survey of 3-mcpd in foods - Food Standards Australia New Zealand

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3-MCPD is one of a group of chemical contaminants known as ... 3-MCPD levels in acid-HVP are now significantly lower than those originally found in the 1980s ...
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 carrying out a survey to investigate the levels of 3-monochloropropane-1,2-diol (3-MCPD) in selected foods and beverages. The following paper presents the background to, and the aims of, this survey, as well as details of the methodology being followed. A full list of the samples included in the survey is also provided. Background 3-MCPD is one of a group of chemical contaminants known as chloropropanols. It was 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 as soups, prepared meals, savoury snacks, gravy mixes and bouillon cubes. Although 3-MCPD levels in acid-HVP are now significantly lower than those originally found in the 1980s,1-3 subsequent work by both the JFSSG and the UK food industry has indicated4,5 that the contaminant can also occur in several other foods and ingredients as a result of processing, storage conditions or migration from certain food contact materials. Studies have also shown6 that elevated levels of 3-MCPD can occur in some types 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 given in Annex I. Studies have shown that 3-MCPD is carcinogenic in rats when given in high doses over 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 the SCF's advice, the UK Food Advisory Committee (FAC) has recommended8 that industry should take steps to ensure that 3-MCPD is undetectable (i.e. less than 0.01 mg/kg) in foods and, where technologically feasible, in food ingredients as well. Discussions are currently underway on a proposal from the European Commission which, for the first time, sets EU-wide limits for 3-MCPD in certain foods. As currently drafted, the proposal sets a maximum limit of 0.01 mg/kg for "products containing acid-HVP" and "soy sauce". However, limits may eventually be set for a wider 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’s recommended 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 consumer exposure 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 the Guidelines for Planning and Reporting JFSSG Surveys.9 Further details are given below. Sampling strategy The survey has been targeted at those products which either have been shown to contain 3-MCPD or which it has been suggested may contain the contaminant as a result of processing or storage conditions. These are outlined below: • foods such as salami, some cheeses (parmesan, processed cheeses and cheese alternatives) and some cereal products subjected to high temperature processing which 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 and lecithin) which could theoretically act as precursors to 3-MCPD; • products containing malt and/or malt extracts, some of these ingredients having previously 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 is due 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 cookin-sauces) (34 products) By including such a wide range of food commodities, it has not been possible to cover the whole market for each one. Instead, for those commodities where 3-MCPD is more likely to be found, a larger number of samples has been allocated. Where the results for any commodity subsequently indicate that 3-MCPD is found at elevated levels in several of the products sampled, further samples will be taken to increase the coverage of the market. For each commodity, a range of samples has been allocated, including brand leaders, own-brand products and, where appropriate, take-away products. An attempt has been made to reflect market share, where possible, using information from recent issues of the publications, Retail Business and Consumer Goods UK.10,11 In the case of own-brand products, these have been taken mainly from Tesco, Asda, Sainsbury, and Safeway, the leading supermarket chains in the UK, with the distribution of products broadly reflecting the relative market share of these four retailers.12 A full list of the samples included in the survey (with brand details) is given in Annex II. Sampling has been undertaken by MAFF Central Science Laboratory (CSL), with the samples being purchased between November 1999 and January 2000 and the majority being bought from supermarket and other retail outlets (e.g. independent butchers and take-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 the level of 3-MCPD in foods. In addition, it has been assumed that branded products do not vary with retailer and thus, for ease of sample collection, branded products have been purchased only from the four main supermarkets. For most products, a minimum sample size of 200 g (or one pack) has been purchased, while for products which require 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 any contamination of the sample and must keep records to show that these procedures have been 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 a photograph/photocopy reference for all samples. Samples have been stored as recommended by the manufacturer or frozen if stability is in question. For the majority of products, each sample will be homogenised and divided into three subsamples. One of these subsamples will be used for analysis and the others stored appropriately. Of the two stored subsamples, one will be available for analysis by the company or retailer if required. In the case of products which require some form of cooking or heating, at least one additional unopened pack (with the same batch or sell-by date) will be stored appropriately and will be available for use should an analysis of the "raw" sample subsequently be required. "Cooked" products will be homogenised (using a kitchen blender) 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 be analysed "as consumed" to enable a more accurate estimate of dietary exposure to be obtained. As a consequence, the following products will require some form of further preparation: • • • • • • • • •

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 a saucepan. Powdered soups and drinks will be made up with boiling water or hot milk, as appropriate. Detailed procedures for preparing these foods, based mainly on manufacturers' instructions, have been drawn up in association with CSL and are attached at Annex III. Analysis A detailed workplan, which sets out the analytical batch protocol and the Internal Quality Control (IQC) procedures to be used by CSL during this survey, is attached at Annex IV. The UKAS-accredited method used for the analysis of 3-MCPD is attached at Annex V.

