Vitamin Analysis — EN 12821 / EN 14122 / HPLC and LC-MS/MS

What Is Vitamins?

Vitamin analysis is a group of analysis that requires a separate method for each vitamin. Fat-soluble vitamins (A, D, E, K) are measured by HPLC after saponification and extraction, and water-soluble ones (group B, C) are measured by HPLC or LC-MS/MS after appropriate extraction. Microbiological determination methods are used for some vitamins such as B12 and folate.

Purpose of Vitamins

The purpose is to verify vitamin claims on the nutrition label, control the level of additives in fortified products, and monitor vitamin loss throughout shelf life.

Which Products Is Vitamins Applied To?

Vitamin takviyeleri
Fortified milk and beverages
Baby food and follow-on formulas
breakfast cereals
Foods for special medical purposes

In Which Sectors Is Vitamins Used?

FoodAgricultureRetailPharmaceuticalsBiotechnology

Standards Related to Vitamins

TS EN 12821 (D vitamini — HPLC)
TS EN 12822 (E vitamini — HPLC)
TS EN 12823-1/-2 (A vitamini — HPLC)
TS EN 14122 (B1 vitamini — HPLC)
TS EN 14152 (B2 vitamini — HPLC)
TS EN 14130 (C vitamini — HPLC)
TS EN 14164 (B6 vitamini)

Vitamins Conditions

Light and oxygen: Many vitamins are sensitive to light and oxygen; sample preparation is done in the dark
saponification: Required for separation of fat-soluble vitamins from the matrix
Vitamer separation: Some vitamins have more than one active form; Which one is measured is reported

Equipment for Vitamins

HPLC (with UV, fluorescence and DAD detectors)
LC-MS/MS
Saponification and extraction devices
Microbiological determination infrastructure
Light-protected glass material

Sample Requirements for Vitamins

The sample should be sent protected from light and at appropriate temperature.
The list of vitamins to be analyzed should be determined in advance
Declared vitamin levels must be reported
Since each vitamin is analyzed separately, sufficient samples are required

Duration of Vitamins

Each vitamin is a separate analysis and the duration increases in direct proportion to the number of vitamins in the list. Incubation time is added for vitamins that require microbiological determination.

Vitamins Procedure

1The vitamins and methods to be analyzed are determined.
2The sample is prepared by protecting it from light.
3For fat-soluble substances, saponification and extraction are applied.
4Appropriate extraction is performed for water-soluble substances.
5Chromatographic measurement or microbiological determination is performed.
6Results are reported by vitamin.

Vitamins Acceptance Criteria

Acceptance criteria are label declaration and (EU) 1169/2011 tolerance guidelines. In order to make a vitamin declaration, a certain proportion of the reference intake value must be met within the scope of (EC) 1924/2006; Additionally, the provisions of (EC) 1925/2006 apply to enrichment.

Evaluation of Results

Vitamin content decreases during shelf life; Therefore, it is a common practice to add more than the declared level in production. The label declaration is expected to be met at the end of shelf life, so it makes sense to evaluate it together with the stability study.

Vitamins Report

Sample description and list of analyzed vitamins
Standard and method applied for each vitamin
Measured vitamer form
Results, detection limits and measurement uncertainties
Comparison with label declaration

Accreditation

EUROLAB holds internationally recognised accreditation obtained from accreditation bodies party to the IAF and ILAC multilateral recognition arrangements, under ISO/IEC 17025 for laboratory testing, ISO/IEC 17020 for inspection and surveillance, and ISO/IEC 17021-1 and ISO/IEC 17065 for system and product certification. This chain ensures the reports and certificates issued are accepted worldwide.

Whether this test can be offered within accredited scope depends on your product and the standard applied; since accreditation is method-based, the scope is confirmed in writing at the quotation stage.

Scope and authorisations

Related Regulations

Regulation (EU) 1169/2011 on Food Information to Consumers
Regulation (EC) 1925/2006 on the Addition of Vitamins and Minerals to Food
Nutrition and Health Declarations Regulation (EC) 1924/2006

Food Testing — What Does This Testing Field Cover?

The section below describes the field that the Vitamins test belongs to; the conditions and acceptance criteria of this particular test are given in the sections above. The scope specific to your product is confirmed in writing at the quotation stage.

Food testing shows that a product is safe for consumption and contains what its label states. The family works along three axes: microbiological safety, chemical safety and nutritional composition. All three require different laboratory infrastructure, a different body of methods and a different turnaround time.

