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

An isocratic reversed-phase packed capillary high-performance Liquid chroma tography method for the determination of all-trans-retinol, all-trans-retin oic acid and 13-cis-retinoic acid has been developed, utilizing on-column f ocusing large volume injection and operation at 50 degrees C. The focusing mobile phase consisted of acetonitrile-ammonium acetate (0.5% in water)-wat er (45:5:50). The eluting mobile phase consisted of acetonitrile-ammonium a cetate (0.5% in water)-acetic acid (94.9:5:0.075). In addition to high wate r content, pH contributed to additional selective focusing of the retinoic acids when using the non-eluting mobile phase. Injection volumes up to 100 mu l 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 reduce d from 45 to 25 min. Furthermore, increasing the temperature from 25 to 70 degrees 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 concentratio n limit of detection of 5 ng ml(-1), was found using on-column UV detection at 360 nm. (C) 1999 Elsevier Science B.V. All rights reserved.