🍎 Vitamin C (L-ascorbic acid) in foodstuffs — titration method according to Tillmans (2,6-dichlorophenolindophenol) for colourless and slightly coloured products and spectrophotometric method with extraction of the excess dye with xylene for strongly coloured products; PN-A-04019

Food chemistry PN-A-04019

Overview

WHAT THE STANDARD COVERS. Scope from the PKN catalogue card, in full: “Two methods for determining the vitamin C content of foodstuffs are given, depending on the degree of colouring of the product. For colourless and slightly coloured products the titration method according to Tillmans is presented, and for strongly coloured products the spectrophotometric method. The way of preparing samples of various kinds of foodstuffs for testing is specified”. According to the article by A. Mazurek and M. Włodarczyk-Stasiak (University of Life Sciences in Lublin, 2013) both methods use 2,6-dichlorophenolindophenol: in the spectrophotometric method ascorbic acid is oxidised quantitatively in an acidic medium by an excess of the dye to dehydroascorbic acid, the excess dye is extracted with xylene and determined spectrophotometrically at a wavelength of 500 nm. We have not read the text of PN-A-04019 itself.

STATUS (PKN card as of 08.11.2026). Card PN-A-04019:1998 (Polish version) without a withdrawal label — the standard is CURRENT: published 18-02-1998, 6 pages, price group C, Food, Agriculture and Forestry Sector, KT 200 Food Concentrates, Starch and Dietary Products, ICS 67.050; title “Foodstuffs -- Determination of vitamin C content”. “Replaces: PN-A-75101-11:1990 - Polish version, PN-A-86030:1978 - Polish version, PN-R-87020:1992 - Polish version, PN-A-79033:1985 - Polish version”. The card has no “Implements” field — it does not indicate an ISO or EN document that the standard adopts. Searching “PN-A-04019” in the PKN catalogue returns only this card — there is no other edition or foreign-language version in the catalogue. The 2013 article states that the standard was introduced on the basis of ISO 6557/2:1984; we have not compared the text of the Polish standard with the ISO text. ISO page (read 08.11.2026): ISO 6557-2:1984 “Fruits, vegetables and derived products — Determination of ascorbic acid content — Part 2: Routine methods” — published, edition 1 of 1984-11, 4 pages, ISO/TC 34/SC 3, ICS 67.080.01, stage 90.93; last reviewed and confirmed in 2023, this version is current.

ACCORDING TO THE TEXT OF ISO 6557-2:1984 — THE DOCUMENT ON WHICH, ACCORDING TO THE ARTICLE, THE STANDARD WAS BASED (full French text; this is not the text of PN-A-04019). ISO scope: fruits, vegetables and derived products — method A, titrimetric with 2,6-dichlorophenolindophenol, which may be used only in the absence of certain interfering substances (2.6), and method B, spectrometric with the same dye after extraction with xylene, for products in strongly coloured solutions. Method A (clause 2): ascorbic acid is extracted from the sample with an oxalic acid solution of 2 % (m/m) or with a solution of metaphosphoric acid and acetic acid (15 g of metaphosphoric acid in 40 ml of glacial acetic acid and 200 ml of water, to 500 ml; usable for 7–10 days in a refrigerator) and titrated with the dye solution to a salmon-pink colour. The dye solution is stored in a brown bottle in a refrigerator and — since the dye decomposes over time — a fresh one is prepared periodically; ascorbic acid standard solution 1 g/l (50 mg weighed to the nearest 0.01 mg, in a 50 ml flask, made up with extraction solution). Preparation: remove stones and seed cavities, mix thoroughly, filter; thaw frozen products in a closed vessel and add the exuded liquid before mixing. Test portion 10–100 g (in the text: weighed to the nearest 0.1 mg); volume of extraction solution in millilitres numerically 1 to 5 times the mass of the test portion in grams; filter, discarding the first millilitres; final ascorbic acid concentration in the test solution 0.1–1 mg/ml. Standardisation of the dye: 5 ml of standard solution with 5 ml of extraction solution is titrated rapidly to a salmon-pink colour persisting for at least 5 s, three times, reading to the nearest 0.1 ml; blank with 5 ml of extraction solution; the blank result is subtracted, and the titre is expressed in milligrams of ascorbic acid equivalent to 1.0 ml of dye. Determination: three portions of filtrate, each containing about 2 mg of ascorbic acid, titrated rapidly to the same end point; blank with the same volume of extraction solution; three determinations on the same sample. Result in mg per 100 g of product = (V0 − V1) × m1 / m0 × 100, where m0 — mass of the test portion in the portion for titration (g), m1 — titre of the dye (mg of ascorbic acid per 1.0 ml), V0 and V1 — volume of dye used for the sample and for the blank (ml); the result is the mean of three determinations. Interfering substances (2.6): iron, copper, tin, reductones, sulfhydryl compounds, sulphites and sulphur dioxide; reductones occur especially in products that have been excessively heated or stored for a long time. Check: 2 drops of 0.05 % methylene blue solution to 10 ml of a mixture of equal volumes of test solution and extraction solution — decolourisation within 5–10 s indicates interfering substances; tin cannot be detected in this way — for it, 5 drops of 0.05 % indigo carmine solution to 10 ml of test solution with 10 ml of hydrochloric acid diluted 1 + 3. Method B (clause 3): the same extraction solution, test solution 0.05–0.5 mg/ml; acetate buffer of pH 4 (300 g of anhydrous sodium acetate, 700 ml of water and 1000 ml of glacial acetic acid); xylene checked for purity (after shaking with decolourised dye the xylene layer must not be coloured after 10 min, otherwise the xylene is distilled). Into a 25 ml centrifuge tube with a glass stopper: 1–5 ml of test solution, an equal volume of buffer, immediately dye in excess, mix, 10 ml of xylene, shake vigorously for 6–10 s, centrifuge, transfer the xylene layer to a cell and measure the absorbance at 500 nm; blank — absorbance of xylene alone. Calibration curve: four tubes with 0.2; 0.4; 0.6 and 0.8 ml of dye. Result by the same formula, where V0 — volume of dye added, V1 — volume of excess dye read from the curve; two determinations, whose difference must not exceed 3 % of the mean value (3.6). Corrections (3.7): with pigments passing into xylene — hydroquinone correction (a second absorbance after adding hydroquinone solution, subtracted from the first); with products heated or stored for a long time, and in some cases also with natural products (e.g. blackcurrant juice) — a control test with 40 % formaldehyde, which corrects for the action of reducing substances not related to ascorbic acid. Test report (clause 4): method, result, operations not specified in the standard or regarded as optional and incidents that may have influenced the result, data identifying the sample.