Reporting of results It is intended that the results will be reported in the Food Safety Information Bulletin and in an accompanying Food Surveillance Information Sheet in early Autumn, although publication may be delayed if it proves necessary to take further samples of any commodity. The FAC will also have the opportunity to comment on the results as part 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 samples analysed will be included in the Food Surveillance Information Sheet. Companies or retailers whose products have yielded unusual or unexpected results or results which give rise for concern, but where there is no immediate safety issue, will be sent their own results approximately seven weeks prior to the publication of the Bulletin article and invited to make a brief statement. The statement will be included in the Food Surveillance Information Sheet. The Guidelines9 currently state that all results quoted should be the "best estimate value" ± 2 standard deviations. While the results of most JFSSG surveys are reported in this way, the results of surveys carried out by the Working Party on Food Authenticity 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 by the FAC at its meeting on 23 March. How the results of the 3-MCPD survey are subsequently 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 protein 1992. CSL Report FD 93/17. 3. JFSSG (1999). Survey of 3-monochloropropane-1,2-diol (3-MCPD) in acidhydrolysed vegetable protein. Food Surveillance Information Sheet No. 181. 4. JFSSG (1999). Survey of 3-monochloropropane-1,2-diol (3-MCPD) in soy sauce and similar products. Food Surveillance Information Sheet No. 187. 5. Food Advisory Committee (1999). Further update on 3-MCPD in foods and food ingredients (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 Committee for Food. Food Science and Techniques, Thirty-sixth Series. Office for Official Publications of the European Community, Luxembourg. 8. Food Advisory Committee (1999). Recent developments on 3-MCPD in food and food 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 Intelligence on Retailing, London. 11. Consumer Goods UK (various editions, 1998 onwards). Corporate Intelligence Group, 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/black tea and other methylxanthine containing beverages. Abstract Paper Am. Chem. 217: U49 Part 1.

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

HVP Since the 1980s, it has been recognised that the procedure used to manufacture the savoury food ingredient acid-HVP can result in the formation of 3-MCPD. Most acid-HVP is produced using strong hydrochloric acid, and 3-MCPD is formed as a result of the high temperature chlorination of lipids present in the protein starting materials. Surveys carried out by MAFF in 19901 and 19922 showed that levels of up to 100 mg/kg were quite common in acid-HVP at that time. Since then, however, levels in HVP used in the UK have declined markedly, as was shown by the results of a recent JFSSG survey of acid-HVP3 in which 3-MCPD was undetectable (less than 0.01 mg/kg) or was found at low levels in over 75% of the 50 samples analysed. Roasted cereals, dark malts and dark malt extracts Information from the UK brewing and malting industries indicates that 3-MCPD levels of up to 0.3-0.4 mg/kg can occur in roasted cereals and dark speciality malts which are used to add colour and flavour to most dark beers and some lagers. Extracts derived from these ingredients, which are used to flavour certain foods and drinks, may also contain 3-MCPD levels of over 0.1 mg/kg. Despite extensive work by these industries to reduce the levels of 3-MCPD in these ingredients, they have been unable to make significant reductions without adversely affecting their flavour characteristics. However, due to the low levels of use of these ingredients, the concentrations of 3-MCPD in the final product are below 0.01 mg/kg. Fermented sausages Certain types of fermented sausage such as salami have also been shown to contain levels of 3-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 casings industry is carrying out work at European level to determine the contribution of the casings to the 3-MCPD content of salami and, like other users of epichlorohydrin-based wet strength resins (see below), has already started to use higher grade resins with much lower levels of 3-MCPD. Soy sauces Following the finding of high levels of 3-MCPD in Chinese soy sauce (6-124 mg/kg) in several Member States, in September 1999, the JFSSG carried out a survey of 40 samples of soy sauce and similar products available on the UK market.4 The results showed that around two-thirds of the samples contained 3-MCPD at levels very close to or below the FAC's recommended limit of 0.01 mg/kg. However, the remaining one third of the samples contained levels above 0.01 mg/kg, the highest being 30 mg/kg. These products were imported from China and Hong Kong, Taiwan and the Philippines, indicating that higher levels are not restricted to any one country of origin. Several grades of soy sauce are manufactured in the Far East, including the traditional fermented product as well as lower grades which can contain acid-HVP or which may be produced using an acid treatment. It is known that such acid treatments can generate very large amounts of 3-MCPD. They can also occur during the manufacture of acid-HVP. While many 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 in other parts of the world may not yet have made these changes. Domestic cooking and formation of 3-MCPD in food In 1998, the JFSSG funded a study of the effect of cooking on 3-MCPD levels in foods. The results of this work6 show that elevated levels of 3-MCPD can occur in toasted bread (0.020.32 mg/kg), with slightly lower concentrations in some grilled cheeses (0.08-0.12 mg/kg) and fried batters (0.07-0.09 mg/kg). In contrast, 3-MCPD is undetectable or present only at very low levels in cooked meat, gravy, sauces and stocks. Food contact materials Information from the packaging industry and others indicates that very low levels of 3-MCPD may migrate into food and beverages due to its presence in certain types of epichlorohydrin-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 to develop higher grade "third generation" resins which have significantly lower levels of 3-MCPD, and these are increasingly being used in the above applications. With the increase in the availability and use of these resins, 3-MCPD exposure from this source will continue to decrease.

