1、BRITISH STANDARD Fatty food - Determination of pesticides and polychlorinated biphenyls (PCBs) Part 4. Determination, confirmatory tests, miscellaneous The European Standard EN 1528-4 : 1996 has the status of a British Standard ICs 67.040 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPY
2、RIGHT LAW BS EN 1528-4 : 1997 BS EN 1628-4 : 1997 Committees responsible for this British Standard The preparation of this British Standard was entmsed to Technical Panel Awl-B, Food analysis - Horizontal methods, upon which the following bodies were representid Associon of Public Analysts Departmen
3、t of kade and Industry (Laboratory of the Government Chemist) Food and Drink Federaion Institute of Food Science and Technology ihishy of Agriculture Fisheries and Food Royal Society of Chemisbry This British Standard, having been prepared under the direction of the Consumer Products and Services Se
4、ctor Board, was pubiished under the authority of the Standards Board and comes into effect on 16 June 1997 Q BSI 1997 Amendments issued since publication The foilowing ES1 references reiate to the work on this standard: Commiaee reference Awl43 Draft for comment 9450478 DC ISBN O 680 27382 2 STD-BSI
5、 BS EN 1528-4-ENGL 1777 m 1b24bb7 Ub223bL 15T BS EN 1528-4 : 1997 Contents page Committees responsible hide front cover Nationai foreword ll Foreword 2 Text of EN 1528-4 3 O BSI 1997 1 STD-BSI BS EN 1528-q-ENGL 2997 M Lb24bb9 Ob223b2 07b M BS EN 1528-4 : 1997 National foreword This British Standard
6、has been prepared by Technical Committee AW/-B and is the Enghsh ianguage version of EN 1528-4 : 1996 Fatty food - Determination of pesticides and polychlorinated biphenyls WBs) Part 4 : Determination, confirmatmy tests, m.iscellanecyus published by the European Committee for Sta,nda,rWon (CEN). dis
7、cussions in which the United Kingdom took an active part. EN 1528-4 was produced as a result of international Cross-references Publication referred to EN 15281 : 1996 EN 15282 : 1996 EN 15283 : 1996 Corresponding British Standard BS EN 15281 : 1997 Fatty food - Determination of pesticides and polych
8、lorinated biphmyis (PCBS) Part 1: General BS EN 15282 : 1997 Fatty food - Determination of pesticides and polychlorinated biphenyls o Part 2 : Extmction of fat, pesticides and PCBS, and DB-1701,0V-225, m, Stationary phases should be coated onto the support with care, the ratio depending on the suppo
9、rthase combination chosen. Newly filied columns should be conditioned for at least 24 h at a temperature near the maximum recommended operating temperature with the type of stationary phase used, and should then be tested for their eciency and selectivity at the required operating ternperahre using
10、standard mixhires of pesticides. The end of the column should always be disconnected from the detector during conditioning. Pure, dry nitrogen (oxygen-free, especialiy when using an electron capture detector (ECD), or an argohewne mixture (in the case of a pulsed ECD), should be used as carrier gas
11、for packed columns. The flow rate depends on the size and type of column used. Genem, gas flow rates should be controlled as accurately as possible. Molecular sieve filters should be installed for ail gas supplies and regenerated regularly. Finailx GC conditions (column length, stationary phase type
12、, injector, detector and column temperatures, gas flow rates, etc.) should be such that the separation of the pesticides and FCBs likely to be present is as complete as possible. Capillary GC has a separation power superior to that of packed columns. This technique is recommended especially in the c
13、ase of complex extra - SE-54 (equivalent to DB-5, CP Sil 8, BP-5, SPB-5, etc.); - OV-17 (equivalent to OVll, OV-22, SP-2250, DG710, DB 608, etc.); - DB 13Ol(equivalent to DB624); - DB17Ol(equivalent to OV-1701, CP Si lWB, BP-10, SPB-7, etc.); etc.); - WAX (equivalent to DBWAX, CP-WAX-52-CB, Carbowax
14、 20 M, etc.)2) - OV 225 (equivalent to DB-225, SIL MB, SPB-2330, A test for separation efficiency of capillary columns, is given in 7.2 of EN 15281 : 1996. 4.1.3 Iqiection techniques Various injection techniques are useful such as: a) Grob splitJess iqjection. b) On-column injection. c) Programmed T
15、emperature Vaporization (PTQ injection. The applicability of these techniques depends on the apparatus used and on special requirements. 4.2 Preliminary tests Determine the linear dynamic range of detector response under the actual GC conditions used by injecting dilute standard solutions. Inject in
16、to the gas chromatograph an appropriate volume (between 1,0 pl and 10 pl depending on the system) of the purified extracts obtained according to the analytical method used. The chromatogram so obtained should enable both the identity and the approximate concentration of the compounds present in the
