HPLC method development, validation and impurity characterization for an antitumor Hsp90 inhibitor—PU-H71 (NSC 750424)

• An HPLC method for a potent antitumor Hsp90 inhibitor, PU-H71, was developed.
• Presence of TFA in both diluent and eluent is critical to HPLC method's performance.
• The HPLC method was validated to be stability-indicating per ICH guidelines.
• Total of 20 related compounds were identified through HRMS and MS/MS fragments.
• Sulfur oxidation and hydrolysis of methylene dioxy group are observed degradations.

An HPLC method for the assay of the heat shock protein 90 inhibitor, PU-H71 (NSC 750424), has been developed and validated. The stress testing of PU-H71 was carried out in accordance with ICH guidelines Q1A (R2) under aqueous, acidic, alkaline, oxidative, thermolytic and photolytic conditions. The separation of PU-H71 from its impurities and degradation products was achieved within 50 min on a Mac-Mod ACE 3 C18 column (150 mm × 4.6 mm i.d., 3 μm) with a gradient mobile phase comprising 20–95% acetonitrile in water, with 0.1% trifluroacetic acid in both phases. LC–quadrupole TOF/MS was used to obtain accurate mass data on various components as well as on their fragments for characterization of impurities and degradation products. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.1–0.3 mg/mL, r ≥ 0.9998), accuracy (recovery 99.7–101.1%), precision (intra-lab RSD ≤ 1.39%, inter-lab RSD ≤ 0.91%), sensitivity (LOD 0.08 μg/mL), and ruggedness. The developed method was suitable for the assay and stability monitoring of PU-H71 drug substance.

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