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The comparison of the stability indicating  two HPLC methods and their application for the determination of bosentan in coated tablets 

Anna M. Zielińska ,  Sławek Wicherkiewicz ,  Wojciech Łuniewski ,  Katarzyna Sidoryk ,  Edyta Pesta ,  Andrzej Kutner 

Pharmaceutical Research Institute, Rydygiera 8, Warsaw 01-793, Poland


Nowadays, there is an increasing need for fast HPLC separations characterized by high efficiency and good resolution. The ultra-high pressure liquid chromatography (UHPLC) has been considered suitable to meet this challenge, but it was found that this method also has serious disadvantages. Currently, the range of applications of the UHPLC in the analysis of pharmaceutical substances and dosage forms is discussed in the literature [1- 2].  In this study we investigated the consequences of shortening the analysis time. Bosentan, a non-peptide antagonist of human endothelin receptors, was chosen as an example due to its therapeutic importance and lack of analytical methods described. Two high-performance liquid chromatography methods were compared, both in the reversed phase, with UV detection at 220 nm by performing the validation of the methods and comparing the resulting performance characteristics. The first separation (method A) has been achieved on Kinetex column, 2.6m C18 100A, 150 x 4.60 mm, the second – fast (method B) employed Kinetex column, 1.7m XB-C18 100A  50 x 3.0 mm. Both methods were performed with a buffered mobile phase containing 0.1% of triethylamine (TEA) in water brought to the pH 2.5 with phosphoric acid and methanol as the solvent A and acetonitrile as the solvent B. Gradient program was used and flow rate 0.8 ml/min and 0.4 ml, for the methods A and B respectively. The methods were validated according to the ICH guidelines for specificity, precision on the specified and LOQ limits, intermediate precision, accuracy, linearity (correlation coefficient=0.999) and robustness. The robustness was confirmed using  four factors: the mobile phase pH, the flow rate of the mobile phase, column temperature and the other batch of the column. The limits of detection and quantification were established. Both validated methods  fulfilled  the acceptance criteria. The method B was 3.5 times faster than the method A, but the method A showed much better sensitivity (LOQ 0.0132 and 0.1505 mg/mL for method A and B, respectively) and resolution (Rs between compound B and bosentan 3.39 and 1.75 for method A and B, respectively).  This  lowered sensitivity limits the  utility of the method B, especially in the analyses of low levels of active substances (e.g. bioanalysis, validation of the cleaning procedures).

The project was supported  by The National Centre for Research and Development, under the contract No. UDA-POIG.01.03.01-14-062/09-00.


[1]   D. Guillarme, D.T.T. Nguyen, S. Rudaz. J.L. Veuthey, Method transfer for fast liquid chromatography in pharmaceutical analysis: Application to short columns packed with small particle. Part I: Gradient experiments, Eur. J. Pharm. Sci. 68 (2008) 430-440.

[2]   R. Kormany, J. Fekete, D. Guillarme, S. Fekete, Reliability of computer-assisted method transfer between several column dimension packed with 1.3-5 mm core-shell particles and between various instruments, J. Pharm. Biomed. Anal. 94 (2014) 188-195.


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Submitted: 2014-04-04 09:29
Revised:   2014-05-02 19:35