QUANTIFICATION OF PYRITINOL IN SOLID PHARMACEUTICAL FORMULATION BY HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY-ULTRAVIOLET DETECTION AND SELECTIVITY EVALUATION BY MASS SPECTROMETRY

Abstract

A high-throughput method for pyritinol quantification in solid pharmaceutical formulation was developed. Chromatography was performed on silica gel 60 F₂₅₄ HPTLC plates using the mixture dichloromethane – methanol – formic acid 9:1:1 (v/v/v) as mobile phase. Detection was carried out by UV absorbance at 300 nm. Calibration showed a polynomial regression with a determination coefficient (R²) of 0.9992. For chromatography, repeatability (relative standard deviation, RSD) and intermediate precision (RSD) in matrix were 0.4% and 3.0%, respectively. Recoveries of spiked samples at three levels ranging from 98.5 to 101.9% with intermediate precisions of RSD 3.7 to 4.7%. Limits of detection and quantification were 0.6 and 2.0 μg mL⁻¹ (6 and 20 ng/band), respectively. The method capacity to detect degradation products and/or byproducts within routine conditions of analysis was evaluated through forced degradation processes. Selectivity was evaluated determining the peak purity by UV-spectrophotometry, which showed correlation coefficients (r) > 0.9997. Additionally, peak identity and purity was confirmed by mass spectrometry. The mass spectra showed just pyritinol ions at m/z 369 [M + H]⁺ and 391 [M + Na]⁺ being acquired directly from the sample bands by an elution-based interface. Considering the validation results, reduced analysis cost, accelerated analysis time, and high throughput capacity, this simple, yet reliable planar chromatographic method is a good alternative for pyritinol analysis in pharmaceutical formulations.

Keywords:

• high-performance thin-layer chromatography
• mass spectrometry
• planar chromatography
• pyrithioxin
• pyritinol

ACKNOWLEDGMENTS

The authors want to thank the Chilean Government as well as the University of Concepcion, Chile, for the doctoral fellowship granted, Merck (Darmstadt, Germany) for support regarding plate material, Laboratorio Chile (Santiago, Chile) for providing a secondary standard substance, CAMAG (Muttenz, Switzerland) for support of quantitative instrumental equipment, Dr. Heinrich Luftmann, University of Münster, Germany, for providing the ChromeXtractor and Professor Dr. Wolfgang Schwack, University of Hohenheim, Stuttgart, Germany, for the excellent working conditions at the Institute of Food Chemistry.

Notes

^a Percentage of the content labeled.

^b Mean of three different determinations on different plates and days.

^c Standard deviation and relative standard deviation (RSD).

^a Mean of the same sample measured in sextuplicate.

^b Standard deviation.