ANNEX II: LIST OF SAMPLES cereals Class Biscuits

1 1 1 1 1

code C01 C02 C03 C04 C05

Brand McVitie's McVitie's McVitie's Fox's Burton's

Digestive Rich Tea Ginger Nuts Crunch Creams Jammie Dodgers

Type

Retail Outlet J Sainsbury J Sainsbury J Sainsbury Tesco Tesco

Malted biscuits

1 1 1 1 1

C06 C23 C08 C09 C10

Tesco Safeway Sainsbury's Sainsbury's Crawford's

Malted milk Malted milk Morning coffee Malted milk Thin Arrowroot

Tesco Safeway J Sainsbury J Sainsbury J Sainsbury

Savoury crackers

1 1 1 1 1 1 1 1 1 1

C11 C12 C13 C14 C15 C16 C17 C18 C19 C20

McVitie's Jacob's Jacob's McVitie's Jacob's McVitie's Jacob's Tesco Sainsbury's Asda

Mini Cheddars original Twiglets original Cream Crackers Krackawheat Ritz Tuc Cornish Wafers Cream Crackers Cream Crackers 50% less fat cream crackers

J Sainsbury J Sainsbury J Sainsbury J Sainsbury J Sainsbury J Sainsbury Tesco Tesco J Sainsbury Asda

Toasted biscuits

1 1 1 1 1 1 1 1 1 1

C21 C22 C24 C25 C26 C27 C28 C29 C30 C33

Sainsbury's BN garlic toast Tesco Jacob's Jacob's Pogen Ryvita Jacquet Asda Safeway

Les Mignons French onion flavour nibbles Bite size Garlic Toasties baked Melba toast Continental Pecan Toast Continental Raisin Toast Krisprolls Original Rye Crispbread Toast Francais French toast Dutch crispbakes

J Sainsbury Tesco Tesco Tesco Tesco Tesco Tesco J Sainsbury Asda Safeway

Corn snacks

1 1 1 1 1 1 1 1 1 1

C31 C32 C34 C35 C36 C37 C38 C39 C40 C74

Sainsbury's M&S Tesco Golden Wonder Walker's Doritos cheese KP Skips Golden Wonder Phileas Fogg Safeway

Original Tortillas Golden Cheese Tasters Rico's Corn Snacks Tangy Cheese flavour Nice 'n' Spicy flavour Nik Naks Monster Munch pickled onion Corn Chips Movie bag tangy cheese flavour Prawn cocktail flavour Skips Cheesy flavour Wotsits Authentic Tortilla Chips Onion rings

J Sainsbury M&S Tesco Tesco Tesco Tesco Tesco Tesco Tesco Safeway

Breads

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

C41 C42 C43 C44 C45 C46 C47 C48 C49 C50 C51 C52 C53 C54 C55 C56 C57 C58 C59 C60

Safeway Tesco M&S Allinson Kingsmill Hovis Hovis Warburton Kingsmill Mighty White Mother's Pride Sainsbury's Safeway Asda Sainsbury's Tesco Soreen Sainsbury's Tesco Safeway

Bakery wholemeal loaf Wholemeal medium sliced loaf Wholemeal medium sliced loaf Wholemeal thick cut loaf Country Gold loaf Wholemeal thick sliced loaf Granary thick sliced loaf Wholemeal toastie sliced loaf Square thick sliced white loaf Medium sliced softgrain loaf Medium sliced Scottish plain white loaf Crumpets Crumpet Crumpet Fruit malt loaf Medium sliced malted brown loaf Fruity malt loaf Ciabatta loaf Plain Naan bread Wholemeal Pitta bread

Safeway Tesco M&S J Sainsbury J Sainsbury J Sainsbury J Sainsbury J Sainsbury J Sainsbury J Sainsbury Tesco J Sainsbury Safeway Asda J Sainsbury Tesco J Sainsbury J Sainsbury Tesco Safeway