WHAT THE SCIENTIFIC PUBLICATION SAYS ABOUT THIS STANDARD (Mazurek, Włodarczyk-Stasiak, Current Issues in Pharmacy and Medical Sciences 26(4), 2013, pp. 443–447). PN-A-04019:1998 does not allow the total vitamin C content (the sum of ascorbic and dehydroascorbic acid) to be determined, only ascorbic acid, because dehydroascorbic acid does not react with 2,6-dichlorophenolindophenol; other reducing substances present in the sample reduce the dye and raise the result. In reference material BCR-431 with a certified value of 483 mg/100 g (sum of both acids) the spectrophotometric method according to the standard gave 438 mg/100 g — a statistically significant difference — and the HPLC method gave 403.8 mg/100 g of ascorbic acid and 73.8 mg/100 g of dehydroascorbic acid: the result of the standard was thus lower than total vitamin C and higher than ascorbic acid alone. In apple and grapefruit juices and in blackcurrant nectar the results of the two methods differed significantly; they agreed only in orange juice; the authors noted the largest difference in blackcurrant nectar. According to the article, ignoring dehydroascorbic acid gives large errors especially in stored fruits and vegetables, in which the proportion of this acid increases. The article also states that CEN withdrew EN 14130:2003 (determination of vitamin C by HPLC) in 2010 without replacement — consistent with the PKN card of PN-EN 14130:2004 (withdrawn 21-11-2010, with no successor indicated; read 08.11.2026).

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, read 08.11.2026; “Laboratories” tab on labcoda.pl, 08.11.2026; current scope documents on the PCA website, read 08.11.2026). The number A-04019 appears in 8 records at 6 Polish laboratories: AB 285, AB 311, AB 452, AB 705 (1 each) and AB 486 and AB 552 (2 each); it does not appear at laboratories of other countries. Counted across all fields of the records and all forms of notation of the number; NOT included in the set are 16 records with the number R-04019 — this is a different standard, PN-R-04019 on available manganese in soil. Notations: “PN-A-04019:1998” without a point (AB 285, AB 552 — 2), “PN-A-04019:1998 pkt. 2” (AB 311), “PN-A-04019:1998, pkt. 2” (AB 452), “PN-A-04019:1998 pkt. 2.” (AB 486 — 2), “PN-A-04019:1998 p. 2” (AB 705). All 8 records appear in the current PCA scope documents in the same wording: AB 285 — issue 28 of 13.01.2026, AB 311 — 27 of 11.06.2026, AB 452 — 25 of 30.10.2025, AB 486 — 29 of 23.07.2026, AB 552 — 26 of 21.08.2026, AB 705 — 21 of 06.05.2026. The “Laboratories” tab (“PN-A-04019”) shows the same 6 laboratories with the same number of records (8) (tab query on labcoda.pl, 08.11.2026).

