Fast and selective HPLC-DAD method for determination of pholcodine and related substances

Ana Petkovska, Hristina Babunovska, Marina Stefova


Quality control of pharmaceuticals requires development of fast, efficient and reliable methods for determination of active compounds as well as known and very often unknown impurities within defined concentration ranges. In this work, a simple and rapid HPLC-UV-DAD method for identification and quantification of pholcodine process related impurities and some degradation products was developed and validated. Pholcodine and its five structural analogues such as morphine, codeine, thebaine, oripavine, and papaverine were separated in less than 10 minutes using reversed phase LiChrospher C-8 column. For optimal chromatographic performance with reproducible retention times, gradient elution with 2% ammonium hydroxide in water and acetonitrile was used. The method was validated by establishing its selectivity, specifity, sensitivity, linearity, intra- and inter-day precision and robustness. All tested parameters confirmed that the method is suitable for determination of pholcodine and its five impurities in pharmaceutical drug samples. The results obtained from real sample analysis give support to the suitability of the proposed method for the purpose of quality control.


pholcodine; impurities; validation; HPLC-DAD; opium alkaloids

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R. Nageswara Rao, V. Nagaraju, An overview of the recent trends in development of HPLC methods for determination of impurities in drugs, J. Pharm. Biomed.Anal. 33, 335–377 (2003).

European Pharmacopoeia, Seventh Edition, Monograph Volume 2, EDQM of the Council of Europe, Strasburg, 2010, pp. 2674–2675.

The United States Pharmacopoeia, XV, 15th revision, Mack Publishing Company, 1955, pp. 168–187.

O.M. Denk, A.I. Gray, G.G. Skleleren, D.G. Watson, Isolation and identification of three potential impurities of pholcodine bulk drug substance, J. Pharm. Pharmacol. 52, 819–829 (2000).

Clark’s analysis of drugs and poisons in pharmaceuticals, body fluids and postmortem materials, Third Edition, Monograph Volume 2, Pharmaceutical Press, 2004 pp. 845–1617.

B. Rembrang, L. Krenn, B. Kopp, G. Buchbauer, A. Nikiforov, Principal content analysis of opium alkaloids contents for origin determination, Pharmazie 49,766–768 (1994).

M. E. Bosh, A.R. Sanchez, F. Sanchez Rojas, C. Bosch Ojeda, Morphine and its metabolites: Analytical methodologies for its determination, J. Pharm. Biomed.Anal. 43, 799–815 (2007).

M. Gergov, I. Ojanpera, E. Vuori, Simultaneous screening for 283 drugs in blood by liquid chromatography-ion spray tandem mass spectrometry with multiple reaction monitoring, J.Chromatogr. B 795, 41–53 (2003).

International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals of Human Use, Q2(R1): Validation of Analytical Methods: Text and Methodology, November (2005).



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