BS EN ISO 15302:2007 – Animal and vegetable fats and oils — Determination of benzo[a]pyrene content — Reverse-phase high-performance liquid chromatography method

1 Scope
This International Standard specifies a method for the determination of benzo[a]pyrene in crude or refined edible oils and fats by reverse-phase high-performance liquid chromatography (HPLC) using fluorimetric detection in the range from 0,1 µg/kg to 10 µg/kg.
2 Normative reference
The following standard contains provisions, which through reference in this text, constitute provisions of this International Standard. At the time of publication the edition indicated was valid. All standards are subject to revision m and parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent edition of the standard indicated below. Members of IEC and ISO maintain registers of currently valid International Standards.
ISO 661:1989, Animal and vegetable fats and oils — Preparation of test sample
3 Principle
Adsorption of a suitable amount of sample on an alumina column, followed by elution with light petroleum of any benzo[a]pyrene present, and subsequent analysis of the eluate by HPLC using a fluorimetric detector.
4 Reagents
All reagents shall be of recognized analytical grade. Where analytical grade solvents other than the recommended ones are used, a full blank analysis shall be carried out and the results of this blank analysis reported.
4.1 Water, double distilled, filtered through a membrane filter of 0,45 µm pore size; deionized water obtained by purifying demineralized water systems may also be used.
4.2 Light petroleum (boiling point range between 40 °C and 60 °C), or hexane, redistilled over potassium hydroxide pellets (4 g/l).
4.3 Acetonitrile, suitable for HPLC.
4.4 Tetrahydrofuran, suitable for HPLC.
4.5 Toluene, suitable for HPLC.
4.6 Sodium sulfate, granular, anhydrous.
4.7 Alumina, activity grade 4, prepared from neutral aluminium oxide, activity grade super 1, deactivated by the addition of 10 ml distilled water to 90 g of alumina.
CAUTION — THE REACTION IS EXOTHERMIC AND PRESSURE MAY BUILD UP.
Shake the container for about 15 min and allow the contents to equilibriate for 24 h. Store the alumina in a closed vessel at ambient temperature.
4.8 Benzo[a]pyrene, 99,0 % purity.
CAUTION — BENZO[a]PYRENE IS A KNOWN CARCINOGEN. CARRY OUT ALL WORK WITH IT IN A FUME HOOD, WEARING GLOVES TO MINIMIZE EXPOSURE.
4.9 Benzo[a]pyrene solutions 1)
4.9.1 Stock solution
Weigh, to the nearest 0,1 mg, about 12,5 mg of benzo[a]pyrene in a 25 ml graduated flask. Dissolve it in toluene (4.5) and fill to the mark.
This solution contains about 0,5 mg/ml benzo[a]pyrene and should be stored in the dark at 4 °C when it is stable for 6 months at least.
4.9.2 Standard solutions
Prepare two standard solutions containing approximately 0,2 µg/ml and 0,01 µg/ml of benzo[a]pyrene respectivelym by diluting aliquots of the stock solution (4.9.1) with acetonitrile (4.3).
BS EN ISO 15302:2007 – Animal and vegetable fats and oils — Determination of benzo[a]pyrene content — Reverse-phase high-performance liquid chromatography method