10
effect (+I) is very high when compared with other ionising. The basis of peak shape, symmetry, retention time and peak tailing, 10% Tetramethylammonium hydroxide aqueous was decided as the buffer preparation to be used. Different percentage of 10 Tetramethylammonium hydroxide and organic solvents were analysed and according to experiments with acetonitrile and methanol, excellent retention time, column pressure and peak tailing were observed with methanol. For this reason, acetonitrile was selected as an organic modifier. After many trials, based on the peak shape, peak symmetry, retention time and peak tailing, at 0.4 ml/min flow rate were selected.
Detection of Wavelength
UV spectrum of spiraeoside and their pacebo peaks were scanned between 200 nm – 600 nm by photo-diode array detector. Wavelength at 365 nm was found to be optimum for all analysed peaks.
pH adjustment of the buffer
Different tests on pH of the Tetramethylammonium hydroxide buffer were made to achieve the optimum pH at which all peaks related with APIs and placebo separated well. Based on peak shape, peak tailing and theoretical plate count, suitable pH of the buffer was found as 2.5.
The optimised chromatographic conditions are a isocratic study of buffer (Tetra methyl ammonium hydroxide, at pH 2.5) and acetonitrile at 365 nm as detection wavelength, 0.4 mL/min flow rate, 35ºC column temperature, 25ºC tray temperature and 100 mL injection volume. The typical HPLC chromatograms (Figure 2) represent the spiraeoside peak could be detected.
Validation of the Method
The developed method was validated for as per ICH Q2 (R1) guidelines [13] and validation of compendial procedures from USP [14] for various parameters such as specificity, filter effect and carry over effect.
Specificity
The peak purity indices for the gel solutions were determined with PDA detector under optimised chromatographic conditions. Peak purity indices were found as (purity angle < purity threshold) (Figure 3) indicating that no additional peaks were co-eluting with the placebo sample. Baseline resolution was achieved for all investigated compounds.
Conclusion
The proposed RP-HPLC method for identification of spiraeoside was found specific and selective according to validation studies. The method was validated as per ICH guidelines [13] and validation of compendial procedures from USP [14]. The developed method can be used for the routine analysis of identification of spiraeoside in pharmaceutical gel formulations.
References
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2. L. Pobłocka-Olech, DANIEL GŁÓD1, MARIA E.ŻEBROWSKA2, MAŁGORZATA SZNITOWSKA2, and MIROSLAWA KRAUZE-BARANOWSKA1, “TLC determination allium cepa,” Acta Pharm, vol. 66, pp. 543–554, 2016.
3. G. I. Vysochina, T. A. Kukushkina, and E. S. Vasfilov, “Biologically Active Substances in Filipendula ulmaria (L.) Maxim. Growing in the Middle Urals,” Chem. Sustain. Dev., vol. 21, pp. 369–374, 2013.
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7. Y. Algul, Derya; Uzuner, “RP-HPLC ANALYSIS OF HYPEROSIDE IN A CREAM FORMULATION Derya Algul * , Yasemin Uzuner,” Indo Am. J. Pharm. Res., vol. 6, no. 5, 2016.
Figure 2. HPLC chromatogram of gel sample
8. M. Geetha, M. Saravanakumar, and P. S. Devi, “Hplc Analysis of Quercetin and Cyanidin From Onion Peel
( Allium Cepa L .,),” Am. J. Pharmtech Res., no. November 2011, 2011.
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Figure 3. HPLC peak purity plot of gel sample
14. “Validation of compendial procedures.” [Online]. Available:
https://hmc.usp.org/ sites/default/files/documents/HMC/GCs-Pdfs/c1225.pdf. [Accessed: 23-Jun-2017].
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