Development of a novel, fast, simple, non-derived rp-hplc method for simultaneous estimation of benzocaine and tibezonium iodide from mucoadhesive dosage form as well as human saliva and its validation

The current study aimed at development and validation of a simple, accurate, precise, sensitive, robust and specific high performance liquid chromatography (HPLC) method for simultaneous estimation of benzocaine (BZN) with tibezonium iodide (TIB) from combined dosage form as well as from human saliva. Instrumental conditions were developed on an auto-sampler Agilent® 1260 Infinity series RP-HPLC machine. The method was validated in accordance with ICH harmonized guidelines Q2 (R1) by using acetonitrile and phosphate buffer as mobile phase in a volumetric proportion of 70:30 which was flowing through C₁₈ Agilent® column. Flow of mobile phase was maintained at 1 mL/min at 35 °C and elute was detected 318 nm. The peaks for BZN and TIB were separated at 2.29 and 4.15 min, respectively. Linearity range for such were 0.24-20.16 and 0.14-10.08 μg/mL, respectively. Linear coefficient of regression (R2) for BZN and TIB were 0.9987 and 0.9993, respectively. The relative standard deviation (RSD) values for both drugs were less than 2.0% indicating the accuracy and precision. The limit of detection (LOD) and limit of quantification (LOQ) outcomes for BZN were 56 and 187 ng/mL, respectively. For TIB, it were 38 and 127 ng/mL, respectively. The drugs separation against placebo justified the specificity of the method. It was concluded that the developed and validated method could be potentially for in vitro as well as in human salivary extracts of formulated dosage forms.