Development and
validation of a sensitive LCMS/MS method for the simultaneous quantification of
Azelnidipine and Chlorthalidone in human plasma
Phaneendra Rao P.G. and Sreenivasa Rao Battula
Res. J. Chem. Environ; Vol. 26(7); 85-93;
doi: https://doi.org/10.25303/2607rjce085093; (2022)
Abstract
Highly resolved, selective and sensitive liquid chromatography/tandem mass spectrometry
(LC–MS/MS) method was optimised for the simultaneous quantification and recently
approved combined medication for the treatment of hypertension viz. azelnidipine
and chlorthalidone in spiked human plasma. The study utilizes nifedipine as internal
standard. In the extraction of analytes from spiked plasma, acetonitrile was used
to induce protein precipitation and then liquid – liquid extraction was carried
using ethyl acetate solvent followed by the LCMS-MS analysis. The analytes were
separated on Inertsil ODS C18 (4.6 mm×100 mm, 5 μm) column at room temperature with
65:35 (v/v) ratio of acetonitrile and 0.1% formic acid as the mobile phase at 0.5
mL/min flow rate. Detection was performed on a triple quadrupole mass spectrometer
employing electrospray ionization technique, operating in multiple reaction monitoring
with the transitions of m/z 583 → 331, 339 → 321 and 347 → 328 respectively for
azelnidipine, chlorthalidone and nifedipine.
The analytes were detected at a retention time of 1.72 min, 2.62 min and 3.12 min
respectively for azelnidipine, chlorthalidone and nifedipine and the analysis was
completed within run time of 5 min. The analysis was carried in the calibration
range of 5-500 ng/mL with a very sensitive detection limit of 1.5 ng/mL. The method
was validated as per FDA guidelines for bioanalytical method validation and the
results in all the validation parameters were in the acceptable limit. Results proved
that the method was selective, sensitive, accurate, precise and stable and hence
can be applicable for the routine analysis of azelnidipine and chlorthalidone in
biological samples.
