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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Baseline levels of melamine in food items sold in Canada. I. Dairy products and soy-based dairy replacement products a
a
a
a
Sheryl A. Tittlemier , Benjamin P.-Y. Lau , Cathie Ménard , Catherine Corrigan , a
a
a
Melissa Sparling , Dean Gaertner , Xu-Liang Cao & Bob Dabeka
a
a
Food Research Division, Banting Research Centre 2203D, Health Canada, Ottawa, Ontario, Canada, K1A 0L2 Published online: 17 Aug 2010.
To cite this article: Sheryl A. Tittlemier , Benjamin P.-Y. Lau , Cathie Ménard , Catherine Corrigan , Melissa Sparling , Dean Gaertner , Xu-Liang Cao & Bob Dabeka (2010): Baseline levels of melamine in food items sold in Canada. I. Dairy products and soy-based dairy replacement products, Food Additives & Contaminants: Part B: Surveillance, 3:3, 135-139 To link to this article: http://dx.doi.org/10.1080/19440049.2010.502654
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
Food Additives and Contaminants: Part B Vol. 3, No. 3, September 2010, 135–139
Baseline levels of melamine in food items sold in Canada. I. Dairy products and soy-based dairy replacement products Sheryl A. Tittlemier*y, Benjamin P.-Y. Lau, Cathie Me´nard, Catherine Corrigan, Melissa Sparling, Dean Gaertner, Xu-Liang Cao and Bob Dabeka Food Research Division, Banting Research Centre 2203D, Health Canada, Ottawa, Ontario, Canada, K1A 0L2
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(Received 30 April 2010; final version received 13 June 2010) A variety of dairy and soy-based dairy replacement products (n ¼ 246) purchased from Canadian retail outlets were analysed for baseline levels of melamine (MEL) using a sensitive LC–MS/MS method (method quantification limit ¼ 4 mg/kg). MEL was infrequently detected; only 14% of the items analysed contained quantifiable levels of MEL. The concentrations observed, aside from one recalled sample of candy, ranged from 0.00435 to 0.276 mg/kg, and were at least 10 times lower than the 2.5 mg/kg interim standard for melamine in products containing milk and milk-derived ingredients established by Health Canada. The consumption of foods containing these low levels of MEL does not constitute a health risk for consumers. Keywords: LC/MS; additives, general
Introduction In September 2008, it became public that melamine (MEL) had been deliberately added to diluted milk in China to pass non-specific protein assays. An unusual increase in reported kidney failure and kidney stone formation in infants in Beijing, Gansu province, and other locations in China prompted study into MEL contamination (Gossner et al. 2009). Subsequent food surveys found MEL concentrations of up to 4700 mg/kg (Wu et al. 2009) and 6196 mg/kg (Gossner et al. 2009) in Chinese infant formula and milk powder, respectively. High concentrations of MEL were also discovered in non-dairy creamer (6694 mg/kg) and animal feed (21,000 mg/kg) (Gossner et al. 2009). In response to this incident, many countries began testing their domestic and imported food products for MEL contamination. Canadian health authorities surveyed a number of infant formula and found low levels of MEL in 76% of the samples analysed (Tittlemier et al. 2009). Concentrations of MEL ranged from 4.31 to 346 mg/kg (median ¼ 16 mg/kg), which were orders of magnitude lower than those in the adulterated infant formula in China. These low levels of MEL were not attributed to intentional misuse or adulteration, as were the high levels observed in Chinese formula, and were below Health Canada’s interim standard of 0.5 mg/kg for melamine in infant
