Simultaneous Determination of Paracetamol and Orphenadrine Citrate in Rat Plasma using LC–MS/MS Spectrometry Method: Pharmacokinetics and Drug-Drug Interaction Profile Studies

Herein, it is the first developed and fully validated LC-MS/MS method for simultaneous determination of paracetamol (PAR) and orphenadrine citrate (ORP) in rat plasma after oral administration in respect of therapeutic drug monitoring, bioequivalence, pharmacokinetic (PK) and drug-drug interaction (DDI) studies. PAR and ORP were extracted from plasma sample using ACN-induced protein precipitation method. The separation procedure was carried out on an Agilent Eclipse Plus ODS (4.60 x 100 mm, 3.50 μm) column using water-enriched gradient mobile phase consisting of 6 mM ammonium formate plus 0.1% formic acid and ACN at a flow rate of 0.5 mL min -1 . The mass spectrometry parameters were optimized and multiple reaction monitoring (MRM) in positive ion mode of two transitions was utilized for quantification of precursor to production at m/z 152 → 110 & 65 for PAR as [M+2H] 2+ , 270.3 → 181 for ORP as [M + H] + . According to US-FDA bioanalytical requirements, the proposed method was verified in terms of linearity, selectivity, recovery, accuracy, precision, matrix effects, and stability. The method was linear in the range of 1-5000 and 1-500 ng mL -1 with detection limits (S/N of 3) of 0.12 and 0.07 ng mL -1 for PAR and ORP, respectively. The selectivity and high sensitivity of the method succeeded in the study of PK parameters and DDI between PAR and ORP after oral dose administration. The synergistic action between the two drugs was confirmed by studying variable parameters like K a , T max , C max , AUC 0-24 , MRT, and Cl/F. PAR affects the absorption of ORP and decreases its C max but increases its T max in accordance with elevating AUC 0–24 and reducing Cl/F for both drugs. The current PK research is a useful tool for determining the DDI of both medicines and might be used in therapeutic drug monitoring and bioequivalence studies.