1、 Reference numbers ISO/TS 27106:2009(E) IDF/RM 217:2009(E) ISO and IDF 2009TECHNICAL SPECIFICATION ISO/TS 27106 IDF/RM 217 First edition 2009-12-01 Cheese Determination of nisin A content by LC-MS and LC-MS-MS Fromage Dtermination de la teneur en nisine A par CL-SM et CL-SM-SM ISO/TS 27106:2009(E) I
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7、 749 09 47 Fax + 32 2 733 04 13 E-mail copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2009 All rights reservedISO/TS 27106:2009(E) IDF/RM 217:2009(E) ISO and IDF 2009 All rights reserved iiiForeword ISO (the International Organizat
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15、ct of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO/TS 27106 IDF/RM 217 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published j
16、ointly by ISO and IDF. ISO/TS 27106:2009(E) IDF/RM 217:2009(E) iv ISO and IDF 2009 All rights reservedForeword IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide. IDF membership comprises National Committees in every member country as well a
17、s regional dairy associations having signed a formal agreement on cooperation with IDF. All members of IDF have the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for mi
18、lk and milk products. The main task of Standing Committees is to prepare International Standards. Draft International Standards adopted by the Standing Committees are circulated to the National Committees for endorsement prior to publication as an International Standard. Publication as an Internatio
19、nal Standard requires approval by at least 50% of IDF National Committees casting a vote. In other circumstances, particularly when there is an urgent market requirement for such documents, a Standing Committee may decide to publish an other type of normative document which is called by IDF: Reviewe
20、d method. Such a method represents an agreement between the members of a Standing Committee and is accepted for publication if it is approved by at least 50 % of the committee members casting a vote. A Reviewed method is equal to an ISO/PAS or ISO/TS and will, therefore, also be published jointly un
21、der ISO conditions. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO/TS 27106 IDF/RM 217 was prepared by the International Dairy Federation (IDF)
22、and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF and ISO. All work was carried out by the Joint ISO-IDF Action Team on Food additives and vitamins of the Standing Committee on Analytical methods for additives and contam
23、inants under the aegis of its project leader, Mr. T. Berger (CH). TECHNICAL SPECIFICATION ISO/TS 27106:2009(E) IDF/RM 217:2009(E) ISO and IDF 2009 All rights reserved 1Cheese Determination of nisin A content by LC-MS and LC-MS-MS 1 Scope This Technical Specification specifies a method for the quanti
24、tative determination of the nisin A content in cheese. The method is suitable for measuring low levels of nisin A with a quantification limit of 1 mg/kg. NOTE Nisin is a peptide produced by some bacteria (e.g. Lactococcus lactis subsp. Lactis) inhibiting or destroying other microorganisms. It is wid
25、ely used as a natural preservative for foods, e.g. vegetables, cheese, meat, and cacao. In cheese making, nisin is used to prevent late blowing. Its use is restricted to maximum levels in the final product. Nisin appears in two forms, nisin A and nisin Z, which differ in one amino acid. This method
26、is applicable to the determination of nisin A only. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 nisin A content mass fraction of substance determined by the procedure specified in this Technical Specification NOTE The nisin A content is e
27、xpressed in milligrams per kilogram. 3 Principle The sample is grated and extracted with dilute formic acid at 80 C. After ultracentrifugation, interfering proteins are separated by means of filtration through an ultrafiltration (UF) membrane. In the purified extract, nisin A is separated using a po
28、lymeric stationary phase and detected using mass spectrometry (with MS-MS as an option). 4 Reagents and reference substances Use only reagents of recognized analytical grade and distilled water or water of at least equivalent purity, unless otherwise specified. 4.1 Bovine serum albumin (BSA) stock s
29、olution. Dissolve 10 mg of BSA (purity 96 % mass fraction), in 10 ml water. 4.2 Bovine serum albumin (BSA) buffer solution. Mix 80 ml water with 20 ml of acetonitrile (4.6), 0,5 ml of formic acid (4.3), 0,01 ml of trifluoracetic acid (4.5) and 1 ml of BSA stock solution (4.1). 4.3 Formic acid (HCOOH
30、). 4.4 Formic acid solution, HCOOH= 5 g/l. Pipette 0,41 ml of formic acid (4.3) into a 100 ml one-mark volumetric flask (5.12). Make up to the mark with water and mix. ISO/TS 27106:2009(E) IDF/RM 217:2009(E) 2 ISO and IDF 2009 All rights reserved4.5 Trifluoracetic acid (CF 3 COOH). 4.6 Acetonitrile
31、(CH 3 CN), “pure”. 4.7 Methanol (CH 3 OH). 4.8 Nisin A, of purity 95 % mass fraction 1) . 5 Apparatus Usual laboratory equipment and, in particular, the following. 5.1 Laboratory centrifuge, capable of producing a radial acceleration of at least 3 000g. 5.2 Ultracentrifuge, capable of producing a ra
