Method Development and Validation of LC-MS/MS-Based Assay: Quantitation of Leniolisib in Rat Plasma and its Application in Pharmacokinetics Study

Introduction: Leniolisib is a kinase inhibitor containing a pyridine moiety. It is generally administered orally and is widely used for the treatment of activated phosphoinositide 3- kinase delta syndrome (APDS). However, it may cause side effects such as headache, sinusitis, and atopic dermatitis. Leniolisib levels can be quantified in rat plasma using the LC-MS method. Methods: The proposed method was carried out using a Zorbax SB C18 column (150 mm × 4.6 mm, 3.5 μm) at ambient temperature. The injection volume was 10 μL, with a flow rate of 1 mL/min, and samples were maintained at ambient temperature. The total run time was less than 7 minutes. The mobile phase was prepared by mixing methanol and buffer in a 30:70 ratio. The buffer was prepared by dissolving 1 mL of triethylamine (TEA) in 1 L of HPLC-grade water, and the pH was adjusted to 2.5 using formic acid. Results and Discussion: We investigated key analytical parameters, including system suitability, specificity, biological matrix screening, sensitivity, linearity, matrix effects, precision and accuracy, analyte recovery, and ruggedness. Furthermore, pharmacokinetic studies were also conducted. Conclusion: We established a simple, cost-effective, rugged, and sensitive LC-MS method for the determination of Leniolisib, using Sorafenib as the internal standard. The sample run time was shorter than that of previously reported methods, making this approach more efficient. The total chromatographic runtime was 5.00 min, with retention times of 2.424 min for Leniolisib and 4.575 min for Sorafenib. The method was validated over a dynamic linear range of 12.5-100 ng/mL for Leniolisib, with a correlation coefficient (r²) of 0.999. Intra-batch and inter-batch precision (%CV) across five levels (LLOQ, LQC, MQC, HQC, and ULOQ) was less than 15%, in accordance with USFDA guidelines. The method was further validated according to ICH recommendations and demonstrated sufficient robustness to produce reproducible results under a variety of chromatographic conditions. Moreover, the use of readily available reagents and standards allowed this bioanalytical strategy to be easily adapted for multiplex quantification of other analyte combinations.