🍎 Sweeteners and preservatives (acesulfame K, aspartame, saccharin, sorbic acid, benzoic acid) — HPLC

Food Chemistry PN-EN 12856

Overview

STATUS (as of 03.11.2026). PN-EN 12856:2002 — Polish version (published on 06.05.2002), implements EN 12856:1999 [IDT]; the PKN catalogue card has no note of withdrawal, and in the iTeh catalogue EN 12856:1999 has the status “Published”. The title of the standard concerns the sweeteners acesulfame K, aspartame and saccharin; sorbic acid and benzoic acid are named in the standard as substances that this method also allows to be determined.

SCOPE (clause 1). An HPLC method for the determination of acesulfame K, aspartame and saccharin; it also allows the determination of caffeine, sorbic acid and benzoic acid in foodstuffs. Interlaboratory tests were carried out for acesulfame K in marzipan, yoghurt, fruit yoghurt, orange juice beverage, cola, cream and jam, for aspartame in marzipan, fruit yoghurt, orange juice beverage, orange-flavoured beverage, cola, jam and preparation for flan, and for sodium saccharin in marzipan, yoghurt, fruit yoghurt, orange juice, orange juice beverage, cola, cream and jam.

PRINCIPLE (clause 3). The sample is extracted or diluted with water; if necessary, the solution is purified on a solid-phase extraction column or with Carrez reagents. The sweeteners are separated on a reversed-phase HPLC column and determined spectrometrically at 220 nm.

SAMPLE PREPARATION (6.1). Clear liquid products (e.g. lemonades, cola, beverages): 20 ml of sample are diluted with water in a 100 ml volumetric flask and filtered through a 0.45 µm membrane filter. Cloudy liquid products (e.g. juices, flavoured milk drinks): 2 ml of Carrez solution I and then 2 ml of Carrez solution II are added to 20 ml of sample with 50 ml of water, the mixture stands for 10 min, is made up to 100 ml and filtered through a fluted filter paper (discarding the first 10 ml of filtrate) and through a 0.45 µm filter. Jams, preserves and marmalades (except fruit curds): about 20 g of sample with about 60 ml of water are held for 20 min in an ultrasonic bath at 40 °C and, after cooling, clarified with Carrez reagents. Semi-solid and solid products (e.g. curd cheese desserts, yoghurt products, delicatessen salads; except custard powder): 10–20 g of sample with about 50 ml of water, ultrasound for 20 min at 40 °C and Carrez clarification; for very complex matrices additionally solid-phase extraction — 2 ml of filtrate on a C18 cartridge activated with 3 ml of methanol and 20 ml of water, elution with about 20 ml of mobile phase. Custard powder: about 10 g in a 500 ml flask with about 400 ml of water and 6 ml of each Carrez solution. The temperature shall not exceed 40 °C, since aspartame can be degraded. If the fat-free insoluble matter exceeds about 3 g, centrifugation (10 min, at least 1400 g) is advisable, washing the settled matter twice with water and combining the supernatants in the volumetric flask.

IDENTIFICATION AND DETERMINATION (6.2, 6.3). The sweeteners are identified by their retention times against the standard, by simultaneous injection of the standard solution and the sample, or by adding the standard to the sample and recording an absorption spectrum. Equal volumes of sample and standard solutions are injected; the interval between successive injections of the standard solutions should not be less than 15 min. In the case of interferences, washing the column with a mixture of 50 parts by volume of mobile phase and 50 parts of acetonitrile is recommended. The determination is carried out by the external standard method or from a calibration graph whose linearity is checked.

CALCULATION AND RESULT (clause 7). With the external standard, w (mg/kg) or ρ (mg/l) = A1 × V1 × m1 × F / (A2 × V2 × m0) × 1000; from the calibration graph — C × F × V1 / m0; the factor F depends on the purification (column — 10, Carrez clarification — 1). The result for saccharin (sodium saccharin or saccharin as free imide), acesulfame K and aspartame is reported without a decimal place; the conversion factor from sodium saccharin to saccharin as free imide is 0.7593.

