Establishment and Validation of LC-MS/MS Technique for Lenacapavir Quantification in Rat Plasma, with Application to Pharmacokinetic Assessment.

Abstract

Background: Establishing and validating a sensitive and accurate LC-MS method for quantifying Lenacapavir in rat plasma was the primary objective of this study. D₆- Lenacapavir was used as the internal standard, and the validation procedure adhered to the protocols specified by the Food and Drug Administration of the United States. Method: This article presents an overview of the bioanalytical LC-MS method, utilizing a Waters Symmetry C18 column, 150mm x 4.6mm, 3.5µm and an organic mobile phase comprising acetonitrile and 0.1% formic acid buffer in a ratio of 20:80. Results: The calibration curve for Lenacapavir exhibited a linearity range of 5–100 ngmL^-1 (r² = 0.9999). Liquid-liquid extraction was employed to recover Lenacapavir from rat plasma, resulting in recovery percentages of 98.97%, 99.51%, and 99.49% at three different concentration levels. Lenacapavir remained stable during storage under various conditions (three freeze-thaw cycles, benchtop, autosampler, short-term, and long-term storage). Pharmacokinetic analysis yielded key parameters, including a half-life of 120 hrs and a time to reach a maximum concentration of 4hrs. Lenacapavir and IS were identified using proton adducts in the LC-MS analysis at m/z 969.32/509.15 and 975.28/515.07, respectively, by employing positive mode multiple reaction monitoring. Conclusion: This comprehensive evaluation demonstrates that the method meets stringent criteria for system specificity, linearity, and accuracy, all well within the predefined acceptance limits. Its adaptability for the precise determination of Lenacapavir positions it as an invaluable tool in the field of bioanalysis, expanding its clinical utility.