1、October 2006DEUTSCHE NORM English price group 10No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.100.30!,ym“98658
2、74www.din.deDDIN 10482-2Determination of the annatto content of cheese Part 2: High performance liquid chromatography methodBestimmung des Annattogehaltes in Kse Teil 2: Hochleistungsflssigchromatographisches Verfahrenwww.beuth.deTranslation by DIN-Sprachendienst.In case of doubt, the German-languag
3、e original should be consulted as the authoritative text.Document comprises 12 pagesDIN 10482-2:2006-10 2 Contents Page Foreword.2 1 Scope .3 2 Normative references .3 3 Terms and definitions 3 4 Abbreviations3 5 Principle.3 6 Reagents3 7 Apparatus and ancillaries 5 8 Sampling6 9 Procedure 6 10 Eval
4、uation7 11 Precision9 12 Test report. 9 Annex A (informative) Specimen chromatogram10 Annex B (informative) Results of interlaboratory testing 11 Bibliography.12 Foreword This standard has been prepared by Technical Committee Chemische und physikalische Untersuchung von Milch und Milchprodukten of t
5、he Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee). DIN 10482 consists of the following parts, under the general title Determination of the annatto content of cheese: Part 1: Photometric method Part 2: High performance liquid chromat
6、ography method DIN 10482-2:2006-10 3 1 Scope This standard specifies a method of determining annatto (E160b) by high performance liquid chromatography (HPLC) 1 after solid phase extraction (SPE). It is suitable for cheese. 2 Normative references The following referenced documents are indispensable f
7、or the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. DIN EN ISO 707, Milk and milk products Guidance on sampling 3 Terms and definitions For the purpose of
8、this standard, the follow term and definition applies. 3.1 annatto (E160b) a natural yellow dyestuff contained in the ripe seed coat of the achiote tree (Bixa orellana) NOTE Depending on the method of extraction, the dyestuff extracted from the seed coat is composed of the carotenoid bixin (C25H30O4
9、) and/or norbixin (C24H28O4), both of which may occur as cis- or trans-isomers 2. 4 Abbreviations SPE: Solid Phase Extraction HPLC: High Performance Liquid Chromatography 5 Principle The cheese sample is extracted by shaking it with acetone containing hydrochloric acid. After removing fat and -carot
10、ene by solid phase extraction (SPE), the annatto is eluted. After elution from the SPE column with a suitable solvent, the annatto constituents cis- and trans-bixin and cis- and trans-norbixin in the resultant sample solution are separated by HPLC analysis and detected spectrophotometrically at 460
11、nm. They are identified on the basis of their retention times and determined quantitatively by regression analysis. 6 Reagents 6.1 General Analytical grade reagents shall be used and the water used shall be double-distilled or water of at least equivalent purity. 6.2 Reagents for extracting annatto
12、from cheese 6.2.1 Sea sand, as filtration aid. DIN 10482-2:2006-10 4 6.2.2 Acetone containing hydrochloric acid, prepared by adding 2 ml of 2 mol/l hydrochloric acid, HCl, to 200 ml of 100 % (m/m) acetone, C3H6O. 6.2.3 100 % (m/m) absolute ethanol, C2H6O. 6.2.4 Particle-free gaseous nitrogen, for st
13、ripping the acetone containing hydrochloric acid. Its pressure shall be finely regulated with a reducing valve. 6.2.5 Acetone. 6.3 SPE reagents 6.3.1 n-Hexane. 6.3.2 (1+1) mixture of n-hexane/diethyl ether, prepared by mixing 10 ml of n-hexane and 10 ml of diethyl ether. 6.3.3 (7+3) mixture of metha
14、nol/glacial acetic acid, prepared by mixing 70 ml of methanol and 30 ml of glacial acetic acid. Use a fresh solution for each analysis. 6.4 HPLC reagents 6.4.1 Acetonitrile, suitable for HPLC analyses. 6.4.2 5 % (V/V) acetic acid, C2H4O2, prepared by mixing 5 ml of glacial acetic acid with 95 ml of
15、water. 6.5 (75+25) (V/V) acetonitrile/glacial acetic acid mixture as the HPLC mobile phase, prepared by mixing 250 ml of 5 % acetic acid (6.4.2) and 750 ml of acetonitrile (6.4.1). 6.6 Reagents for photometric annatto determination CAUTION Take precautions to prevent the inhalation or swallowing of
16、trichloromethane and wear suitable protective clothing and gloves to prevent it from coming into contact with the skin. 6.6.1 Trichloromethane, suitable for photometric determinations. 6.6.2 2,5 % (m/m) potassium hydroxide solution, KOH, prepared by dissolving 2,5 g of potassium hydroxide in 97,5 g
17、of water. 6.7 Standard substances1)Examples of standard substances that may be used are Dragoco Annatto Yellow (about 0,5 % solution of bixin in vegetable oil) supplied by Dragoco in Holzminden, Germany, and water-soluble Annatto-A-160-WS (0,53 % to 0,60 % norbixin in 2,5 % potassium hydroxide solut