formula and sole-source nutrition products. An experts committee convened by the World Health Organization suggested that low concentrations (i.e. less than 1 mg/kg) in foods may be considered as ‘‘baseline’’ levels (World Health Organization 2009) and therefore, not a health concern. This present survey was conducted to examine Canadian dairy products and soy-based dairy replacement products sold in Canada for the presence of MEL. This work was also performed to provide information on the extent to which the general Canadian population, in addition to infants, was exposed to MEL via the consumption of food. Materials and methods Samples Domestic and imported dairy products and soy-based dairy substitutes (n ¼ 117) were purchased from national chain grocery stores in Ottawa, Canada, between September 30 and October 2, 2008. Similar milk- and soy-containing items originating from Asia (n ¼ 91) were purchased from smaller grocery stores in Ottawa on October 16, 2008. The bulk of the items were obtained via convenience sampling, where every unique item containing milk, milk powder, soy or soy powder as a major ingredient was purchased for analysis. The main types of items purchased and analysed are listed in Table 1; further details on
*Corresponding author. Email: [email protected] yCurrent address: Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main Street, Winnipeg, Manitoba, Canada, R3C 3G8. ISSN 1939–3210 print/ISSN 1939–3229 online ß 2010 Taylor & Francis DOI: 10.1080/19440049.2010.502654 http://www.informaworld.com
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Table 1. Information on the product groups and food items purchased from Canadian markets and analysed for melamine. Product group
Types of food items in product group purchased and analysed
Milk
Liquid cow’s milk, liquid goat’s milk, whole milk, partially skim milk, skim milk, chocolate milk, lactose free milk, organic milk, evaporated milk, condensed milk, milk powder Soft cheese, 0–% milk fat (MF), yogurt, plain yogurt, fruit-flavoured yogurt, organic yogurt, soy yogurt Flavoured yogurt drinks (1–1.5% MF), soy drinks, soy coffee creamer, soy milk, coffee drinks, milk tea, nutritional supplement, milk shakes Instant soy drink, malt drink, chocolate drink, milk tea Instant soybean cereal, fruit yogurt flavoured rice cereal Ice cream, ready-to-eat custard Milk candy, coffee milk candy, red bean milk candy, milk candy with nougat Biscuits with milk cream
Yogurt Beverages
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Powdered drink mixes Powdered cereal mixes Desserts Candy Cookies
Table 2. Summary results of the analysis of milk-containing Canadian Total Diet study composite food samples for melamine (MEL).
Food item type Milk, whole Milk, 2% Milk, 1% Milk, skim Milk, evaporated Milk, chocolate 1% Milk, butter 1% Cream Ice cream Yogurt
No. of samples analysed
No. of samples MEL 4 LOD
4 4 4 4 4 3 3 4 4 4
0 0 0 0 2 0 0 0 0 2
individual food items are listed in Supplemental Table 1. Samples of individual products were frozen, refrigerated or stored at room temperature until analysis. All samples were stored in the same manner in the laboratory as they were stored in the retail outlets. In addition, all samples were analysed prior to the expiry date indicated on their packaging. Composite milk and yogurt samples (n ¼ 38, listed in Table 2) were also analysed in this study. These composite samples were prepared for the Canadian Total Diet Study (TDS) from individual items purchased each year from 2004 through 2007 in a selected Canadian city. The Canadian TDS is a market basket survey that samples various food items from four different grocery stores and fast food restaurants in a selected Canadian city over a 5-week period each year (Conacher et al. 1989). Foods are prepared as for consumption, and replicate food items from the various grocery stores or restaurants visited are combined and homogenised to form a composite sample. Composite milk and yogurt samples were stored in chemically cleaned polypropylene bottles and lids, or glass jars with plastic lids and polytetrafluoroethylene (PTFE) lid liners at 20 C until analysis.
