A BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR QUANTIFICATION OF GLYCOPYRROLATE AND NEOSTIGMINE IN RAT PLASMA BY LC-MS AND ITS APPLICATION TO PHARMACOKINETIC STUDY

Background: After surgery, non-depolarizing neuromuscular blocking medications include
neostigmine (NEO) and glycopyrrolate (GLY). Numerous traditional approaches, such as HPLC and
UPLC procedures, are established for the quantification of GLY and NEO; nevertheless, they lack
sensitive and specific analysis, especially in complex matrices. Using the LC/MS approach, this work
develops a bioanalytical method for quantifying both drugs in rat plasma and applies it to
pharmacokinetic studies. Methodology: The plasma was extracted using acetonitrile, and Rivastigmine
was employed as an internal standard. An MRM method with positive ions was used for multiple
reactions. A C18 column and a mobile phase - 70:30 mixture of acetonitrile and buffer was utilised at a
flow rate of 1 ml/min. Plasma vortex for 10 minutes and centrifuged at 4000 rpm at 20°C. Validation
and stability studies are conducted according to the ICH guidelines. The pharmacokinetic study by
WinNonlin (Version 5.2) software. Results and Discussion: Rt for Glycopyrrolate and Neostigmine at
1.838 and 2.800min. GLY has a precision (%CV) of 0.45 at HQC and 3.57 at LQC. NEO had a precision
(%CV) of 1.13 at HQC and 2.79 at LQC. From 2 to 40 ng/mL of GLY and 10 to 200 ng/mL of NEO,
the standard curves showed a linear relationship. LOD and LOQ for both drugs were 3pg/mL and
10pg/mL. Conclusion: A simple, affordable, reliable, and sensitive approach for quantifying GLY and
NEO in rat plasma using LC-MS, with Rivastigmine serving as the internal standard, was developed,
validated, and successfully applied in the pharmacokinetic study of rat plasma.