The Methoxylated, Highly Conjugated C(40) Carotenoids, Spirilloxanthin and Anhydrorhodovibrin, Can Be Separated Using High Performance Liquid Chromatography with Safe and Environmentally Friendly Solvents

High performance liquid chromatography (HPLC) is a frequently used technique in carotenoid research. So far, however, little attention has been paid to the fact that many of the organic solvents used in HPLC separation of highly apolar C(40) carotenoids impose a significant threat to both health (especially for women) and the general laboratory environment. Here, we developed a solvent combination capable of allowing high-resolution HPLC separation of the C(40) carotenoid, spirilloxanthin, and all of its biosynthetic precursors beginning with phytoene, using relatively safe, environmentally friendly solvents. We show that separation of spirilloxanthin and its precursors anhydrorhodovibrin and lycopene using modern ultra-high performance chromatography (UHPLC) poses particular problems for apolar carotenoid separation, due to the long residence times in the sample delivery system, which facilitates carotenoid aggregation. We resolved these problems by developing the solvent delivery combination acetone/acetonitrile/isopropanol/methanol (65/30/5/2 (v/v/v/v)), which allows excellent column separation using the safe isocratic solvent system methanol/tetrahydrofuran (98/2 (v/v)). We also demonstrate that the development strategy for optimizing a solvent system for carotenoid separation can be well-described by the use of the average dielectric constant of the total sample delivery solvent, and present a formal method for analysis of the efficiency of separation.