The most distinctive feature of the field is TIME PRESSURE. A steel specimen can wait on a shelf; a raw milk or fresh meat sample cannot. The time the sample was taken, the transport temperature and how long it takes to reach the laboratory directly determine the validity of the result. In food microbiology the cold chain record is therefore an inseparable part of the test report.

On the microbiological side there are two different questions and they must not be confused. DETECTION of a pathogen is a qualitative question: is Salmonella present in the sample or not? ENUMERATION of indicator organisms is a quantitative question and an indicator of production hygiene. The absence of a pathogen in a product does not show that hygiene is good; if the total mesophilic aerobic count is high, there is a problem in the process.

On the chemical side, pesticide residues, mycotoxins, heavy metals, additives, veterinary drug residues and process contaminants (acrylamide, furan, PAH) are measured. Multi-residue methods in this area (QuEChERS-based LC-MS/MS and GC-MS/MS) can screen hundreds of active substances in a single analysis; limits are defined separately for each product–substance pair.

On the composition side, protein, fat, moisture, ash, fibre, sugar profile, fatty acid composition, vitamin and mineral content are determined. These values are the basis of the nutrition declaration on the label and the tolerance ranges are defined in legislation — the difference between the declared value and the measured value must stay within that tolerance.

There is NO CE marking on food, and this shapes how the field works. Conformity is demonstrated through operating a HACCP-based food safety system, complying with labelling rules and not exceeding contaminant, residue and microbiological limits. A test result produces evidence not for a certificate but for a functioning system.

Allergen management carries particular weight. The presence of an undeclared allergen in a product is one of the most frequent causes of food recalls. Analysis here runs together with cleaning validation: it is shown that the line has been cleaned adequately after a production run containing an allergen and that the risk of cross-contamination is under control.

Voluntary schemes have become effectively mandatory in this field. Retail chains require their suppliers to hold a certificate under a GFSI-recognised scheme (BRCGS, IFS, FSSC 22000); without it, getting onto the shelf is practically impossible.

Method groups in this field

Pathogen detection
Detection of Salmonella, Listeria monocytogenes, Campylobacter, Cronobacter and Shiga toxin-producing E. coli; culture-based and molecular methods.
Indicator counts
Total mesophilic aerobic count, Enterobacteriaceae, coliforms and E. coli, coagulase-positive staphylococci, yeasts and moulds, lactic acid bacteria, sulphite-reducing Clostridia.
Pesticide and veterinary residues
Multi-residue screening by LC-MS/MS and GC-MS/MS with QuEChERS extraction.
Mycotoxins and contaminants
Aflatoxins, ochratoxin A, deoxynivalenol, patulin; heavy metals (ICP-MS), acrylamide and PAH compounds.
Nutritional composition
Kjeldahl protein, fat and fatty acid profile, moisture, ash, dietary fibre, sugar profile, vitamin and mineral analyses.
Allergens and authenticity
Gluten, milk, egg, nut and soy allergen analyses; molecular methods for species identification and adulteration detection.

Commonly confused in this field

Sampling is half the result. A sample transported at the wrong temperature or delayed gives an invalid result even if it is analysed correctly in the laboratory.
The absence of a pathogen does not show that hygiene is good; indicator counts give separate information and the two are read together.
A result from a sample taken from a lot can be generalised to the whole lot only if the sampling plan was set up statistically.
Limits are defined in separate regulations by product group and are updated regularly; a conformity decision based on an outdated limit table may be wrong.
For exports, the limits of the destination country may differ from EU limits; the same product may be compliant in one market and non-compliant in another.

Which decisions these results support

Verification of the HACCP plan and process monitoring
The basis for the nutrition declaration and allergen information on the label
Health certificates before export and compliance with destination market limits
Supplier and lot-based acceptance control
Shelf-life studies and determining storage conditions
Producing evidence for audits under GFSI-recognised schemes

Related Tests

Frequently Asked Questions About Vitamins

If the vitamin degrades at the end of its shelf life, will the label be inappropriate?

The declaration is expected to be valid throughout its shelf life. Therefore, the margin of loss is predicted in the formulation and verified by the stability study. The value measured only on the day of production is not a sufficient basis.

Can you measure all vitamins in one analysis?

Some vitamin groups can be combined in a single method, but there is no single analysis for all of them. Each vitamin requires its own extraction and measurement condition; A list is given item by item at the bidding stage.