Recent Trends in Beverage Testing – Vitamins

Driven by the global COVID-19 pandemic, consumers increasingly seek food and beverages that support immunity and health. As a result, the global functional beverages market is expected to grow to $158 billion in 2023.¹ Waters' HPLC and UPLC solutions enable reliable, accurate, and efficient testing methods to ensure that finished beverage products meet manufacturers' specifications and nutritional label claims.

Popularity of functional beverage products to support well-being and immune health

Today's food and beverage manufacturers must continually innovate to meet increasing consumer demand for products that support lifestyle and health goals. Six out of ten global consumers choose products that deliver health, immune, or fitness benefits.

The functional beverages market includes a variety of vitamin- and mineral-fortified drinks, including flavored waters, plant-based drinks, dairy-based beverages, sports drinks, and energy drinks. In particular, flavored waters continue to grow in popularity and product diversity as health-conscious consumers seek low- or no-sugar alternatives to sports drinks, energy drinks, and traditional carbonated sodas.

When enhancing beverages with vitamins, laboratory testing is required in a number of areas, including:

• Product development
• Ensuring vitamin stability throughout product lifecycle
• Confirming finished products meet label claims

Clear and accurate labeling is critical to support consumer choice and meet regulatory requirements, and manufacturers must be confident in the testing of added vitamins, minerals, and other relevant ingredients.

Complexity and challenges surrounding vitamin testing and analysis

Legacy analytical methods for vitamin testing in food and beverages have been limited to targeting a small suite of vitamins, resulting in the need to employ several analytical methods and a greater investment in multiple, and often different, analytical technologies to carry out multi-vitamin testing. As a result, laboratory staff requires extensive training on these varying methods and technologies.

There are many challenges associated with vitamin testing, including vitamin stability, which can be impacted by factors such as exposure to light or heat, this can have implications for the preparation and storage of calibration standards and samples. Often, a beverage may contain additional ingredients that can interfere with vitamin testing methods, so a high degree of analytical selectivity is required for accurate testing.

In a case study, Improving standards for vitamin analysis using mass detection, Amway's Dr.Chandra discusses the benefits of introducing mass detection, such as the ACQUITY QDa Mass Detector into routine vitamin testing workflows. Within, Dr. Chandra reports:

• The constant need to seek new analytical technologies to deliver selectivity and detection improvements to analytical workflows for the routine testing of multivitamins
• The ACQUITY QDa Mass Detector has helped harmonize Amway's vitamin analysis methods
• The ACQUITY QDa Mass Detector also simultaneously reduces the impact of co-elutions from other product ingredients when confirming lower concentrations of vitamins, which are challenging to measure with optical detection

HPLC flexibility enables accurate analysis of multiple, diverse vitamins

High Pressure Liquid Chromatography (HPLC) is frequently used to measure the concentration of vitamins in beverages. The flexibility provided through the array of column stationary phases and detectors makes it a useful technique when analyzing multiple vitamins, which are diverse in structure, chemical properties, and added concentration values.

Reversed phase chromatography is the typical HPLC column stationary phase used for the separation of water-soluble vitamins as well as fat-soluble vitamins, using water with either methanol or acetonitrile as the mobile phase. Additives and buffers such as formic acid and ammonium formate are often added to improve peak shapes, retention, and selectivity. Some legacy methods running on silica C₁₈ columns would use ion-pairing reagents to improve the retention and selectivity of the targeted water-soluble vitamins.

Modern reversed-phase chemistries such as the ACQUITY UPLC HSS T3 can retain and separate several water-soluble vitamins without the need of ion-pairing reagents. The application note, Rapid Analysis of 10 Water-soluble Vitamins, Caffeine, and Six Common Food Dyes Using ACQUITY UPLC with UV Detection, provides example retention and separation for several water-soluble vitamins without the use of ion-pairing reagents.

An alternative approach to reversed-phase chromatography for the retention and separation of polar analytes is the use of hydrophilic interaction liquid chromatography (HILIC), an HPLC technique often used for the analysis of polar compounds. The application note, ACQUITY UPLC Analysis of Water-Soluble Vitamins, provides an example HILIC separation using the ACQUTY UPLC BEH Amide Column.

When using HILIC for the analysis of vitamins in beverages, sample dilution with an organic solvent such as acetonitrile may be required to reduce the 'strength' of the water-based sample injection diluent with a 'weaker' solvent closer to the mobile phase starting conditions used in HILIC.

How UPLC drives increased laboratory efficiency, productivity, and cost-reduction

Some common goals when modernizing existing vitamin testing methods or creating a new testing method include:

• Accelerating sample turnaround time
• Reducing the number of testing methods
• Reducing the cost of analysis
• Reducing solvent consumption and waste

The use of Ultra Performance Liquid Chromatography (UPLC), which utilizes sub 2 µm column technology enables laboratories to transfer current methods or consolidate existing HPLC methods to meet the above goals.

A case study, Manufacturer Reduces Vitamin QC Analysis Times by Up to 90%, discusses the laboratory and business benefits gained by replacing traditional HPLC technology with ACQUITY UPLC H-Class technology. The case study highlights benefit to laboratory productivity and efficiency including:

• Consolidation of test procedures into fewer methods
• A 90% decrease in analysis time
• A 70-80% decrease in solvent use

In addition to the use of UPLC, to further increase productivity, techniques such as parallel column regeneration offer a solution to increase throughput and reduce analysis time. An example of applying parallel column regeneration to a vitamin method is outlined in the application note, Increasing Sample Throughput Using Parallel Column Regeneration for the Analysis of Water Soluble Vitamins by Hydrophilic Interaction Liquid Chromatography (HILIC). In parallel column regeneration the re-equilibration segment is excluded from the injection cycle, this means that the analysis time is shortened, increasing productivity while still maintaining the separation.

Enhancing B vitamin analysis with MaxPeak High Performance Surface Technology

Strong interactions between metal surfaces of HPLC or UPLC flow paths and certain analytes can result in poor peak shapes, analyte loss, inconsistent peak response and other LC issues which impacts the quality of data and reliability of methods.

To address these interactions Waters has recently launched the ACQUITY PREMIER Solution which incorporates MaxPeak High Performance Surface (HPS) technologies. The application brief, Enhancing the LC-MS/MS Analysis of B-group Vitamins with MaxPeak High Performance Surface Technology, highlights improvements for B vitamin analysis in areas such as:

• Peak shape
• Peak tailing
• Peak response
• Carry-over

Protecting brands with standardized and reliable beverage testing workflows

Waters' instrument and service solutions enable beverage testing laboratories to create efficient testing methods, supporting beverage manufacturers in:

• Developing new functional beverage products to meet changing consumer preference
• Ensuring finished products meet label claims for added vitamins
• Minimizing overage of expensive ingredients such as vitamins

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