Joint Food Safety and Standards Group

SURVEY OF 3-MCPD IN FOODS

Introduction

The Joint MAFF/DH Food Safety and Standards Group (JFSSG) is currently carryingout a survey to investigate the levels of 3-monochloropropane-1,2-diol (3-MCPD) inselected foods and beverages. The following paper presents the background to, andthe aims of, this survey, as well as details of the methodology being followed. A fulllist of the samples included in the survey is also provided.

Background

3-MCPD is one of a group of chemical contaminants known as chloropropanols. Itwas first identified as a contaminant of acid-hydrolysed vegetable protein (acid-HVP),an ingredient which for many years has been used to flavour savoury foods such assoups, prepared meals, savoury snacks, gravy mixes and bouillon cubes. Although3-MCPD levels in acid-HVP are now significantly lower than those originally found inthe 1980s,1-3 subsequent work by both the JFSSG and the UK food industry hasindicated4,5 that the contaminant can also occur in several other foods and ingredientsas a result of processing, storage conditions or migration from certain food contactmaterials. Studies have also shown6 that elevated levels of 3-MCPD can occur in sometypes of cooked products. A summary of the possible sources of 3-MCPD in the diet,which includes the results of work carried out by both the JFSSG and industry, is givenin Annex I.

Studies have shown that 3-MCPD is carcinogenic in rats when given in high dosesover prolonged periods and, on this basis, the EC Scientific Committee for Food (SCF)has advised7 that levels should be undetectable in food products. In line with theSCF's advice, the UK Food Advisory Committee (FAC) has recommended8 thatindustry should take steps to ensure that 3-MCPD is undetectable (i.e. less than 0.01mg/kg) in foods and, where technologically feasible, in food ingredients as well.

Discussions are currently underway on a proposal from the European Commissionwhich, for the first time, sets EU-wide limits for 3-MCPD in certain foods. Ascurrently drafted, the proposal sets a maximum limit of 0.01 mg/kg for "productscontaining acid-HVP" and "soy sauce". However, limits may eventually be set for awider range of foodstuffs.

Project aims

The survey has three main aims:

• to check that selected foods on the UK market are complying with the FAC’srecommended limit for 3-MCPD of 0.01 mg/kg*

• to expand the data currently available on levels of 3-MCPD in foods and beverages(including cooked products) and, thus, to facilitate the estimation of consumerexposure to the contaminant

• to inform future EC discussions on limits for 3-MCPD in foods

Methodology

The survey is being carried out, and the results will be reported, in accordance with theGuidelines for Planning and Reporting JFSSG Surveys.9 Further details are givenbelow.

Sampling strategy

The survey has been targeted at those products which either have been shown tocontain 3-MCPD or which it has been suggested may contain the contaminant as aresult of processing or storage conditions. These are outlined below:

• foods such as salami, some cheeses (parmesan, processed cheeses and cheesealternatives) and some cereal products subjected to high temperature processingwhich have previously been shown to contain 3-MCPD;

• products with a combination of high fat and salt levels and a low pH;

• products with a long shelf-life;

• products containing acid-HVP;

• products containing mono or di-glycerides of fatty acids (such as emulsifiers andlecithin) which could theoretically act as precursors to 3-MCPD;

• products containing malt and/or malt extracts, some of these ingredients havingpreviously been shown to contain 3-MCPD; and

• products in contact with wet-strength epichlorohydrin resins.

Based on this list, the survey comprises 250 samples from the following categories:

• Cereals - biscuits (including malted and toasted products); bread; breakfast cereals;corn snacks; and savoury crackers (73 products)

* A separate survey of 3-MCPD in 60 food ingredients is also being carried out by RHM Technology Ltd and isdue to be completed by March 2000.

• Dairy - creams (aerosols and dessert toppings); low-fat cheeses; parmesan cheeses;and processed cheeses and cheese alternatives (31 products)

• Meats - chargrilled burgers; cooked meats; salami; and smoked bacon (56 products)

• Miscellaneous - battered and breaded products; cakes, pastries and fruit pies;confectionery; pizzas; pork pies; and various drinks (beer, tea and malted drinks)(56 products)

• Soups and sauces - canned, fresh and powdered soups and sauces (table and cook-in-sauces) (34 products)

By including such a wide range of food commodities, it has not been possible to coverthe whole market for each one. Instead, for those commodities where 3-MCPD ismore likely to be found, a larger number of samples has been allocated. Where theresults for any commodity subsequently indicate that 3-MCPD is found at elevatedlevels in several of the products sampled, further samples will be taken to increase thecoverage of the market. For each commodity, a range of samples has been allocated,including brand leaders, own-brand products and, where appropriate, take-awayproducts. An attempt has been made to reflect market share, where possible, usinginformation from recent issues of the publications, Retail Business and ConsumerGoods UK.10,11 In the case of own-brand products, these have been taken mainly fromTesco, Asda, Sainsbury, and Safeway, the leading supermarket chains in the UK, withthe distribution of products broadly reflecting the relative market share of these fourretailers.12 A full list of the samples included in the survey (with brand details) isgiven in Annex II.

Sampling has been undertaken by MAFF Central Science Laboratory (CSL), with thesamples being purchased between November 1999 and January 2000 and the majoritybeing bought from supermarket and other retail outlets (e.g. independent butchers andtake-away outlets) in the Leeds/York area. In concentrating on one geographical area,it has been assumed that the location of the retail outlets will have no effect on thelevel of 3-MCPD in foods. In addition, it has been assumed that branded products donot vary with retailer and thus, for ease of sample collection, branded products havebeen purchased only from the four main supermarkets. For most products, a minimumsample size of 200 g (or one pack) has been purchased, while for products whichrequire some form of cooking or heating, a minimum weight of 400 g (or two packs)has been taken.

Logging and storage

The Guidelines9 state that the contractor must follow Good Laboratory Practice (GLP)or United Kingdom Accreditation Scheme (UKAS) procedures during handling,storage, homogenisation and subsampling of survey samples to preclude anycontamination of the sample and must keep records to show that these procedures havebeen followed.

In line with these procedures, CSL have recorded the type of product (brand name),date and place of sampling, date of manufacture, batch number, best before date and aphotograph/photocopy reference for all samples. Samples have been stored asrecommended by the manufacturer or frozen if stability is in question. For themajority of products, each sample will be homogenised and divided into threesubsamples. One of these subsamples will be used for analysis and the others storedappropriately. Of the two stored subsamples, one will be available for analysis by thecompany or retailer if required.

In the case of products which require some form of cooking or heating, at least oneadditional unopened pack (with the same batch or sell-by date) will be storedappropriately and will be available for use should an analysis of the "raw" samplesubsequently be required. "Cooked" products will be homogenised (using a kitchenblender) and divided into three subsamples as above.

Cooking procedures

As requested by the FAC, all of the foods and beverages included in the survey will beanalysed "as consumed" to enable a more accurate estimate of dietary exposure to beobtained. As a consequence, the following products will require some form of furtherpreparation:

• battered and breaded products• chargrilled burgers• cook-in sauces• crumpets• pizzas• smoked bacon• soups• malted drinks• tea infusions (from tea bags)

Most of the foods will be grilled, microwave cooked, oven baked or heated in asaucepan. Powdered soups and drinks will be made up with boiling water or hot milk,as appropriate. Detailed procedures for preparing these foods, based mainly onmanufacturers' instructions, have been drawn up in association with CSL and areattached at Annex III.

Analysis

A detailed workplan, which sets out the analytical batch protocol and the InternalQuality Control (IQC) procedures to be used by CSL during this survey, is attached atAnnex IV. The UKAS-accredited method used for the analysis of 3-MCPD is attachedat Annex V.

