Retinoid quantification by HPLC/MS(n).
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.


METHODS
125: 4133-4144; Wagner et al. 1992.


METHODS
116: 55-66). This paper describes an HPLC/mass spectrometry/mass spectrometry product ion scan (HPLC/MS(n)) 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 MS(n) functions set at optimal collision-induced dissociation energy (30% to 32%) for all MS(n) 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/MS(n) procedure possessing high sensitivity and specificity for retinoids.
McCaffery
Peter
P
University of Massachusetts, E.K. Shriver Center, Waltham, MA, USA. peter.mccaffery@umassmed.edu
Evans
James
J
Koul
Omanand
O
Volpert
Amy
A
Reid
Kevin
K
Ullman
M David
MD
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007536:Isomerism; D008099:Liver / Q000737:chemistry; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012160:Retina / Q000737:chemistry; D012176:Retinoids / Q000032:analysis* / Q000737:chemistry*; D012680:Sensitivity and Specificity; D014212:Tretinoin / Q000032:analysis / Q000737:chemistry
D012176:Retinoids; D014212:Tretinoin