Breakfast cereals

1 1 1 1 1 1 1 1 1 1 1 1 1

C61 C62 C63 C64 C65 C66 C67 C68 C69 C70 C71 C72 C73

Kelloggs Kelloggs Kelloggs Kelloggs Weetabix Weetabix Nestle Quaker Sainsbury's Weetabix Nestle Kelloggs Tesco

Cornflakes Bran Flakes Rice Krispies All-Bran Plus Weetabix Alpen Shredded Wheat Sugar Puffs Cornflakes Ready Brek oats Cheerios Frosties Pingu Crisp shaped cereal

J Sainsbury J Sainsbury J Sainsbury J Sainsbury J Sainsbury J Sainsbury J Sainsbury Tesco J Sainsbury Tesco Tesco J Sainsbury Tesco

total

73

Page 1

1 1 1

dairy

Class

code Brand

Description

Retail Outlet

Processed 1 Da01 Dairylea cheese 1 Da02 Tesco spreads 1 Da03 Primula 1 Da04 Laughing Cow

Big Cheese Spread Smooth and Creamy medium fat light soft cheese cheese spread triangles

J Sainsbury Tesco Tesco Tesco

Processed 1 Da05 Safeway cheese 1 Da06 Tesco slices 1 Da07 Kraft Dairylea 1 Da08 Kraft Singles 1 Da09 Asda 1 Da10 Golden Vale 1 Da11 Kraft Dairylea

Reduced Fat Cheese Singles Singles Dairylea thick slices Singles 50% less fat Singles Cheese Strings Strip Cheese original

Safeway Tesco Tesco Tesco Asda Tesco Asda

parmesan

1 1 1 1 1 1

Da13 Da14 Da15 Da16 Da17 Da12

Sainsbury's Buitoni Tesco Asda Castelli Safeway

Grated Parmesan medium fat hard cheese Grated Parmesan cheese Fresh grated Parmesan Parmigiano Reggiano Fresh grated Parmesan Fresh shavings Grated Italian medium fat hard cheese

J Sainsbury Tesco Tesco Asda Tesco Safeway

Low fat cheeses/ cheese alternatives

1 1 1 1 1 1 1 1 1

Da18 Da19 Da20 Da21 Da22 Da23 Da24 Da25 Da26

Tesco Asda Sainsbury's Safeway Asda Kraft Dairylea Flora Heinz Kraft

Fresh grated half fat full flavoured cheese Half fat mild cheese Be good to yourself Mature 16% fat cheese Medium half fat cheese Half fat mild grated cheese Dairylea light half fat cheese spread Medium mature real alternative to cheddar Weight Watchers 95% fat free low fat alternative to Red Leicester Philadelphia Light medium fat soft cheese

Tesco Asda J Sainsbury Safeway Asda J Sainsbury J Sainsbury J Sainsbury J Sainsbury

Creams (aerosols/ topping)

1 1 1 1 1

Da27 Da28 Da29 Da30 Da31

Anchor Anchor Elmlea Nestle Asda

Light Swirls UHT Delissimo UHT Whipped Squirty Tip Top dessert topping (canned) 35% less fat real dairy cream

Tesco Tesco Asda Tesco Asda

total 31

Page 2

meats

Class

code

Brand

Description

Retail Outlet

chargrilled burgers

1 1 1 1 1 1 1 1 1

Me01 Me02 Me03 Me04 Me05 Me06 Me07 Me09 Me10

Tesco Sainsbury Dalepak Linda Macartney Campbells Frozen Food Tesco Asda Mc Donalds Burger King

Chargrilled beefburgers (frozen) Chargrilled beefburgers (frozen) flame grilled quarter pounders (frozen) Flame grilled veg burgers American Fast Food Diner Burgers in buns Vegetarian char grilled quarter pounder Prime beef chargrilled quarter pounders Beefburgers Chargrilled beefburgers

Tesco J Sainsbury Tesco Tesco Asda Tesco Asda York York

smoked bacon

1 1 1 1 1 1 1 1 1 1

Me12 Me13 Me14 Me15 Me16 Me17 Me18 Me19 Me20 Me21

Tesco Sainsbury's Asda Danepak Wall's Safeway Tulip butcher butcher butcher

Smoked rindless back British Thick Cut Smoked Back Bacon Smoked rindless back Classic dry cure Smoked rindless back Smoked Rindless Tulip smoked back Smoked back Smoked back Smoked back

Tesco J Sainsbury Asda Tesco Tesco Safeway Safeway York market Scott Old Pork Butcher

salami

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Me22 Me23 Me24 Me25 Me26 Me27 Me28 Me29 Me30 Me31 Me32 Me33 Me34 Me35 Me36 Me37 Me39

Tesco Tesco Pepperami Sainsbury's Waitrose Waitrose Asda Landwirt Nolke Agraimpex Tesco Deli 1 Tesco Deli 1 Sainsbury's Deli 2 Sainsbury's Deli 2 Asda Waitrose Deli 3 Safeway Safeway

