Design and Optimization of LC-MS Bioanalytical Method for Efonidipine and Chlorthalidone Using Quality by Design Strategy

ABSTRACT
Objective
The Quality by Design (QbD) technique was used in this work to design and validate a novel LC-MS method for
the simultaneous measurement of Efonidipine Hydrochloride, Ethanolate, and Chlorthalidone in artificially
generated human plasma. To guarantee the method achieved the right analytical performance, the parameters of
flow rate, heat block temperature, and mobile phase composition in methanol were optimised in an analytical
science, systematic approach employing a central composite design. Resolution and retention time were two of
the analytical method's most important characteristics.
Methods
The chromatographic separation was achieved using a Shimadzu capcell pak C18 (150×4.6mm, 3µm), and a
mobile phase consisting of Phosphate buffer (pH 3.0) (40:35:25% v/v/v): methanol and acetonitrile. The mobile
phase was allowed to flow at a rate of 1.0 mL/min. A mass spectrometer coupled with an electrospray ionization
(ESI) source operating in the positive ion was used for detection. Data were obtained in the multi-reaction
monitoring (MRM) acquisition mode. The best recovery and maximum matrix cleanliness were obtained from the
liquid-liquid extraction sample preparation.
Results
The retention time for Efonidipine and Chlorthalidone were 6.38 and 8.60 min respectively. Sample extraction
was performed using liquid-liquid extraction (LLE), and this technique produced very pure extracts with good
recovery rates. A linear calibration curve was found in the range of 18.75-65.61 µg/mL for Efonidipine and 60-
210 µg/mL for Chlorthalidone with a correlation coefficient r² > 0.99. The validation process confirmed the LC�MS analytical method's selectivity, sensitivity, linearity, accuracy, and precision for both Chlorthalidone and
Efonidipine. Bench-top stability, freeze-thaw stability, short-term stability, long-term stability, and autosampler
stability data showed good recovery and proved the method's stability.