Validated LC-MS/MS Methods for Quantification of AC02 and Porcine ACTH1-39 in Human Plasma: Application to a Phase I Study.

Background/Objectives: AC02 is a novel 39-amino-acid adrenocorticotropic hormone (ACTH) analogue designed for the treatment of infantile spasms. To support its clinical study, in which porcine ACTH1-39 served as the positive-control drug, reliable methods for the determination of AC02 and porcine ACTH1-39 in human plasma were required. Reported analytical methods for ACTH analogues are mainly immunoassays, which are easily affected by cross-reaction and the hook effect, necessitating a more selective analytical approach. Methods: Two LC-MS/MS methods were developed for the determination of ACTH analogues AC02 and porcine ACTH1-39 in human plasma. Human ACTH1-39 was included as a selectivity marker to confirm that endogenous ACTH does not interfere with the quantification of AC02. Based on the distinct concentration requirements and matrix challenges, two sample-preparation procedures were established: micro-solid-phase extraction coupled with protein precipitation for porcine ACTH1-39 (LLOQ 0.100 ng/mL), and acid-mediated protein precipitation for AC02 (LLOQ 0.500 ng/mL). The [M+6H]6+ ions were selected as precursor ions, and the corresponding 5+ fragment ions, formed by loss of the C-terminal phenylalanine, were used for MRM detection. Results: Despite a mass difference of only 0.98 Da between AC02 and human ACTH1-39, which are indistinguishable by mass spectrometry, baseline chromatographic separation was achieved. Both methods were fully validated in accordance with current bioanalytical guidelines. Conclusions: The validated methods were successfully applied to the phase I clinical study of AC02 and porcine ACTH1-39, enabling reliable quantification of the drug candidate and its active comparator, porcine ACTH1-39.