Abstract. Purpose: Compounds chemically related to analyte as surrogate reference standards in quantitative HPLC and the impact of internal standard on such applications have been investigated. Method: A simple reversed-phase isocratic HPLC method with UV detection was developed and validated. The solutes were paracetamol (principal analyte), caffeine (internal standard) and candidate surrogate reference standards (aspirin, benzoic acid and phenacetin). The chromatographic conditions were Zorbax C-18 column, methanol/2.5% ethanoic acid (2:3) mobile phase and UV detection at 257nm. The relationship between signal intensities and concentrations of a pair of analyte and candidate surrogate reference standard was used to determine a constant (Sα) which was later used in a derived equation to evaluate the content of nine brands of paracetamol tablets. Results: The retention times of the solutes were 8.1 ± 0.03 min (aspirin), 11.7 ± 0.05 min (benzoic acid), 4.7 ± 0.02 min (caffeine), 3.0 ± 0.01 min (paracetamol) and 11.1 ± 0.06 min (phenacetin). Sα for the various candidate surrogate reference standards were: 18.23 ± 0.048(aspirin), 11.66 ± 0.251(benzoic acid) and: 1.15 ± 0.051(phenacetin). The assay values with each of the candidate surrogate reference standards either met the monograph requirements of the United States Pharmacopoeia and National Formulary (2004) or the British Pharmacopoeia (2007) or both. Effect of internal standard was void. Conclusion: Surrogate reference standards were successfully applied to the assay of paracetamol tablets. The proposed method can potentially be used in routine quantitative HPLC applications once the HPLC method is developed and Sα is also determined with previously available chemical reference standard.

Keywords: Surrogate reference, surrogate constant, pharmacopoeia, HPLC, analyte