Max MEL (mg/kg)
Min MEL (mg/kg)
0.00720
0.00640
0.00551
0.00507
Sample analysis All samples were analysed as purchased; they were not prepared as for consumption. For example, powdered drink mixes were sub-sampled and directly analysed, as opposed to mixing with water as per the preparation instructions. Thus, the MEL concentrations provided in this paper are for the retail products, rather than for prepared food items. However, solid food items were homogenised using a food processor or a coffee grinder to obtain a homogenous sample prior to analysis. All samples were processed using liquid and solid phase extraction and analysed by combined liquid chromatography–tandem mass spectrometry (Xu et al. 2009). Sample preparation, sample analysis and data analysis were all performed using the methods described by Tittlemier et al. (2009). Briefly, each sample was fortified with 13C3-MEL internal recovery standard (Wellington Laboratories, Guelph, ON, Canada), left to equilibrate for a specified period of time, and extracted with a 1.0 N HCl solution. Solid samples were mixed using a hand held homogeniser. Samples were then centrifuged, aliquotted and mixed with dichloromethane. The aqueous top layer was
Downloaded by [Pennsylvania State University] at 11:09 10 May 2013
Food Additives and Contaminants: Part B cleaned using mixed mode cation exchange/reversed phase solid phase extraction (Oasis MCX, 150 mg, 30 mm, 6 cc; Waters Corp., Milford, MA, USA). Sample eluate was evaporated to dryness and reconstituted in 90:10 (v/v) acetonitrile/H2O and mixed well. The reconstituted extract was filtered, diluted by a factor of five and spiked with 15N3,13C3-MEL internal performance standard (Cambridge Isotope Laboratories, Andover, MA, USA). Samples were mixed well and stored at room temperature until instrumental analysis. Samples were chromatographed on a hydrophilic interaction (HILIC) column using a binary mobile phase of aqueous ammonium formate and formic acid, and formic acid in acetonitrile. The analysis of MEL and stable isotope labelled internal standards was performed using multiple reaction monitoring (MRM) in the positive ion electrospray mode. The transition of m/z 127 ! 85 was used for quantitation of MEL; m/z 127 ! 68 and m/z 127 ! 43 were used for confirmation. The analogous transitions of m/z 130 ! 87 and 130 ! 44, and m/z 133 ! 89 and 133 ! 45 were monitored for the internal standards 13C3-MEL and 15 N3,13C3-MEL, respectively.
Data analysis Analyte peaks were positively identified as MEL if the retention time of analytes in samples and blanks were within 0.1 min of the retention times of 13C3-MEL and 15 N3,13C3-MEL, the peak had a signal to noise ratio greater than 9:1, and the ratio of the peak height of the quantitation transition to the peak height of the primary confirmation transition (m/z 127 ! 68) was within 20% of the average ratio in calibration standards. MEL was quantitated using a seven-point calibration curve (ranging from 0.10 to 100 ng/ml; r2 4 0.99) of standards prepared in 90:10 (v/v) ACN/H2O. A 1/X weighted linear curve was used to plot the MEL response factor versus the MEL concentration in the calibration standards. The MEL response factor was calculated as the ratio of the peak height of the MEL quantitation MRM transition to the peak height of the 15 N3,13C3-MEL quantitation MRM transition. Concentrations of MEL in samples were blank and recovery corrected by subtracting the concentration of MEL in the associated reagent blank and dividing by the percentage of 13C3-MEL internal recovery standard present in each sample.
Quality control procedures Reagent blanks consisting of 5 g H2O were processed along with every 10 samples to monitor the background concentration of MEL. Solvent blanks of 90:10
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(v/v) ACN/H2O were run throughout the instrumental analysis to monitor for carryover. Two in-house reference materials, one liquid and one powdered infant formula each known to contain approximately 25 ng/g MEL, were processed as QC samples along with every 40 samples. Duplicate analyses were performed on a sample randomly selected from every group of 20 extracted. In addition, a solution of MEL (50 ng/ml) prepared using a source independent of the calibration standards was also ran during instrumental analysis to monitor stability of the standard calibration on a day-to-day basis. This method used to analyse MEL in dairy products and soy-based dairy replacement products was also used to analyse two samples in an international proficiency test. A milk powder and a powdered baking mix were analysed as part of the test organised by the Institute for Reference Materials and Measurements {Breidbach, 2010 3190/id}, and the results reported had associated satisfactory z-scores of 0.4 and 0.0, respectively.
