Vitamin K1 – Analysis in Food for Special Medical Purposes (FSMP)

Improved sample preparation optimises the determination of phylloquinone (vitamin K1)

June 2021. Finally, times of non-reproducible and too low results for vitamin K1 in Food for Special Medical Purposes (FSMP) belong to the past. A simple modification of CEN EN method 14148:2003 allows for quantitative and reproducible extraction of fortified vitamin K1 from FSMP products containing ≤ 25g fat/100g product, even if micro-encapsulated vitamin K1 has been used during production. Instead of the time-consuming enzymatic digestion, ultrasonic treatment is used to apply thermal and mechanic energy to the sample. The obtained validation data show that sometimes, solving analytical challenges can be that easy.

Function and analysis of vitamin K

Vitamin K1 structure with vitamin K-rich foods

The fat-soluble vitamin K plays an important role in blood coagulation, bone metabolism, and the regulation of cellular growth. Thus, its daily nutritional intake is essential for human beings. Also, in case of special medical nutrition requirements, a sufficient supply of vitamin K has to be ensured. Consequently, FSMP products are fortified with vitamin K, usually in form of vitamin K1. For analysis, method CEN 14148:2003 is commonly used for vitamin K1 quantification in foodstuff. This method comprises enzymatic digestion with lipase followed by lipophilic extraction in order to remove fat from the sample. The extract is then analysed by high-performance liquid chromatography (HPLC), post-column derivatisation enables the fluorimetric detection of vitamin K1.^[1]

Quantification of micro-encapsulated vitamin K1

Lately, producers of FSMP, food supplements and functional food have discovered micro-encapsulation as a beneficial technology to stabilise bioactive compounds with a coating made from edible polymers like acacia gum, maltodextrin, or starch in their products. These small particles (ca. 5‑50 µm) are less prone to oxidation caused by elevated temperature, light, and atmospheric oxygen. Moreover, micro-encapsulation may help to stabilise fat-soluble substances like vitamin K1 in an aqueous solution.^[2]

However, the bioactive compound is not only "protected" from environmental influences, but also from being analytically captured. Accurate and precise vitamin K1 analysis, especially in low-fat FSMP products and even in products without encapsulation, has so far been a major challenge for laboratories. Thus, frequently varying and too low results were observed.^[3]

The solution: ultra-efficient ultrasonic treatment

Therefore, Eurofins developed a new sample preparation within the CEN EN method 14148:2003 to determine reliably and quantify vitamin K1. The sample treatment is suitable even for those products where vitamin K1 is micro-encapsulated.

Beforehand, different approaches like enzymatic digestion, supposedly suitable for the respective polymer encapsulation as well as hydrochloric acid treatment were tested without success. Since intensive grinding was known to improve recoveries of vitamin K1, mechanic treatment in an ultrasonic bath was tested, and finally, this approach paved the way towards reliable results.

The ultrasonic treatment is now combined with the CEN EN method 14148:2003 to replace the enzymatic treatment with lipase. In addition, this change shortened the analysis time required for sample preparation by 1.5 hours.

Validation data prove suitability in practice

Full validation was performed for five FSMP samples and two reference materials. The obtained relative standard deviations of six-fold sample preparation and vitamin K1 determination ranged between 1.0 and 9.5%. Spiking recoveries were well within the range of 80 – 110%, as accepted by the FDA reference guidelines for the respective concentration range [4].

In the reference materials, Nist 1869 (infant milk formula on soy base) and Nist 3280 (vitamin tablet) 108 ± 1.0% and 95 ± 4.3% of the certified values were found, respectively, proving that vitamin K1 is not degraded by the applied modification.

Do you have any further questions about vitamin K1 analysis?

Further information on the development of the new preparation method for FSMP samples can be found in our poster "Vitamin K1 quantification in FSMP products containing ≤ 25 g fat/100 g". If you have any questions about the analysis of FSMP products, please contact your personal account manager or our expert Tobias Spiess directly.

References

[1] CEN EN 14148:2003 Determination of vitamin K1 by HPLC.
[2] Corrêa-Filho, L.C., Maldao-Martins, M., Alves, V.D. 2019. Advances in the Application of Microcapsules as Carriers of Functional Compounds for Food Products. Applied Sciences. Vol. 9 (571), p.1-18.
[3] Delmonte, P., Barrientos, St., Rader, J.I. 2013. Modifications of AOAC Official Method 999.15 to Improve the Quantification of Vitamin K-1 in Complex Formulated Nutritional Products. Food Composition and Additives. Vol. 96 (1), p. 91-101.
[4] U.S. Food & Drug Administration (FDA). 2019. Quantitative Method Acceptability Criteria. Guidelines for the Validation of Chemical Methods for the FDA Foods and Veterinary Medicine Program. 3^rd Edition, Appendix 2, p. 23.