18、ion) supplied by Hansen in Denmark. 6.8 Stock and standard solutions1)a) Bixin stock solution, prepared by making 500 l of Dragoco Annatto Yellow up to 50 ml with trichloro-methane (6.6.1). 1) The information relating to manufacturers designations in subclauses 6.7 and 6.8 is given for the convenien
19、ce of the user of this standard and does not constitute an endorsement by DIN of the manufacturer or the product name. Equivalent products may be used if they can be shown to lead to the same result. DIN 10482-2:2006-10 5 b) Norbixin stock solution, prepared by making 500 l of water-soluble Annatto-
20、A-160-WS up to 50 ml with 2,5 % potassium hydroxide solution (6.6.2). If kept in the dark at 4 C, the stock solution will be stable for six days. Prepare five standard solutions in each case of bixin and norbixin with contents of 0,1 g/ml to 5 g/ml by diluting 20 l, 100 l, 200 l, 500 l and 1 000 l o
21、f the bixin and the norbixin stock solutions with methanol/ glacial acetic acid mixture (6.3.3) to a final volume of 10 ml. Determine the exact contents photometrically and use the solutions directly for quantifying the HPLC result. If a mixed standard is used, no more than 500 l of the particular s
22、tandard solution shall be used since separation effects may occur. 7 Apparatus and ancillaries In addition to standard laboratory equipment, the following shall be used. 7.1 Mortar and pestle. 7.2 Sealable centrifuge tubes, of nominal capacity 100 ml, suitable for the centrifuge (7.3). 7.3 Centrifug
23、e, capable of being operated at an effective centrifugal acceleration of 1 000 g. 7.4 Glass funnel, having a diameter of about 10 cm. 7.5 Fluted filter paper, moistened with acetone (6.2.5) prior to filtration. 7.6 Tapered flask, of nominal capacity 100 ml, suitable for the rotary evaporator (7.7).
24、7.7 Rotary evaporator with temperature-controlled water bath, capable of being maintained at a temperature of 40 C. 7.8 Ultrasonic bath. 7.9 Volumetric flasks, of nominal capacities 10 ml and 50 ml. 7.10 Membrane filter, having a pore size of 0,45 m, for filtering the sample solution (9.1.2). 7.11 V
25、ariable-volume piston-operated pipette, for volume ranges of 20 l to 100 l and 100 l to 1 000 l. 7.12 Analytical balance. 7.13 Reducing valve, suitable for steel nitrogen bottles. 7.14 One-mark bulb pipettes, of nominal capacities 2 ml, 10 ml, 20 ml and 50 ml. 7.15 Peleus ball or aspirette, suitable
26、 as a suction aid for one-mark bulb pipettes (7.14). 7.16 Pasteur pipettes. 7.17 Water bath or heating block, suitable for heating a 100 ml tapered flask and capable of being maintained at a temperature of 40 C. 7.18 Solid phase extraction apparatus. DIN 10482-2:2006-10 6 7.19 Solid phase extraction
27、 column, containing a solid phase functionalized with aminopropyl radicals, e.g. Separtis IST Isolute NH22 g/6 ml column (Separtis GmbH, Grenzach-Whylen, Germany)2). 7.20 Spectrophotometer, for determining the absorbance at a wavelength of 488 nm, suitable for cells having a path length of 1 cm. 7.2
28、1 HPLC analytical system, comprising the following. 7.21.1 Pulsation-free HPLC pump, suitable for generating a steady liquid flow rate of 1,0 ml/min. 7.21.2 Sample injection valve, for injecting volumes of 10 l, or autosampler. 7.21.3 Chromatographic column, suitable for separating bixin and norbixi
29、n isomers, e.g. C18reversed phase column, with a particle size of 5 m, a diameter of 4 mm and a length of 250 mm (e.g. Hypersil ODS2)2). 7.21.4 Column oven, suitable for the chromatographic column, capable of being maintained at 40 C 1 C. Elution may be performed at other temperatures provided it ca
30、n be shown that equivalent results are obtained. 7.21.5 Variable UV/VIS detector, for measurements at a wavelength of 460 nm, or photodiode array detector for measurements at 460 nm. 7.21.6 Electronic integrator or computer-aided data-processing system, for data integration and evaluation. 8 Samplin
31、g As specified in DIN EN ISO 707. 9 Procedure 9.1 Preparation of sample solution CAUTION Take precautions to prevent the inhalation or swallow acetone, acetone containing hydrochloric acid, diethyl ether, n-hexane, methanol and glacial acetic acid and wear gloves to prevent these reagents from comin
32、g into contact with the skin. Perform all extractions in a fume cupboard. 9.1.1 Cheese extraction Store the cheese samples at 20 C prior to analysis and crumble or grate the pieces of cheese or cheese rind before testing. Grind 5 g of the cheese sample or edible3)cheese rind to be tested in a mortar
33、 with 15 g of sea sand and transfer the resultant mixture quantitatively to a sealable 100 ml centrifuge tube. Add 20 ml of acetone containing hydrochloric acid and shake vigorously for ten minutes, then centrifuge for five minutes at 1 000 g. 2) The information relating to apparatus and manufacture
34、rs designations in subclauses 7.19 and 7.21.3 is given for the convenience of the user of this standard and does not constitute an endorsement by DIN of the manufacturer or product name. Equivalent products may be used if they can be shown to lead to the same result. 3) The term “edible” is used as