17、extracts to be established 4.3 Determination Make sure that all measurements are performed within the linear dynamic range of the system. Prepare at least two standar solutions of the pesticides or PCB congeners identified in the solvent to be used for the final extract (usually light petroleum omhe
18、xane). Their concentrations should encompass the probable concentration expected in the final extracts. Then iiiject quai volumes of the final extracts obtained and of the two or more standard solutions into the gas chromatograph. It is essential that the injection of the purified portions of the sa
19、mple extracts is preceded and followed by injection of the standard solutions. Measure the peak mas or peak heights. The results obtained from any two injections of the same standard ) SE30 . Carbowax 20 M are examples of suitable products available commercially. This information is given for the co
20、nvenience of users of this European Standard and does not constitute an endorsement by CEN of these products. Q BSI 1997 - STD-BSI BS EN L529-q-ENGL L717 BI Lb27bb7 Ob223b7 b78 Page 6 EN 16284 : 1996 solution should not Mer more than approximately 5 % from each other. Inclusion of an intend standard
21、 is useful (see clause 4 of EN 15283 : 199s). It is necessary to ensue that the standard materiais and samples are dissolved in the same solvent, otherwise varying evaporation profiles will result, which could lead to changes in the retention times and peak areas or heghts. For example, increases in
22、 peak heghts of 35 % have been observed for PCB congeners on changing from isctan e to toluene. The contents of individual PCB congeners should not be added together to obtain the total PCBs since such a value is meaningless. There is also no point in c8LTying out other extrapoiations to a fictitiou
23、s total content of PCBs (e.g. calculated as Clophen A 60 3, since these are generally based on the incorrect assumption that the PCB distribution pattern in the sample is exactly the same as that of the industrial PCB commercial product. A determination is only possible if the mean of recoveries fro
24、m multiple determinations for the substance concerned is in the range 70 % to 110 % for individuai determinations. Compliance with this condition has to be checked periodicaliy by repeated measurements of recovery from samples containing known additions of the relevant sandard material. 5 connrmator
25、y tests i 6.1 General When analyses are performed for regulatory purposes it is especiauy important that confinnatory tests are carried out before reporhng aversely on samples containjng residues of pesticides not usually associated with that commodity or where maximum residue limits (MRLS) appear t
26、o have been exceeded. Contamination of samples with non-pesticidal chemicals occurs from time to time, and in some chromatographic methods these compounds can have simiar properties to pesticides and could therefore be misidentified as such. Examples in gas chromatography include the responses of EC
27、D to phthalate esters and of phosphorusspecific detectors to compounds containing sulfur. Confirmabry tests can be divided into two types: quantitative tests are necessary when MRh appear to be exceeded, whilst qualitative conrmation of identity is also needed in these cases and when atypical residu
28、es are encountered. Qualitative tests can involve chemical reactions or separations where some loss of the residue occurs. Particular problems occur in confirmation when MRIs are set at or about the lunit of determination. The need for confirmaiory tests can depend upon the type of sample or its kno
29、wn history. in many substrates, certain residues are nearly always found For a series of samples of similar origin it could only be necessary to confirm the identity of residues in the initial samples. Simiarly, when it is known that a particular pesticide has been applied to the sample material the
30、re could be little need for confirmation of identi, although a random proportion of samples should be confirmed. Where control samples are available, these should be used to check the presence of possible interfering substances. In quantitative confirmation at least one aiteniative procedure shouid
31、be used and the lower result reported. In qualitative confirmation, an altemative technique using diferent physiochemical properties is desirable. The necessary steps to positive identification are a matter of judgement for the analyst and particular attention should be paid to the choice of a metho
32、d which will eliminate the effect of interfering compounds. The chosen method will depend upon the availability of suitable apparatus and expertise within the testuig iabomtory. As guidance to the anayst a number of altemative procedures for confirmation are given in 6.2 to 6.9. 6.2 Alternative GC c
33、olumns The resuits obtained in the primary anaipis should be quantitatively and qualitaively confirmed using at least one altemative column containing a stationary phase of different polarity. The quantitative results obtained should be within 20 % of the primary analysis and the lower figure should
