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Latest BIS methods for vitamin determination in diverse food matrices

Wednesday, 08 October, 2025, 16 : 00 PM [IST]

Disha Zanwar

Vitamins are essential micronutrients required by the human body in small but consistent amounts to sustain various chemical and physiological functions. They cannot be synthesised by humans in sufficient quantity for normal metabolic requirements with the notable exception of Vitamin D. They are widely distributed in natural food sources and can easily be incorporated into diets to meet daily needs. These are diverse organic compounds with a wide range of structural and chemical properties. Vitamins are necessary for healthy cell function, growth, and development. Deficiency of these micronutrients causes a variety of clinical disorders including anemia, growth retardation, weakness and neurological illnesses. Conversely, maintaining adequate vitamin levels has a significant positive impact on overall health.

There are two main classes of vitamins based on their water affinity: hydro-soluble and fat-soluble. Hydro-soluble vitamins are composed by the group-B vitamins and vitamin C. The fat-soluble class is formed by vitamins A, D, E and K. This classification is important because it provides insights into the likely pathways of absorption—whether through lipid (fat) absorption or via hydrophilic (water-soluble) nutrient pathways. Additionally, it helps determine the appropriate techniques for extracting these compounds from foods or biological tissues, as well as the methods used for their analysis.

The fortification of foods such as milk, infant foods, fruit juices, and cereals has played a crucial role in addressing concerns related to micronutrient deficiencies. Accurate information on vitamin content in food is essential for assessing dietary adequacy and ensuring optimal nutrient intake. In this context, vitamin analysis becomes important, as sensitive and reliable analytical methods are vital for verifying the declared vitamin content on food labels, and meeting both regulatory and health standards for human consumption. One of the most popular approaches towards the determination of vitamins in food products were the microbiological methods. Unfortunately, such assays are outdated, extremely time-consuming, expensive and inaccurate. The instrumentation-based methods, on the other hand, offers a significantly more sensitive, versatile, robust, accurate and precise platform for the determination of vitamins, either of their different bioactive forms individually, or in total and converted into one stable form.

The Bureau of Indian Standards (BIS) has developed various Indian Standards for determining vitamin content in foodstuffs. Recently, BIS revised the Indian Standards for the estimation of Vitamin B5 (Pantothenic Acid), Vitamin B9 (Folic Acid), Vitamin B12, and Vitamin D (Refer Table 1). These updated standards are the outcome of a collaborative Research & Development (R&D) project focused on validating test methods for vitamins in selected food matrices. The project was led by CSIR–Central Food Technological Research Institute (CFTRI), Mysuru, in collaboration with five participating laboratories: Nestlé India Limited Laboratory Services (Moga), Envirocare Labs Pvt. Ltd. (Thane), Eureka Analytical Services Pvt. Ltd. (Bengaluru), Eurofins Analytical Services India Pvt. Ltd. (Bengaluru), and Microchem Silliker Pvt. Ltd. (Navi Mumbai).

The earlier versions of these standards, developed nearly five decades ago relied on microbiological assays and colorimetric methods. These traditional techniques are now considered outdated due to their time-consuming nature and limitations in sensitivity and accuracy. With advances in analytical technology, it became essential to update the standard methods to align with modern, highly sensitive chromatographic techniques such as HPLC and LC-MS/MS, which offer improved precision and reliability. While laboratories have already been using such methods based on AOAC, EN, and other international references these are often specific to particular food matrices. Given the diversity of food matrices (e.g., aqueous, fatty, fibrous), suitable sample preparation, extraction, and processing techniques are critical for broad applicability. Therefore, the R&D project included a multi-laboratory validation process, ensuring that the revised methods meet the Standard Method Performance Requirements (SMPR) and are applicable across a wide range of food products.

Table 1: Recently published Indian Standards on Vitamins Determination in Foodstuffs

Sr No	Indian Standard Number	Indian Standard Title	Applicable Food Matrix
1	IS 9840: 2025	Determination of Vitamin B5 pantothenic acid in foodstuffs by ultra-high performance liquid chromatography and tandem mass spectrometry UHPLC-MS/MS - Method of Test	Infant formula and adult nutritional; infant & breakfast cereals; beverages; fruits and vegetable; nuts and nut products.
2	IS 7234: 2025	Determination of Vitamin B9 Folates in Foodstuffs by Ultra-High-Performance Liquid Chromatography - Tandem Mass Spectrometry - Method of Test	Infant formula and adult/paediatric nutritional formula, cereals and pulses, fruits and vegetables and nut and nut products.
3	IS 7529: 2025	Determination of Vitamin B12 in Foodstuffs by HPLC- UV - Method of Test	Infant formulas; adult/paediatric formulas; infant and breakfast cereals; pulses; fruits and vegetables; fortified rice kernels; spirulina; beverages; Vitamin premixes; nuts and nut products.
4	IS 19308: 2025	Determination of Vitamin B12 in Foods Using Liquid Chromatography-Tandem Mass Spectrometry LC-MS/MS - Method of Test	Fortified atta (flour), almonds and similar matrices.
5	IS 5835:2025	Determination of Vitamin D in Fortified Vegetables Oils by ultra-high performance liquid chromatography and tandem mass spectrometry UHPLC-MS/MS Method of Test	Fortified vegetables oils.