Reporting of results

It is intended that the results will be reported in the Food Safety Information Bulletinand in an accompanying Food Surveillance Information Sheet in early Autumn,although publication may be delayed if it proves necessary to take further samples ofany commodity. The FAC will also have the opportunity to comment on the results aspart of a wider review of the issue in September 2000.

In line with the Guidelines9, the brand names of the products and a full list of samplesanalysed will be included in the Food Surveillance Information Sheet. Companies orretailers whose products have yielded unusual or unexpected results or results whichgive rise for concern, but where there is no immediate safety issue, will be sent theirown results approximately seven weeks prior to the publication of the Bulletin articleand invited to make a brief statement. The statement will be included in the FoodSurveillance Information Sheet.

The Guidelines9 currently state that all results quoted should be the "best estimatevalue" ± 2 standard deviations. While the results of most JFSSG surveys are reportedin this way, the results of surveys carried out by the Working Party on FoodAuthenticity are reported as though they were to be used for enforcement purposes (i.e.all benefit of doubt is given to the manufacturer). This issue is due to be considered bythe FAC at its meeting on 23 March. How the results of the 3-MCPD survey aresubsequently reported will be dependent on the outcome of this review.

REFERENCES

1. MAFF (1991). Survey of hydrolysed vegetable proteins for chlorinated propanols.CSL Report FD 91/6.

2. MAFF (1993). Survey of chlorinated propanols in hydrolysed vegetable protein1992. CSL Report FD 93/17.

3. JFSSG (1999). Survey of 3-monochloropropane-1,2-diol (3-MCPD) in acid-hydrolysed vegetable protein. Food Surveillance Information Sheet No. 181.

4. JFSSG (1999). Survey of 3-monochloropropane-1,2-diol (3-MCPD) in soy sauceand similar products. Food Surveillance Information Sheet No. 187.

5. Food Advisory Committee (1999). Further update on 3-MCPD in foods and foodingredients (FdAC/Contaminants/37). Paper presented to the FAC on 23 April 1998.

6. MAFF (1999). The effects of cooking on the level of 3-monochloropropane-1,2-diol(3-MCPD) in foods. CSL Report FD 98/60.

7. European Commission (1997). Opinion on 3-monochloropropanediol (3-MCPD),expressed on 16 December 1994. pp. 31-33 in Reports of the Scientific Committeefor Food. Food Science and Techniques, Thirty-sixth Series. Office for OfficialPublications of the European Community, Luxembourg.

8. Food Advisory Committee (1999). Recent developments on 3-MCPD in food andfood ingredients. FAC Press Release 5/99

9. JFSSG (1998). Guidelines for Planning and Reporting JFSSG Surveys, Version 3.

10. Retail Business: Market Survey (various editions pre-1998). Corporate Intelligenceon Retailing, London.

11. Consumer Goods UK (various editions, 1998 onwards). Corporate IntelligenceGroup, Retail Intelligence, London.

12. Food Industry News, October 1999, Beacon Publishing, East Sussex.

13. Personal communication from Conrad Astil, Unilever.

14. Engelhardt U.H. et al. (1999). Antioxidative phenolic compounds in green/blacktea and other methylxanthine containing beverages. Abstract Paper Am. Chem. 217:U49 Part 1.

ANNEX I: POSSIBLE SOURCES OF 3-MCPD IN THE DIET

HVPSince the 1980s, it has been recognised that the procedure used to manufacture the savouryfood ingredient acid-HVP can result in the formation of 3-MCPD. Most acid-HVP isproduced using strong hydrochloric acid, and 3-MCPD is formed as a result of the hightemperature chlorination of lipids present in the protein starting materials. Surveys carried outby MAFF in 19901 and 19922 showed that levels of up to 100 mg/kg were quite common inacid-HVP at that time. Since then, however, levels in HVP used in the UK have declinedmarkedly, as was shown by the results of a recent JFSSG survey of acid-HVP3 in which3-MCPD was undetectable (less than 0.01 mg/kg) or was found at low levels in over 75% ofthe 50 samples analysed.

Roasted cereals, dark malts and dark malt extractsInformation from the UK brewing and malting industries indicates that 3-MCPD levels of up to0.3-0.4 mg/kg can occur in roasted cereals and dark speciality malts which are used to addcolour and flavour to most dark beers and some lagers. Extracts derived from theseingredients, which are used to flavour certain foods and drinks, may also contain 3-MCPDlevels of over 0.1 mg/kg. Despite extensive work by these industries to reduce the levels of3-MCPD in these ingredients, they have been unable to make significant reductions withoutadversely affecting their flavour characteristics. However, due to the low levels of use of theseingredients, the concentrations of 3-MCPD in the final product are below 0.01 mg/kg.

Fermented sausagesCertain types of fermented sausage such as salami have also been shown to contain levels of3-MCPD of up to 0.1 mg/kg. This may be due to the formation of 3-MCPD within the meat(due to the interaction between fat and salt in the product, coupled with its long shelf-life)and/or due to the presence of 3-MCPD in the resins used in the sausage casings. The casingsindustry is carrying out work at European level to determine the contribution of the casings tothe 3-MCPD content of salami and, like other users of epichlorohydrin-based wet strengthresins (see below), has already started to use higher grade resins with much lower levels of3-MCPD.

Soy saucesFollowing the finding of high levels of 3-MCPD in Chinese soy sauce (6-124 mg/kg) in severalMember States, in September 1999, the JFSSG carried out a survey of 40 samples of soy sauceand similar products available on the UK market.4 The results showed that around two-thirdsof the samples contained 3-MCPD at levels very close to or below the FAC's recommendedlimit of 0.01 mg/kg. However, the remaining one third of the samples contained levels above0.01 mg/kg, the highest being 30 mg/kg. These products were imported from China and HongKong, Taiwan and the Philippines, indicating that higher levels are not restricted to any onecountry of origin.

Several grades of soy sauce are manufactured in the Far East, including the traditionalfermented product as well as lower grades which can contain acid-HVP or which may beproduced using an acid treatment. It is known that such acid treatments can generate verylarge amounts of 3-MCPD. They can also occur during the manufacture of acid-HVP. Whilemany European manufacturers have made changes to their HVP manufacturing processes over

the last 3-4 years which have significantly reduced 3-MCPD levels, some manufacturers inother parts of the world may not yet have made these changes.

Domestic cooking and formation of 3-MCPD in foodIn 1998, the JFSSG funded a study of the effect of cooking on 3-MCPD levels in foods. Theresults of this work6 show that elevated levels of 3-MCPD can occur in toasted bread (0.02-0.32 mg/kg), with slightly lower concentrations in some grilled cheeses (0.08-0.12 mg/kg) andfried batters (0.07-0.09 mg/kg). In contrast, 3-MCPD is undetectable or present only at verylow levels in cooked meat, gravy, sauces and stocks.

Food contact materialsInformation from the packaging industry and others indicates that very low levels of3-MCPD may migrate into food and beverages due to its presence in certain types ofepichlorohydrin-based wet strength resins used in paper (e.g. tea bag paper, coffee filters,absorbent meat padding) and cellulose casings. Work has been carried out by the industry todevelop higher grade "third generation" resins which have significantly lower levels of3-MCPD, and these are increasingly being used in the above applications. With the increase inthe availability and use of these resins, 3-MCPD exposure from this source will continue todecrease.