32、dial acceleration of 20 800g. 5.3 Ultrafiltration membrane, of pore size 30 kD 2) . 5.4 Membrane filter, of pore size 0,22 m 3) . 5.5 Balance, capable of weighing to the nearest 10 mg, with a readability of 1 mg. 5.6 Analytical balance, capable of weighing to the nearest 0,1 mg, with a readability o
33、f 0,01 mg. 5.7 Water bath, capable of shaking and maintaining a temperature of 80 C 2 C. 5.8 Ultrasonic bath, capable of shaking and maintaining a temperature of 80 C 2 C. 5.9 Cheese grater, with openings of size approximately 2 mm. 5.10 LC-MS equipment. 5.10.1 Elution gradient pumping system, capab
34、le of operating at 0,25 ml/min. 5.10.2 Manual or automatic injector, capable of injecting volumes of 5 l. 5.10.3 Column heater, capable of maintaining a column temperature of 40 C 2 C. 5.10.4 Column, reversed phase, PLRP-S, 300 4 ) , 3 m, 150 mm 2 mm. 5.10.5 Mass spectrometer detector, capable of op
35、erating in ion mode ESI+ at m/z 839,6. 5.11 LC-MS-MS equipment (optional). 1) Ambicin N is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of this product. 2) Millipo
36、re Centricon YM-30 is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of this product. 3) Millipore Millex-GV PVDF 0,22 m is an example of a suitable product availabl
37、e commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of this product. 4) PLRP-S, 300 is the trade name of a product supplied by Polymer Lab Ltd. This information is given for the convenience of users of this docu
38、ment and does not constitute an endorsement by ISO or IDF of the product named. Equivalent products may be used if they can be shown to lead to the same results. ISO/TS 27106:2009(E) IDF/RM 217:2009(E) ISO and IDF 2009 All rights reserved 35.11.1 Elution gradient pumping system, capable of deliverin
39、g a flow rate of 0,2 ml/min. 5.11.2 Manual or automatic injector, capable of injecting volumes of 10 l. 5.11.3 Column heater, capable of maintaining a column temperature of 30 C 2 C. 5.11.4 Column for reversed phase chromatography, PLRP-S, 300 4) , 3 m, 150 2 mm. 5.11.5 Mass spectrometer detector, c
40、apable of operating in ion mode ESI+ MS-MS at m/z 672/672, 672/811, 672/649, 840/840. 5.12 One-mark volumetric flasks, of capacity 100 ml, ISO 1042 2class A. 6 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or sto
41、rage. Sampling is not part of the method specified in this Technical Specification. A recommended sampling method is given in ISO 707 IDF 50 1 . 7 Procedure 7.1 Preparation of the nisin A standard solution 7.1.1 Nisin A standard stock solution, nA= 100 mg/l. Weigh, to the nearest 0,01 mg, 10,00 mg n
42、isin A (4.8) into a 100 ml one-mark volumetric flask (5.12). Make up to the mark with formic acid solution (4.4) and mix. Prepare the nisin A standard stock solution daily. 7.1.2 Nisin A standard working solution, nA= 300 g/l. Pipette 300 l of nisin A standard stock solution (7.1.1) into a 100 ml on
43、e-mark volumetric flask (5.12). Make up to the mark with BSA buffer solution (4.2) and mix. The standard working solution thus obtained contains 300 g of nisin A per litre. Prepare the nisin A standard working solution daily. 7.2 Extraction of the test portion Before weighing, grate the test sample
44、with the cheese grater (5.9). Weigh, to the nearest 0,01 g, 2,50 g of test sample into a 100 ml one-mark volumetric flask (5.12). Add 70 ml of water and 0,5 ml of formic acid (4.3) and mix. Put the flask either in the water bath (5.7) at 80 C and shake it for 30 min or in the ultrasonic bath (5.8) a
45、t 80 C and shake it for 10 min. After cooling to room temperature, make up to the mark with water and mix. NOTE Soft cheese can be grated after freezing. ISO/TS 27106:2009(E) IDF/RM 217:2009(E) 4 ISO and IDF 2009 All rights reserved7.3 Filtration of the test portion with UF membrane Pipette approxim
46、ately 1,5 ml of the extract into a 1,5 ml tube (e.g. Eppendorf) and centrifuge using the ultracentrifuge (5.2) at 20 800g for 10 min. Determine the tare of the collecting container for the ultrafiltration membrane (5.3) on the analytical balance (5.6). Place the collecting container on the ultrafilt
47、ration membrane and set the analytical balance to zero. Filter 0,6 ml to 0,7 ml of the centrifuged extract through a membrane filter (5.4) into the tared ultra filtration membrane. Weigh the amount of the extract on the ultrafiltration membrane using the analytical balance. Centrifuge the ultrafiltr
48、ation membrane in the laboratory centrifuge (5.1) at 3 000g for 45 min. Determine the gross mass of the filter container. Supplement the net mass of filtrate with water to the original amount weighed with a correction for the peptide distribution in the ultrafiltration membrane. NOTE Generally, the
49、amount of water added is approximately 15 % of the extract used for centrifugation. Consult the information provided by the membrane manufacturer. Transfer the filtrate thus obtained into an HPLC vial and measure. 7.4 LC-MS and LC-MS-MS determination 7.4.1 Elution solvents for LC-MS Use the following elution solvents: Eluant A: pipette 2,5 ml of formic acid (4.3) and 0,05 ml of trifluoracetic acid (4.5) into 500 ml water. Eluant B: mix 350 ml of acetonitrile (4.6) with 150 ml water, 2,5 ml of
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