Kabanos pack of 2 Chorizo Sarta Doux Pepperami salami German salami 10 slices Hungarian salami Ardennes pepper salami Asda salami Milano German mini salami Kabanos French saucisson Italian Milano salami Strafino salami Traditional chorizo Asda German salami German salami German salami Danish salami

Tesco Tesco Safeway J Sainsbury Waitrose Waitrose Asda Asda Asda Tesco Tesco J Sainsbury J Sainsbury Asda Waitrose Safeway Safeway

cooked meats

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Me38 Me40 Me41 Me53 Me54 Me42 Me43 Me47 Me48 Me50 Me51 Me52 Me55 Me56 Me57 Me58 Me59 Me60 Me61 Me62

Sainsbury's Tesco B. Matthews Asda Safeway Sainsbury's Asda Tesco Tesco Safeways Matthews Matthews Asda Sainsbury's Rustlers M&S Safeway Asda Safeway Asda

Smoked ham Tesco British Ham Norfolk smoked ham Oak Smoked Ham Wafer Thin Smoked Ham Hot 'n' spicy chicken drumsticks Chicken satay Flamegrilled tikka chicken dippers Roast in store chinese basted chicken Chicken satay selection Char-grill chicken breast 200g Tikka chargrill Marinated turkey fillets Scottish smoked salmon Anchovies fillets in pure olive oil Flame Cooked BBQ Rib Sandwich Chicken tikka mini fillets Hot & Spicy wings Mini chicken tikka satay Cooked pork cocktail sausages Mini shish kebab

J Sainsbury Tesco Tesco Asda Safeway J Sainsbury Asda Tesco Tesco Safeway J Sainsbury Tesco Asda J Sainsbury J Sainsbury M&S Safeway Asda Safeway Asda

total

56

Page 3

miscellaneous

Class ready to eat meat

1 1 1 1 1

code Ms01 Ms02 Ms03 Ms04 Ms05

Brand Pork farms Sainsbury's M&S Dickinson & Morris Tesco

Description Pork lattice pie Crusty bake mini pork pies Melton Mowbray cured pork pies Melton Mowbray pork pie Traditional pork pie

Retail Outlet Tesco J Sainsbury M&S Tesco Tesco

Pizza

1 1 1 1 1 1

Ms06 Ms07 Ms08 Ms09 Ms10 Ms11

Sainsbury's Tesco Tesco Safeway Pizza Hut Dominos

Fresh pizzeria pepperoni Classico garlic mushroom Thin & crispy cheese & tomato (chilled) Thin & crispy cheese & tomato (frozen) Country Feast Vegetarian Supreme

J Sainsbury Tesco Tesco Safeway Pizza Hut Dominos

Battered & breaded

1 1 1 1 1 1 1 1 1 1 1 1

Ms12 Ms13 Ms14 Ms15 Ms16 Ms17 Ms18 Ms19 Ms20 Ms21 Ms27 Ms31

Sainsbury's M&S McDonald's Young's Tesco Asda Bernard Matthews Bird's Eye Fish (take-away) Fish (take-away) KFC Tryton Foods

Chicken breast nuggets Flame grilled tangy barbecue style coated chicken Chicken nuggets Scottish Island Scampi Salmon crunchies with broccoli Traditional style Yorkshire Puds Golden Fishies 100% Cod fillet fish fingers Haddock Cod Chicken portions Aunt Bessie Sunday Best Yorkshire Puddings

J Sainsbury M&S McDonald's Tesco Tesco Asda J Sainsbury Tesco

KFC Safeway

Cakes, pastries fruit pies

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Ms22 Ms23 Ms24 Ms25 Ms26 Ms28 Ms29 Ms30 Ms32 Ms33 Ms34 Ms35 Ms36 Ms37 Ms38

Mr Kipling Cadbury's McVities Sainsbury's Tesco Safeway M&S Asda Sainsbury's Ottoman Mr Kipling Sainsbury's Asda Safeway Tesco

Cream Slices (frozen) Luxury chocolate gateau (frozen) American Dream Colorado Choc & Crunch Cream Pie (frozen) Hand finished Black Forest Gateau (frozen) Strawberry cheesecake (frozen) 2 Chocolate pies (frozen) Cream Cakes Apple Sponge Sandwich (chilled) ASDA Brand Chilled Cake Bakery family apple pie Six sweet waffles 6 Country slices Apple Danish trellis (store baked pastry) Fresh Donuts (store baked pastry) American style cookies (Smartie cookie)2 (store baked pastry) Eccles cakes (store baked pastry)

