determine pharmacokinetics and disposition in various ocular tissues following topical (TOP), intracameral (IC) and intravitreal (IVT) administration of cefuroxime to rabbits.Following TOP, IC and IVT dosing plasma and various ocular tissues (aqueous humor (AH), vitreous humor (VH), conjunctiva, trabecular mesh (TM), lens and retina-choroid (RC)) were collected and analyzed to understand the disposition of cefuroxime. Postintravenous administration plasma samples were collected to determine the systemic pharmacokinetics.Post-TOP dosing cefuroxime concentrations were observed only in conjunctiva up to 48 h. IC administration showed cefuroxime concentrations in AH up to 8 h; in conjunctiva, TM and plasma, the concentration lasted up to 4 h and in RC and VH till 1 h. IVT administration of cefuroxime showed concentrations in all ocular tissues (up to 8 h) and lasted up to 48 h except in conjunctiva and RC.There was evidence that the mechanism(s) of cefuroxime entry into the eye by via IVT, IC and TOP routes is clearly different. The present ocular tissue data may aid clinicians for considering appropriate choice in the treatment of post-operative ocular complications due to bacterial infections including endophthalmitis.

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.

Validated LC-ESI-MS/MS method for the determination of tunicamycin in rat plasma: Application to a pharmacokinetic study.

A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of tunicamycin in rat plasma as per regulatory guideline. Chromatography of tunicamycin and the IS in the processed plasma samples was achieved on an X-Terra phenyl column using a binary gradient (mobile phase A, acetonitrile and mobile phase B, 5 mm ammonium formate) elution at a flow rate of 0.6 ml/min. LC-MS/MS was operated under the multiple reaction monitoring mode using the electrospray ionization technique in positive ion mode and the transitions of m/z 817.18 → 596.10, 831.43 → 610.10, 845.29 → 624.10, 859.23 → 638.10 and 309.24 → 163.20 were used to quantitate homologs A-D and the IS, respectively. The total chromatographic run time was 4.5 min. The correlation coefficient (r2 ) was >0.99 for all homologs with accuracy 90.7-107.4% and precision 0.74-15.1%. The recovery of homologs was 78.6-90.2%. No carryover was observed and the matrix effect was minimal. Tunicamycin four homologs were found to be stable on the bench-top for 6 h, for up to three freeze-thaw cycles, in the injector for 24 h and for 1 month at -80°C. The applicability of the validated method has been demonstrated in a rat pharmacokinetic study.

Simultaneous determination of bendamustine and γ-hydroxybendamustine in mice dried blood spots and its application in a mice pharmacokinetic study.

A selective, sensitive and rapid mice dried blood spot (DBS) method has been developed and validated for the simultaneous quantification of bendamustine (BM) and γ-hydroxy-bendamustine (HBM) as per regulatory guidelines using an LC-MS/MS. Quality control, calibration curve and study sample DBS cards were sonicated with 5% formic acid in water before extraction with ethyl acetate enriched with internal standard (I.S.). The organic layer was evaporated and residue was reconstituted in 0.1% formic acid in acetonitrile for LC-MS/MS analysis. Chromatographic resolution of both analytes (BM and HBM) and the I.S. (loperamide) was achieved on an Atlantis dC18 column using 0.2% formic acid:acetonitrile (25:75, v/v) as an eluant delivered at a constant flow-rate of 0.5 mL/min. The total chromatographic run time was 3.2 min. The MS/MS ion transitions monitored were m/z 358.0 → 228.0, 374.0 → 338.0 and 477.0 → 210.0 for BM, HBM and the I.S, respectively. The assay was linear in the range of 5.65-2544 ng/mL for both BM and HBM. The within-run and between-run accuracy and within-run and between-run precision were in the range of 0.96-1.00 and 1.36-9.94%, respectively for BM; 0.88-1.03 and 4.57-11.7%, respectively for HBM on mice DBS cards. Stability studies showed that both analytes were stable at room temperature for 7 days and at -80 °C for 55 days on DBS cards. The validated DBS method has been applied to a pharmacokinetic study in mice.

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.

A simple, specific and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the quantitation of second generation antiandrogens and their active metabolites namely apalutamide, enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma. The method involves extraction of apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 along with internal standard (IS) from 100 µL mice plasma through a simple protein precipitation process. The chromatographic analysis was performed on a Waters Alliance HPLC system using a gradient mobile phase (comprising 10 mM ammonium acetate and acetonitrile in a flow-gradient) and X-Terra Phenyl column. The UV detection wave length was set at λmax 250 nm. Apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 and the IS eluted at 13.6, 11.4, 9.68, 6.11, 6.93 and 4.69 min, respectively with a total run time of 15 min. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 209 - 5215 ng/mL (r 2=0.998). The intra- and inter-day precisions were in the range of 0.56-13.5 and 1.04-13.9%, respectively. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.

A novel dried blood spot LC-MS/MS method for the quantification of apalutamide in mouse whole blood: Application to pharmacokinetic study in mice.

A simple, sensitive and rapid assay method has been developed and validated for the estimation of apalutamide on mouse dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The method utilizes liquid extraction of apalutamide from 3 mm punched disks from DBS cards (spiked or study samples). The extracted sample was chromatographed on an Atlantis dC18 column using gradient elution with 0.2% formic acid and acetonitrile at a flow rate of 1.00 mL/min. The total run time was 3.0 min. The MS/MS ion transitions monitored were m/z 478 → 450 for apalutamide and m/z 481 → 453 for the IS (apalutamide-d3 ). Method validation was performed as per regulatory guidelines. The assay was linear in the range of 0.95-2030 ng/mL. The intra- and inter-day precisions were in the ranges of 2.37-8.53 and 6.76-11.5%, respectively. Stability studies showed that apalutamide was stable on DBS cards for one month. This novel method has been applied to analyze the DBS samples of apalutamide obtained from a pharmacokinetic study in mice.