1、June 2013 Translation by DIN-Sprachendienst.English price group 11No 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 6
2、7.140.20!%:o$“2237601www.din.deDDIN 10785Analysis of coffee and coffee products Determination of acrylamide Methods using HPLC-MS/MS and GC-MS after derivatization,English translation of DIN 10785:2013-06Untersuchung von Kaffee und Kaffee-Erzeugnissen Bestimmung von Acrylamid Verfahren mittels HPLC-
3、MS/MS und mittels GC-MS nach Derivatisierung,Englische bersetzung von DIN 10785:2013-06Analyse de caf et des drivs Dosage de lacrylamide Mthodes CLHP-MS/MS et CG-MS aprs derivatisation,Traduction anglaise de DIN 10785:2013-06SupersedesDIN 10785:2012-10www.beuth.deDocument comprises 20 pagesIn case o
4、f doubt, the German-language original shall be considered authoritative.10.14 DIN 10785:2013-06 2 A comma is used as the decimal marker. Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus .5 6 Sampling .6 7 Procedure .6 8 Calibration 9 9 Evaluation 10
5、 10 Procedure characteristics and precision . 10 11 Measurement uncertainty 11 12 Test report . 11 Annex A (informative) Performance characteristics . 12 Annex B (informative) Examples of absorber materials. 13 Annex C (informative) Examples of recommended columns and analysis conditions 14 Bibliogr
6、aphy . 20 DIN 10785:2013-06 3 Foreword This document has been prepared by Working Committee NA 057-05-09 AA Kaffee of the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee) in DIN. Attention is drawn to the possibility that some of the
7、elements of this document may be the subject of patent rights. DIN and/or DKE shall not be held responsible for identifying any or all such patent rights. Warning Users of this standard must be familiar with practical work in laboratories. This standard does not point out all risks which might occur
8、 during the use of this method. It is the responsibility of the user to make sure that all safety precautions necessary are taken and that all national safety regulations are obeyed. Amendments This standard differs from DIN 10785:2012-10 as follows: a) the incorrect and imprecise provisions in 8.2
9、have been amended and additional information given. Previous editions DIN 10785: 2012-10 DIN 10785:2013-06 4 1 Scope This standard specifies methods for determining the acrylamide content in coffee and coffee products by means of extraction, clean-up by solid-phase extraction and determination by HP
10、LC-MS/MS and GC-MS. It is applicable to roasted coffee, soluble coffee, coffee substitutes and coffee products. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated referenc
11、es, the latest edition of the referenced document (including any amendments) applies. DIN ISO 3696, Water for analytical laboratory use Specification and test methods 3 Principle The coffee sample is extracted with water or, in the case of soluble products, dissolved in water. A clean-up by solid ph
12、ase extraction is employed to remove interfering matrix compounds. Two alternative methods can be used for the determination: high performance liquid chromatography with mass spectrometric detection (HPLC-MS/MS) or gas chromatography with mass spectrometric detection (GC-MS) following bromination of
13、 acrylamide. In both cases isotopic labelled internal standards are used. 4 Reagents Warning In view of the health risks when working with acrylamide, appropriate preventive and protection measures shall be taken, such as using a fume cupboard, aspirating acrylamide-containing solutions only with a
14、pipette, and avoiding skin and eye contact or inhalation of acrylamide-containing vapour. If available, reagents of “residue analysis grade” or “analytical reagent grade” shall be used. The level of impurities in the reagents that contribute to the blank should be negligibly small. The blank shall b
15、e checked regularly. 4.1 Water, of grade 1 according to DIN ISO 3696; MS grade is recommended. 4.2 Operating gases of high purity, suitable for GC and mass spectrometry according to the instructions of the manufacturer of the analytical instruments. 4.3 Solvents, such as methanol, ethyl acetate, ace
16、tonitrile, n-hexane; MS grade is recommended. 4.4 Acrylamide, C3H5NO, reference compound. 4.4.1 Acrylamide stock standard solution ( = 1 000 g/ml), weigh (0,10 0,001) g acrylamide into a 100 ml one-mark volumetric flask and swirl it in 30 ml of water in order to dissolve the acrylamide. Then fill up
17、 to the mark with water ( = 1 000 g/ml). The stock solution is stable for at least three months when stored protected from light at a maximum of 6 C. 4.4.2 Working solution for calibration, ( = 10 g/ml), transfer (1,0 0,001) ml of the stock standard solution (4.4.1) into a 100 ml one-mark volumetric