34、 be reported, since the higher gure could have been enhanced by interference from CO- extracted material Further quantitative confirmation is required if the results diner by more than 20 Oh, except when the MRL is set at or about the limit of determination when a variation of up to 100 % would be a
35、cceptable. In choosing the altemative column material, consideration should be given to separating any other pesticide or PCB residues or interfering compounds known to have retention times on the primary column identical to that of the residue detected. The ateniative column may be a packed column
36、or, preferably, a capillary column whose difering resolving power can be utilized. whilst the use of an altemative gas chromatographic column might not always give positive confirmation, it wil often quickly disprove a suspected identity In either case, further confirmation is required to idenhfy th
37、e residue. 3, Clophen A 60 is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. O BSI 1997 STD=BSI BS EN L528-q-ENGL 2777 Lb2qbbY Ob223bA 50q Page 6
38、EN 1628-4 : 1996 6.3 Alternative GC detectors When pesticides containing several chemical elements are present, detectors showing enhanced response to these elements may be used. Detectors such as flame photomec (sulfur, phosphorus and tin), aikali flame ionization hosphonis and nitrogen) and cotdom
39、etridconductivity (nitrogen, sulfur and halogens) can give valuable additional information on residues The sulfur/phosphorus response ratio obtained by using a fame photometric detector can give usefui information in the case of phosphorothioas. 6.4 Thin layer chromatography (TLC) In some instances,
40、 confirmation of gas chromatographic nws is most conveniently achieved by TLC. Identication is based on two criteria, %value and visuahation reaction. The scientific literature con- numerous references to the technique. An ILTPAC Report on Pesticides 2 reviews the technique and serves as a convenien
41、t introduction. The quantitative aspects of thin layer chromatography are, however, limited. A further extension of this technique involves the removal of the area on the piate corresponding to the is the volume of filtrate after clean-up, in is the initial volume (here: 250 mi), in millilitres. mil
42、lilitreq 6.7 Further processing For further processing of the residue after evaporation derived from 6.6, methods B and D (see clauses 6 and 8 of EN 15283 : 1996) have proven suitable, see table 1. When samples need to be analysed only for organochlorine and non polar organophosphorus compounds, it
43、is advantageous to use method D since it involves little work and time. By using method B, the more polar organophosphorus compounds and several metabolites can be determjned additionally Further, the extracts are cleaned up more intensively so that method B is appropriate also for the analysis of m
44、atmiah from which CMo E removes oniy a portion of the lipids. Table 1. Organochlorine and organophosphorus compounds including several metabolites recovered at levels exceeding 70 % (marked thus +) or not recovered (marked thus -) in recovery experiments following further extract clean-up 1 Compound
45、 aldrin (HHDN) y-chlordane chiorfenson O, p-TDE (DDD) O, p-TDE (DDD) O, p-DDE p, p-DDE O, p-DDT p, p-DDT dieldrin (HEOD) a-endodan +ndodan endosulfan sulphate endrin fenson a-HCH y-HCH (IIndane) Food and Agncuiture Organization of the United Nations (FAO); World Health Organization BtOra, Y, Vitorov
46、ic, S. u., “hier, K-E!, and Klisenko, MA: Pure Appl.) Chem., 53, 1039- 1049 (1981) Cochrane, W.P: Chemical derivatisation techniques in pesticide anaiysis, advances and applications; ACS Symposium Series 136, American Chemical Society, Washington, D.C., S. 231 - 249,1980. Pesticide Analytical Manual
47、, Food and Drug Admjnistmtion, Washington D.C. USA, Vol.1, Chapter 6, Section 652. Hutzinger, O., and Safe, E: Mass Spectrometry of Pesticides and Poilutants, CRC Press, 1973. Sphon, JA, and Brumley, W.C.: Biochemical Application of Mass Spctrometq Editors: Waller, G.R., Donner, O.C.; John Wdey film
48、 thickness 0,23 pm) 110 “C isothermal for 2 min programmed to rise at 6 “C/min from 110 “C to 245 “C isothermal at245“C for2min temperature 350 “C Detector Electron capture detector, hector Programmable temperature vaporizer cm PTVprogram “ime(min) minus 0,15 Split open minus OJO 0,20 Split close 0,
49、25 PTV temperature 250 “C 2900 Split open 4,oo PTV temperature 40 “C PTV temperature 40 “C Split flow rate 50 mlmin O BSI 1997 B.1.2 Operating conditions 2 Column Fused silica capillary DB1701(30 m long, 0,53 mm i.d; film thickness 1,0 pm) 80 “C isothermal for 1 min programmed to rise at 30 Wmin from 80 “C to 150 “C and at 5 Wmin from 150 “C to 280 “C temperature 280 “C Column temperature Detector Electron capture detector, hector Programmable temperature vaporizer (m PTVProgram %e(min) . minus 0,15 minus 0,lO Split open 0,20 Spl