Results and discussion MEL was infrequently detected in the dairy products and soy-based dairy substitutes analysed in this study. Only 14% of the individual food items (Table 3) and 11% of the TDS dairy composites (Table 2) contained quantifiable levels of MEL (method quantitation limit (MQL) ¼ 0.004 mg/kg). MEL was more often found in individual food items that contained milk (21%), as opposed to the soy-based dairy product substitutes (2%). Aside from one candy sample, all concentrations of MEL were at least 10 times lower than the 2.5 mg/kg interim standard for melamine in products containing milk and milk-derived ingredients (Health Canada 2009). The one sample that contained MEL above Health Canada’s interim standard was a candy that was a product of China; it contained 7.29 mg/kg of MEL. This was not considered to be a new violative result, because a recall of this particular brand of candy was initiated by the Canadian Food Inspection Agency just prior to the September 2008 time period, in which this sample was collected. Of the 32 samples that contained MEL above the MQL, 22 were self-identified as being a product of a particular country on their packaging. The median MEL concentration in the 13 products that indicated they were a product of China or Taiwan (0.0513 mg/kg) was significant greater than in those nine that indicated they were product of Canada or the USA (0.00825 mg/ kg) (Mann–Whitney rank sum test; p ¼ 0.045). There are few data in the peer reviewed literature regarding baseline levels of melamine in dairy products or soy-based dairy replacement products. Most of the
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Table 3. Summary results of the analysis of soy- and milk-containing dairy and dairy substitute food items marketed in Canada for melamine (MEL).
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Food item type
n analysed
n 4 MQL
11 2 1 1 1 47 12 2 35 1 12 4 1 38 1 6 1 23 9
3 0 0 1 0 1 4 2 11 0 3 0 0 1 0 0 0 0 2
Candy Cheese, soft Cookies Custard Ice cream Milk Milk, condensed and evaporated Milk, powder Milk-containing beverage Milk-containing cereal mix Milk-containing drink mix Soy-based cheese substitute Soy-based sour cream substitute Soy-containing beverage Soy-containing cereal mix Soy-containing drink mix Soy-containing yogurt Yogurt Yogurt beverage
Max MEL (mg/kg)
Min MEL (mg/kg)
7.29
Geometric mean MEL (mg/kg)
0.638
0.0792
0.00742 0.0307 0.0122 0.282
0.0175 0.00528 0.00435
0.0254 0.00802 0.0489
0.0213
0.00765
0.00920
0.00696
0.00712
0.0160
0.00663
0.00728
Table 4. Concentrations of melamine (MEL) quantitated in Chinese dairy products.
Food item Liquid milk Powdered infant milk Powdered soy milk Liquid milk Milk tea Raw liquid milk Semi-finished liquid milk Flavoured liquid milk Ice cream, liquid milk, powdered milk Japanese bun with cream Cream bun Creamed corn crepe
n analysed
–a –
n4 MQL
Max MEL (mg/kg)
1 3 1
33.8 mg/L 23.63 31.73 0.28b 11.07b 2.19 1.89 1.74 6175 4.0 37.0 13.6
1 6 1 – – 2 3 5 105 1 4 1
2 3 5 4 1 4 1
Min MEL (mg/kg)
Geometric mean MEL (mg/kg)
1.32
8.5
2.09 1.67 0.08 0.01
2.14 1.81 0.36
0.8
– 8
Reference (Chen and Yan 2009) (Chen and Yan 2009) (Chen and Yan 2009) (Miao et al. 2009) (Miao et al. 2009) (Sun et al. 2010) (Sun et al. 2010) (Sun et al. 2010) (Xu et al. 2009) (Fujita et al. 2009) (Fujita et al. 2009) (Fujita et al. 2009)
Notes: aNot provided in reference. b Not specified in reference whether this value is a mean, maximum, or minimum.
