(M0930-01-07) Development and Validation of a Sensitive Method for Quantification of Acalabrutinib and ACP-5862 in Human Plasma Using HPLC‑MS/MS

Purpose: Acalabrutinib is a second-generation Bruton's tyrosine kinase (BTK) inhibitor approved for the treatment of B-cell malignancies, including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Designed for improved selectivity over first-generation BTK inhibitors like ibrutinib, acalabrutinib minimizes off-target effects, reducing adverse events. ACP-5862 is the primary active metabolite of acalabrutinib, formed via CYP3A metabolism. It retains significant BTK inhibitory activity, contributing to overall drug efficacy. Understanding the pharmacokinetics and pharmacodynamics of both acalabrutinib and ACP-5862 is crucial for optimizing dosing strategies and managing drug-drug interactions, particularly in patients receiving concurrent CYP3A modulators. This paper describes the development and validation of a 2-in-1 assay for quantifying acalabrutinib and ACP-5862 in human plasma

Methods: A 20-µL sample of human K₂EDTA plasma was spiked with an isotope-labeled internal standard (IS), acalabrutinib-d4 and ACP-5862-d4, then get extracted by protein precipitation using acetonitrile (MeCN). The supernatant was further diluted using 10mM ammonium bicarbonate in water and the mixture was injected into a Waters CSH Phenyl-Hexyl column, with mobile phases composed of ammonium bicarbonate, formic acid (FA), water, and MeCN. Detection was conducted using a Sciex Triple Quad 6500 MS/MS system in positive ion mode, monitoring acalabrutinib, acalabrutinib-d4, ACP-5862, and ACP-5862-d4 at ion transitions of 466.1→372.2, 470.1→376.2, 482.1→388.3, and 486.1→388.3.

Results: To achieve symmetric peaks for both acalabrutinib and ACP-5862, multiple columns and mobile phases were tested. The XSelect Premier CSH Phenyl-Hexyl 2.1 × 50 mm, 2.5 μm column was selected as the optimal choice, using a mobile phase consisting of ammonium bicarbonate, FA, water, and MeCN. This combination provided the best peak shape with no observed carryover. Acalabrutinib has a pyridopyrimidine core and multiple amide groups, resulting in superior charging efficiency in positive ion mass spectrometry. Protein precipitation using MeCN, followed by dilution, was selected as the extraction procedure due to its simplicity and the cleanliness of the sample extracts. The entire extraction process takes only 20 minutes, with sufficient sensitivity and signal-to-noise ratio achieved at 1.00 ng/mL. The method was validated over a range of 1.00 to 1000 ng/mL, with a dilution QC at 5000 ng/mL for both acalabrutinib and ACP-5862. Excellent precision and accuracy were achieved, with inter-assay accuracy ranging from -3.7% to 8.3% and precision ranging from 3.7% to 7.7%. Carryover, specificity, selectivity, matrix effect, and stability were assessed following M10 industry guidance and met the acceptance criteria. Selectivity and matrix effects were evaluated using eight individual plasma lots (six normal, one lipemic, and one hemolyzed), with no significant interference observed. In-matrix stability for 89 days has been established, with additional tests planned. Furthermore, the method was also validated in human plasma using lithium heparin as an anticoagulant, yielding comparable results.

Conclusion: A simple, high throughput, and sensitive method for quantification of acalabrutinib and ACP-5862 in human plasma was developed and validated.