35、defined in ZZulV 3. DIN 10482-2:2006-10 7 Filter the supernatant through a fluted filter paper moistened with acetone and collect the filtrate in a 100 ml tapered flask. Rinse the fluted filter paper with acetone and then repeat the extraction two more times. If the upper rim of the filter paper has
36、 a slight yellow coloration after filtration, use scissors to cut it off as a strip about 1 cm wide. Rinse the strip with drops of acetone until the colour disappears and transfer the resultant extract to the 100 ml tapered flask. Evaporate the extracts in the tapered flask down on a rotary evaporat
37、or in vacuo at 40 C. Remove residual water by adding 10 ml of ethanol (6.2.3) and evaporating down again. Strip off residual solvent with nitrogen for 15 minutes at 40 C. Now perform the solid phase extraction (SPE) on the residue in the flask. 9.1.2 Solid phase extraction (SPE) During extraction, p
38、articularly if it is vacuum-aided, ensure that the eluates leave the column dropwise at as constant a volumetric rate as possible. 1) Condition the stationary phase by rinsing it with 15 ml of n-hexane. 2) Take up the acetone extraction residue in the 100 ml tapered flask in 2 ml of acetone and, aft
39、er treating it for a short time in an ultrasonic bath, transfer the solution to the SPE column. To ensure quantitative transfer, add a further 2 ml of acetone to the tapered flask, repeat the ultrasonic treatment and add this solution to the column. 3) To remove fat and -carotene, rinse the SPE colu
40、mn, first with 10 ml of n-hexane/diethyl ether (6.3.2) and then with 2 ml of acetone. 4) To isolate the annatto dyes bixin and norbixin, pass 8 ml of the (7+3) mixture of methanol and glacial acetic acid (6.3.3) through the SPE column and collect the eluate in a 10 ml volumetric flask. Make the elua
41、te up to the mark with the same solvent and filter it through a membrane filter (7.10) prior to the determination to obtain the sample solution for the HPLC analysis. The solution is photosensitive and will be stable only for one day. NOTE The precision data of this standard are regarded as equivale
42、nt to that of the photometric method specified in DIN 10482-1 and the user is free to choose which method to adopt. 9.2 Determination Determine the annatto content by HPLC analysis, applying 10 l of sample solution to the column. In addition to free cis- and trans-norbixin acids, the samples may als
43、o contain cis- and trans-bixin. Detect the annatto constituents at a wavelength of 460 nm and calculate the annatto content by regression analysis. 10 Evaluation 10.1 Calculation of concentration of HPLC standard solutions Calculate the concentrations of the HPLC standard solutions, STD, in mg/l, to
44、 two decimal places using equation (1): %1cm1STD00010EA= (1) DIN 10482-2:2006-10 8 where Ais the absorbance found for norbixin and bixin at a wavelength of 488 nm in the methanol/glacial acetic acid mixture (6.3.3); %1cm1E is the absorbance (2 931) of bixin and norbixin at a wavelength of 488 nm in
45、a 1 % methanol/ glacial acetic acid solution (6.3.3) in a cell having a path length of 1 cm; the absorbance is linear in the range 0 to 1,45; 10 000 is the factor for converting %1cm1E (which is for a concentration expressed in 10 g/l) to a sample solution concentration expressed in mg/l. The photom
46、etric determination shall be preceded by a zeroing of the photometer (7.20) at a wavelength of 488 nm with air as the reference (i.e. with no cell in the beam path). The methanol/glacial acetic acid solution (6.3.3) is used as reference for the HPLC standard solutions. 10.2 HPLC integration mode ana
47、lysis Perform the integration by comparing the integrated peak areas of the sample and standard solutions, treating the peak areas of cis- and trans-bixin and of cis- and trans-norbixin as sums. 10.3 Calibration of HPLC analytical system Prepare the regression graph by multipoint calibration, perfor
48、ming a single determination for each of the HPLC standard solutions, with an injection volume of 10 l. Linear least-squares regression analysis for the pairs of values for the integrated area y of the standard peak, in units of area, and concentration of the standard, xSTD, in mg/l, of the standard
49、solutions with the concentration as independent variable using equation (2) yields the coefficients a and b: y = axSTD+ b (2) where a is the slope of the regression line; b is the intercept of the regression line on the axis. 10.4 Calculation of annatto content Calculate the annatto content, w, in mg/kg, using equation (3): MaVbyw=MLp)(3) where yPis the peak area of the sample; M is the initial sample mass, in g (here, 5 g); VMLis the elution volume from the solid phase extraction, in ml (here, 10 ml); DIN 10482-2:2006-10 9 a i