WHAT THE LABORATORIES TEST (database copy, complete items — 8). Titration method — 7 records at 5 laboratories: AB 311 — foodstuffs: soft drinks, juices, sweets, food concentrates, cereal products, dietary supplements, food for particular nutritional uses, (5.0–100000) mg/100 g; AB 486 — light-coloured foodstuffs: soft drinks, sweets, food concentrates, (2.5–2500) mg/100 g or 100 ml, and light-coloured dietary supplements, (15–100000) mg/100 g or 100 ml; AB 552 — milk and milk products, cereal grains and cereal and flour products, confectionery and bakery products, fruit, vegetables, mushrooms and their products, including vegetable-meat products, soft drinks, food concentrates, coffee, tea, cocoa, fruit and herbal teas, foods for particular nutritional uses, (1.2–500) mg/100 g and mg/100 ml, and dietary supplements, (1.2–100000) mg/100 g and mg/100 ml; AB 705 — juices, nectars and soft drinks, (0.3–44.0) mg/100 g; AB 452 — juices, nectars, drinks, syrups, fruit, vegetable and fruit-vegetable purées, “as L-ascorbic acid”, (1–130) mg/100 g, by potentiometric titration. Spectrophotometric method — 1 record: AB 285 — fruit and vegetable products, ascorbic acid (vitamin C), (8.9–315.0) mg/100 g or mg/100 ml. Four laboratories with the titration method cite point 2 of the standard; AB 285 (spectrophotometry) and AB 552 cite the standard without a point. Which method point 2 of the Polish standard describes we have not checked — in ISO 6557-2 clause 2 is the titration method.

ALONGSIDE THIS STANDARD. Records in which the name of the tested characteristic refers to vitamin C or ascorbic acid number 28 in the copy, at 19 Polish laboratories (read 08.11.2026). Apart from these 8: PN-EN 14130:2004 (HPLC) — 10 records at 7 laboratories, even though according to the PKN card the standard has been withdrawn since 21-11-2010 without a successor (it has a separate entry in the Knowledge Base); the remaining 10 — laboratories’ own procedures with liquid chromatography, the methodology of the IŻŻ publishing house of 1997 (titration method) and general notations “Standards” or “Standards/procedures” in flexible scopes. Three laboratories have both routes: AB 452 has, besides PN-A-04019, an HPLC procedure in which vitamin C is “the sum of L(+) ascorbic acid and dehydroascorbic acid”, AB 486 — a procedure with liquid chromatography, AB 552 — PN-EN 14130:2004.

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. What we measure: the result of this standard is L-ascorbic acid, not total vitamin C — dehydroascorbic acid does not react with the dye (2013 article); in stored products its proportion increases, so the “vitamin C” result according to this standard may be markedly lower than the result of a method determining the sum of both acids (e.g. the AB 452 procedure). The result according to PN-A-04019 and the result of a summative method are not interchangeable, although both are called “vitamin C content” in the scopes. Choice of method: according to the card the titration method is for colourless and slightly coloured products; AB 486 records the condition “light-coloured”, and AB 552 applies the titration method also to coffee, tea, cocoa and teas — how the laboratory assesses the colour does not follow from the scope. Measurement: reducing substances (in ISO 6557-2: reductones from products excessively heated or stored for a long time, sulfhydryl compounds, sulphites, sulphur dioxide, iron, copper, tin) decolourise the dye just as ascorbic acid does and raise the result — in reference material BCR-431 the result of the spectrophotometric method was higher than the content of ascorbic acid alone; ISO provides for a check with methylene blue and indigo carmine and for corrections with hydroquinone and formaldehyde — whether and how the Polish standard provides for them we have not read. Dilution: the solution for titration is to contain 0.1–1 mg/ml of ascorbic acid (ISO 6557-2) — for dietary supplements with a range up to 100000 mg/100 g (i.e. up to 100 g per 100 g) this requires multiple dilution, and any error in the factor passes directly into the result. Calculation: the dye titre m1 is a measurement of this method — standardisation with the standard solution, with subtraction of the blank (ISO 2.4.3.2); the dye decomposes, so a titre from another day or from an old solution gives a result shifted by the same factor in all samples. ISO 6557-2 gives the result only in mg per 100 g of product; some scopes also give mg per 100 ml — with such a result one needs to know whether the volume was measured or the sample was weighed, and ISO gives no conversion.

