1、ICS 67.220.10Untersuchung von Gewrzen und wrzenden Zutaten Bestimmung des Capsaicinoidgehaltes inPaprikapulvern, Paprikaoleoresinen, Chilipulvern und Chilioleoresinen Verfahren mitHochleistungsflssigchromatographie (HPLC)In keeping with current practice in standards published by the International Or
2、ganization for Standardization(ISO), a comma has been used throughout as the decimal marker.Ref. No. DIN 10234 : 2003-02English price group 08 Sales No. 010810.03DEUTSCHE NORM February 200310234Continued on pages 2 to 5. No part of this translation may be reproduced without the prior permission ofDI
3、N Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).Translation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text.Determining the cap
4、saicinoid content ofpowdered paprika and chilli peppers by highperformance liquid chromatography (HPLC)ForewordThis standard has been prepared by Technical Committee Gewrze und wrzende Zutaten of theNormenausschuss Lebensmittel und landwirtschaftliche Produkte (Foodstuffs and Agricultural ProductsSt
5、andards Committee).1 ScopeThis standard specifies a routine method of determining capsaicinoid contents of greater than 10 mg/kg inpowdered paprika and paprika oleoresins by high performance liquid chromatography. If the extract issuitably diluted, it can also be used for examining chilli powders an
6、d chilli oleoresins.2 Normative referencesThis standard incorporates, by dated or undated reference, provisions from other publications. Thesenormative references are cited at the appropriate places in the text, and the titles of the publications arelisted below. For dated references, subsequent ame
7、ndments to or revisions of any of these publicationsapply to this standard only when incorporated in it by amendment or revision. For undated references, thelatest edition of the publication referred to applies.DIN 10230 Preparation of ground samples of spices and condiments for analysisISO 5725-1 :
8、 1994 Accuracy (trueness and precision) of measurement methods and results Part 1: Gen-eral principles and definitionsISO 5725-2 : 1994 Accuracy (trueness and precision) of measurement methods and results Part 2: Basicmethods for the determination of repeatability and reproducibility of a standard m
9、eas-urement method3 ConceptCapsaicinoid contentThe sum of the capsaicin, dihydrocapsaicin and nordihydrocapsaicin contents, in mg/kg, determined by themethod described in this standard.Page 2DIN 10234 : 2003-024 PrincipleThe sample is extracted with an acetonitrile/water mixture under reflux and an
10、aliquot of the solution obtainedis analysed by high performance liquid chromatography using fluorescence detection.5 Reagents5.1 GeneralUnless otherwise specified, analytical grade reagents shall be used and the water used shall be distilled or ofequivalent purity.The following reagents shall be use
11、d.5.2 Nonivamid (pelargonic acid vanillylamide, PAVA), C17H27NO3, having a molar mass of 293,4 g/mol and apurity of greater than 97 % (m/m), determined by HPLC, used as standard substance.CAUTION. This substance is a strong irritant and must be handled with caution.5.3 Natural capsaicinoid mixture (
12、capsaicin, dihydrocapsaicin and nordihydrocapsaicin) as reference sub-stance to aid in peak assignment.5.4 Solvents5.4.1 Acetonitrile.5.4.2 Water.5.4.3 99 % to 100 % (m/m) acetic acid.5.5 Eluent, prepared by mixing 50 parts by volume of acetonitrile, 50 parts by volume of water and 0,5 partsby volum
13、e of acetic acid and degassing.5.6 Solvent mixture, for standard solutions and sample extraction, prepared by mixing 50 parts by volumeof acetonitrile and 50 parts by volume of water.5.7 Standard solutions5.7.1 Stock solution, prepared by dissolving (70 t 0,1) mg of PAVA in 100 ml of solvent mixture
14、.5.7.2 Standard solution, prepared by diluting 5 ml of stock solution to 250 ml using the solvent mixture.5.7.3 Dilution series, prepared from standard solution for calibration purposes. Table 1 shows an exampleof a suitable dilution series. The linear calibration range shall encompass at least five
15、 dilution steps.Table 1: Dilution series6 ApparatusIn addition to standard laboratory equipment, the following shall be used.6.1 One-mark bulb pipettes, one having a nominal capacity of 5 ml and the other being of larger capacityfor collecting the sample extract when extracting oleoresins as describ
16、ed in subclause 7.3.2.6.2 Volumetric flasks, of nominal capacities 20 ml, 50 ml, 100 ml, 250 ml and 500 ml.6.3 Boiling aid (boiling chips or beads).6.4 Cellulose fluted filter, having a diameter of 18,5 cm, a mass of about 75 g/m2and a thickness of about0,20 mm.Page 3DIN 10234 : 2003-026.5 Separatin
17、g system6.5.1 High performance liquid chromatograph.6.5.2 Fluorescence detector, having an excitation wavelength of 280 nm and an emission wavelength of320 nm.6.5.3 Solvent degassing system.6.6 Columns6.6.1 Analytical column, having an internal diameter of 4 mm to 4,6 mm and a length of 250 mm, pack
18、ed withoctadecylsilane-modified silica gel (RP-18, end-capped) of particle size 5 m, capable of separating the baselines of nordihydrocapsaicin and capsaicin.6.6.2 Precolumn, suitable for the analytical column, having an internal diameter of 4 mm to 4,6 mm and alength of 4 mm, packed with the same s
19、tationary phase as in subclause 6.6.1, suitable for a flow rate of1 ml/min.6.7 Recorder or integrator.6.8 Injection valve, with 20 l sample loop or any other system operating with the same accuracy.7 Procedure7.1 CalibrationInject 20 l of each of the PAVA solutions in the dilution series spaced out
20、in time at the same interval as usedfor analysing the test solutions. Measure the peak areas and prepare a calibration graph by plotting theconcentration of PAVA in the standard solution against the peak area and by calculation.7.2 Sample preparationPrepare unground samples prior to the analysis as
