🍎 Determination of cyclamate in foodstuffs by high performance liquid chromatography — sodium cyclamate is extracted from the sample with water (liquid products and products giving a clear solution — dilution to about 400 mg/l; semi-solid products, chocolate and fat emulsions — clarification with Carrez reagents, for chocolate and mayonnaise after defatting with light petroleum), 20 ml of the solution is converted with sulfuric acid and sodium hypochlorite into N,N-dichlorocyclohexylamine, extracted with n-heptane and determined on a reversed phase (C18, methanol–water 80:20) with UV detection at 314 nm; external standard calibration, result as cyclohexylsulfamic acid in mg/kg or mg/l without decimal places, PN-EN 12857

Food chemistry PN-EN 12857

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of SIST EN 12857:2000, i.e. EN 12857:1999 (English text; the file is a scan — pages read as images): EN title page, contents, foreword and Clauses 1–9 (up to the test report, p. 7 of the EN). The sample does not contain Annexes A (determination of active chlorine in hypochlorite solutions), B (figures — example chromatogram), C (precision data) and D (bibliography); we know their titles from the contents.

ACCORDING TO THE TEXT OF EN 12857:1999. The standard was prepared by CEN/TC 275 “Food analysis — Horizontal methods” (secretariat DIN) and approved by CEN on 16.04.1999; national standards had to adopt it by October 1999 at the latest. Scope (1): an HPLC method for the determination of sodium cyclamate in foodstuffs; validated in an interlaboratory study according to ISO 5725:1986 on lemonade, an orange juice drink, fruit yoghurt and spray cream; according to experience the method is also suitable for gherkins, canned cherries, pineapple, orange nectar, apricot and blackberry jam, cherry nectar, hard candies, fruit and strawberry yoghurts, fruit quark, rice dessert with apples and raisins, chocolate and vanilla pudding powder, vanilla ice cream and passion fruit–orange ice cream. Reference (2): EN ISO 3696 (water for analytical use). Principle (3): sodium cyclamate is extracted from the sample with water, converted into N,N-dichlorocyclohexylamine and determined by HPLC on a reversed-phase column with UV detection at 314 nm. Reagents (4): analytical grade, water at least grade 1 according to EN ISO 3696; HPLC-grade methanol and n-heptane; light petroleum boiling at 40–60 °C; anhydrous sodium sulfate (washed with n-heptane if necessary); sodium carbonate solution 50 g/l; sodium hypochlorite solution with 1,7 % active chlorine (diluted from the commercial product, chlorine content checked regularly, e.g. according to Annex A); sulfuric acid 50 % (mass fraction); standard solutions — about 898 mg sodium cyclamate in 200 ml (cyclohexylsulfamic acid 800 mg/200 ml), dilutions of 0,25; 1; 2,5; 5; 10 and 20 ml to 100 ml give 10, 40, 100, 200, 400 and 800 mg/l of the acid; footnote to 4.8: conversion factor from sodium cyclamate to cyclohexylsulfamic acid 0,8909; Carrez solution No. 1 — 15 g potassium hexacyanoferrate(II) in 100 ml; No. 2 — 30 g zinc sulfate in 100 ml; mobile phase — methanol and water 80 + 20 (by volume), filtered (e.g. 0,45 µm) and degassed, with a small change of the ratio if necessary; EDTA conditioning solution 10 g/l; cellulose powder (≥ 99,9 %, acid-washed). Apparatus (5): chromatograph with UV detector at 314 nm (preferably diode array); reversed-phase column, e.g. RP C18, 5 µm, 250 mm × 4 mm, with an RP C18 precolumn (recommended especially for solid samples); column suitability criterion — baseline separation of the cyclamate peak; if a diode array detector or measurement at a second wavelength shows interferences, other chromatographic conditions must be chosen; ultrasonic bath, homogenizer, water bath at 60 °C, centrifuge of at least 1400 g, separating funnels, fluted filters, optionally phase-separating filters. Preparation of solutions (6.1): liquid products and products giving a clear solution (e.g. clear juices, filtered gherkin brine, hard candies) — dilution with water to about 400 mg/l cyclamate or directly to derivatization; semi-solid products (e.g. dairy products, desserts, spray cream, cloudy juices, jams) — about 15 g in a 100 ml flask with 80 ml water, 10 min in the ultrasonic bath, 1–2 ml each of Carrez solutions No. 1 and No. 2, made up with water, filtered discarding the first 10 ml; chocolate — about 15 g melted at 60 °C, fat extracted twice with 25 ml light petroleum with centrifugation, the remaining petroleum evaporated, 30 ml water, transferred to a 100 ml flask, 1 ml each of Carrez No. 1 and No. 2, 20 °C, made up, filtered; fat emulsions (e.g. mayonnaise) — about 15 g with 2,5 g cellulose, twice 25 ml light petroleum, 40 ml water, then as for chocolate; with an insoluble fat-free residue above about 3 g, centrifugation and washing the residue twice before filtration is recommended; 20 ml is taken for derivatization. Derivatization (6.2): warning — chlorine is formed, work in an efficient fume hood; 20 ml of sample or standard solution in a separating funnel with 1 ml sulfuric acid, 10,0 ml n-heptane and 2,5 ml sodium hypochlorite is shaken vigorously for 1 min, the lower aqueous layer is discarded, the heptane layer washed with 25 ml sodium carbonate solution (30 s), dried with 1 g sodium sulfate (about 7 g for emulsions, or a phase-separating filter) and filtered; derivatized solutions are stable for 24 h at +4 °C. Identification (6.3): by retention time against the standard, co-injection or addition of standard to the sample, and spectrum recording in a suitable wavelength range; interval between standard injections at least 15 min, between samples sufficiently long (e.g. 30 min); for the column of 5.1.2 the verified conditions are: flow 1,0 ml/min, injection 20 µl, 314 nm; daily conditioning: 10 min water, 30 min EDTA solution, 10 min water. Determination (6.4): external standard method — peak areas or heights against the standard of the closest value, or a calibration curve (concentration of cyclohexylsulfamic acid in mg/l), with a linearity check. Calculation (7): w or ρ = A1 · V1 · m1 · F / (A2 · V2 · m0) · 1000 (external standard; V1 and V2 each 100 ml, m1 — mass of cyclamate in the standard in mg, m0 — mass or volume of the test portion, F — factor for additional dilutions) or w or ρ = ρcs · V1 · F / m0 (calibration curve); result in mg/kg or mg/l of cyclohexylsulfamic acid, without decimal places; note in 7.3: conversion factor from sodium cyclamate to cyclohexanesulfamic acid 0,8906. Precision (8): data from the interlaboratory study (Annex C) may not apply to other concentration ranges and matrices; repeatability r and reproducibility R: lemonade (mean 435,9 mg/l) — r 16,7, R 38,1 mg/l; orange juice drink (178,3 mg/l) — r 15,4, R 24,4 mg/l; spray cream (280,9 mg/kg) — r 26,0, R 49,7 mg/kg; fruit yoghurt (647,6 mg/kg) — r 42,4, R 168,4 mg/kg. Test report (9): sample identification, reference to the standard, date and method of sampling (if known), dates of receipt and testing, results with units, observations and operations not covered by the method.

