An improved high performance liquid chromatography–diode array detection–mass spectrometry method for determination of carotenoids and their precursors phytoene and phytofluene in human serum

An improved high performance liquid chromatography–diode array detection–mass spectrometry method was developed for determination of various carotenoids and their precursors phytoene and phytofluene in human serum. A polymeric C30 column and mobile phase of (A) methanol/acetonitrile/water (84:14:4, v/v/v) and (B) dichloromethane (100%) were employed with the gradient condition of 100% A and 0% B initially, raised to 10% B at 4 min, 18% B at 12 min, 21% B at 17 min, 30% B at 20 min and maintained until 25 min and increased further to 39% B at 28 min, 60% B at 40 min and returned to 100% A and 0% B at 45 min. A total of 30 carotenoids, including 6 all-trans forms, 20 cis-isomers, 2 β-carotene epoxides, phytoene and phytofluene, were resolved within 45 min at a flow-rate of 1 mL/min, column temperature 25 °C and detection wavelengths 450, 348 and 286 nm. Identification of carotenoids was carried out by comparing retention behavior, absorption and mass spectral data with those of reference standards, isomerized standards and reported values. An internal standard parared was found appropriate for quantitation of all the carotenoids. The developed method provided high sensitivity with low detection and quantitation limits (2–14 and 6–43 ng/mL), high recovery (91–99%), and small intra-day and inter-day variations (0.14–6.01% and 0.31–7.28%). Application of the developed method to Taiwan subjects supplemented with carotenoid-rich capsules revealed β-carotene plus its cis isomers as well as epoxide derivatives to be present in largest amount (1069.8–2783.1 ng/mL) in serum, followed by lutein plus its cis isomers (511.6–2009.5 ng/mL), phytofluene plus its cis isomer (515.0–1765.0 ng/mL), lycopene plus its cis isomers (551.1–1455.1 ng/mL), β-cryptoxanthin plus its cis isomers (458.0–965.0 ng/mL), all-trans-zeaxanthin (110.0–177.0 ng/mL), phytoene (41.8–165.0 ng/mL) and all-trans-α-carotene (37.5–95.9 ng/mL).