ANNEX II: LIST OF SAMPLES

cereals

Class code Brand Type Retail OutletBiscuits 1 C01 McVitie's Digestive J Sainsbury

1 C02 McVitie's Rich Tea J Sainsbury1 C03 McVitie's Ginger Nuts J Sainsbury1 C04 Fox's Crunch Creams Tesco1 C05 Burton's Jammie Dodgers Tesco

Malted biscuits 1 C06 Tesco Malted milk Tesco1 C23 Safeway Malted milk Safeway1 C08 Sainsbury's Morning coffee J Sainsbury1 C09 Sainsbury's Malted milk J Sainsbury1 C10 Crawford's Thin Arrowroot J Sainsbury

Savoury crackers 1 C11 McVitie's Mini Cheddars original J Sainsbury1 C12 Jacob's Twiglets original J Sainsbury1 C13 Jacob's Cream Crackers J Sainsbury1 C14 McVitie's Krackawheat J Sainsbury 1 C15 Jacob's Ritz J Sainsbury1 C16 McVitie's Tuc J Sainsbury1 C17 Jacob's Cornish Wafers Tesco1 C18 Tesco Cream Crackers Tesco1 C19 Sainsbury's Cream Crackers J Sainsbury1 C20 Asda 50% less fat cream crackers Asda

Toasted biscuits 1 C21 Sainsbury's Les Mignons French onion flavour nibbles J Sainsbury1 C22 BN garlic toast Bite size Garlic Toasties baked Tesco1 C24 Tesco Melba toast Tesco1 C25 Jacob's Continental Pecan Toast Tesco1 C26 Jacob's Continental Raisin Toast Tesco1 C27 Pogen Krisprolls Tesco1 C28 Ryvita Original Rye Crispbread Tesco1 C29 Jacquet Toast Francais J Sainsbury1 C30 Asda French toast Asda1 C33 Safeway Dutch crispbakes Safeway

Corn snacks 1 C31 Sainsbury's Original Tortillas J Sainsbury1 C32 M&S Golden Cheese Tasters M&S1 C34 Tesco Rico's Corn Snacks Tangy Cheese flavour Tesco1 C35 Golden Wonder Nice 'n' Spicy flavour Nik Naks Tesco1 C36 Walker's Monster Munch pickled onion Tesco1 C37 Doritos cheese Corn Chips Movie bag tangy cheese flavour Tesco1 C38 KP Skips Prawn cocktail flavour Skips Tesco1 C39 Golden Wonder Cheesy flavour Wotsits Tesco1 C40 Phileas Fogg Authentic Tortilla Chips Tesco1 C74 Safeway Onion rings Safeway

Breads 1 C41 Safeway Bakery wholemeal loaf Safeway1 C42 Tesco Wholemeal medium sliced loaf Tesco1 C43 M&S Wholemeal medium sliced loaf M&S1 C44 Allinson Wholemeal thick cut loaf J Sainsbury1 C45 Kingsmill Country Gold loaf J Sainsbury1 C46 Hovis Wholemeal thick sliced loaf J Sainsbury1 C47 Hovis Granary thick sliced loaf J Sainsbury1 C48 Warburton Wholemeal toastie sliced loaf J Sainsbury1 C49 Kingsmill Square thick sliced white loaf J Sainsbury1 C50 Mighty White Medium sliced softgrain loaf J Sainsbury1 C51 Mother's Pride Medium sliced Scottish plain white loaf Tesco1 C52 Sainsbury's Crumpets J Sainsbury 11 C53 Safeway Crumpet Safeway 11 C54 Asda Crumpet Asda 11 C55 Sainsbury's Fruit malt loaf J Sainsbury1 C56 Tesco Medium sliced malted brown loaf Tesco1 C57 Soreen Fruity malt loaf J Sainsbury1 C58 Sainsbury's Ciabatta loaf J Sainsbury1 C59 Tesco Plain Naan bread Tesco1 C60 Safeway Wholemeal Pitta bread Safeway

Breakfast 1 C61 Kelloggs Cornflakes J Sainsburycereals 1 C62 Kelloggs Bran Flakes J Sainsbury

1 C63 Kelloggs Rice Krispies J Sainsbury1 C64 Kelloggs All-Bran Plus J Sainsbury1 C65 Weetabix Weetabix J Sainsbury1 C66 Weetabix Alpen J Sainsbury1 C67 Nestle Shredded Wheat J Sainsbury1 C68 Quaker Sugar Puffs Tesco1 C69 Sainsbury's Cornflakes J Sainsbury1 C70 Weetabix Ready Brek oats Tesco1 C71 Nestle Cheerios Tesco1 C72 Kelloggs Frosties J Sainsbury1 C73 Tesco Pingu Crisp shaped cereal Tesco

total 73

Page 1

dairy

Class code Brand Description Retail Outlet

Processed 1 Da01 Dairylea Big Cheese Spread J Sainsburycheese 1 Da02 Tesco Smooth and Creamy medium fat light soft cheese Tescospreads 1 Da03 Primula cheese spread Tesco

1 Da04 Laughing Cow triangles Tesco

Processed 1 Da05 Safeway Reduced Fat Cheese Singles Safewaycheese 1 Da06 Tesco Singles Tescoslices 1 Da07 Kraft Dairylea Dairylea thick slices Tesco

1 Da08 Kraft Singles Singles Tesco1 Da09 Asda 50% less fat Singles Asda1 Da10 Golden Vale Cheese Strings Tesco1 Da11 Kraft Dairylea Strip Cheese original Asda

parmesan 1 Da13 Sainsbury's Grated Parmesan medium fat hard cheese J Sainsbury1 Da14 Buitoni Grated Parmesan cheese Tesco1 Da15 Tesco Fresh grated Parmesan Parmigiano Reggiano Tesco1 Da16 Asda Fresh grated Parmesan Asda1 Da17 Castelli Fresh shavings Tesco1 Da12 Safeway Grated Italian medium fat hard cheese Safeway

Low fat 1 Da18 Tesco Fresh grated half fat full flavoured cheese Tescocheeses/ 1 Da19 Asda Half fat mild cheese Asdacheese 1 Da20 Sainsbury's Be good to yourself Mature 16% fat cheese J Sainsburyalternatives 1 Da21 Safeway Medium half fat cheese Safeway

1 Da22 Asda Half fat mild grated cheese Asda1 Da23 Kraft Dairylea Dairylea light half fat cheese spread J Sainsbury1 Da24 Flora Medium mature real alternative to cheddar J Sainsbury1 Da25 Heinz Weight Watchers 95% fat free low fat alternative to Red Leicester J Sainsbury1 Da26 Kraft Philadelphia Light medium fat soft cheese J Sainsbury

Creams 1 Da27 Anchor Light Swirls UHT Tesco(aerosols/ 1 Da28 Anchor Delissimo UHT Tescotopping) 1 Da29 Elmlea Whipped Squirty Asda

1 Da30 Nestle Tip Top dessert topping (canned) Tesco1 Da31 Asda 35% less fat real dairy cream Asda

total 31

Page 2

meats

Class code Brand Description Retail Outlet

chargrilled 1 Me01 Tesco Chargrilled beefburgers (frozen) Tescoburgers 1 Me02 Sainsbury Chargrilled beefburgers (frozen) J Sainsbury

