A Comprehensive Report on the HPLC-ESI-MS/MS Method Development and Validation for Quantifying Cabotegravir in K3EDTA-Human Plasma, Employing Cabotegravir-D5 as an Internal Standard

Abstract

Aiming to comprehend the plasma concentration of cabotegravir, a meticulously crafted bioanalytical method, aligned with the guidelines set by the US Food and Drug Administration (FDA), was devised and validated. This involved the utilization of cabotegravir D5 as an isotopic internal standard. The mobile phase consisted of methanol and 5 mM ammonium acetate in water, in an 80:20 ratio, v/v—Zorbax SB-C18 (50 × 2.1 mm, 5 μm) served as the chromatographic column. Cabotegravir and cabotegravir D5 were identified using proton adducts at m/z 409.20/370.20 and 409.20/144.8, respectively, employing the positive mode multiple reaction monitoring. Cabotegravir exhibited linearity within concentration ranges of 2 to 1000 ng/mL (r2 = 0.999). The recovery of cabotegravir from plasma by Liquid-liquid extraction yielded an average %CV of 7.71 at four quality control levels. Intra-day precision displayed accuracy of 100, 106, 97, 97, and 103%, respectively, whereas accuracy for inter-day precision was 100, 103, 98, 100, and 100% across LLQC, LQC, MQC-1, MQC-2, and HQC levels. The stability of cabotegravir remained consistent under diverse conditions, including five freeze-thaw cycles, benchtop, autosampler, and short-term, and long-term storage. This evaluation verifies that the method aligns with predefined acceptance limits and positions it as an indispensable tool in bioanalysis, significantly expanding its clinical utility.