STATUS (as of 03.10.2026). In the PKN search (term “12857”, 03.10.2026) the only result is PN-EN 12857:2002 (Polish version, “Introduces: EN 12857:1999 [IDT]”), without a withdrawal note. Card: publication date 06.05.2002, 15 pages, KT 235 Food Analysis, ICS 67.050, no withdrawal note. In the iTeh catalogue (read 03.10.2026) EN 12857:1999 has the status “Published”, stage 90.93 (“Decision to confirm”), with a review completion date of 30.09.2026; the field “Withdrawal Date 31-Oct-1999” on this card is the deadline for withdrawing conflicting national standards, not a withdrawal of EN 12857.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, load up to 17.09.2026, read 03.10.2026). The number 12857 appears in 4 records at 4 laboratories: AB 377, AB 400, AB 451, AB 583. In the current PCA documents (read 03.10.2026; AB 451 issue No. 26 of 24.11.2025, AB 377 issue No. 29 of 12.12.2025, AB 583 issue No. 22 of 29.01.2026, AB 400 issue No. 37 of 07.08.2026) the number appears in 4 lines at the same 4 laboratories; in each document the number of page markers equals the number of PDF pages, without repetitions; a search of all PCA documents downloaded from the BIP on 03.10.2026 found no laboratories outside the database copy. Form “PN-EN 12857:2002” (AB 451, AB 583), “PN - EN 12857:2002” with spaces (AB 377) and “PN-EN 12857” without year together with procedure PB-262 in a flexible accreditation scope (AB 400). None of these items is suspended. The “Laboratories” tab (term “PN-EN 12857”) shows 3 laboratories — AB 400, AB 451, AB 583 — checked by a tab query on the labcoda.pl production server on 03.10.2026; AB 377 writes the designation with spaces around the hyphen, so it is not matched by prefix. The database copy contains no other reference documents for cyclamate (search for “cyklamin”, 03.10.2026).