1 Me03 Dalepak flame grilled quarter pounders (frozen) Tesco1 Me04 Linda Macartney Flame grilled veg burgers Tesco1 Me05 Campbells Frozen Food American Fast Food Diner Burgers in buns Asda1 Me06 Tesco Vegetarian char grilled quarter pounder Tesco1 Me07 Asda Prime beef chargrilled quarter pounders Asda1 Me09 Mc Donalds Beefburgers York1 Me10 Burger King Chargrilled beefburgers York

smoked bacon 1 Me12 Tesco Smoked rindless back Tesco1 Me13 Sainsbury's British Thick Cut Smoked Back Bacon J Sainsbury1 Me14 Asda Smoked rindless back Asda1 Me15 Danepak Classic dry cure Tesco1 Me16 Wall's Smoked rindless back Tesco1 Me17 Safeway Smoked Rindless Safeway1 Me18 Tulip Tulip smoked back Safeway1 Me19 butcher Smoked back York market1 Me20 butcher Smoked back Scott1 Me21 butcher Smoked back Old Pork Butcher

salami 1 Me22 Tesco Kabanos pack of 2 Tesco1 Me23 Tesco Chorizo Sarta Doux Tesco1 Me24 Pepperami Pepperami salami Safeway1 Me25 Sainsbury's German salami 10 slices J Sainsbury1 Me26 Waitrose Hungarian salami Waitrose1 Me27 Waitrose Ardennes pepper salami Waitrose1 Me28 Asda Asda salami Milano Asda1 Me29 Landwirt Nolke German mini salami Asda1 Me30 Agraimpex Kabanos Asda1 Me31 Tesco Deli 1 French saucisson Tesco1 Me32 Tesco Deli 1 Italian Milano salami Tesco1 Me33 Sainsbury's Deli 2 Strafino salami J Sainsbury1 Me34 Sainsbury's Deli 2 Traditional chorizo J Sainsbury1 Me35 Asda Asda German salami Asda1 Me36 Waitrose Deli 3 German salami Waitrose1 Me37 Safeway German salami Safeway1 Me39 Safeway Danish salami Safeway

cooked meats 1 Me38 Sainsbury's Smoked ham J Sainsbury1 Me40 Tesco Tesco British Ham Tesco1 Me41 B. Matthews Norfolk smoked ham Tesco1 Me53 Asda Oak Smoked Ham Asda1 Me54 Safeway Wafer Thin Smoked Ham Safeway1 Me42 Sainsbury's Hot 'n' spicy chicken drumsticks J Sainsbury1 Me43 Asda Chicken satay Asda1 Me47 Tesco Flamegrilled tikka chicken dippers Tesco1 Me48 Tesco Roast in store chinese basted chicken Tesco1 Me50 Safeways Chicken satay selection Safeway1 Me51 Matthews Char-grill chicken breast 200g J Sainsbury1 Me52 Matthews Tikka chargrill Marinated turkey fillets Tesco1 Me55 Asda Scottish smoked salmon Asda1 Me56 Sainsbury's Anchovies fillets in pure olive oil J Sainsbury1 Me57 Rustlers Flame Cooked BBQ Rib Sandwich J Sainsbury1 Me58 M&S Chicken tikka mini fillets M&S1 Me59 Safeway Hot & Spicy wings Safeway1 Me60 Asda Mini chicken tikka satay Asda1 Me61 Safeway Cooked pork cocktail sausages Safeway1 Me62 Asda Mini shish kebab Asda

total 56

Page 3

miscellaneous

Class code Brand Description Retail Outletready to eat 1 Ms01 Pork farms Pork lattice pie Tescomeat 1 Ms02 Sainsbury's Crusty bake mini pork pies J Sainsbury

1 Ms03 M&S Melton Mowbray cured pork pies M&S1 Ms04 Dickinson & Morris Melton Mowbray pork pie Tesco1 Ms05 Tesco Traditional pork pie Tesco

Pizza 1 Ms06 Sainsbury's Fresh pizzeria pepperoni J Sainsbury1 Ms07 Tesco Classico garlic mushroom Tesco1 Ms08 Tesco Thin & crispy cheese & tomato (chilled) Tesco1 Ms09 Safeway Thin & crispy cheese & tomato (frozen) Safeway1 Ms10 Pizza Hut Country Feast Pizza Hut1 Ms11 Dominos Vegetarian Supreme Dominos

Battered & 1 Ms12 Sainsbury's Chicken breast nuggets J Sainsburybreaded 1 Ms13 M&S Flame grilled tangy barbecue style coated chicken M&S

1 Ms14 McDonald's Chicken nuggets McDonald's1 Ms15 Young's Scottish Island Scampi Tesco1 Ms16 Tesco Salmon crunchies with broccoli Tesco1 Ms17 Asda Traditional style Yorkshire Puds Asda1 Ms18 Bernard Matthews Golden Fishies J Sainsbury1 Ms19 Bird's Eye 100% Cod fillet fish fingers Tesco1 Ms20 Fish (take-away) Haddock1 Ms21 Fish (take-away) Cod1 Ms27 KFC Chicken portions KFC1 Ms31 Tryton Foods Aunt Bessie Sunday Best Yorkshire Puddings Safeway

Cakes, 1 Ms22 Mr Kipling Cream Slices (frozen) J Sainsburypastries 1 Ms23 Cadbury's Luxury chocolate gateau (frozen) J Sainsburyfruit pies 1 Ms24 McVities American Dream Colorado Choc & Crunch Cream Pie (frozen) J Sainsbury

1 Ms25 Sainsbury's Hand finished Black Forest Gateau (frozen) J Sainsbury1 Ms26 Tesco Strawberry cheesecake (frozen) Tesco1 Ms28 Safeway 2 Chocolate pies (frozen) Safeway1 Ms29 M&S Cream Cakes Apple Sponge Sandwich (chilled) M&S1 Ms30 Asda ASDA Brand Chilled Cake Asda1 Ms32 Sainsbury's Bakery family apple pie J Sainsbury1 Ms33 Ottoman Six sweet waffles Tesco1 Ms34 Mr Kipling 6 Country slices J Sainsbury1 Ms35 Sainsbury's Apple Danish trellis (store baked pastry) J Sainsbury1 Ms36 Asda Fresh Donuts (store baked pastry) Asda1 Ms37 Safeway American style cookies (Smartie cookie)2 (store baked pastry) Safeway1 Ms38 Tesco Eccles cakes (store baked pastry) Tesco

Confectionery 1 Ms39 Kit Kat Real milk chocolate fingers Tesco1 Ms40 Twix Milk chocolate covered caramel + biscuit Tesco1 Ms41 Mars bar Milk chocolate with soft nougat + caramel Tesco1 Ms42 Tunnock's caramel wafer Tesco1 Ms43 Maltesers Tesco

Beer 1 Ms44 Newcastle Brown Ale Tesco1 Ms45 Guinness Original Tesco1 Ms46 Mackeson's Tesco1 Ms47 Murphy's Draught Stout Tesco1 Ms48 Sainsbury's Sainsbury's Irish stout J Sainsbury

Tea bags 1 Ms49 Tesco Premium tea bags Tesco1 Ms50 Sainsbury's Sainsbury's Red Label J Sainsbury1 Ms51 Brooke Bond BB Choicest blend Tesco1 Ms52 Tetley Tetley tea bags Tesco1 Ms53 Premier Typhoo Tesco

Malted drink 1 Ms54 Ovaltine Ovaltine Tesco1 Ms55 Sainsbury's Malted drink J Sainsbury1 Ms56 SKBeecham Horlicks original Tesco

56 total

Page 4

soups and sauces

Class Code Brand Description Retail OutletCanned 1 So01 Heinz Cream of Tomato Tesco

1 So02 Baxter's Mushroom potage with woodland mushrooms Tesco1 So03 Campbell's Consomee clear beef J Sainsbury1 So04 Sainsbury's Cream of Mushroom J Sainsbury1 So05 Tesco Cream of Chicken Soup Tesco

powder 1 So07 Batchelor's Cup-a-soup Cream of mushroom with croutons Tescocup 1 So09 Tesco Chicken and vegetable soup in a cup Tesco

1 So11 Tesco Minestrone Cup-a-soup Tesco1 So12 Sainsbury's Oxtail soup in a cup J Sainsbury1 So13 Safeway Roast chicken soup in a mug Safeway1 So16 Safeway Slim choice Minestrone soup in a mug Safeway

packet 1 So10 Knorr French onion Tesco1 So15 Batchelors Cream Of Garden Vegetable Soup Tesco1 So14 Asda Cream of chicken soup Asda

fresh 1 So17 New Covent Garden Fresh spicy corn chowder J Sainsbury1 So18 Sainsbury's Fresh soup winter vegetable J Sainsbury1 So19 Tesco Fresh beef and tomato soup Tesco1 So20 Asda Fresh soup minestrone Asda1 So08 Safeway Minestrone soup Safeway

sauces 1 So21 Heinz Tomato ketchup Tesco(table & 1 So22 HP HP sauce Tescocook-in) 1 So23 Crosse & Blackwell Branston sandwich pickle Tesco