WHAT WE DO NOT GIVE HERE. The example chromatographic conditions of Annex A (including the composition of the mobile phase from A.5), the chromatograms (Annex B) and the full precision data (Annex C) — they are outside the available text of the standard. Maximum permitted levels of sweeteners and preservatives in food.

Method principle

The sample is diluted or extracted with water and, if necessary, purified with Carrez reagents or on a solid-phase extraction column with a C18 phase. Acesulfame K, aspartame and saccharin are separated on a reversed-phase HPLC column in a mobile phase of phosphate buffer and acetonitrile and determined spectrometrically at 220 nm, preferably with a diode array detector. The content is calculated by the external standard method or from a calibration graph; the same method allows caffeine, sorbic acid and benzoic acid to be determined.

Applications

• Acesulfame K, aspartame and saccharin in foodstuffs (clause 1)
• Clear beverages (lemonades, cola) and cloudy ones (juices, flavoured milk drinks) (6.1.1, 6.1.2)
• Jams, preserves and marmalades and semi-solid and solid products, e.g. curd cheese desserts and yoghurt products (6.1.3, 6.1.4)
• Custard powder (6.1.5)
• Caffeine, sorbic acid and benzoic acid — determination by the same method (clause 1)
• Checking the use of sweeteners and preservatives — food additives permitted only under the conditions of the list in Annex II (Regulation (EC) No 1333/2008, Article 4(1), Annex I)

Key parameters

Standard

Standard number

PN-EN 12856:2002

Title (PL)

Artykuły żywnościowe — Oznaczanie zawartości acesulfamu-K, aspartamu i sacharyny — Metoda wysokosprawnej chromatografii cieczowej

Title (EN)

Foodstuffs — Determination of acesulfame-K, aspartame and saccharin — High performance liquid chromatographic method

Step-by-step procedure

1. Clear beverages

   Dilute 20 ml of sample with water in a 100 ml flask and filter through a 0.45 µm membrane filter (6.1.1).

2. Cloudy beverages

   Mix 20 ml of homogenised sample with 50 ml of water in a 100 ml flask with 2 ml of Carrez solution I, then with 2 ml of Carrez solution II, shake, let stand for 10 min, make up to the mark with water, filter through a fluted filter paper (discard the first 10 ml) and through a 0.45 µm filter (6.1.2).

3. Jams and solid products

   Weigh to the nearest 1 mg about 20 g of jam (or 10–20 g of a semi-solid or solid product) into a 100 ml flask, add about 60 ml (or 50 ml) of water and hold for 20 min in an ultrasonic bath at 40 °C — not higher, since aspartame is degraded; after cooling, clarify with Carrez reagents as above (6.1.3, 6.1.4).

4. Additional purification and centrifugation

   For very complex matrices, apply 2 ml of filtrate to a C18 cartridge activated with 3 ml of methanol and 20 ml of water and elute with about 20 ml of mobile phase. If the fat-free insoluble matter exceeds about 3 g, centrifuge for 10 min at at least 1400 g, wash the settled matter twice with water and combine the supernatants (6.1.2–6.1.4).

5. Chromatography

   Inject equal volumes of sample and standard solutions, with an interval between injections of the standard solutions of not less than 15 min; identify by retention times or absorption spectra, determine by the external standard method or from a calibration graph (6.2, 6.3).

6. Calculation

   Calculate w (mg/kg) or ρ (mg/l) from the external standard equation or from the calibration graph; F = 10 after the cartridge, 1 after Carrez clarification; report the result without a decimal place (clause 7).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Acetonitrile — in the classification notified to ECHA: highly flammable (H225), harmful if swallowed, in contact with skin and if inhaled (H302, H312, H332), causes serious eye irritation (H319)
• The available text of the standard contains no safety clause — other health and safety rules are set by the laboratory

Frequently asked questions