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Food Testing >> Resources >> Navigating Vitamin K2 Testing: Key Challenges and Emerging Technologies

Navigating Vitamin K2 Testing: Key Challenges and Emerging Technologies

Introduction

Vitamin K plays a crucial role in various physiological processes, from supporting proper blood clotting to contributing to bone and cardiovascular health¹. As interest in this essential nutrient grows, ensuring accurate testing and reliable measurement of vitamin K levels has become increasingly important for the infant formula, food, beverage, and dietary supplement industries.

This white paper provides an in-depth look at the significance of vitamin K in the body, highlighting the latest testing techniques used to assess its presence and functionality. Additionally, it offers troubleshooting strategies for addressing aberrant results, ensuring precise and trustworthy data.

The Role of Vitamin K

Vitamin K is a fat-soluble vitamin discovered in 1943 by the Danish researcher Henrik Dam². Vitamin K exists in nature in several forms. Plants synthesize phylloquinone, or vitamin K1, which is the predominant form in the diet derived from leafy green vegetables. Vitamin K2 consists of a family of menaquinones, with menaquinone-4 (MK4) and menaquinone-7 (MK7) among the most nutritionally significant. MK4 is found in butter, egg yolks, cheese, and organ meats. MK7 is much less prevalent in the U.S. diet, found in certain hard cheeses and in the Japanese fermented soybean food called natto, which is the highest known dietary source of K2³.

While vitamin K1 is included in many multivitamins due to its critical role in blood coagulation, the increased awareness and usage of MK7 vitamin K2 dietary supplements has risen significantly over the past several years due to vitamin K2’s known role in the activation of vitamin K-dependent proteins for the proper utilization of calcium in the body, which is critical for optimal bone mineralization and reducing arterial calcification. MK7 is commonly found in bone, heart, and brain health dietary supplements, as well as sports supplements and immune health products. Vitamin K2 packs a punch for bone and heart health with a small daily intake⁴. Typical inclusion levels are in the 100 to 300 mcg range⁵. Because of this, and it being a fat-soluble nutrient, analytical testing for MK7 has its challenges. Other critical factors that play a role in vitamin K2 testing are its source, stability, and specific degradation factors.

Testing Methodologies

Various testing methodologies are available for analyzing vitamin K, with the optimal approach depending on the product type and specific testing requirements. Below are two methods well-suited for a range of sample matrices.

High Performance Liquid Chromatography with Fluorescence Detection (HPLC-FLD)

For products such as infant formula that require reference methods to use the same highly reproducible procedure, the fluorescence method is preferred as it is the standard set by the AOAC (Association of Official Analytical Chemists). In the fluorescence method, the vitamin K requires derivatization by post column reduction, a chemical modification that allows the compound to be detected by fluorescence. This method is much more selective than the UV detection method. Fluorescence also greatly reduces the time it takes to analyze a sample as it does not require multiple rounds of purification to be analyzed.

Tandem Mass Spectrometry

Tandem mass spectrometry (or MSMS for short) for vitamin K2 testing is optimal when it comes to premixes and finished products that need to test for a very small amount of vitamin K2 with the actual mass of the MK7 compound detected through a fully automated process. MSMS’s testing advantage is due to its high sensitivity, specificity, and accuracy.

An advanced technology that incorporates MSMS is Supercritical Fluid Extraction-Mass Spectrometry. This revolutionary analytical technique that combines extraction, separation, and detection into one automated process.

Advantages of SFE-SFC-MSMS testing for vitamin K2 is the ability to detect the one correct MK7 atom configuration, called an isomer, that is nutritionally active. Synthetically produced MK7 often contains the wrong isomers, and its vitamin K2 activity is very low or non-existent, which will significantly impact your test results. On the other hand, MK7 naturally produced by bacterial fermentation contains only the correct isomer.

Navigating Unexpected Results

Given the challenges with vitamin K2 testing, what should you do if you have tested your vitamin K2 MK7 product and you’re not getting the results you expect?

Start by reviewing the test method used. Mass spectrometry is the most precise technique, capable of detecting even the smallest inclusion levels.

Next, consider product stability:

Was the sample exposed to light during testing?
Are there formulation components that could degrade MK7?
Positively charged minerals like zinc can reduce vitamin K2 potency.
High pH liquid formulations can also compromise MK7 stability, leading to lower test results.

Finally, assess the source of your vitamin K2:

Is it synthetically produced and missing its nutritionally active isomer?
Naturally sourced vitamin K2 is the only form guaranteed to maintain its full nutritional benefits

Thorough testing and strategic formulation refinements are key to maintaining potency and achieving consistent, reliable results.

The Eurofins Advantage

Selecting the right laboratory for analyzing your premix or finished product is essential to ensuring accurate vitamin K2 potency and expected levels. At Eurofins, our rigorous testing protocols and advanced methodologies safeguard sample integrity, preventing degradation and delivering precise results.

We provide testing across all stages of product development—from bulk ingredients to premixes and finished formulations, including capsules, tablets, soft gels, and beverages. With advanced technology and rigorous laboratory protocols, Eurofins is your trusted partner in delivering accurate, reliable results with confidence

Reach out today to connect with an expert.