1 So24 Lea and Perrin's Worcestershire sauce Tesco1 So25 Asda White wine and garlic with double cream Asda1 So26 Van den Berg Chicken Tonight creamy curry J Sainsbury1 So27 Homepride Barbecue cook-in-sauce J Sainsbury1 So28 Colman's Sauce for Cottage Pie with rich gravy J Sainsbury1 So29 Asda pasta sauce Asda1 So30 Discovery Creole Recipe Sauce J Sainsbury1 So31 Sharwood's Tikka Masala medium-hot J Sainsbury1 So32 Ragu Chunky onion and garlic sauce for bolognese J Sainsbury1 So33 Sainsbury's Mushroom Bolognese sauce J Sainsbury1 So35 Tesco Rogan josh with tomato onion and garkic Tesco1 So36 Safeway Italian Sauce (bolognese) for pasta Tomato with onions & garlic Safeway

total 34

Page 5

ANNEX IIa: LIST OF ADDITIONAL 50 SAMPLES

CEREALSClass code Brand Type Retail Outlet

Biscuits 1 C075 Sainsbury Economy Digestives J Sainsbury1 C076 Tesco Value Digestives Tesco1 C077 Asda Digestive Asda1 C078 Safeway Digestive Safeway1 C079 Sainsbury's Ginger Snaps J Sainsbury1 C080 Tesco Ginger Nuts Tesco1 C107 McVitie's 25% Reduced Fat Digestives J Sainsbury

Malted biscuits 1 C081 Asda Malted milk Asda1 C082 Co-op Malted milk Co-op

Savoury crackers 1 C083 Asda Cream Crackers 50% Less fat Asda1 C084 Safeway Cream Crackers Safeway 1 C085 Sainsbury's Lightly Salted Crispy Crackers J Sainsbury1 C086 Tesco 50% Lower Fat Cream Crackers Tesco 1 C087 Safeway Half Fat Cream Crackers Safeway1 C088 Tesco Cornish Wafers Tesco1 C089 Sainsbury's Cornish Wafers J Sainsbury

Toasted biscuits 1 C090 Tesco French Toast Tesco1 C091 Asda Melba Toast Asda1 C092 Asda Dutch Crispbakes Asda1 C093 Tesco Dutch Crispbakes Tesco1 C094 Rakusen's Traditional Matzos J Sainsbury1 C095 Morrisons Original Crispbread Morrisons1 C096 Morrisons Crispwheat Morrisons

Breads 1 C097 Tesco Granary Malted Brown Bread Tesco1 C098 Sainsbury's Storebaked Harvest Grain J Sainsbury1 C099 Thomas the Baker Wholemeal Thomas, Malton 1 C100 Woodhead The Bakers Wholemeal Woodhead The Bakers, Malton1 C101 Allinsons Hi-Bran J Sainsbury1 C102 Mothers Pride Scottish Plain Traditional White Bread Tesco1 C103 Asda Sliced wholemeal loaf Asda1 C104 Sainsbury's Sliced wholemeal loaf J Sainsbury1 C105 Warburtons Malted Danish Sliced Bread J Sainsbury1 C106 Morrisons Superlife Medium Sliced Wholemeal Morrisons

DAIRYClass code Brand Type Retail Outlet

Processed 1 Da32 Sainsbury's Cheddar Cheese Food Slices J Sainsburycheese 1 Da33 Asda Cheese Food Slices Asdaslices 1 Da34 Rowson Processed Cheese Food Slices Morrisons

Low fat 1 Da35 Safeway 16% Half Fat Mature Cheese Safewaycheeses/cheese

MEATSClass code Brand Type Retail Outlet

salami 1 Me69 Independent delicat'n Pepper salami Lawrie's, York1 Me70 Independent delicat'n Danish salami Derek Fox butchers 1 Me71 Independent delicat'n Salami The Bakery, Norton.

cooked meats 1 Me63 Sainsbury's Scottish Smoked Salmon J Sainsbury1 Me64 Tesco Scottish Smoked Salmon Tesco1 Me65 Tesco Finest salted Anchovies Tesco1 Me66 John West Flat Fillets of Anchovies in Olive Oil Morrisons1 Me67 Safeway Roast Chicken Hot 'n' Spicy Drumsticks Safeway1 Me68 Morrisons Chicken satay kebabs Morrisons

Cakes, 1 Ms57 Morrisons Store baked raspberry jam d'nuts Morrisonspastries 1 Ms58 Sainsbury's Store baked sugar doughnuts J Sainsburyfruit pies 1 Ms59 Safeway Store baked jam doughnuts Safeway

1 Ms60 Woodhead The Baker Store baked jam doughnuts Woodhead The Baker

total 50

Page 1

ANNEX III: COOKING PROCEDURES

Introduction

The following foods and drinks require some form of "cooking" (i.e. foods will be grilled,microwave cooked, oven baked, heated in a saucepan; powdered soups and drinks will bemade up with boiling water or hot milk, as appropriate):

Cereals• Crumpets

Meat• Chargrilled burgers (except McDonalds and Burger King samples)• Bacon

Miscellaneous• Pizzas (except Dominos and Pizza Hut samples)• Battered and breaded products (except KFC, McDonalds and fish take-away samples)• Tea infusions (made from tea bags)• Malted drinks

Soups and sauces• Apart from the table sauces, all of these samples (soups and cook-in-sauces) will be

cooked.

In general the manufacturers' cooking instructions will be followed as stated on the packets.However, if the food appears to be becoming overcooked, cooking will cease. In addition, ifthe food appears to be raw, then the samples will be cooked for longer than recommended.Oven-cooked samples will be turned as necessary to ensure even cooking. Checks will bemade to ensure that the grill positions used cook evenly (e.g. the grill pan will be covered withwhite bread which will be toasted to show hot or cold spots.) If the cooking is not even theproducts will be rotated on the grill during cooking. The grill temperature cannot be adjustedby the oven controls. Therefore, grilling will be carried out using a "medium" shelf position(second rack down, 7 cm from the grill element).

In order to avoid cross-contamination all cooking implements, baking trays, cups, grill pans,saucepans, spatulas and spoons will be cleaned between samples.

Detailed procedures

Cereals

Crumpets

Manufacturers' recommendations: Only one of the packets contains detailed instructions. Therecommendation is to use a pre-heated grill (moderate heat). Grill for 4 minutes, turninghalfway through grilling.

Method: The grill will be pre-heated for 5 minutes. The crumpets will be evenly spaced on thegrill at a "medium" shelf position, 7 cm from the grill, and toasted for the recommended timeuntil golden brown, turning half way.

Meats

Meats will be cooked by grilling in preference to frying, oven baking or microwave cooking(except when specified in manufacturers' instructions) because:

• Grilling is applicable to a wider range of samples;• The temperature is more easily controlled;• The heat is likely to be more evenly distributed;• Addition of fat or oil is avoided.

Chargrilled burgers

Manufacturers' recommendations: Cooking (grilling) instructions on 6 packets of frozenburgers suggest using a medium grill for between 10 and 25 minutes; 5 samples fall within therange 10 to 20 minutes and one states 25 minutes. Instructions on a microwave burger statethat the burger and bun should be cooked from frozen at 650W for 2 minutes and allowed tostand for a further 30 seconds.

