50: Effect of structural variations between surrogate reference standards and target analyte on assay values in isocratic HPLC-UV quantitation

Abstract: The study explores the use of surrogate reference standards for HPLC-UV quantification of paracetamol in tablets, and the effect of the relative structural variations of the surrogate candidates on assay values. A cost-effective isocratic RP-HPLC method was developed, utilizing a mobile phase of methanol and water (65:35 v/v) at pH 3.10, with a C-18 Copsil column and UV detection at 235 nm. A mixture comprising the target analyte (paracetamol) and surrogate reference standards (4-aminophenol, caffeine, acetanilide, and aspirin) was analyzed at a flow rate of 1.5 mL/min. The study established surrogate constant (S∞) for all the surrogate standards and employed in a derived equation to evaluate paracetamol content. Multiple t-test comparative study of the assay values obtained using the surrogate reference standards and pharmacopoeial methods were all statistically comparable, except when aspirin was used as a surrogate, which yielded results significantly different from pharmacopoeial methods and other surrogates, yet within acceptable limits. In conclusion, the closer the structural similarity of the surrogate reference to the target analyte, the smaller the mean difference of assay values. In the selection of surrogate reference standards for RP-HPLC external calibration, structural analogs to the target are recommended to enhance the accuracy of assay results.