Development of an Advanced HPLC-MS/MS Method for the Determination of Carotenoids and Fat-Soluble Vitamins in Human Plasma

Abstract

The concentration of carotenoids and fat-soluble vitamins in human plasma may play a significant role in numerous chronic diseases such as age-related macular degeneration and some types of cancer. Although these compounds are of utmost interest for human health, methods for their simultaneous determination are scarce. A new high pressure liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) method for the quantification of selected carotenoids and fat-soluble vitamins in human plasma was developed, validated, and then applied in a pilot dietary intervention study with healthy volunteers. In 50 min, 16 analytes were separated with an excellent resolution and suitable MS signal intensity. The proposed HPLC-MS/MS method led to improvements in the limits of detection (LOD) and quantification (LOQ) for all analyzed compounds compared to the most often used HPLC-DAD methods, in some cases being more than 100-fold lower. LOD values were between 0.001 and 0.422 µg/mL and LOQ values ranged from 0.003 to 1.406 µg/mL, according to the analyte. The accuracy, precision, and stability met with the acceptance criteria of the AOAC (Association of Official Analytical Chemists) International. According to these results, the described HPLC-MS/MS method is adequately sensitive, repeatable and suitable for the large-scale analysis of compounds in biological fluids.

Keywords: carotenoids; fat-soluble vitamins; high antioxidant diet; human plasma; pilot human study; tandem mass spectrometry.

Figure 1

Chromatogram of working standard solutions obtained by high pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. Peaks: (1) retinol; (2) 25-hydroxycholecalciferol; (3) retinol acetate; (4) α-tocotrienol; (5) cholecalciferol; (6) astaxanthin; (7) lutein; (8) zeaxanthin; (9) cantaxanthin; (10) E-β-apo-8′-carotenal; (11) cryptoxanthin; (12) 13-Z-β-carotene; (13) α-carotene; (14) β-carotene; (15) 9-Z-β-carotene; and (16) 5-Z-lycopene.

Figure 2

Effect of solvent used for reconstitution of samples. (a) Chromatogram of sample reconstituted in methyl tert-butyl ether (MTBE); (b) chromatogram of sample reconstituted in MeOH.