J Sainsbury J Sainsbury J Sainsbury J Sainsbury Tesco Safeway M&S Asda J Sainsbury Tesco J Sainsbury J Sainsbury Asda Safeway Tesco

Confectionery

1 1 1 1 1

Ms39 Ms40 Ms41 Ms42 Ms43

Kit Kat Twix Mars bar Tunnock's Maltesers

Real milk chocolate fingers Milk chocolate covered caramel + biscuit Milk chocolate with soft nougat + caramel caramel wafer

Tesco Tesco Tesco Tesco Tesco

Beer

1 1 1 1 1

Ms44 Ms45 Ms46 Ms47 Ms48 Sainsbury's

Newcastle Brown Ale Guinness Original Mackeson's Murphy's Draught Stout Sainsbury's Irish stout

Tesco Tesco Tesco Tesco J Sainsbury

Tea bags

1 1 1 1 1

Ms49 Ms50 Ms51 Ms52 Ms53

Premium tea bags Sainsbury's Red Label BB Choicest blend Tetley tea bags Typhoo

Tesco J Sainsbury Tesco Tesco Tesco

Malted drink

1 1 1

Ms54 Ovaltine Ms55 Sainsbury's Ms56 SKBeecham

Ovaltine Malted drink Horlicks original

Tesco J Sainsbury Tesco

56

total

Tesco Sainsbury's Brooke Bond Tetley Premier

Page 4

soups and sauces Class Canned 1 1 1 1 1

Code So01 So02 So03 So04 So05

Brand Heinz Baxter's Campbell's Sainsbury's Tesco

Description Cream of Tomato Mushroom potage with woodland mushrooms Consomee clear beef Cream of Mushroom Cream of Chicken Soup

Retail Outlet Tesco Tesco J Sainsbury J Sainsbury Tesco

powder cup

1 1 1 1 1 1

So07 So09 So11 So12 So13 So16

Batchelor's Tesco Tesco Sainsbury's Safeway Safeway

Cup-a-soup Cream of mushroom with croutons Chicken and vegetable soup in a cup Minestrone Cup-a-soup Oxtail soup in a cup Roast chicken soup in a mug Slim choice Minestrone soup in a mug

Tesco Tesco Tesco J Sainsbury Safeway Safeway

packet

1 So10 Knorr 1 So15 Batchelors 1 So14 Asda

French onion Cream Of Garden Vegetable Soup Cream of chicken soup

Tesco Tesco Asda

fresh

1 1 1 1 1

Fresh spicy corn chowder Fresh soup winter vegetable Fresh beef and tomato soup Fresh soup minestrone Minestrone soup

J Sainsbury J Sainsbury Tesco Asda Safeway

Tomato ketchup HP sauce Branston sandwich pickle Worcestershire sauce White wine and garlic with double cream Chicken Tonight creamy curry Barbecue cook-in-sauce Sauce for Cottage Pie with rich gravy pasta sauce Creole Recipe Sauce Tikka Masala medium-hot Chunky onion and garlic sauce for bolognese Mushroom Bolognese sauce Rogan josh with tomato onion and garkic Italian Sauce (bolognese) for pasta Tomato with onions & garlic

Tesco Tesco Tesco Tesco Asda J Sainsbury J Sainsbury J Sainsbury Asda J Sainsbury J Sainsbury J Sainsbury J Sainsbury Tesco Safeway

So17 So18 So19 So20 So08

New Covent Garden Sainsbury's Tesco Asda Safeway

sauces 1 So21 Heinz (table & 1 So22 HP cook-in) 1 So23 Crosse & Blackwell 1 So24 Lea and Perrin's 1 So25 Asda 1 So26 Van den Berg 1 So27 Homepride 1 So28 Colman's 1 So29 Asda 1 So30 Discovery 1 So31 Sharwood's 1 So32 Ragu 1 So33 Sainsbury's 1 So35 Tesco 1 So36 Safeway

total

34

Page 5

ANNEX IIa: LIST OF ADDITIONAL 50 SAMPLES

CEREALS Class Biscuits

1 1 1 1 1 1 1

code C075 C076 C077 C078 C079 C080 C107

Brand Sainsbury Tesco Asda Safeway Sainsbury's Tesco McVitie's

Type Economy Digestives Value Digestives Digestive Digestive Ginger Snaps Ginger Nuts 25% Reduced Fat Digestives

Retail Outlet J Sainsbury Tesco Asda Safeway J Sainsbury Tesco J Sainsbury

Malted biscuits

1 1

C081 C082

Asda Co-op

Malted milk Malted milk

Asda Co-op

Savoury crackers

1 1 1 1 1 1 1

C083 C084 C085 C086 C087 C088 C089

Asda Safeway Sainsbury's Tesco Safeway Tesco Sainsbury's

Cream Crackers 50% Less fat Cream Crackers Lightly Salted Crispy Crackers 50% Lower Fat Cream Crackers Half Fat Cream Crackers Cornish Wafers Cornish Wafers

