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1、INTERNATIONAL STANDARD ISO 11027 First edition 1993-07-15 Pepper and pepper oleoresins - Determination of piperine content - Method using high-performance liquid chromatography Poivres, okorbines de poivres - Determination de Ia teneur en pipbine - Methode par Chromatographie en Phase liquide 2 haut
2、e Performance Reference number ISO 11027:1993(E) ISO 11027:1993(E) Foreword ISO (the International Organization for Standardization) is a worldwide federation of national Standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical
3、 committees. Esch member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with
4、 the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of th
5、e member bodies casting a vote. International Standard ISO 11027 was prepared by Technical Committee lSO/TC 34, Agricultural food products, Sub-Committee SC 7, Spices and condimen ts. Annex A of this International Standard is for information only. 8 ISO 1993 All rights reserved. No patt of this publ
6、ication may be reproduced or utilized in any form or by any means, electronie or mechanical, including photocopying and microfilm, without per- mission in writing from the publisher. International Organization for Standardization Case Postale 56 l CH-l 211 Geneve 20 l Switzerland Printed in Switzerl
7、and ii -,-,- INTERNATIONAL STANDARD ISO 11027:1993(E) Pepper and pepper oleoresins - Determination of piperine content - Method using high-performance liquid chromatography 1 Scope This International Standard specifies a method for the determination, by high-performance liquid chro- matography, of t
8、he piperine content of peppers (Piper nigrum Linnaeus), whole or powdered, as weil as their extracts (oleoresins). This method enables the Separation and, if necessary, the determination of the other alkaloids of pepper (isochavicine, isopiperine and piperittin). 2 Normative references The following
9、 Standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publi- cation, the editions indicated were valid. All Standards are subject to revision, and Parties to agreements based on this International Standard are encour
10、aged to investigate the possibility of applying the most re- cent editions of the Standards indicated below. Members of IEC and ISO maintain registers of cur- rently valid International Standards. ISO 28253 981, Spices and condiments - Prepa- ration of a ground Sample for analysis. ISO 5564:1982, Bl
11、ack pepper and white pepper, whole or ground - Determination of piperine con ten t - Spectrophotometric method. 3 Principle 3.1 Ground pepper Extraction with ethanol under reflux, then determi- nation of piperine by high-performance liquid chro- 2 matography (HPLC), in accordance with the procedure
12、described in this International Standard. 3.2 Whole pepper Preparation by grinding the Sample, then extraction of the powder obtained, followed by determination of piperine by HPLC, in accordance with the procedure described in this International Standard. 3.3 Oleoresins of pepper Dilution of the ol
13、eoresin in ethanol, then determi- nation of piperine by HPLC, in accordance with the procedure described in this International Standard. 4 Reagents Use only reagents of recognized analytical grade and distilled or demineralized water or water of equivalent purity. 4.1 Reference substance Piperine of
14、 at least 98 % purity, determined by the spectrometric method described in ISO 5564. 9 WARNING - This product should be handled with care as it is strongly irritating. 4.2 Solvents 4.2.1 Ethanol 4.2.2 Acetoni , 96 % (VW). trile WARNING - This product should be handled with care as it is lachrymatory
15、. 4.2.3 Acetic acid, 1 % (VW) aqueous Solution. -,-,- ISO 11027:1993(E) 4.3 Elution solvent Mix 52 volumes of acetic acid Solution (4.2.3) and 48 volumes of acetonitrile (4.2.2). 5 Apparatus Usual laboratory equipment and, in particular, the fol- lowing. 5.1 Pipettes, of 2 ml, 5 ml and 10 ml capacit
16、y. 5.2 Volumetric flasks, of 25 ml, 50 ml and 100 ml capacity (made of antiactinic glass or wrapped in alu- minium feil). 5.3 Filtration diaphragms, non-adsorbing. 54 . Separation System 5.4.1 Chromatograph, for high-performance liquid chromatography. 5.4.2 Pumping System, which enables a flow rate
17、which is constant or programmed at high pressure to be obtained and maintained. 5.4.3 Degassing Systems, for solvents. 5.4.4 Suitable detector System, adjustable to a wavelength of 343 nm. 5.5 Recorder or integrator, the Performance of which is compatible with the apparatus as a whole. 5.6 Column Ma
18、terial: stainless steel or glass Length: 10 cm to 25 cm Inside diameter: 0,4 cm to 0,5 cm Stationary Phase: grafted silica with derived octadecyl functional group, C,a, particle size 5 Pm max. 5.7 Injection System Injection valve with 10 1 loop or any other System giving the same injection accuracy.
