🍎 Determination of caffeine content in regular and decaffeinated green and roasted coffee beans and in regular and decaffeinated coffee extract (instant coffee) by HPLC — extraction of 1 g (or 0.5 g of extract) with water at 90 °C for 20 min in the presence of magnesium oxide, purification of an aliquot of the filtrate on a small column of silica modified with phenyl groups, separation on a C18 column with methanol/water 30 + 70 at 40 °C, UV detection 254–280 nm (caffeine maximum 272 nm), result in g caffeine per 100 g dry matter; PN-ISO 10095:1997 (withdrawn 01.08.2018)

Food chemistry PN-ISO 10095

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of ISO 10095:1992 (first edition, 1992-01-15, English text) — PN-ISO 10095:1997 adopts it as equivalent [EQV] (PKN card). The sample covers the whole document: foreword, Clauses 1–11 and Annex A (bibliography), 6 pages in total. The sample text is a scan with character recognition errors; we read numbers and units from the context of sentences.

ACCORDING TO THE TEXT OF ISO 10095:1992. The standard was prepared by ISO/TC 34 “Agricultural food products”, Subcommittee SC 15 “Coffee”. Scope (1): determination of caffeine content by HPLC in regular and decaffeinated green and roasted coffee beans and in regular and decaffeinated coffee extract powder; the reference method (UV absorption spectrometry) is ISO 4052:1983. References (2): ISO 1447:1978 (moisture of green coffee, routine method), ISO 3726:1983 (loss in mass of instant coffee at 70 °C under reduced pressure), ISO 6673:1983 (loss in mass of green coffee at 105 °C). Principle (3): extraction of caffeine from a test portion with water at 90 °C in the presence of magnesium oxide; filtration and purification of an aliquot on a silica microcolumn modified with phenyl groups; determination by HPLC with UV detection. Reagents (4): HPLC-grade methanol; ammonia solution 0.3 mol/l with methanol 90 + 10; elution solvent methanol/water/acetic acid 75 + 25 + 1; mobile phase methanol/water 30 + 70 (600 ml of methanol to 2 l, filtered through 0.45 µm; changing the methanol concentration shifts the retention time); ethanol/water 1 + 4; magnesium oxide; caffeine stock solution 0.5 g/l (125 mg in a 250 ml amber flask, in ethanol/water; stable for a month in a refrigerator); standard A 0.010 g/l (2 ml to 100 ml) — for decaffeinated products, standard B 0.05 g/l (5 ml to 50 ml) — for regular ones, both prepared on the day of use. Apparatus (5): HPLC with UV detector 254–280 nm — a wavelength close to 280 nm is preferred, caffeine absorption maximum 272 nm; C18 column, preferably with spherical particles, at least 5000 theoretical plates (plate number from the standard peak); the conditions of the standard were chosen for a 110 mm × 4.6 mm column — another column may require another mobile phase; 3 ml purification column with phenyl silica, particles about 40 µm; mill for roasted coffee, cooled grinder for green coffee, sieve 630 µm, filters 0.45 µm, water bath 90 ± 1 °C with stirring, balance 0.0001 g, 250 ml screw-cap bottle. Sampling (6): green coffee in bags according to ISO 4072, instant coffee according to ISO 6670. Preparation (7): grinding to pass the sieve. Procedure (8): dry matter is calculated from moisture according to ISO 1447 or ISO 6673 (green coffee) or ISO 3726 (instant); test portion 1 g of green or roasted coffee, 0.5 g of extract; into the bottle 4 ± 0.5 g of magnesium oxide and 100 g of water, weighed to 0.1 g; 20 min at 90 °C with stirring, cooling and reweighing — the masses must be equal, otherwise the extraction is repeated with a new test portion; filtrate; the column is activated with 5 ml of methanol and 5 ml of water, not allowed to dry out; 2.0 ml of filtrate (regular coffee) or 10.0 ml (decaffeinated) is applied; washed with 2 × 2.5 ml ammonia/methanol, about 20 ml of air blown through; caffeine eluted with 7.5 ml of elution solvent into a flask and made up; the column can be regenerated with methanol, at most 10 times; HPLC — flow 0.5–1.5 ml/min, column 40 °C (a higher temperature improves separation but should not exceed 60 °C), injection of 10 µl of test solution and the same of standard; Beer’s law normally holds up to 0.0250 g/l, otherwise a calibration curve is needed. Results (9): g caffeine per 100 g dry matter, from the peak areas of sample and standard, the standard concentration, test portion and dry matter — separate formulae for regular products (standard B) and decaffeinated products (standard A). Precision (10): interlaboratory test of ISO/TC 34/SC 15 in 1987, 18 laboratories, design according to ISO 5725; Table 1 — repeatability and reproducibility limits (g/100 g): roasted coffee — two content levels, 0.07 and 0.34 and 0.04 and 0.12 (levels illegible in the scan), decaffeinated roasted coffee (< 0.1 g/100 g) 0.01 and 0.02, instant coffee 0.09 and 0.36, decaffeinated instant coffee (< 0.3 g/100 g) 0.02 and 0.03. Test report (11): method, results, details not specified in the standard and incidents that may have influenced the result.