Asda Safeway J Sainsbury Tesco Safeway Tesco J Sainsbury

Toasted biscuits

1 1 1 1 1 1 1

C090 C091 C092 C093 C094 C095 C096

Tesco Asda Asda Tesco Rakusen's Morrisons Morrisons

French Toast Melba Toast Dutch Crispbakes Dutch Crispbakes Traditional Matzos Original Crispbread Crispwheat

Tesco Asda Asda Tesco J Sainsbury Morrisons Morrisons

C097 C098 C099 C100 C101 C102 C103 C104 C105 C106

Tesco Sainsbury's Thomas the Baker Woodhead The Bakers Allinsons Mothers Pride Asda Sainsbury's Warburtons Morrisons

Granary Malted Brown Bread Storebaked Harvest Grain Wholemeal Wholemeal Hi-Bran Scottish Plain Traditional White Bread Sliced wholemeal loaf Sliced wholemeal loaf Malted Danish Sliced Bread Superlife Medium Sliced Wholemeal

Tesco J Sainsbury Thomas, Malton Woodhead The Bakers, Malton J Sainsbury Tesco Asda J Sainsbury J Sainsbury Morrisons

Breads

1 1 1 1 1 1 1 1 1 1

DAIRY Class

code

Brand

Type

Retail Outlet

Processed cheese slices

1 1 1

Da32 Da33 Da34

Sainsbury's Asda Rowson

Cheddar Cheese Food Slices Cheese Food Slices Processed Cheese Food Slices

J Sainsbury Asda Morrisons

Low fat cheeses/ cheese

1

Da35

Safeway

16% Half Fat Mature Cheese

Safeway

MEATS Class

code

Brand

Type

Retail Outlet

salami

1 1 1

Me69 Me70 Me71

Independent delicat'n Independent delicat'n Independent delicat'n

Pepper salami Danish salami Salami

Lawrie's, York Derek Fox butchers The Bakery, Norton.

cooked meats

1 1 1 1 1 1

Me63 Me64 Me65 Me66 Me67 Me68

Sainsbury's Tesco Tesco John West Safeway Morrisons

Scottish Smoked Salmon Scottish Smoked Salmon Finest salted Anchovies Flat Fillets of Anchovies in Olive Oil Roast Chicken Hot 'n' Spicy Drumsticks Chicken satay kebabs

J Sainsbury Tesco Tesco Morrisons Safeway Morrisons

Cakes, pastries fruit pies

1 1 1 1

Ms57 Ms58 Ms59 Ms60

Morrisons Sainsbury's Safeway Woodhead The Baker

Store baked raspberry jam d'nuts Store baked sugar doughnuts Store baked jam doughnuts Store baked jam doughnuts

Morrisons J Sainsbury Safeway 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 be made 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, if the 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 be made to ensure that the grill positions used cook evenly (e.g. the grill pan will be covered with white bread which will be toasted to show hot or cold spots.) If the cooking is not even the products will be rotated on the grill during cooking. The grill temperature cannot be adjusted by 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. The recommendation is to use a pre-heated grill (moderate heat). Grill for 4 minutes, turning halfway through grilling.

Method: The grill will be pre-heated for 5 minutes. The crumpets will be evenly spaced on the grill at a "medium" shelf position, 7 cm from the grill, and toasted for the recommended time until 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 frozen burgers suggest using a medium grill for between 10 and 25 minutes; 5 samples fall within the range 10 to 20 minutes and one states 25 minutes. Instructions on a microwave burger state that the burger and bun should be cooked from frozen at 650W for 2 minutes and allowed to stand 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 other and the edge of the pan. The pan will be placed at a "medium" shelf position, 7 cm from the grill. The burgers will be grilled for the recommended time and be turned over half way through this time. The cooking time will be varied as necessary according to whether the burgers appear to be becoming over-cooked or are under-cooked. As the samples are to be bulked and homogenised the effects of slight variations in cooking between individual burgers will be minimised. In the case of the microwave burger, both burgers from the packet will be cooked in the bun as directed. Both the burger and bun will subsequently be homogenised and subsampled prior to analysis to reflect the level of 3-MCPD in the product "as consumed". Bacon Manufacturers' recommendations: Only a few samples have cooking instructions, and these recommend a medium grill for 4 to 5 minutes. Method: A whole pack of each sample (approx. 200g) will be grilled in two batches by placing half of the rashers on a grill pan equidistant from each other and the edge of the pan. The grill will be pre-heated for 10 minutes. The rashers will be grilled for 2 minutes on each side using a "medium" shelf position, 7 cm from the grill. Again, if any rashers appear to be cooking faster, then they will be rotated. The cooking time will be varied, if necessary, due to the thickness of the rashers and the samples will be monitored for signs of burning.