19、 5.8 Test sieve, of aperture size 500 Pm. 6 Calibration method WARNING - Throughout the Operation, it is imperative that the solutions are not exposed to light (for instance wrap the flasks in aluminium feil) and are used as quickly as possible because of the instability of the piperine solutions. 6
20、.1 Reference solution Prepare for immediate use a 1 g/l stock Solution of the piperine (4.1) in ethanol (4.2.1). 6.2 Calibration curve From the reference Solution (6.1), prepare at least three Standard solutions of piperine with concen- trations ranging from 0,05 g/l to 0,2 g/l. Inject each Solution
21、 into the chromatograph (5.4.1). Repeat the determination at least once. Measure the peak areas and repeat the tests if the results deviate by more than 5 %. Plot the calibration curve, i.e. the mass of piperine in- jected versus the peak area. Define the mean slope of the curve. 6.3 Calculation of
22、the response factor, K Calculate the response factor K using the following formulae: K = m/A m =mxP, where m is the mass of piperine, in milligrams; A is the area of the piperine peak, in integrator units; P, is the purity of the reference piperine (4.1); m is the corrected mass of piperine, in mill
23、i- grams. 7 Sampling lt is important that the laboratory receive a Sample which is truly representative and has not been dam- aged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 948).
24、1) ISO 948:1980, Spices and condiments - Sampling. 2 ISO 11027:1993(E) 8 Preparation of test Sample WARNING - It is imperative to operate away from light because of the instability of the piperine solutions. 8.1 Peppers in powder form 8.1.1 Check that all of the test Sample Passes through the 500 Pm
25、 sieve (5.8). If this is not the case, grind the powder in accordance with ISO 2825 until the required particle size is achieved. 8.1.2 From the test Sample prepared as in 8.1 .l, weigh, to the nearest 0,Ol g, 0,5 g to 1 g, and trans- fer it to a 100 ml round-bottom flask (5.2) fitted with a reflux
26、condenser. Add 50 ml of ethanol (4.2.1) and bring it to the boil. Maintain for 3 h under reflux. Allow the Solution to cool. Filter the Solution and col- lect it in a 100 ml one-mark volumetric flask (5.2). Rinse the extraction flask and filter with successive portions of ethanol (4.2.1). Add the wa
27、sh liquids to the 100 ml volumetric flask and make up to the mark with more ethanol. 8.2 Whole peppers 8.2.1 Grind the test Sample in accordance with ISO 2825 until the powder obtained Passes entirely through the 500 Pm sieve (5.8). 8.2.2 Using the test Sample prepared as in 8.2.1, proceed as descri
28、bed in 8.1.2. 8.3 Oleoresins of peppers Thoroughly homogenize the test Sample of oleoresin. 9 Procedure 9.1 Test Portion 9.1.1 Peppers, whole or ground Take with the pipette (5.1) 10 ml of the extraction Solution, obtained as described in 8.1.2 or 8.2.2, and transfer it to a 25 ml one-mark volumetri
29、c flask (5.2). Make up to the mark with the elution solvent (4.3). NOTE 1 This dilution may be modified, if necessary, by the analyst depending on the assumed piperine content of the Sample and the sensitivity of the detector. 9.1.2 Oleoresins of pepper Weigh into a 100 ml one-mark volumetric flask,
30、 to the nearest 0,l mg, 0,2 g of oleoresin (see 8.3) and make up to the mark with ethanol (4.2.1). Pipette 10 ml of this mixture into a 50 ml one-mark volumetric flask and make up to the mark with the elution solvent (4.3). NOTE 2 This dilution may be modified, if necessary, by the analyst depending
31、 on the assumed piperine content of the Sample and the sensitivity of the detector. 9.2 Determination The final dilution shall be clear. If this is not the case, filter it on the filtration diaphragm (5.3). Inject the test Solution into the chromatograph (5.4.1). On the chromatogram obtained, measur
32、e the peak of the piperine (see typical chromatograms given in an- nex A). Carry out two tests and repeat the determination if the results deviate by more than 5 %. 10 Calculation 10.1 Peppers, whole or ground Calculate the piperine content, as a percentage by mass, using the following formula: AxKx
33、+x Fx 100 where A mx is the units; is the mass of the Sample, in mil ligram s; area of the piperine peak, in integrator K is the response factor determined for the reference substance. NOTE 3 The ratio 25/10 is related to the dilution pro- posed in 9.1 .l and should be modified accordingly when anot
34、her dilution is used. i 10.2 Oleoresins of pepper Calculate the piperine content, as a percentage by mass, using the following formula: AxKx$x+)xlOO where A, 4 and K have the same meaning as in 10.1. NOTE 4 The ratio 50/10 is related to the dilution pro- posed in 9.1.2 and should be modified accordi
35、ngly when another dilution is used. -,-,- ISO 11027:1993(E) 11 Repeatability Take as the result for the response factor K and the piperine content, the mean value of several (at least three) determinations carried out on the same test Sample. These different values should not differ from their mean
36、by more than 5 %. b) the apparatus System; c) the characteristics of the column (material, di- mensions, packing, stationary Phase); d) the characteristics of the detector (optional) and the operating conditions; e) the characteristics of the mobile Phase (flow rate and nature); 12 Test report The t
37、est report shall include the following information: a) reference to this International Standard; f) identification of the test Sample (quantity injected and final dilution); g) results obtained. -,-,- ISO 11027:1993(E) Annex A (informative) Typical chromatograms FigureA.l Shows typical chromatograms
38、, taken under the operating conditions given in this International Standard. Reference piperine Piperine Oleoresin of pepper Figure A.1 -,-,- ISO 11027:1993(E) UDC 633.841543544 Descriptors: agricultural products, food products, seasonings, spices, pepper, Chemical analysis, determination of content, high Performance liquid chromatography. Price based on 5 pages