Method: Samples will be cooked from frozen using an electric grill pre-heated for 10 minutes.The contents of a packet of burgers will be placed on a grill pan equidistant from each otherand the edge of the pan. The pan will be placed at a "medium" shelf position, 7 cm from thegrill. The burgers will be grilled for the recommended time and be turned over half waythrough this time. The cooking time will be varied as necessary according to whether theburgers appear to be becoming over-cooked or are under-cooked. As the samples are to bebulked and homogenised the effects of slight variations in cooking between individual burgerswill be minimised.

In the case of the microwave burger, both burgers from the packet will be cooked in the bun asdirected. Both the burger and bun will subsequently be homogenised and subsampled prior toanalysis to reflect the level of 3-MCPD in the product "as consumed".

Bacon

Manufacturers' recommendations: Only a few samples have cooking instructions, and theserecommend a medium grill for 4 to 5 minutes.

Method: A whole pack of each sample (approx. 200g) will be grilled in two batches by placinghalf of the rashers on a grill pan equidistant from each other and the edge of the pan. The grillwill be pre-heated for 10 minutes. The rashers will be grilled for 2 minutes on each side usinga "medium" shelf position, 7 cm from the grill. Again, if any rashers appear to be cookingfaster, then they will be rotated. The cooking time will be varied, if necessary, due to thethickness of the rashers and the samples will be monitored for signs of burning.

Miscellaneous

Pizzas

Manufacturers' recommendations: The recommendations are to preheat the oven (typically200-220°C) and to place the pizza onto the top oven shelf, with cooking times varying from 8to 20 minutes.

Method: Pizzas will be cooked singly as recommended (for frozen pizzas, without defrosting)in an electric oven pre-heated for 10 minutes. They will be placed directly on the oven shelf inthe stated position and rotated if one half is cooking more rapidly than the other.

Battered and breaded products

Manufacturers' recommendations: The recommendations are to preheat the oven (typically200-230°C ) and to place the sample on a baking tray, with most cooking times varying from12 to 25 minutes (4 minutes for Asda Yorkshire puddings).

Method: Frozen foods (chicken pieces, scampi, fish pieces, Yorkshire puddings) will becooked as directed on the packet in an electric oven pre-heated for 10 minutes. The fullcontents of a packet will be placed evenly on a baking tray in the stated position in the oven,and cooked for the recommended time. Each food will be cooked separately.

Fish fingers will be grilled as directed on the packet using an electric grill pre-heated for 10minutes. The full contents of a packet will be placed evenly on a baking tray in the statedposition on the grill and cooked for the recommended time.

Tea infusions

The method for preparing tea infusions (see below) is based on information provided by the teaindustry,13 and was previously adopted for the compositional analysis of a "typical" consumerbrew.14 It is being used in preference to the manufacturer’s recommendations. (NB: Thewater will be checked to ensure it does not contain a detectable level of 3-MCPD.)

1. Place a tea bag in a 250 ml ceramic cup and add 235 ml of boiling water.2. After 15 seconds, stir 3 times.3. Leave to infuse for a further 20 seconds, remove the bag from cup squeezing on the side

for before complete removal.4. Prepare a second infusion using steps 1-3 and combine both infusions.

Malted drinks

Manufacturers' recommendations: The recommendations are to mix 3-4 teaspoons (25 g) ofmalted drink granules with around 200 ml of hot milk (not boiling) and to stir constantly untilthe granules have dissolved.

Method: Malted drinks will be prepared as directed on the packet using fresh semi-skimmedmilk (previously found to be free of 3-MCPD). Approximately two cups will be made

separately for each sample and then combined. At least one unopened jar will be kept shouldthe powder need to be analysed.

Soups and sauces

Canned and fresh soups

Manufacturers' recommendations: The manufacturers' conventional cooking technique (i.e.cooking on the hob) for canned and fresh soups recommends heating gently with continuousstirring. Three samples are instructed to be cooked for 3 to 4 minutes whilst two samples areto be cooked for 5 to 6 minutes. The remaining samples have no cooking times given.Samples should not be allowed to boil.

Method: A whole can of soup will be poured into a medium saucepan and warmed on anelectric hotplate (pre-heated for 5 minutes) at a medium setting, with constant stirring for 4minutes. Samples will be removed from the heat if they appear to begin to boil.

Cup-a-soups

Manufacturers' recommendations: The manufacturers' recommendations are for soup powdersto be emptied into a mug and boiling water added with constant stirring until dissolved. All thesoup powders recommend adding 200 ml of boiling water, except one sample (190 ml).

Method: One sachet of sample will be placed in a ceramic cup and the label-stated volume offreshly boiled water added and stirred with a teaspoon for 30 seconds. In order to have around500 g of sample, two sachets will be made up independently and then mixed together.

Other packet soups

Manufacturers' recommendations: Manufacturers recommend emptying the contents of thepacket into a saucepan of water, bringing to the boil on the cooking hob followed bysimmering for 5 minutes with occasional stirring.

Method: Samples will be placed into a medium saucepan and the label-stated volume of coldwater added (typically 900 ml). The soups will be heated on a pre-heated electric hotplateuntil boiling and then simmered for 5 minutes as stated on the packet, with stirring every 30seconds.

Cook-in sauces

Manufacturers' recommendations: These sauces are generally used to complement meat disheswhereby the meat is lightly cooked until brown and the sauce added and subsequently cooked.Typical cooking times for these sauces on the hob range from 20 to 30 minutes.

Method: Sauces will be poured into a medium saucepan and simmered on a pre-heated electrichotplate at a low setting with stirring for 30 seconds every 5 minutes. Cooking will be for thetime specified on the packet. No meat or other ingredients mentioned in the manufacturers'recipe will be added to the sauce.

ANNEX IV: CSL WORKPLAN

The method used will be that validated by CSL on behalf of MAFF. For foods and ingredientsnot analysed during the validation project, minor variations developed as part of CSL’scommercial and R&D work will be applied, if necessary (see Annex V for UKAS-accreditedmethod).

Analytical batch protocol

A total of 250 samples will be analysed for 3-MCPD. The samples have been divided into 5classes:

• Cereals - Sub-classes: biscuits (including malted and toasted products); bread; breakfastcereals; corn snacks; and savoury crackers (73 products)

• Dairy - Sub-classes: creams (aerosols and dessert toppings); low-fat cheeses; parmesancheeses; and processed cheeses and cheese alternatives (31 products)

• Meats - Sub-classes: chargrilled burgers; cooked meats; salami; and smoked bacon (56products)

• Miscellaneous - Sub-classes: battered and breaded products; cakes, pastries and fruit pies;confectionery; pizzas; pork pies; and various drinks (beer, tea and malted drinks) (56products)

• Soups and sauces - Sub-classes: canned, fresh and powdered soups and sauces (table andcook-in-sauces) (34 products)

Food samples will be analysed in classes defined in the purchasing plan. The extractionprocedure required will be as described in the validated method (Annex V).

The classes and extraction procedures are:

Class Sample Procedure as forCereals all samples cerealsDairy cheeses meats/cheeseDairy creams oils and fatsMeats all samples meats/cheeseMiscellaneous beer, tea, malted drinks soupsMiscellaneous others meats/cheeseSoups & sauces soups soupsSoups & sauces sauces soups/meats as appropriate

Analysis

Reference materials

Each laboratory extraction batch will comprise:

• Between 10 and 20 food samples.

• A cross-class reference material to be carried through the entire project. This will be a soysauce characterised in-house and containing 0.030 mg/kg 3-MCPD.

• A food-class reference material appropriate to the class of sample. The reference materialsfor the 5 food classes will be:

• Cereals: Breadcrumbs containing 0.030 mg/kg 3-MCPD characterised in themethod validation trial.

• Dairy: Cheese alternative containing 0.035 mg/kg 3-MCPD characterised underan EC scientific co-operation (SCOOP) project being co-ordinated by the UK.

