Advancing vitamin analysis in foods

Researchers at Teagasc and Sapienza University of Rome are developing and validating methods for the measurement of fat-soluble vitamins and water-soluble vitamins in foods.

Vitamins are a vital component of our diet, essential for the normal functioning of the human body; prolonged deficiencies can lead to serious health issues. They have widely varying chemical and physiological functions and are broadly distributed in food sources.

Vitamins A, D, E and K are fat-soluble vitamins (FSVs), and play an essential role in a number of physiological processes, such as metabolic health, vision, bone health and immune response. The body stores FSVs in the liver, fat tissue and skeletal muscle, and reserves can stay in the body for months.

Vitamins B and C, on the other hand, are water-soluble vitamins (WSVs). WSVs are well known for their role in energy metabolism as well as in the maintenance of healthy muscles, skin, eyes, hair and liver. Unlike FSVs, they do not stay in the body for long and have limited storage, and are rapidly excreted by the kidneys (vitamin B12 is the exception to this, as it can be stored in the liver for many years). Due to the limited storage of WSVs in the body, they require routine intake.

Limited vitamin research capabilities in Ireland

Accurate measurements are critical for determining the vitamin content of foods, the bioavailability of vitamins from food, and vitamin stability during handling, processing, storage and preparation. This information can be used to estimate population intakes, to establish dietary requirements, for governmental food fortification strategies and to research the relationships between diet and health.

In the food industry, the accurate measurement of the vitamin content of foods is required to ensure foods meet the specifications required by manufacturers and are within limits laid down by EU and international legislation.

At present, there are limited analytical methods for measuring vitamins in laboratories within Ireland, leading to a major gap in both analytical and research capability. The exception to this is the emerging capacity to measure vitamin K at Teagasc, and the measurement of vitamin D at University College Cork.

Teagasc’s George Hull – a Research Leaders 2025 and Marie Skłodowska-Curie Fellow – is leading a project to bridge the gap in knowledge and produce two improved methods for measuring vitamins. He is doing this by exploring the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) – a powerful analytical technique.

The challenges of vitamin extraction

The analysis of vitamins is challenging due to their unstable nature. Many factors contribute to this, such as exposure to heat, light and air. They also have complex chemistries and a range of compounds and metabolites that define each vitamin. 

WSVs can be present in free form or bound to proteins or polysaccharides that are present in the food matrix, or they can be phosphorylated (attached to a phosphate group). To release bound or phosphorylated vitamins, acid hydrolysis or enzymatic digestion is required to enable the quantification of the total WSV content of food items.

FSVs can be present in free form, esterified (combined with an acid) or complexed (containing several groups of vitamins) to the food matrix. Alkaline digestion or enzymatic digestion is therefore required to release the bound forms of the FSVs and enable the quantification of the total FSV content of the food item.

Methods for vitamin analysis

The official international analytical chemistry reference methods for vitamins – those used to obtain and process information about composition and structure – are based on high pressure liquid chromatography (HPLC) techniques with ultraviolet (UV) or fluorescence detection (FLD). For WSVs, some microbiological assays are also used.

These techniques use old technology, with some of these methods having been established more than 25 years ago. For most of the vitamins, the official reference methods require a different method, which makes the analysis of vitamins very expensive and time consuming.

Research-performing organisations and private sector companies performing vitamin analyses routinely use official reference methods, as they are considered the gold standard and give a high level of assurance in the result obtained. However, the expense and time-consuming nature of these methods greatly impedes the vitamin research capabilities in Ireland.

LC-MS/MS has many advantages over HPLC and microbial assays. Concerning the latter, even though microbial assays are inexpensive and rapid, they have low specificity and sensitivity when compared to HPLC and LC-MS/MS. Moreover, microbial assays for WSVs require an individual method for each vitamin.

LC-MS/MS combines the chromatographic separation capabilities of HPLC with the identification capabilities of mass spectrometry, and thus has the ability to use isotopically labelled (those in which atoms are replaced by their stable isotopes) internal standards. Due to this, LC-MS/MS has a higher level of specificity, sensitivity, accuracy and precision when compared to HPLC. This therefore enables the ability to measure a greater number of vitamins in a single method, and provides a greater level of assurance in the result obtained.

Through this project, George and the wider project team aim to produce two LC-MS/MS methods – one for WSVs and one for FSVs – that are capable of measuring all the major vitamin forms and are compliant with the standards required to produce an official reference method.

FUNDING

This research is funded by the Research Leaders 2025 programme (cofunded by Teagasc and the European Union’s Horizon 2020 research and innovation programme) under the Marie Skłodowska-Curie grant agreement number 754380.