reported data is associated with adulterated food items produced in China, since that is where the deliberate addition of MEL to milk occurred. All concentrations observed in products examined in this study, apart from the recalled candy, were lower than those concentrations reported in Chinese domestic dairy products (Table 4). The majority of concentrations reported in Chinese dairy and milk-containing products in other studies were greater than 1 mg/kg. Aside from the recalled candy, the highest concentration of MEL detected in this study was 0.282 mg/kg. It should be kept in mind that the data on MEL in Chinese domestic dairy products and milk-containing
items in Table 4 are not necessarily representative of baseline levels of MEL in these foods. Samples in these studies may have been selected for analysis based on the fact that they were likely to contain adulterated milk (Fujita et al. 2009). However, a comparison of MEL concentrations in Canadian and Chinese domestic milk products strongly suggests that for the few instances in which sub-ppm levels of MEL were found in products sold in Canada that these sub-ppm levels were not due to misuse or adulteration. Over 90% of the Chinese dairy products sampled in the fall of 2008, such as liquid milk and yogurt and powdered milk products
Food Additives and Contaminants: Part B contained greater than 1 mg/kg MEL (Gossner et al. 2009). The relatively low levels of MEL concentrations found in products sold in Canada are orders of magnitude lower than those reported in Chinese products, and are consistent with the designation as baseline levels (World Health Organization 2009).
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Summary Canadians are infrequently exposed to low levels of MEL in dairy products and soy-based dairy replacement products. The concentrations observed in products examined in this survey, aside from one recalled sample of candy, were at least 10 times lower than the 2.5 mg/kg interim standard for melamine in products containing milk and milk-derived ingredients (Health Canada 2009). Therefore, it is considered that the consumption of foods containing these low levels of MEL does not constitute a health risk for consumers.
Acknowledgement Karen Pepper and John Moisey assisted with sampling. ST organised and managed the study and wrote the manuscript. BPYL performed the LC–MS/MS analyses. CM, CC, DG and MS prepared the samples for instrumental analysis. XLC and BD provided samples from the Canadian Total Diet Study.
References Breidbach A, Bouten K, Kroger K, Ulberth F. 2010. Capabilities of laboratories to determine melamine in food. Results of an international proficiency test. Anal Bioanal Chem. 396:503–510. Chen Z, Yan X. 2009. Simultaneous determination of melamine and 5-hydroxymethylfurfural in milk by
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capillary electrophoresis with diode array detection. J Agric Food Chem. 57:8742–8747. Conacher HBS, Graham RA, Newsome WH, Graham GF, Verdier P. 1989. The Health Protection Branch Total Diet Program: An Overview. Can Inst Food Sci Technol J. 22:322–326. Fujita M, Kakimoto K, Nagayoshi H, Konishi Y, Uchida K, Osakada M, Okihashi M, Obana H. 2009. Determination of melamine in Chinese-made processed food. Shokuhin Eiseigaku Zasshi. 50:131–134. Gossner CME, Schlundt J, Ben Embarek P, Hird S, Lo-FoWong D, Beltran JJO, Teoh KN, Tritscher A. 2009. The melamine incident: implications for international food and feed safety. Environ Health Perspect. 117:1803–1808. Health Canada. 2009. Available from: http://www.hcsc.gc.ca/fn-an/securit/chem-chim/melamine/qa-melamineqr-eng.php#8. Accessed January 6, 2010. Miao H, Fan S, Wu YN, Zhang L, Zhou PP, Li JG, Chen HJ, Zhao YF. 2009. Simultaneous determination of melamine, ammelide, ammeline, and cyanuric acid in milk and milk products by gas chromatography–tandem mass spectrometry. Biomed Environ Sci. 22:87–94. Sun H, Wang L, Ai L, Liang S, Wu H. 2010. A sensitive and validated method for determination of melamine residue in liquid milk by reversed phase high-performance liquid chromatography with solid-phase extraction. Food Control. 21:686–691. Tittlemier SA, Lau BPY, Me´nard C, Corrigan C, Sparling M, Gaertner D, Pepper K, Feeley M. 2009. Melamine in infant formula sold in Canada: Occurrence and Risk Assessment. J Agric Food Chem. 57:5340–5344. World Health Organization. 2009. Toxicological and Health Aspects of Melamine and Cyanuric Acid. Geneva: WHO. Wu YN, Zhao YF, Li JG. 2009. A survey on occurrence of melamine and its analogues in tainted infant formula in China. Biomed Environ Sci. 22:95–99. Xu XM, Ren YP, Zhu Y, Cai ZX, Han JL, Huang BF, Zhu Y. 2009. Direct determination of melamine in dairy products by gas chromatography/mass spectrometry with coupled column separation. Anal Chim Acta. 650:39–43.