18、 flask and then fill up to the mark with water. This solution shall be stored protected from light at a maximum of 6 C and shall be freshly prepared every working day. Depending on the working range more dilution steps according to 4.4.2 might be necessary. DIN 10785:2013-06 5 4.5 D3-acrylamide (acr
19、ylamide-2,3,3-d3) internal standard, C3H2D3NO, reference compound. 4.5.1 Stock standard solution (internal standard), the stock standard solution (internal standard) shall be prepared analogously to the stock standard solution in 4.4.1. Alternatively, a commercially available solution with a mass co
20、ncentration, , of 1 000 g/ml can be used. The information of the manufacturer regarding the stability of the solution shall be observed. 4.5.2 Working solution (internal standard), the working solution of the internal standard is prepared analogously to 4.4.2 from commercially available or self-prep
21、ared stock solutions (4.5.1). NOTE 1 For HPLC-MS/MS the solutions according to 4.4.1 to 4.5.2 can be prepared using the HPLC eluent as a solvent. When using GC-MS all standards according to 4.4.2 and 4.5.2 shall be subjected to the derivatization step according to 7.5.1. NOTE 2 Instead of D3-acrylam
22、ide it is also possible to use 13C3acrylamide for the preparation of the internal standard solution. However in the following clauses the procedure and calculation are described for D3-acrylamide only. 4.6 Saturated bromine water, saturate distilled water with bromine in a 100 ml one-mark volumetric
23、 flask until a phase of bromine is formed at the bottom of the flask (around 3,5 % of bromine at 4 C). Acidify the bromine water to a pH of about 1 using concentrated hydrobromic acid, (HBr, with a specific gravity of 1,48 g/cm3). Stored at 4 C and protected from light, the solution can be used for
24、about 4 weeks. 4.7 Potassium bromide, KBr, p.a. 4.8 Sodium thiosulfate (pentahydrate), Na2S2O3 5 H2O, p.a. 4.9 Triethylamine, (C2H5)3N, p.a. 4.10 Sodium sulfate (anhydrous, granular), Na2SO4, p.a. 4.11 Carrez solution I, dissolve 10,6 g of potassium hexacyanoferrate trihydrate K4Fe(CN)6 3 H2O p.a. i
25、n 100 ml of water. Stored at 4 C and protected from light, the solution is stable for 6 months. 4.12 Carrez solution II, dissolve 21,9 g of zinc acetate dihydrate Zn(CH3COO)2 2 H2O p.a. in 100 ml of water. Stored at 4 C and protected from light, the solution is stable for 6 months. 4.13 Borate buffe
26、r, pH 8,6. Mix 68 ml of a 0,1 molar sodium borate solution (20,12 g Na2B4O7per litre of water) and 32 ml of 0,1 molar hydrochloric acid, c(HCl) = 0,1 mol/l, in a 100 ml one-mark volumetric flask. 5 Apparatus 5.1 General Standard laboratory apparatus and, in particular, apparatus according to 5.2 to
27、5.15 are required. Apparatus and parts of the apparatus which can come into contact with the sample and extract shall be free of residues which can cause blank values. Preferably glassware or equipment made of stainless steel or PTFE (polytetrafluoroethylene) shall be used. 5.2 Analytical balance, c
28、apable of weighing to an accuracy of 0,1 mg. 5.3 Coffee mill, suitable for grinding roasted coffee beans. DIN 10785:2013-06 6 5.4 Glassware, for collecting and storing the extracts, preferably made of brown glass, as sample vials for manual or automatic use, equipped with an inert seal (e.g. vials w
29、ith PTFE coated septum). 5.5 Ultrasonic bath, capable of being maintained at 40 C. 5.6 Laboratory centrifuge, suitable for 15 ml and 50 ml centrifugal tubes and with a minimum g-force of 2 000 g. 5.7 Centrifuge tubes, nominal capacities of 15 ml and 50 ml. 5.8 One-mark volumetric flasks, nominal cap
30、acities of 20 ml and 100 ml. 5.9 Pipettes, glass or automatic, suitable for measuring volume ranges of standard and sample extract dilutions. 5.10 Glass or polypropylene cartridges, with sorbents for the solid phase extraction (SPE), and for the clean-up of extracts in 7.3.2 and/or 7.5.1 (examples a
31、re given in Table B.1). 5.11 High performance liquid chromatograph (for the test procedure according to 7.4), equipped with ESI and mass spectrometric detector (HPLC-MS/MS); gas supply as specified by the manufacturer. 5.12 HPLC column (for the test procedure according to 7.4), suitable for acrylami
32、de chromatography (examples are given in Table C.1). 5.13 Gas chromatograph (for the test procedure according to 7.5) with mass spectrometric detector (GC-MS), and operating gas supply (4.3) as specified by the manufacturer. 5.14 GC column (for the test procedure according to 7.5) capillary column,
33、suitable for acrylamide chromatography (examples given in Table C.3). 5.15 Membrane filter, syringe filter (e.g. cellulose acetate filters (0,45 m), suitable for filtration of the sample eluate obtained by solid phase extraction before injection into the chromatographic system. 6 Sampling Until a DI
