Do You Know How Much Vitamin A is in that Cod Liver Oil You Produced?

The National Institutes of Health (NIH) defines Vitamin A as the name of a group of fat-soluble retinoids, primarily retinol and retinyl esters. This vitamin is involved in immune function, supports cell growth, and plays a critical rule in the normal functioning of the heart, eyes, lungs, and other organs.  The NIH notes on its website the Recommended Dietary Allowance (RDAs) of Vitamin A, and it warns that there are health consequences for both deficiency and excessive amounts of the vitamin in the human body.

Cod liver oil is recognized by the National Library of Medicine as a source of Vitamin A and can be obtained from eating fresh cod liver or by taking supplements.  Fortune Business Insights notes that the “global cod liver oil market is projected to grow from $88.75 million in 2022 to $162.86 million by 2029.”

It is crucial for supplement manufacturers, including those who provide cod liver oil capsules, to have multiple quality control procedures in place.  In food or supplement manufacturing where the product is a complex mixture, UV-Visible derivative spectroscopy offers a fast and easy quality control check. Complex samples don’t have to be worked out or components separated for quality testing. The sample can be measured as-is. For production management, this could eliminate the need for additional analysis steps and save time overall, as food scientists can utilize UV-Visible derivative spectroscopy for determining the vitamin A content in cod liver oil, a complex sample consisting of multiple chromophores.

UV-Visible derivative spectroscopy

UV-Visible absorption spectroscopy is a robust, non-destructive technique for quantifying chromophores in solution. It is a highly useful analytical technique most frequently used for the quantification of analytes within a given solution-phase sample. The method is centered around the ability of molecules to absorb light in the UV-Visible region of the electromagnetic spectrum, which can incite electronic transitions between the ground and excited states. As electronic structure and the associated transition probability are unique to a given molecule, the measured absorption spectrum will provide information specific to the analyte of interest. Through Beer’s law (eq. 1), where A is the measured absorbance as a function of wavelength, c is the concentration of the analyte, l is the pathlength and ε is the extinction coefficient as a function of wavelength, the measured absorption is shown to be linearly proportional to the analyte concentration. This allows for a simple and quick quantitative analysis.

UV-Visible absorption bands are typically broad due to a variety of factors,¹ and as a result can be complicated to interpret when multiple chromophores are present in solution (Figure 1). While Beer’s law (eq. 1) describes the correlation between absorbance and concentration of one chromophore, when multiple chromophores are measured in a single sample the resulting absorption spectrum is additive. Equation 2 describes the relationship between the measured absorbance and the absorption related to each chromophore, where A_T,λ is the total absorbance and A_1,λ, A_2,λ, and A_n,λ are the absorbances related to each individual component. For samples with significant spectral overlap, determining the contributions from each chromophore can be difficult or, in the case of principle component analysis, require a certain degree of mathematical fitting and inherent estimation.

To combat these shortcomings, derivative spectroscopy can be employed as a relatively simple method for analyzing the UV-Visible spectra of complex systems. In this method, the nth derivative (1st, 2nd, etc.) is taken of the collected UV-Visible spectrum and graphed as a function of wavelength. These resulting spectra can help better resolve overlapping spectral features ^2-5 and are used in a variety of application spaces, such as food science or pharmaceutical analysis. ^3,5-8 Though previously obtained through complicated experimental procedures, like wavelength modulation, modern software is able to mathematically calculate the derivative spectra quickly and without need of additional calculations.⁹

To demonstrate the use of derivative spectroscopy for complex matrices, the vitamin A content within cod liver oil, a common supplement, was analyzed. Cod liver oil is not only known to contain appreciable amounts of vitamin A, also referred to as retinol, but can also contain a variety of other substances, including vitamin D isomers.^13,14 Retinol is known to absorb in the UV-Visible range,^11,12 while at the same time some of the other components present in cod liver oil are also absorptive and include features in the same spectral region as retinol. As a result, the UV-Visible absorption spectrum of cod liver oil is expected to include overlapping absorption features.

An experiment was conducted using a UV-Vis Spectrophotometer, where the absorption spectra of a cod liver oil sample and retinol standard solutions were collected. The resulting data was further processed to obtain first and second derivative spectra. These resulting spectra were then used to qualitatively and quantitatively analyze the vitamin A content in cod liver oil.

By using derivative spectroscopy, complications from overlapping absorption bands could be avoided, and the vitamin A concentration was found to be in good agreement with the anticipated concentration.

Conclusion

UV-Visible derivative spectroscopy can be used to better resolve overlapping spectral features and accurately assess concentration levels. While derivative spectroscopy can aid in qualitative analysis, there are methods by which derivative spectra can be quantitatively analyzed as well. This technique is relevant in a variety of application spaces, such as food science or pharmaceutical analysis.

In depth details about the experiment procedures and results, including the spectra and charts, are available in the application note Analysis of Vitamin A Within Cod Liver Oil: Use for Derivatives in UV-Visible Absorption Techniques.

Resources & References

• Application note: Analysis of Vitamin A Within Cod Liver Oil: Use for Derivatives in UV-Visible Absorption Techniques
• On-demand webinar recording: UV-Visible Derivative Spectroscopy: Theory and Applications
• Additional Website Resources for UV-Vis Spectrophotometers

Note, the references below are used throughout the application note as well as the above article:

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