• Meats: Salami containing 0.025 mg/kg 3-MCPD characterised in-house.• Miscellaneous (battered/breaded products, cakes/pies, pizzas and confectionery):

Salami as above.• Miscellaneous (beer, malted drinks and tea infusions): Aqueous solution

containing 0.01 mg/kg 3-MCPD to be characterised in-house.• Soups and sauces: Soup containing 0.020 mg/kg 3-MCPD to be characterised in-

house.

Each batch of samples will include the analyses of a deuterated 3-MCPD internal standard (IS)and two reference materials (a cross-class reference and a food-class reference). The referencematerials will identify any "drift" in the analytical results, while the deuterated IS willautomatically compensate for the recovery of 3-MCPD. Comparing the IS with an externalstandard will allow recovery data to be calculated.

Reporting of results

Results will be expressed on a wet (as is) basis. The units will be mg/kg with a reporting limitof 0.01 mg/kg.

IQC criteria

• Reagent blanks must not contain 3-MCPD at a detectable level (0.005 mg/kg). If the blankvalue exceeds 0.005 mg/kg the batch will be repeated.

• Results for the food-class and cross-class reference materials must fall within 2 standarddeviations of the mean value determined by the characterisation procedure (if necessaryupdated with additional data obtained during the project). Where the result for the food-class reference material exceeds 2 standard deviations, the batch will be repeated. Wherethe result for the cross-class reference material exceeds 2 but is lower than 3 standarddeviations, the batch will be considered acceptable if the result for the food-class referencematerial is satisfactory.

Determination of 3-monochloropropanediol in food and food ingredients using

mass spectrometric detection.

1. Scope

This method is suitable for the determination of 3-monochloropropanediol (3-MCPD)

in hydrolysed vegetable protein (HVP), soups and stocks, stock cubes, malt extract,

salami, fish, cheese, flour, starch, cereals and bread using mass spectrometric

detection. The method uses d5-3-monochloropropanediol (d5-3-MCPD) as an internal

standard. The concentration of 3-MCPD is expressed as mg/kg.

2. Normative References

ISO 3696:1987 Water for analytical use - Specifications and test methods.

3. Principle

To extract 3-MCPD, internal standard is added, followed by saline solution,

and the mixture is blended to a homogeneous consistency. After sonication,

the contents of an Extrelut™ refill pack are added and mixed thoroughly. The

sample plus Extrelut™ are transferred to a glass chromatography column and

the non-polar components are eluted using a mixture of hexane and diethyl

ether. The 3-MCPD is eluted using diethyl ether and the sample extract

concentrated to a small volume. A portion of the concentrated sample extract

is then taken for derivatisation and analysis.

4. Reagents and Materials

During the analysis, unless otherwise stated, use only reagents of recognised

analytical grade and water of at least grade 3 as defined in ISO 3696.

Heptafluorobutyrylimidazole (HFBI) should be stored frozen.

4.1 3-Monochloropropanediol (CAS 96-24-2)

4.2 d5-3-Monochloropropanediol

4.3 Sodium sulphate, anhydrous (CAS 7757-82-6)

4.4 Heptafluorobutyrylimidazole (CAS 32477-35-3)

4.5 Extrelut™ 20 refill packs

4.6 Ethyl acetate (CAS 141-78-6)

4.7 2,2,4-Trimethylpentane (CAS 540-84-1)

4.8 Hexane + Diethyl ether (90 + 10)

To prepare 1 l of hexane + diethyl ether (90 + 10), add 100 ml of diethyl ether

to 900 ml hexane and mix well.

4.9 5 mol/l sodium chloride solution

To prepare 5 mol/l sodium chloride solution, weigh out 290 g of sodium

chloride and dissolve in 1 litre of water.

4.10 Preparation of Standard Solutions

4.10.1 3-MCPD stock solution - A

Weigh approximately 25 mg 3-MCPD (4.1) and make up to 25 ml in a

volumetric flask with ethyl acetate (4.6) to give a solution (A) of

approximately 1000 µg/ml.

4.10.2 Intermediate 3-MCPD standard solution - B

Prepare 10 ml intermediate solution (B) by dilution of solution A (4.10.1) to

exactly 100 µg/ml in ethyl acetate (4.6).

4.10.3 3-MCPD spiking solution - C

Pipette 2 ml of intermediate standard solution B (4.10.2) into a 100 ml

volumetric flask and dilute to volume with ethyl acetate (4.6) to give a solution

containing 2 µg/ml (C).

4.10.4 d5-3-MCPD internal standard stock solution - D

Weigh approximately 25 mg d5-3-MCPD (4.2) and make up to 25 ml in a

volumetric flask with ethyl acetate (4.6) to give a solution (D) of

approximately 1000 µg/ml.

4.10.5 d5-3-MCPD working internal standard solution - E

Prepare 100 ml working internal standard solution (E) by dilution of internal

standard stock solution D (4.10.4) to exactly 10 µg/ml in ethyl acetate (4.6).

4.10.6 3-MCPD calibration solutions

Dispense volumes of the 100 µg/ml solution (B) into 25 ml volumetric flasks

and make up to volume with 2,2,4-trimethylpentane (4.7) to give 0.00, 0.05,

0.10, 0.50, 1.00 and 2.00 µg/ml 3-MCPD.

NOTE 1: By taking 0.1 ml volumes of the calibration solutions to the

derivatisation vials the standards need not be subjected to evaporation. The

table above shows the weight of 3-MCPD present in the vial when derivatised.

5. Apparatus

5.1 Mass spectrometer with gas chromatograph

The following conditions have been found to be suitable:

Injector/ transfer line temperatures 270 °C.

Programme: 50 °C for 1 minute, ramp at 2 °C/minute to 90 °C, ramp to baking

temperature 270 °C, hold for 10 minutes. Helium carrier at 1 ml/minute.

Make splitless injections of 1.5 µl with a 40 second splitless period.

Monitor the following ions in selected ion monitoring mode:

m/z453 3-MCPD291 3-MCPD289 3-MCPD275 3-MCPD253 3-MCPD257 d5-3-MCPD

NOTE 2: High-performance mass spectrometers, including modern ion-trap

instruments, may be operated in full scan mode. In this case peak area

measurements must be made on reconstructed chromatograms of the ions

shown above.

The 3-MCPD peak will be eluted at 10 to 15 minutes. An isomeric

chloropropanol, 2-MCPD, eluting just after 3-MCPD (about 1 peak width

between the two) may be seen in some samples. 2-MCPD has a spectrum

almost identical to 3-MCPD but is lacking the ion at m/z 453.

5.2 GC column, 30 m x 0.25 mm, 0.25 µm film thickness, J & W DB5MS or

equivalent

5.3 Ultrasonic bath

5.4 Glass chromatography column 40 cm length x 2 cm i.d., with sintered glass

disc and tap

5.5 Filter paper (Whatman no. 4 or equivalent)

5.6 Syringe, gas-tight, 1 ml

5.7 Analytical balance, capable of weighing to 4 decimal places

5.8 Centrifuge, capable of attaining 3500 rpm

6. Laboratory Samples

On receipt, samples are given a unique sample number. Perishable samples are

stored at -20 °C. All other samples are stored at room temperature prior to

analysis.

7. Procedure

7.1 Laboratory sample

Dry samples such as stock cubes and cereals should be ground to a fine

consistency. Bread, cheese, salami and fish samples should be minced or

grated to a homogeneous mixture. All samples should be stored in air-tight

containers, frozen if necessary. All samples should be mixed thoroughly before

analysis.

7.2 Test sample

7.2.1 For HVP samples

7.2.1.1 Thoroughly mix the sample. Weigh, to the nearest 0.01 g, an 8 g portion. Add

100 µl of the 10 µg/ml d5-3-MCPD internal standard solution E (4.10.5).

