Development of a Lc–Ms/Ms Method for the Quantification of Toxic Payload Dm1 Cleaved from Bt1718 in a Phase I Study

<b>Background:</b> BT1718 is a novel bicyclic peptide anticancer drug targeting membrane type I matrix metalloproteinase to release its toxic payload DM1. A LC-MS/MS method was validated to quantify DM1 generated from BT1718 in a Phase I/IIa clinical trial. <b>Materials & methods:</b> Plasma samples underwent a reduction reaction to artificially cleave BT1718 into DM1 and its bicycle components. An alkylation step was carried out to stabilize the reaction products, and plasma proteins extracted using acetonitrile. LC-MS/MS analysis utilized a C18 column and Agilent 6460 triple quadrupole mass spectrometer (Agilent, Cheshire, UK). <b>Results:</b> The method was fully validated over a linear range of 200-50,000 ng/ml BT1718, with overall precision ≤10% and accuracy 89-102%. <b>Conclusion:</b> A novel method for quantifying DM1 yielded from BT1718 has been validated and is now being utilized clinically.