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.
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.
AbstractSAFit-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.
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.
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 dC 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-d ). 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.
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.
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.
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