NBN EN 14177 : 2004

Foreword
1 Scope
2 Normative references
3 Principle
4 Reagents
   4.1 General
   4.2 Sodium carbonate solution, mass concentration
        rho(Na[2]CO[3]) approx. 15 g/l
   4.3 Acetic acid, volume concentration varphi(CH[3]COOH)
        approx. 98%.
   4.4 Sodium sulfate anhydrous
   4.5 pH 4 water
   4.6 Absolute ethanol, varphi(CH[3]CH[2]OH) >= 99,7%
   4.7 Ethyl acetate
   4.8 Perchloric acid, varphi(HclO[4]) = 60%
   4.9 Acetonitrile
   4.10 Endogalacturonase enzyme solution, typical activity
        1 400 U/g
   4.11 HPLC mobile phase
   4.12 Patulin stock solution
   4.13 Patulin standard solution
   4.14 Patulin standard solution for calibration
5 Apparatus
   5.1 General
   5.2 Displacement pipettes of 5 ml, 1 ml, 200 micro l and
        50 micro l capacity with appropriate pipette tips
   5.3 Analytical balance
   5.4 UV spectrophotometer, double beam and recording
        suitable for measurement at 250 nm to 350 nm
   5.5 Quartz cells, of optical path length 1 cm
   5.6 Centrifuge, capable of operating at 4 500 g
   5.7 Centrifuge tubes of 50 ml capacity with screw cap lids
   5.8 Rotary evaporator, with water bath set at 40 degree C
   5.9 Filter paper, with pore size 20 micro m to 25 micro m
   5.10 Disposable syringe filters, of 0,2 micro m pore size
        (optional)
   5.11 HPLC apparatus, comprising the following
   5.12 Round bottom flasks, of 250 ml capacity with ground
        glass joint
6 Procedure
   6.1 Preparation of test samples
   6.2 Extraction of patulin
   6.3 Removal of interfering acidic compounds
   6.4 Preparation of the sample test solution
7 Spiking procedure
   7.1 Clear apple juice
   7.2 Cloudy apple juice
   7.3 Apple puree
8 HPLC determination
   8.1 Calibration graph
   8.2 HPLC operating conditions
9 Calculation
10 Precision
   10.1 Interlaboratory test
   10.2 Repeatability
   10.3 Reproducibility
   10.4 Test report
Annex A (informative) Precision data
Bibliography

Describes a method for the determination of patulin in apple juices and apple puree using high performance liquid chromatography (HPLC).