WHAT THE LABORATORIES TEST (items in PCA). Object: food concentrates, non-alcoholic beverages (carbonated, still, juices, syrups), food supplements (AB 377); non-alcoholic beverages, food supplements (AB 451); non-alcoholic beverages, confectionery, food supplements (AB 583); food — flexible scope, “content of food additives” (AB 400). Characteristic and range: cyclamic acid (50–2000) mg/kg in solid samples and (10–800) mg/l in liquids and sodium cyclamate (56–2245) mg/kg and (11–898) mg/l (AB 377); cyclamic acid (18–534) mg/l or mg/kg (AB 451); cyclamate (10–2000) mg/l or mg/kg (AB 583). Technique: HPLC-DAD (AB 377, AB 400), HPLC-UV/VIS (AB 451), HPLC-UV (AB 583).

WHERE AN APPARENTLY CORRECT RESULT IS EASY TO GET. With the quantity: the standard gives the result as cyclohexylsulfamic acid (7.1–7.3), while the standard is weighed as sodium cyclamate; the conversion factor appears twice in the standard with a different fourth digit — 0,8909 in the footnote to 4.8 and 0,8906 in the note to 7.3; AB 377 gives ranges separately for the acid and for sodium cyclamate (e.g. 2000 and 2245 mg/kg). With the matrix: validation was carried out on lemonade, an orange juice drink, fruit yoghurt and spray cream, and the precision data may not apply to other matrices and ranges (1, 8.1); the list of matrices from experience does not mention food supplements, which three laboratories have in their scopes. With preparation: chocolate and mayonnaise require defatting with light petroleum before clarification (6.1.3, 6.1.4), and with a large insoluble residue (above about 3 g) centrifugation is recommended to account for the volume of the residue. With derivatization: the hypochlorite solution must contain 1,7 % active chlorine, checked regularly (4.6); derivatized solutions are stable only for 24 h at +4 °C (6.2). With chromatography: interferences detected with a diode array detector or at a second wavelength require other conditions (5.1.2), and too short an interval between injections risks confusing peaks from earlier samples (6.3). With the range: the precision data cover levels of 178,3–647,6 mg/l or mg/kg; the laboratories give ranges from 10 to 2000 mg/l or mg/kg.

WHAT WE DO NOT GIVE. We do not quote the procedure for determining active chlorine (Annex A), the example chromatogram (Annex B), the detailed precision data (Annex C) or the bibliography — they lie outside the sample. We do not give the maximum permitted levels of cyclamate from food additive legislation.

Method principle

Sodium cyclamate is extracted from the sample with water — after dilution, clarification with Carrez reagents or after defatting with light petroleum — and 20 ml of the solution is acidified with sulfuric acid and oxidized with sodium hypochlorite in the presence of n-heptane, converting cyclamate into N,N-dichlorocyclohexylamine, which passes into the heptane. The heptane layer, washed with sodium carbonate and dried with sodium sulfate, is separated on a reversed-phase column (C18, methanol–water 80:20) and the absorbance is measured at 314 nm. The concentration of cyclohexylsulfamic acid is determined by the external standard method or from a calibration curve, in mg/kg or mg/l.

Applications

• Cyclamate in foodstuffs: validated on lemonade, an orange juice drink, fruit yoghurt and spray cream; according to experience also in fruit preserves, jams, nectars, candies, desserts and ice cream (1)
• Liquid and semi-solid products, chocolate and fat emulsions — separate preparation procedures (6.1)
• Non-alcoholic beverages, food concentrates, confectionery, food supplements, food — items in the accreditation scopes of 4 laboratories in PCA

Key parameters

Standard

Standard number

PN-EN 12857:2002

Title (PL)

Artykuły żywnościowe — Oznaczanie zawartości cyklaminianu — Metoda wysokosprawnej chromatografii cieczowej

Title (EN)

Foodstuffs — Determination of cyclamate — High performance liquid chromatographic method

Step-by-step procedure

1. Sample solution

   Liquids — to about 400 mg/l; semi-solids — 15 g, water, ultrasound, Carrez; chocolate and mayonnaise — defatting with light petroleum (6.1). PN-EN 12857:2002 without a withdrawal note in PKN (03.10.2026).

2. Derivatization

   20 ml + sulfuric acid + n-heptane + hypochlorite, 1 min; washing with sodium carbonate, drying with sodium sulfate; in a fume hood (6.2).

3. Chromatography

   Column conditioning with water and EDTA; injection 20 µl, 1,0 ml/min, 314 nm; identification by retention time and spectrum (6.3).

4. Calibration

   External standard or calibration curve from 10–800 mg/l solutions, linearity check (6.4).

5. Result

   Formula (1) or (2), mg/kg or mg/l of cyclohexylsulfamic acid, without decimal places (7).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Derivatization releases chlorine — only in an efficient fume hood (warning in 6.2)
• Light petroleum, n-heptane and methanol — flammable; hypochlorite and sulfuric acid — corrosive

Frequently asked questions