21、specified in DIN 10230. Before weighing the sample, ensurethat it is thoroughly homogenized, particularly in the case of oleoresins.7.3 Extraction and analysis7.3.1 Powdered test materialWeigh, to an accuracy of 0,01 g, 5 g of powdered paprika into a 100 ml round bottom flask, add 40 ml of solventmi
22、xture and some boiling chips or beads, and extract for two hours under reflux.After cooling and allowing to settle (for about 30 minutes), carefully decant the supernatant extraction solutioninto a 50 ml volumetric flask through a fluted filter previously moistened with the solvent mixture. Take up
23、theextraction residue into the flask in 5 ml of solvent mixture, again allow it to settle (for about five minutes) andfilter it into the volumetric flask. Rinse the filter into the flask with solvent mixture and make up the contentsof the flask to the mark with solvent mixture.7.3.2 OleoresinsWeigh,
24、 to an accuracy of 0,01 g, 5 g of paprika oleoresins into a 100 ml round bottom flask, add 40 ml of solventmixture and a few boiling chips or beads, and extract under reflux for two hours.After cooling and complete separation of the phases (if necessary store sealed overnight in the dark), suck offt
25、he acetonitrile/water phase using a pipette, thereby separating it from floating and settling constituents of theoleoresin, and transfer it to a 50 ml volumetric flask. Make up the contents of the flask to the mark with solventmixture.7.3.3 AnalysisTransfer a portion of the test solutions obtained a
26、s specified in subclauses 7.3.1 or 7.3.2 to a sample containerand inject 20 l into the chromatograph. If the amount of sample injected exceeds the linear calibration range,dilute the test solution appropriately.8 Evaluation8.1 GeneralQuantify capsaicin, dihydrocapsaicin and nordihydrocapsaicin using
27、 the calibration graph obtained as insubclause 7.1, initially expressing the concentrations of these substances as the PAVA concentration. Sinceall the capsaicinoids produce the same molar fluorescence yield because of the identical fluorescing vinillylradical, the concentrations of the individual c
28、apsaicinoids can be calculated taking account of the molar massesand using conversion factors. For this purpose, the molar masses shall be taken as 305,4 g/mol for capsaicin,307,4 g/mol for dihydrocapsaicin and 293,4 g/mol for nordihydrocapsaicin.Page 4DIN 10234 : 2003-028.2 Calculation8.2.1 Content
29、s of individual capsaicinoids as capsaicin, dihydrocapsaicin and nordihydrocapsaicinCalculate the content of the individual capsaicinoid, wn, in mg/kg of sample material, as PAVA using thefollowing equation:(1)whereynis the peak area of the individual component, in integration units (area);m is the
30、slope of the calibration graph, in integration units (area)/(g/ml);b is the intercept of the calibration graph on the y-axis, in integration units (area);E is the initial sample mass, in g;VGis the total volume of test solution, in ml (e.g. 50 ml if the sample is extracted as in subclause 7.3.1 or7.
31、3.2); if the test solution is diluted as in subclause 7.3.3, use the total volume of the sample after dilution.8.2.2 Content of individual componentsCalculate the content of individual capsaicinoid of sample, wi, in mg/kg, using the following equation:(2)where fiis the molar factor for converting th
32、e PAVA concentration to the concentration of the individualcapsaicinoid (1,041 for capsaicin, 1,048 for dihydrocapsaicin and 1,000 for nordihydrocapsaicin).8.2.3 Total contentCalculate the total content, wG, in mg/kg, as the sum of the concentrations of capsaicin, dihydrocapsaicin andnordihydrocapsa
33、icin, using the following equation:(3)Report the result to one decimal place.9 Precision9.1 Interlaboratory testDetails of an interlaboratory test to establish the precision of the method are summarized in Annex B. The valuesobtained are not necessarily applicable to concentration ranges and types o
34、f sample other than those specifiedhere.9.2 Repeatability limit(same operator, same equipment)The absolute difference between two successive results obtained under repeatability conditions will not exceedthe repeatability limit, r, given in table 2 in more than 5 % of cases.9.3 Reproducibility limit
35、(different operators, different equipment)The absolute difference between two individual results obtained under reproducibility conditions will notexceed the reproducibility limit, R, in table 2 in more than 5 % of cases.Table 2: Repeatability and reproducibility limits (values given in mg/kg)RrEmVb
36、yw=Gnn)(inifww =iGwwPage 5DIN 10234 : 2003-0210 Test reportThe test report shall refer to this standard and include the following details:a) identification of sample;b) test results, as in clause 8;c) date of receipt of sample;d) date of testing;e) indication whether the repeatability limit requirem
37、ent was met;f) any procedures not specified in this standard and undertaken optionally, and details of all the circum-stances that may have affected the result(s).Annex AExample of a typical chromatogramFigure A.1 shows the chromatogram of a typical powdered paprika recorded under the conditions spe
38、cified inthis standard. The use of a standard capsaicin mixture makes it easier to identify the peaks of the capsaicinoidsnordihydrocapsaicin, capsaicin and dihydrocapsaicin.Figure A.1Annex BResults of interlaboratory testingThe precision of the method was determined by Technical Committee Gewrze und wrzende Zutaten in aninterlaboratory test involving six laboratories. The statistical results obtained in an evaluation as specified inISO 5725-1 and ISO 5725-2 are summarized in table B.1.Table B.1rsRsrx,Time, in minR
