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Journal of Lipid Research, Vol. 43, 1143-1149, July 2002
Copyright © 2002 by Lipid Research, Inc.

Methods

Retinoid quantification by HPLC/MSⁿ

Peter McCaffery^1,*, James Evans^*, Omanand Koul^*, Amy Volpert, Kevin Reid^* and M. David Ullman^*,

^* University of Massachusetts, E.K. Shriver Center, Waltham, MA
Bedford Veterans Affairs Hospital,, Bedford, MA

¹ To whom correspondence should be addressed. e-mail: peter.mccaffery{at}umassmed.edu

Retinoic acid (RA) mediates most of the biological effects of vitamin A that are essential for vertebrate survival. It acts through binding to receptors that belong to the nuclear receptor transcription factor superfamily (Mangelsdorf et al. 1994). It is also a highly potent vertebrate teratogen. To determine the function and effects of endogenous and exogenous RA, it is important to have a highly specific, sensitive, accurate, and precise analytical procedure. Current analyses of RA and other retinoids are labor intensive, of poor sensitivity, have limited specificity, or require compatibility with RA reporter cell lines (Chen et al. 1995. Biochem. Pharmacol. 50: 1257–1264; Creech Kraft et al. 1994. Biochem. J. 301: 111–119; Lanvers et al. 1996. J. Chromatogr. B Biomed. Appl. 685: 233–240; Maden et al. 1998. Development. 125: 4133–4144; Wagner et al. 1992. Development. 116: 55–66). This paper describes an HPLC/mass spectrometry/mass spectrometry product ion scan (HPLC/MSⁿ) procedure for the analysis of retinoids that employs atmospheric pressure chemical ionization MS. The retinoids are separated by normal-phase column chromatography with a linear hexane-isopropanol-dioxane gradient. Each retinoid is detected by a unique series of MSⁿ functions set at optimal collision-induced dissociation energy (30% to 32%) for all MSⁿ steps. The scan events are divided into three segments, based on HPLC elution order, to maximize the mass spectrometer duty cycle. The all-trans, 9-cis, and 13-cis RA isomers are separated, if desired, by an isocratic hexane-dioxane-isopropanol mobile phase.

This paper describes an HPLC/MSⁿ procedure possessing high sensitivity and specificity for retinoids.

Abbreviations: APCI, atmospheric pressure chemical ionization; CAD, collisional activation decomposition; RA, retinoic acid

Supplementary key words retinol • retina • retinoic acid • didehydroretinoic acid • 4-oxo-retinoic acid • mass spectrometry • atmospheric pressure chemical ionization • high-performance liquid chromatography/mass spectrometry/mass spectrometry product ion scan

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