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Baseline levels of melamine in food items sold in Canada. I. Dairy products and soy-based dairy replacement products a
a
a
a
Sheryl A. Tittlemier , Benjamin P.-Y. Lau , Cathie Ménard , Catherine Corrigan , a
a
a
Melissa Sparling , Dean Gaertner , Xu-Liang Cao & Bob Dabeka
a
a
Food Research Division, Banting Research Centre 2203D, Health Canada, Ottawa, Ontario, Canada, K1A 0L2 Published online: 17 Aug 2010.
To cite this article: Sheryl A. Tittlemier , Benjamin P.-Y. Lau , Cathie Ménard , Catherine Corrigan , Melissa Sparling , Dean Gaertner , Xu-Liang Cao & Bob Dabeka (2010): Baseline levels of melamine in food items sold in Canada. I. Dairy products and soy-based dairy replacement products, Food Additives & Contaminants: Part B: Surveillance, 3:3, 135-139 To link to this article: http://dx.doi.org/10.1080/19440049.2010.502654
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
Food Additives and Contaminants: Part B Vol. 3, No. 3, September 2010, 135–139
Baseline levels of melamine in food items sold in Canada. I. Dairy products and soy-based dairy replacement products Sheryl A. Tittlemier*y, Benjamin P.-Y. Lau, Cathie Me´nard, Catherine Corrigan, Melissa Sparling, Dean Gaertner, Xu-Liang Cao and Bob Dabeka Food Research Division, Banting Research Centre 2203D, Health Canada, Ottawa, Ontario, Canada, K1A 0L2
Downloaded by [Pennsylvania State University] at 11:09 10 May 2013
(Received 30 April 2010; final version received 13 June 2010) A variety of dairy and soy-based dairy replacement products (n ¼ 246) purchased from Canadian retail outlets were analysed for baseline levels of melamine (MEL) using a sensitive LC–MS/MS method (method quantification limit ¼ 4 mg/kg). MEL was infrequently detected; only 14% of the items analysed contained quantifiable levels of MEL. The concentrations observed, aside from one recalled sample of candy, ranged from 0.00435 to 0.276 mg/kg, and were at least 10 times lower than the 2.5 mg/kg interim standard for melamine in products containing milk and milk-derived ingredients established by Health Canada. The consumption of foods containing these low levels of MEL does not constitute a health risk for consumers. Keywords: LC/MS; additives, general
Introduction In September 2008, it became public that melamine (MEL) had been deliberately added to diluted milk in China to pass non-specific protein assays. An unusual increase in reported kidney failure and kidney stone formation in infants in Beijing, Gansu province, and other locations in China prompted study into MEL contamination (Gossner et al. 2009). Subsequent food surveys found MEL concentrations of up to 4700 mg/kg (Wu et al. 2009) and 6196 mg/kg (Gossner et al. 2009) in Chinese infant formula and milk powder, respectively. High concentrations of MEL were also discovered in non-dairy creamer (6694 mg/kg) and animal feed (21,000 mg/kg) (Gossner et al. 2009). In response to this incident, many countries began testing their domestic and imported food products for MEL contamination. Canadian health authorities surveyed a number of infant formula and found low levels of MEL in 76% of the samples analysed (Tittlemier et al. 2009). Concentrations of MEL ranged from 4.31 to 346 mg/kg (median ¼ 16 mg/kg), which were orders of magnitude lower than those in the adulterated infant formula in China. These low levels of MEL were not attributed to intentional misuse or adulteration, as were the high levels observed in Chinese formula, and were below Health Canada’s interim standard of 0.5 mg/kg for melamine in infant