Miscellaneous Pizzas Manufacturers' recommendations: The recommendations are to preheat the oven (typically 200-220°C) and to place the pizza onto the top oven shelf, with cooking times varying from 8 to 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 in the 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 (typically 200-230°C ) and to place the sample on a baking tray, with most cooking times varying from 12 to 25 minutes (4 minutes for Asda Yorkshire puddings). Method: Frozen foods (chicken pieces, scampi, fish pieces, Yorkshire puddings) will be cooked as directed on the packet in an electric oven pre-heated for 10 minutes. The full contents 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 10 minutes. The full contents of a packet will be placed evenly on a baking tray in the stated position 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 tea industry,13 and was previously adopted for the compositional analysis of a "typical" consumer brew.14 It is being used in preference to the manufacturer’s recommendations. (NB: The water will be checked to ensure it does not contain a detectable level of 3-MCPD.) 1. 2. 3. 4.

Place a tea bag in a 250 ml ceramic cup and add 235 ml of boiling water. After 15 seconds, stir 3 times. Leave to infuse for a further 20 seconds, remove the bag from cup squeezing on the side for before complete removal. 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) of malted drink granules with around 200 ml of hot milk (not boiling) and to stir constantly until the granules have dissolved. Method: Malted drinks will be prepared as directed on the packet using fresh semi-skimmed milk (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 should the 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 continuous stirring. Three samples are instructed to be cooked for 3 to 4 minutes whilst two samples are to 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 an electric hotplate (pre-heated for 5 minutes) at a medium setting, with constant stirring for 4 minutes. 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 powders to be emptied into a mug and boiling water added with constant stirring until dissolved. All the soup 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 of freshly boiled water added and stirred with a teaspoon for 30 seconds. In order to have around 500 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 the packet into a saucepan of water, bringing to the boil on the cooking hob followed by simmering for 5 minutes with occasional stirring. Method: Samples will be placed into a medium saucepan and the label-stated volume of cold water added (typically 900 ml). The soups will be heated on a pre-heated electric hotplate until boiling and then simmered for 5 minutes as stated on the packet, with stirring every 30 seconds. Cook-in sauces Manufacturers' recommendations: These sauces are generally used to complement meat dishes whereby 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 electric hotplate at a low setting with stirring for 30 seconds every 5 minutes. Cooking will be for the time 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 ingredients not analysed during the validation project, minor variations developed as part of CSL’s commercial and R&D work will be applied, if necessary (see Annex V for UKAS-accredited method). Analytical batch protocol A total of 250 samples will be analysed for 3-MCPD. The samples have been divided into 5 classes: •

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



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



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



Miscellaneous - Sub-classes: 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 - Sub-classes: canned, fresh and powdered soups and sauces (table and cook-in-sauces) (34 products)

Food samples will be analysed in classes defined in the purchasing plan. The extraction procedure required will be as described in the validated method (Annex V). The classes and extraction procedures are: Class Cereals Dairy Dairy Meats Miscellaneous Miscellaneous Soups & sauces Soups & sauces

Sample all samples cheeses creams all samples beer, tea, malted drinks others soups sauces

Procedure as for cereals meats/cheese oils and fats meats/cheese soups meats/cheese soups 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 soy sauce characterised in-house and containing 0.030 mg/kg 3-MCPD.



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

Cereals: Breadcrumbs containing 0.030 mg/kg 3-MCPD characterised in the method validation trial. Dairy: Cheese alternative containing 0.035 mg/kg 3-MCPD characterised under an 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 inhouse.

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 reference materials will identify any "drift" in the analytical results, while the deuterated IS will automatically compensate for the recovery of 3-MCPD. Comparing the IS with an external standard 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 limit of 0.01 mg/kg. IQC criteria • Reagent blanks must not contain 3-MCPD at a detectable level (0.005 mg/kg). If the blank value exceeds 0.005 mg/kg the batch will be repeated. • Results for the food-class and cross-class reference materials must fall within 2 standard deviations of the mean value determined by the characterisation procedure (if necessary updated with additional data obtained during the project). Where the result for the foodclass reference material exceeds 2 standard deviations, the batch will be repeated. Where the result for the cross-class reference material exceeds 2 but is lower than 3 standard deviations, the batch will be considered acceptable if the result for the food-class reference material 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

3.

Water for analytical use - Specifications and test methods.

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/z 453 291 289 275 253 257

NOTE 2:

3-MCPD 3-MCPD 3-MCPD 3-MCPD 3-MCPD d5-3-MCPD

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 2,2,4trimethylpentane (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.