Determination of retinoids by packed-capillary liquid chromatography with large-volume on-column focusing and temperature optimization

• Published in: Journal of Chromatography A 1999-06, Vol.847 (1), p.59-68
• Main Authors: Molander, P, Gundersen, T.E, Haas, C, Greibrokk, T, Blomhoff, R, Lundanes, E
• Format: Article
• Language: English
• Subjects: Analysis; Biological and medical sciences; General pharmacology; Medical sciences; Pharmacology. Drug treatments; Retinoic acid; Retinoids; Vitamins
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(99)00020-5

An isocratic reversed-phase packed capillary high-performance liquid chromatography method for the determination of all- trans-retinol, all- trans-retinoic acid and 13- cis-retinoic acid has been developed, utilizing on-column focusing large volume injection and operation at 50°C. The focusing mobile phase consisted of acetonitrile–ammonium acetate (0.5% in water)–water (45:5:50). The eluting mobile phase consisted of acetonitrile–ammonium acetate (0.5% in water)–acetic acid (94.9:5:0.075). In addition to high water content, pH contributed to additional selective focusing of the retinoic acids when using the non-eluting mobile phase. Injection volumes up to 100 μl were successfully applied. Operation at elevated temperature provided reduced column backpressure, and allowed higher volumetric flow-rates to be used during sample introduction. Thus the overall analysis time was reduced from 45 to 25 min. Furthermore, increasing the temperature from 25 to 70°C gave a 48% reduction in the reduced plate height. A mass limit of detection of 0.5 ng of the retinoic acids, corresponding to a concentration limit of detection of 5 ng ml −1, was found using on-column UV detection at 360 nm.