STATUS (as of 03.10.2026). PKN card PN-ISO 10095:1997 (Polish version): status “Withdrawn”, publication date 28.10.1997, withdrawal date 01.08.2018, introduces ISO 10095:1992 [EQV], KT 229 “Coffee, Tea and Cocoa”; the card does not name a replacing standard. We did not read the status of ISO 10095:1992 in the iTeh catalogue (since 03.10.2026 the catalogue pages require a browser with scripts). What the withdrawal changes for the result: the card names no successor, so there is no newer PN text against which the method could be compared — laboratories work according to the same document, citing a withdrawn standard.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, data up to 17.09.2026, read 03.10.2026). The number 10095 appears in 4 records at 2 laboratories: AB 285 (1) and AB 381 (3). In the current PCA documents (read 03.10.2026; AB 285 issue no. 28 of 13.01.2026, AB 381 issue no. 29 of 10.06.2026) the number appears at the same 2 laboratories; in every document the number of page markers equals the number of PDF pages, without repetitions; a search of all PCA documents downloaded from BIP on 03.10.2026 found no laboratories outside the database copy. The form “PN-ISO 10095:1997” at both. None of these items is suspended. The “Laboratories” tab (query “PN-ISO 10095”) shows both laboratories — checked by a tab query on the labcoda.pl production server on 03.10.2026.

WHAT THE LABORATORIES TEST (items in PCA). AB 285: roasted and instant coffee — caffeine (0.03–4.40) g/100 g d.m., HPLC-DAD. AB 381: ground, whole-bean and instant coffee — (30–45 000) mg/kg; decaffeinated coffee and coffee drinks — (30–3500) mg/kg; energy drinks — (0.5–400) mg/l; HPLC-UV/DAD.

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. Basis of the result: the standard expresses caffeine in g/100 g dry matter (9), and the dry matter comes from a separate standard (8.1); a result in mg/kg of product (AB 381) is a different basis. Extraction: the mass of the bottle after heating must equal the mass before — otherwise the extraction is repeated with a new test portion (8.3.3). Standard: decaffeinated products are calculated against standard A (0.010 g/l) and from 10 ml of filtrate, regular ones against standard B (0.05 g/l) and from 2 ml (4.8, 8.4.2, 9). Purification column: if it dries out before the sample is applied it must be prepared again; regeneration at most 10 times (8.4.1, Note 4). Linearity: single-point calculation assumes Beer’s law up to 0.0250 g/l — if it deviates, a curve is needed (8.5.2, Note 6). Column: conditions were chosen for a specific 110 × 4.6 mm column; another may require a change of mobile phase (5.2). Scope: the standard covers coffee; coffee drinks and energy drinks (AB 381) lie outside its scope (1). Status: PN-ISO 10095:1997 has been withdrawn since 01.08.2018.

WHAT WE DO NOT GIVE. We do not quote the exact form of the formulae of Clause 9 — they are partly illegible in the scanned sample. We do not summarize ISO 4052, ISO 1447, ISO 3726, ISO 6673, ISO 4072, ISO 6670 or ISO 5725 in this entry. We do not give requirements for caffeine content in decaffeinated coffee or in drinks.

Method principle

Caffeine is extracted from the coffee sample with water at 90 °C in the presence of magnesium oxide. An aliquot of the filtrate is purified on a small column of silica modified with phenyl groups — unwanted compounds are washed off with ammonia in methanol, and caffeine is eluted with a mixture of methanol, water and acetic acid. Caffeine is separated on a C18 column and measured with a UV detector near 272–280 nm; the content is calculated from the peak areas of sample and standard and related to dry matter.

Applications

• Caffeine in regular and decaffeinated green and roasted coffee beans (1)
• Caffeine in regular and decaffeinated coffee extract powder (instant coffee) (1)
• Roasted, ground, whole-bean, instant and decaffeinated coffee as well as coffee and energy drinks — 4 accreditation scope records of 2 laboratories in PCA

Key parameters

Standard

Standard number

PN-ISO 10095:1997

Title (PL)

Kawa — Oznaczanie zawartości kofeiny — Metoda wykorzystująca wysoko sprawną chromatografię cieczową

Title (EN)

Coffee — Determination of caffeine content — Method using high-performance liquid chromatography

Step-by-step procedure

1. Sample preparation

   Grinding to pass the 630 µm sieve; dry matter according to ISO 1447, ISO 6673 or ISO 3726 (7, 8.1). PN-ISO 10095:1997 — withdrawn 01.08.2018 (PKN).

2. Extraction

   Test portion with MgO and 100 g water, 20 min at 90 °C; mass check before and after (8.2, 8.3).

3. Purification

   Phenyl column: activation, application of filtrate, washing with ammonia/methanol, elution of caffeine (8.4).

4. HPLC

   10 µl of test solution and standard, 40 °C, 0.5–1.5 ml/min (8.5).

5. Result

   g caffeine/100 g dry matter; standard B for regular coffee, A for decaffeinated (9).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The standard has no separate safety clause; methanol, ammonia solution and acetic acid are used (4.1–4.3)

Frequently asked questions