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.
Phosphatidylinositol 3‐kinase (PI3K) inhibitors are a novel class of anticancer drugs that are approved to treat various malignancies. We report the development and validation of a HPLC method for the simultaneous quantitation of three PI3K inhibitors, namely copanlisib, duvelisib and idelalisib, in rat plasma as per the regulatory guidelines of the United States Food and Drug Administration. The method involves extraction of copanlisib, duvelisib and idelalisib along with an internal standard (IS; filgotinib) from rat plasma (100 μL) using a liquid–liquid extraction process. The chromatographic separation of the analytes was achieved using step‐wise gradient elution on a Hypersil Gold C18 column. The UV detection wavelength was set at λmax = 280 nm. Copanlisib, duvelisib, idelalisib and the IS eluted at 7.16, 12.6, 11.9 and 9.86 min, respectively, with a total run time of 15 min. The calibration curve ranged from 50 to 5000 ng/mL for all the analytes. Inter‐ and intra‐day precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated for all three analytes, and the results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in rats.
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