Method for the Determination of β-Carotene in Supplements and Raw Materials by Reversed-Phase Liquid Chromatography: Single Laboratory Validation

Joseph Schierle

Bernd Pietsch

Alan Ceresa

Christian Fizet

Edward Waysek

Corresponding author’s e-mail: moc.msd@elreihcS.hpesoJ

Abstract

A single laboratory validation (SLV) study was conducted for a liquid Chromatography (LC) method for the determination of total and all-trans-β-carotene in a variety of dietary supplements, including multivitamin tablets, softgels, capsules, and beadlet raw materials. Extraction variants were developed for the different types of supplements tested based upon the supplement type and level of β-carotene. Water dispersible formulations such as powders, emulsions, tablets, and capsules were enzymatically digested with protease and extracted with dichloromethane–ethanol. Oily suspensions were directly dissolved in dichloromethane–ethanol. After appropriate dilution or concentration, the extracts were chromatographed by using either a reversed-phase C18 column or, in products containing high amounts of α-carotene, a reversed-phase C30 column. The LC systems provided linear responses in the range of 0.1–50 μg β-carotene/mL. The main geometrical isomers of β-carotene (all-trans, 9-cis, 13-cis, and 15-cis) were well separated from each other and from other carotenoids such as α-carotene, cryptoxanthin, lutein, lycopene, and zeaxanthin. Duplicate determinations of total β-carotene performed by 2 technicians in 8 different test materials on 5 different days resulted in relative standard deviations of 1.2–4.4%. Recoveries determined for supplements and beadlet raw material spiked with β-carotene levels of 10 μg to 100 mg/test portion and 0.2–40%, respectively, ranged from 97.5 to 102.1%. On the basis of the accuracy, precision, and recovery results from the SLV study, the method is suggested for a collaborative study on the determination of total and all-trans-β-carotene in dietary supplements.

Abstract

β-carotene is generally regarded as the most commercially important and widely used carotenoid. It is used as a food coloring agent, an antioxidant, and an important and safe pro-vitamin A source (1–3). β-carotene is currently incorporated in a wide variety of dietary supplements, including multivitamin, vitamin A, and antioxidant formulations. Recently, additional carotenoids, including lutein and lycopene, have been the subject of nutritional studies and are now also incorporated, both in combination products and in dietary supplements available to the general public. Spectrophotometry is still a common technique for the analysis of β-carotene in commercial product forms. Schierle et al. (4) reported a spectrophotometric procedure for the determination of total β-carotene in food additives with varying cis-/trans-ratios using an isobestic wavelength. However, spectrophotometric methods cannot differentiate between all-trans-β-carotene and cis-isomers of β-carotene, which may be formed during processing (5–12). Spectrophotometric analytical procedures are also not capable of determining β-carotene in combination products containing other carotenoids such as α-carotene, lutein, or lycopene. β-carotene and carotenoids in general have been intensively studied by liquid chromatography (LC), and procedures have been reported for the separation of β-carotene cis-/trans-isomers (13–18) and for β-carotene in supplements and foods (19, 20). Thus, a chromatographic procedure capable of separating all-trans-β-carotene from the corresponding cis-isomers and from other commercially used carotenoids was deemed necessary to properly determine the β-carotene content in dietary supplements.

References

1. Mathews-Roth MM. Biochemie. 1986;68:875–884.[PubMed]
2. Olson JA. J Nutr. 1986;166:1127–1130.[PubMed]
3. Temple NJ, Basu TK. Nutr Res. 1988;8:685–701.[PubMed]
4. Schierle J, Schellenberger T, Fizet C, Betz R. Eur Food Res Technol. 2002;215:268–274.[PubMed]
5. Sweeny JP, Marsh AC. J Assoc Off Anal Chem. 1970;53:937–940.[PubMed]
6. Panalaks CA, Warthesen JJ, Taoukis PS. J Inst Can Technol Aliment. 1990;3:145–151.[PubMed]
7. Sweeny JP, Marsh AC. J Am Diet Assoc. 1971;59:238–243.[PubMed]
8. Tsukida K, Saiki K, Sugiura M. J Nutr Sci Vitaminal. 1981;27:551–561.[PubMed]
9. Bushway RJ. J Liq Chromatogr. 1985;8:1527–1547.[PubMed]
10. Chandler LA, Schwartz SJ. J Food Sci. 1987;52:669–672.[PubMed]
11. Quackenbusch FW. J Liq Chromatogr. 1987;10:643–653.[PubMed]
12. Godoy HT, Rodriguez-Amaya DB. J Agric Food Chem. 1994;42:1306–1313.[PubMed]
13. Marty C, Berset C. J Agric Food Chem. 1990;38:1063–1067.[PubMed]
14. Tsukida K, Saiki K, Takii T, Koyama Y. J Chromatogr. 1982;245:359–364.[PubMed]
15. Vecchi M, Englert G, Maurer R, Meduna V. Helv Chim Acta. 1981;64:2746–2758.[PubMed]
16. Quackenbusch FW, Smallidge RI. J Assoc Off Anal Chem. 1986;69:767–771.[PubMed]
17. Ben-Amotz A, Lers A, Avron M. Plant Physiol. 1988;86:1286–1291.
18. Schüep W, Schierle J. J AOAC Int. 1997;80:1057–1064.[PubMed]
19. Sundaresan PR. J AOAC Int. 2002;85:1127–1135.[PubMed]
20. Eitenmiller RR, Landen WO Vitamin Analysis for the Health and Food Sciences. CRC Press; Boca Raton, FL: 1999. pp. 3–75. [PubMed][Google Scholar]
21. WHO/FAO. Report of a Joint FAO/WHO expert consultation, Bangkok, Thailand. FAO/WHO; Rome, Italy: 2002. Human Vitamin and Mineral Requirements, Chapter 7: Vitamin A; pp. 87–107. [PubMed]
22. Zechmeister L, Polgar A. J Am Chem Soc. 1944;66:137–144.[PubMed]
23. Emenhiser C, Sander LC, Schwartz SJ. J Agric Food Chem. 1996;44:3887–3893.[PubMed]
24. Mortensen A, Skibsted LH. J Agric Food Chem. 2000;48:279–286.[PubMed]
25. Schierle J, Härdi W, Faccin N, Bühler I In: Carotenoids, Volume 1A, Isolation and Analysis. Britton G, Liaaen-Jensen S, Pfander H, editors. Birkhäuser Verlag; Basel, Switzerland: 1995. pp. 265–272. [PubMed][Google Scholar]
26. Britton G In: Carotenoids, Volume 1B, Spectroscopy. Britton G, Liaaen-Jensen S, Pfander H, editors. Birkhäuser Verlag; Basel, Switzerland: 1995. pp. 13–62. [PubMed][Google Scholar]