7.2.1.2 Add 5 mol/l sodium chloride solution (4.9) to give a final weight of 20 g (HVP

+ salt solution) and blend to a homogeneous mixture using a spatula, crushing

all small lumps.

7.2.1.3 Place in an ultrasonic bath (5.3) for 10 minutes.

7.2.1.4 Proceed to preparation of test portion (7.3).

7.2.2 For soup powders and malt extract samples

7.2.2.1 Thoroughly mix the sample. Weigh to the nearest 0.01 g a 10 g portion of

malt extract or a 5 g portion of soup powder. Add 100 µl of the 10 µg/ml

d5-3-MCPD internal standard solution E (4.10.5).

7.2.2.2 Add 5 mol/l sodium chloride solution (4.9) to give a final weight of 20 g

(sample + salt solution) and blend to a homogeneous mixture using a spatula,

crushing all small lumps.

7.2.2.3 Place in an ultrasonic bath (5.3) for 15 minutes.

7.2.2.4 Proceed to preparation of test portion (7.3).

7.2.3 For salami and cheese samples

7.2.3.1 Thoroughly mix the sample. Weigh, to the nearest 0.01 g, a 20 g portion.

Add 100 µl of the 10 µg/ml d5-3-MCPD internal standard solution E (4.10.5).

7.2.3.2 Add 5 mol/l sodium chloride solution (4.9) to give a final weight of 70 g

(sample + salt solution) and blend to a homogeneous mixture. If necessary to

enable blending add a further 10 g of sodium chloride solution (4.9) solution.

7.2.3.3 Transfer the entire sample to a centrifuge tube and centrifuge at 3500 rpm for

20 minutes.

7.2.3.4 Pour the supernatant layer into a beaker, avoiding the transfer of solid material

and visible fat.

7.2.3.5 Weigh a 20g portion of the supernatant into a 250 ml beaker. Proceed to

preparation of test portion (7.3.2).

7.2.4 For flour, starch, cereals and bread samples

7.2.4.1 Thoroughly mix the sample. Weigh to the nearest 0.01 g a 10 g portion. Add

100 µl of the 10 µg/ml d5-3-MCPD internal standard solution E (4.10.5).

7.2.4.2 Add 5 mol/l sodium chloride solution (4.9) to give a final weight of 40 g

(sample + salt solution) and blend to a homogeneous mixture using a spatula,

crushing all small lumps.

7.2.4.3 Place in an ultrasonic bath (5.3) for 15 minutes.

7.2.4.4 Cover and leave to soak overnight.

7.2.4.5 Proceed to preparation of test portion (7.3.2).

7.3 Test portion

7.3.1 Take 20 g of the prepared test sample (7.2).

7.3.2 Add the contents of an Extrelut™ refill pack (4.5) to the sample and mix

thoroughly with the spatula. Check the bottom of the beaker to ensure

thorough mixing. Add the mixture to the chromatography column (5.4),

vibrate briefly to compact the contents, top with a 1 cm layer of sodium

sulphate (4.3) and leave for 15 to 20 minutes.

7.3.3 Elute the non-polar components with 80 ml hexane + diethyl ether (90 + 10)

(4.8.1). Allow unrestricted flow except for soup powder samples where the

flow should be restricted to about 8 to 10 ml per minute (this is a rapid stream

of drops with visible solvent movement in the column). Close the tap when the

solvent reaches the sodium sulphate layer. Discard the collected solvent.

7.3.4 Elute the 3-MCPD with 250 ml diethyl ether (4.8) at a flow rate of about 8 ml

per minute. This requires a steady and rapid stream of separate drops with

solvent movement in the column just apparent. Collect 250 ml of eluate in a

250 ml volumetric flask.

7.3.5 Add approximately 15 g sodium sulphate (4.3) to the flask and leave for 10 to

15 minutes only.

7.3.6 Filter the eluate into a 250 ml round-bottomed or pear-shaped flask through a

filter paper (5.5).

7.3.7 Concentrate the extract to about 5 ml by rotary evaporation at 35 °C. Do not

allow to dry. Quantitatively transfer the concentrated extract to a 10 ml

volumetric flask with diethyl ether (4.8). Make up to volume with diethyl ether

(4.8).

7.3.8 Add a small quantity of sodium sulphate (4.3) (spatula tip quantity) to the

flask, shake well and leave for 5 to 10 minutes only.

7.3.9 Using a 1 ml gas-tight syringe (5.6) transfer 1 ml of the sample to a 4 ml vial.

7.3.10 Evaporate the solution just to dryness at room temperature under a stream of

nitrogen.

7.3.11 Immediately add 1.0 ml 2,2,4-trimethylpentane (4.7).

7.3.12 Add 0.05 ml heptafluorobutyrylimidazole (4.4), at room temperature, and

immediately seal with a phenolic cap.

7.3.13 Shake using a vortex mixer for a few seconds and heat at 70 °C for 20 minutes.

7.3.14 Allow the mixture to cool at room temperature and add 1 ml distilled water.

7.3.15 Shake on a vortex mixer for 30 seconds, allow the phases to separate then

repeat shaking.

7.3.16 Place a small quantity (spatula tip full) of sodium sulphate (4.3) in a 2 ml vial.

Remove the 2,2,4-trimethylpentane phase to this vial, shake, and stand for 2 to

5 minutes.

7.3.17 Transfer the solution to a new 2 ml vial for GC-MS.

7.4 Blank test

Follow the above procedure for preparation of test portion (7.3.1 - 7.3.19)

substituting 5 mol/l sodium chloride (4.9) solution for the test sample (7.2).

Add 100 µl of the 10 µg/ml d5-3-MCPD internal standard solution E (4.10.5)

at stage 7.3.1

7.5 Calibration standards

To a set of 4 ml vials transfer 0.1 ml of each of the calibration solutions

(4.10.6),10 µl of d5-3-MCPD 10 µg/ml E (4.10.5) and 0.9 ml 2,2,4-

trimethylpentane (4.7). Proceed as in steps 7.3.14 to 7.3.19 above.

7.6 Determination

Standards and samples are to be injected once using the conditions outlined in

5.1. The standards should be injected at random intervals throughout the

analytical batch.

8. Calculation

8.1 Method

8.1.1 Measure the areas of the d5-3-MCPD (m/z 257) and 3-MCPD peaks.

8.1.2 Calculate the ratio of the 3-MCPD (m/z 253) to d5-3-MCPD (m/z 257) peak

areas.

8.1.3 Construct a calibration graph for the standards by plotting the peak area ratio

against the weight in micrograms of 3-MCPD in the vial.

8.1.4 Calculate the slope of the calibration line.

8.1.5 Divide the peak area ratio of the unknowns by the value of the slope to give

the weight of 3-MCPD per vial for the unknown samples.

8.1.6 To correct for the lower quantity of internal standard added to the calibration

standards multiply the weight obtained in (8.1.5) x 10. Divide the weight of

3-MCPD (micrograms) in the vial by the weight of sample (grams) to give the

value in mg/kg as required.

8.2 Final presentation of results

Results are expressed as mg/kg and are quoted to 3 decimal places.

9. Quality Assurance and Control

9.1 Batch protocol

Include a reagent blank in all batches.

9.2 Confirmation of peak identity.

9.2.1 Measure the ratio of the responses at m/z 291, 289, 275, and 253 relative to

the response at m/z 453 for the standards and samples.

9.2.2 At least two of the four ion abundance ratios should be ±20 % of the mean of

the ion abundance ratios of the standards.

9.2.3 Ensure that a minimum library fit of 800 is achieved for a background

subtracted spectrum.

9.2.4 If the signal of the 453 ion is weak, ensure that there is adequate separation of

2-MCPD and 3-MCPD.