WHAT WE DO NOT GIVE. We have not read the text of PN-A-04019:1998: we do not give the numbering of its points, the preparation of samples of “various kinds of foodstuffs” (the card only says that it is in the standard), reagent concentrations, formulae, precision data or differences from ISO 6557-2 — the procedure given above comes from ISO 6557-2:1984 and is marked as such. We do not give the concentration of the dye solution from ISO either — in the text we read, the mass of the dye salt per 200 ml is written in a way that we cannot confirm with a second source. We have not read ISO 6557-1 (reference method) and have not checked its status. We have not read separately the statuses of the cards of the standards replaced by PN-A-04019 (PN-A-75101-11:1990, PN-A-86030:1978, PN-R-87020:1992, PN-A-79033:1985) — we only know that the PN-A-04019 card lists them as replaced. We have not checked citations of the standard in legislation. We have not reviewed the current PCA scope documents of laboratories outside the copy.

Method principle

L-ascorbic acid reduces (decolourises) 2,6-dichlorophenolindophenol. In the titration method according to Tillmans the sample extract in an acidic solution is titrated with a standardised dye solution to a persistent pink colour; in the spectrophotometric method the dye is added in excess, the excess is transferred to xylene and measured at 500 nm, and the dye consumption is read from the calibration curve. Result — mg of ascorbic acid per 100 g of product; dehydroascorbic acid is not determined (PKN card; article by Mazurek and Włodarczyk-Stasiak 2013; procedure — ISO 6557-2:1984, clauses 2–3, not the text of the Polish standard).

Applications

• Colourless and slightly coloured foodstuffs — titration method according to Tillmans (PKN card)
• Strongly coloured foodstuffs — spectrophotometric method with extraction of the excess dye with xylene, 500 nm (PKN card; 2013 article)
• Result: L-ascorbic acid content in mg/100 g (in scopes also mg/100 ml) — without dehydroascorbic acid
• In accreditation scopes: 6 Polish laboratories — juices, nectars, drinks, fruit and vegetable products, sweets, concentrates, cereal products, milk and milk products, dietary supplements, food for particular nutritional uses

Key parameters

Standard

Standard number

PN-A-04019:1998 (Polish version)

Title (PL)

Produkty spożywcze — Oznaczanie zawartości witaminy C

Title (EN)

Foodstuffs — Determination of vitamin C content

Step-by-step procedure

1. Choice of method

   Colourless or slightly coloured product — titration method; strongly coloured — spectrophotometric (PKN card). If interfering substances are suspected — check with methylene blue and indigo carmine (ISO 6557-2, 2.6).

2. Sample preparation and extraction

   Removal of stones, mixing (frozen products thawed in a closed vessel together with the exuded liquid); test portion of 10–100 g with extraction solution in a ratio of 1:1 to 1:5; filtration discarding the first millilitres; solution of 0.1–1 mg/ml (titration) or 0.05–0.5 mg/ml (spectrometry) (ISO 6557-2, 2.4.1–2.4.3.1, 3.4.3.1).

3. Standardisation of the dye

   5 ml of standard solution with 5 ml of extraction solution, rapid titration to a salmon-pink colour persisting for at least 5 s, three times; blank; titre m1 in mg of ascorbic acid per 1.0 ml of dye (ISO 6557-2, 2.4.3.2).

4. Determination

   Titration: three portions of filtrate of about 2 mg of ascorbic acid each, blank (ISO 6557-2, 2.4.3.3–2.4.5). Spectrometry: 1–5 ml of solution + buffer pH 4 + excess dye + 10 ml of xylene, shaking for 6–10 s, centrifugation, absorbance of the xylene layer at 500 nm, calibration curve with 0.2–0.8 ml of dye, two determinations; if necessary, hydroquinone correction or formaldehyde test (ISO 6557-2, 3.4.3.3–3.4.6, 3.7).

5. Calculation and result

   mg/100 g = (V0 − V1) × m1 / m0 × 100, mean of determinations; for spectrometry the difference between two determinations ≤ 3 % of the mean; in the report the method and deviations (ISO 6557-2, 2.5, 3.5–3.6, clause 4). PN-A-04019:1998 — current (PKN card as of 08.11.2026; Polish version).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Xylene has a narcotic effect at high concentrations — all operations with xylene in a fume hood (warning in ISO 6557-2, 3.2.5)
• Formaldehyde 40 %, oxalic acid, glacial acetic acid and hydrochloric acid in corrections and solutions — gloves, goggles, work in a fume hood (general rule; we have not read the text of the Polish standard)
• TECHNICAL NOTE: the result covers only L-ascorbic acid — it is not interchangeable with the result of a method determining the sum of ascorbic and dehydroascorbic acid (2013 article)

Frequently asked questions