Validation and application of a liquid chromatography–tandem mass spectrometric method for the determination of G-856 (Cur-61414) in human plasma using semi-automated solid phase extraction

A liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the determination of G-856 in human plasma to support clinical development. The method consisted of a solid phase extraction for sample preparation and LC–MS/MS analysis in the positive ion mode using TurboIonSpray for analysis. d8-G-856 was used as the internal standard. A linear regression (weighted 1/concentration2) was used to fit calibration curves over the concentration range of 5.00–2000 pg/mL for G-856. There were no significant endogenous interference components in the multiple lots of blank human plasma tested. The accuracy (%Acc) at the lower limit of quantitation (LLOQ) was 98.2% with a precision (%CV) of 5.38%. For quality control samples at 15.0, 800, and 1600 pg/mL, the inter-day %CV was ≤5.03%. Inter-day %Acc ranged from 96.9 to 99.3%. G-856 was stable in human plasma for 184 days at −20 °C and −70 °C storage. G-856 was stable in human plasma at room temperature for up to 16 h and through four freeze/thaw cycles. This validated LC–MS/MS method for determination of G-856 was used to support Phase 1 clinical studies.

► A very sensitive LC–MS method was developed and validated for G-856, hedgehog inhibitor.
► The achievable LLOQ was 5 pg/mL in human plasma.
► Unexpected interference was identified during the clinical study sample analysis.
► Solved the interference issue by partial-validation after the investigation.
► Emphasized the importance of continuous monitoring the method after full validation.