[17] CAROTENOID REVERSED-PHASE HPLC METHODS 185 BC stability is also given by the routine use of antioxidants such as the two well-known radical trapping agents, BHT and vitamin E. This, together with the absence of any sensitizer (to rule out the involvement of singlet oxygen), suggests that the light-induced degradation of this pigment could involve a chain-radical mechanism. Finally, the relative stability of BC was confirmed when the degradative factors were strictly controlled and an- tioxidants were present, suggesting conditions to be used to prolong its storage life. Acknowledgments I thank Dr. George Wolf for a careful review and stimulating discussion of the manu- script. I am grateful to Dr. H. N. Bhagavan (Hoffmann-La Roche, Inc.) for supplying the fl-[14C]carotene. Supported by USDA Grant No. 87-CRCR-I-2593. [17] Carotenoid Reversed-Phase High-Performance Liquid Chromatography Methods: Reference Compendium By NEAL E. CRAFT Introduction High-performance liquid chromatographic (HPLC) methods for the separation of carotenoids were first reported in 1970) A positive pressure of nitrogen gas was applied to a traditional open column system to speed the separation and reduce oxygen exposure. By the standards of today this would constitute low-pressure liquid chromatography. The sophistication of carotenoid HPLC methods has evolved as HPLC columns and equip- ment have improved. The first report of carotenoids separated using high- pressure pumping systems was in 1971, when Stewart and Wheaton2 em- ployed gradient elution normal-phase HPLC to separate 23 different carotenoid peaks from citrus peel using a zinc carbonate column over a period of 5 hr. Their accomplishment constituted a phenomenal improve- ment over previous separations. By comparison, most current HPLC sepa- rations of carotenoids are performed using short (< 20 min) isocratic con- ditions with commercially prepared reversed-phase columns) -6 This is J. P. Sweeney and A. C. Marsh, J. Assoc. Off. Anal. Chem. 53, 937 (1970). 2 I. Stewart and T. Wheaton, J. Chromatogr. 55, 325 (1971). 3 W. Driskell, M. Bashor, and J. Neese, Clin. Chem. 29, 1042 (1983). Copyright© 1992by Academic Press,Inc. METHODS IN ENZYMOLOGY, VOL 213 All rights of reproduction in any form t~served.