Reviewer Name:
Neal Craft
Email:
ncraft@crafttechnologies.comOrganization:
Craft Technologies
Method Reviewed:
LUT‐02
Method Title:
Determination of Lutein and Zeaxanthin Esters and Their Geometric Isomers in
Carotenoid Ester Concentrates Used as Ingredients in Nutritional Supplements:
Validation of a Combined Spectrophotometric‐HPLC Method
Applicable SMPR
2016.004
Summary of Method:
The method uses a combination of spectrophotomety to measure total carotenoids
and normal‐phase HPLC on a saponified sample to estimate the percentage of free
lutein and zeaxanthin content in carotenoid ester concentrates, including their main
geometrical isomers. The unique part of this method is the estimate of a composite‐
specific absorbance due to the differing absorbances of the geometric isomer
distribution. The method is reportedly applicable to carotenoid ester concentrates in
oil suspensions and dosage forms. The sample is first dissolved in hexane–2‐
propanol (95+5, v/v) for spectrophotometric measurement at the maximum
absorption wavelength of ~445 nm. Subsequently, a sample is saponified then
separated using normal‐phase HPLC to determine the relative percentage of the
main geometric isomers of lutein and zeaxanthin.
Method Scope / Applicability
The method is reportedly applicable to carotenoid ester concentrates in oil
suspensions and dosage forms.
General Comments About the
Method
The method principles are sound. Normal‐phase chromatography is an excellent
method to separate xanthophylls and geometric isomers. Spectrophotometry has
been used for decades to estimate carotenoid content. The specific addition in this
method is to account for geometric isomer distribution of the samples and adjust
the calculation of content for the specific isomer content. This should result in a
more accurate estimate of xanthophyll esters. It makes some the assumption that
saponification does not alter the original isomer distribution of the esters.
Method Clarity
The execution of the method is adequately clear. The calculation is more
complicated and makes some assumptions that may not be accurate or
substantiated.
Pros / Strengths
Normal‐phase HPLC is a good method to separate the xanthophylls.
Spectrophotometry is a simple straight‐forward measurement of total carotenoids.
There is an effort to more accurately assess the xanthophyll content.
Cons / Weaknesses
There are assumptions made that could bias the results. Total carotenoids includes
everything that absorbs at 445nm. This could include hydrocarbon carotenes and
more polar xanthophylls which may not be accounted for by the HPLC. It assumes
that the saponification does not alter the isomer distribution which is incorrect. The
calculation of composite‐specific absorbance is theoretically sound but may not be
fully substantiated with foundational data. It assumes a single fatty acid ester and
uses E1% values that are not well documented or generally accepted. The HPLC
conditions are not current technology. Use of neat lutein and zeaxanthin standards
would be beneficial. Method is limited to lutein and zeaxanthin ester products. It
does not include beta‐cryptoxanthin or beadlet products.
General Comments about
Supporting Data
There is a substantial amount of precision , linearity and selectivity data from a
single lab.
Method Optimization
There is no discussion of HPLC or saponification optimization.
Information about the Method Only
General Information
Review of Information in Support of the Method