34、N Standard on sampling is published, the sampling procedure shall be subject to agreement between the interested parties. A representative, thoroughly mixed sample shall be used, which has not been damaged or adulterated during transport or storage. In order to exclude changes in the acrylamide leve
35、ls, the analysis shall be performed shortly after reception of the sample. The samples shall be stored under cool conditions, under the exclusion of light, and shall be exposed to room temperature only for analysis. The date of receipt of the sample as well as the date of roasting or the best-before
36、 date shall be documented along with the date of analysis. 7 Procedure 7.1 General To avoid losses of the analyte it is necessary that the samples are protected from light during extraction and further preparation. For this reason brown glassware shall always be used. Otherwise, the content of the v
37、essels and flasks shall be protected from incident light using aluminium foil. 7.2 Preparation of the extract If necessary, grind the sample in a coffee mill (5.3) and homogenize thoroughly. DIN 10785:2013-06 7 Weigh 2 g of the homogenized sample of roasted coffee, soluble coffee or coffee substitut
38、e or 5 g of liquid coffee beverage to the nearest 1 mg using an analytical balance and transfer it into a 50 ml centrifugal tube. Add 2 ml of n-hexane to the test sample and shake briefly. Then spike the test sample with D3-acrylamide as the internal standard in a concentration corresponding to the
39、expected acrylamide level of the sample. EXAMPLE Weigh 2 g of coffee and add 100 l internal standard solution ( = 10 g/ml), which is equivalent to an acrylamide concentration of 500 g/kg in the coffee sample. Then add 20 ml of distilled water, shake briefly but vigorously, and sonicate (5.5) for 15
40、min at approx. 40 C. Allow a few minutes for precipitation and, in the case of non-sedimenting samples, centrifuge (5.6) for 15 min at 2 000 g to separate suspended solids. Before performing liquid chromatography (7.4) or derivatization and gas chromatographic separation (7.5), take 10 ml from the l
41、ower aqueous phase and use it for a further clean-up according to 7.3. The aqueous phase can be taken through the upper hexane phase using a pipette. If necessary the hexane phase can be removed using a Pasteur pipette. 7.3 Clean-up of the extracts 7.3.1 Carrez precipitation Clean-up the sample prep
42、ared according to 7.2 by Carrez precipitation. Add 1 000 l of Carrez solution I (4.11) and shake. Then add 1 000 l of Carrez solution II (4.12) and shake again. After a short exposure time centrifuge for 4 min at 2 000 g. Decant the supernatant, wash the residue with 2 ml to 3 ml of water, centrifug
43、e and decant again. Combine the aqueous solutions. 7.3.2 Solid phase extraction Clean-up the sample extract from the Carrez precipitation (7.3.1) by solid phase extraction (SPE) using two sequential cartridges with adsorber material (examples are given in Table B.1). The first cartridge contains 500
44、 mg of C18 material, the second cartridge 500 mg of ion exchanger. If appropriate, a combined column can be used. Serial alignment of the columns is also possible. Condition the SPE columns according to the manufacturers instructions successively with methanol and distilled water. Place the complete
45、 sample extract on top of the upper or first SPE column, then add 2 ml to 3 ml of water. Collect the eluate and place it on top of the second or lower conditioned ion exchange column and again add 2 ml to 3 ml of water. By using a light vacuum or pressure, complete elution can be achieved. Collect t
46、he eluate including washing water in a one-mark volumetric flask and fill up to 20 ml with water. 7.4 HPLC-MS/MS measurement 7.4.1 High performance liquid chromatography (HPLC) Prior to the HPLC-MS/MS analysis, add solvent to the cleaned-up extract (7.3.2) in order to make up the desired eluent comp
47、osition and filter through a membrane filter (5.15) before injecting 10 l to 100 l onto the HPLC column. Optimize the device parameters of the HPLC system in accordance with the manufacturers instructions. The chromatographic conditions shall be adjusted to suit the selected column (examples are giv
48、en in Tables C.1 and C.2). The clean-up stages according to 7.3.1 and 7.3.2 are essential for the chromatographic separation of the analyte peaks from the interfering peaks. An example chromatogram is given in Figure C.4. 7.4.2 Identification by mass spectrometry and quantification (HPLC-MS/MS) Detect acrylamide using MS/MS in the positive ionization mode (electrospray ionization, ESI). For identification use the mass transition m/z = 72 55. Acrylamide is identified as present if a signal at the mass track of th
