Analytical Methods for Measuring Astaxanthin

 

Astaxanthin is a member of the carotenoid family, which is comprised of more than 700 natural lipid-soluble pigments that are only produced by phytoplankton, algae, plants, and a limited number of fungi and bacteria. Astaxanthin is quite common in nature, especially in the marine environment, and is probably best known for eliciting the pinkish-red hue in the flesh of salmon and trout, as well as shrimp, lobsters, and crayfish. These animals obtain astaxanthin in their diet from zooplankton, insects, or crustaceans that have accumulated astaxanthin from phytoplankton.

Cyanotech harvests astaxanthin and other productsfrom microalgae grown in Hawaii. Photo courtesy of Cyanotech.

Carotenoids, especially astaxanthin, are distinguished by their capacity to interact with chemically reactive species of oxygen known as singlet oxygen and free radicals. Interestingly, animals have adapted to exploit the potent antioxidant properties of carotenoids. One familiar example is seen in salmon and trout that selectively accumulate astaxanthin from their diet and deposit it in their flesh to protect lipid tissues from peroxidation, a harmful form of oxidation, during the most grueling natural marathon: Swimming upstream for many miles.

A growing body of scientific literature reveals significant evidence that astaxanthin surpasses the antioxidant benefits of beta-carotene, zeaxanthin, canthaxanthin, vitamin C, and vitamin E. Animal cell culture studies have also indicated that astaxanthin can protect skin from the damaging effects of ultraviolet radiation, ameliorate age-related macular degeneration, protect against chemically induced cancers, increase high-density lipoproteins, and enhance the immune system. Astaxanthin is also a natural antiinflammatory that works through multiple pathways to combat inflammation in the body. This is extremely important as scientists discover that many life-threatening diseases such as heart disease, cancer, Alzheimer’s, and stroke have inflammation as a root cause.

This has also become important since the recent discovery that prescription antiinflammatories can have serious side effects. As an antiinflammatory, natural astaxanthin from Haematococcus algae has been shown in various placebo-controlled, double-blind human clinical trials to have beneficial effects on a host of inflammatory conditions, including rheumatoid arthritis, carpal tunnel syndrome, joint and muscle soreness after exercise, and inflammation of the skin from UV radiation and sunburn. Cyanotech Corp. (Kailua Kona, HI) has obtained patents on the use of astaxanthin for some inflammatory conditions, including carpal tunnel syndrome, cold and canker sores, and as an internal and topical sunblock.

Although natural sources of astaxanthin are numerous, nearly all are found in very low concentrations. By far, the green algae Haematococcus pluvialis provides the most concentrated natural source of astaxanthin known, from 10,000 to 40,000 ppm (mg/kg) astaxanthin in addition to other important carotenoids such as beta-carotene, lutein, and canthaxanthin. As a comparison, the flesh of wild Atlantic salmon on average contains 5 ppm of astaxanthin, Coho salmon about 14 ppm, and sockeye salmon, 40 ppm. Since astaxanthin from Haematococcus is typically provided at 4-mg dosages in dietary supplements, each gelcap has the same amount of astaxanthin as 800 g of Atlantic salmon.

ANALYTICAL METHODS

Natural astaxanthin derived from Haematococcus is offered by a number of nutritional supplement ingredient manufacturers in the United States. These companies offer a variety of extracted oleoresin astaxanthin products and stabilized astaxanthin powders. Unfortunately, the analysis methods for astaxanthin and reporting of the astaxanthin content are not standardized. This can lead to confusion in the marketplace and to consumer products with dosages that are lower than what is claimed on product labels.

There are two general methods for analyzing the astaxanthin content of a product: spectrophotometric analysis and high-performance liquid chromatography (HPLC) analysis. In both methods, astaxanthin is extracted from a product into a suitable solvent such as acetone or hexane. In spectrophotometric analysis, the light absorbance of the extract solvent containing the astaxanthin is measured at a wavelength that corresponds to the maximum absorbance for astaxanthin (usually between 470 and 480 nm). The astaxanthin content is then simply calculated by dividing the measured absorbance by the extinction coefficient for pure astaxanthin at the specified wavelength and correcting for dilution of the product sample.

The problem with the spectrophotometric assay method is that in addition to astaxanthin, other carotenoids such as lutein, canthaxanthin, and beta-carotene are falsely included as astaxanthin in the results. Of even greater concern, chlorophyll and degradation products of astaxanthin without health benefits, such as astacene, will also be falsely included as astaxanthin. All of these compounds absorb light at 474 nm and increase the absorbance measurement, which leads to an overstatement of the astaxanthin concentration in the product.

Astaxanthin concentrations determined by spectrophotometric analyses can be overstated by more than 20%. Some manufacturers try to minimize this overstatement by reporting spectrophotometric analysis results as “astaxanthin complex” to indicate that the analysis includes other carotenoids. While this is technically more correct, it does not provide the true level of astaxanthin in a product, nor does it account for the possible presence of chlorophyll or degradation products of astaxanthin. Purchasing astaxanthin from a supplier that uses spectrophotometric analysis means that you can’t be sure if your astaxanthin is really astaxanthin; it will definitely have other carotenoids and it may very well have chlorophyll and the inert astacene as well.

The most technically sound and accurate method for determining the astaxanthin content of a product is by HPLC analysis. Determination of the astaxanthin content of Haematococcus algae by HPLC analysis has been accepted by the U.S. Food and Drug Administration (21 CFR 73.185) and the Canadian Food Inspection Agency (Registration No. 990535); and it is used by Japan’s official analysis agency, the Japan Food Research Laboratory.

HPLC analysis uses column chromatography to separate carotenoids from one another and from chlorophyll and astaxanthin degradation products, and to allow accurate measurement of only astaxanthin. There is, however, a complication: Haematococcus produces astaxanthin primarily as esters with fatty acids attached onto the terminal hydroxyl group(s). Roughly 82% of astaxanthin is present as monoesters, 12% present as diesters, and only 6% is present as free astaxanthin. HPLC analysis of a simple solvent extract of such products would produce many ester-astaxanthin “peaks.” Further, pure monoesters and diesters of astaxanthin are not available to calibrate the analytical results.

This problem is overcome by treating a simple solvent extract of an astaxanthin product with cholesterol esterase, which gently saponifies the esters (or removes the fatty acids from the astaxanthin), yielding free astaxanthin. HPLC analysis of the saponified solvent yields only free astaxanthin peaks, which are easily quantified using pure astaxanthin standards. Beta-carotene, canthaxanthin, lutein, and astacene are clearly separated from the astaxanthin peaks, which can be easily quantified. It is also interesting to note that if the peak heights for beta-carotene, canthaxanthin, lutein, and astacene are added to those for astaxanthin, as would be done with a spectrophotometric analysis, the astaxanthin content would be overstated by 18%.

In conclusion, an HPLC analysis method should be used for accurate determination of the astaxanthin content of products. Astaxanthin content should be reported as percent or total milligrams of free astaxanthin and not as “astaxanthin complex,” which includes other carotenoids and possible degradation products.

 

Gerry Cysewski, PhD, is CEO of Cyanotech Corp. (Kailua Kona, HI), a producer of high-value nutrition and health products from microalgae. For more information about Cyanotech, visit www.cyanotech.com. In addition, a detailed HPLC analysis protocol for astaxanthin analysis is available directly from the author or on the Web at: www.cyanotech.com/pdfs/axbul20.pdf