Determination of relative response factors of the opium alkaloids with HPLC-DAD

In this work, a convenient method for determination of relative UV response factors (RRFs) of morphine, codeine, thebaine, oripavine, noscapine and papaverine by high performance liquid chromatography (HPLC) equipped with a diode array detector (DAD) was presented. Pholcodine was selected as the reference compound for calculating the relative response factors of the alkaloids. The separation of all seven compounds was obtained with optimized gradient elution with high pH value of the mobile phase on a reversed phase column with bidentate C18-C18 bonding technology. The RRFs of the alkaloids were determinate by three different approaches: ‘regression analysis/mass concentration’, ‘regression analysis/molar concentration’and ‘detector sensitivity’ approaches. The ‘regression analysis/molar concentration’ approach gave the accurate approximation of the exact amount of the substance that enters in the detector and the statistically relevant calculation includes several points of different concentrations (at least five), which makes this approach most advantageous one. This method is suitable for quality assessment of the standardised opium dry extract, raw opium and standardised opium tincture by quantitative analysis of not only morphine and codeine as indicated in the respective European Pharmacopoeia monographs, but as well as the major impurities that originate from opium poppy Papaver somniferum L. (Papaveraceae).