formula and sole-source nutrition products. An experts committee convened by the World Health Organization suggested that low concentrations (i.e. less than 1 mg/kg) in foods may be considered as ‘‘baseline’’ levels (World Health Organization 2009) and therefore, not a health concern. This present survey was conducted to examine Canadian dairy products and soy-based dairy replacement products sold in Canada for the presence of MEL. This work was also performed to provide information on the extent to which the general Canadian population, in addition to infants, was exposed to MEL via the consumption of food. Materials and methods Samples Domestic and imported dairy products and soy-based dairy substitutes (n ¼ 117) were purchased from national chain grocery stores in Ottawa, Canada, between September 30 and October 2, 2008. Similar milk- and soy-containing items originating from Asia (n ¼ 91) were purchased from smaller grocery stores in Ottawa on October 16, 2008. The bulk of the items were obtained via convenience sampling, where every unique item containing milk, milk powder, soy or soy powder as a major ingredient was purchased for analysis. The main types of items purchased and analysed are listed in Table 1; further details on
*Corresponding author. Email: [email protected] yCurrent address: Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main Street, Winnipeg, Manitoba, Canada, R3C 3G8. ISSN 1939–3210 print/ISSN 1939–3229 online ß 2010 Taylor & Francis DOI: 10.1080/19440049.2010.502654 http://www.informaworld.com
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S.A. Tittlemier et al.
Table 1. Information on the product groups and food items purchased from Canadian markets and analysed for melamine. Product group
Types of food items in product group purchased and analysed
Milk
Liquid cow’s milk, liquid goat’s milk, whole milk, partially skim milk, skim milk, chocolate milk, lactose free milk, organic milk, evaporated milk, condensed milk, milk powder Soft cheese, 0–% milk fat (MF), yogurt, plain yogurt, fruit-flavoured yogurt, organic yogurt, soy yogurt Flavoured yogurt drinks (1–1.5% MF), soy drinks, soy coffee creamer, soy milk, coffee drinks, milk tea, nutritional supplement, milk shakes Instant soy drink, malt drink, chocolate drink, milk tea Instant soybean cereal, fruit yogurt flavoured rice cereal Ice cream, ready-to-eat custard Milk candy, coffee milk candy, red bean milk candy, milk candy with nougat Biscuits with milk cream
Yogurt Beverages
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Powdered drink mixes Powdered cereal mixes Desserts Candy Cookies
Table 2. Summary results of the analysis of milk-containing Canadian Total Diet study composite food samples for melamine (MEL).
Food item type Milk, whole Milk, 2% Milk, 1% Milk, skim Milk, evaporated Milk, chocolate 1% Milk, butter 1% Cream Ice cream Yogurt
No. of samples analysed
No. of samples MEL 4 LOD
4 4 4 4 4 3 3 4 4 4
0 0 0 0 2 0 0 0 0 2
individual food items are listed in Supplemental Table 1. Samples of individual products were frozen, refrigerated or stored at room temperature until analysis. All samples were stored in the same manner in the laboratory as they were stored in the retail outlets. In addition, all samples were analysed prior to the expiry date indicated on their packaging. Composite milk and yogurt samples (n ¼ 38, listed in Table 2) were also analysed in this study. These composite samples were prepared for the Canadian Total Diet Study (TDS) from individual items purchased each year from 2004 through 2007 in a selected Canadian city. The Canadian TDS is a market basket survey that samples various food items from four different grocery stores and fast food restaurants in a selected Canadian city over a 5-week period each year (Conacher et al. 1989). Foods are prepared as for consumption, and replicate food items from the various grocery stores or restaurants visited are combined and homogenised to form a composite sample. Composite milk and yogurt samples were stored in chemically cleaned polypropylene bottles and lids, or glass jars with plastic lids and polytetrafluoroethylene (PTFE) lid liners at 20 C until analysis.
Max MEL (mg/kg)
Min MEL (mg/kg)
0.00720
0.00640
0.00551
0.00507
Sample analysis All samples were analysed as purchased; they were not prepared as for consumption. For example, powdered drink mixes were sub-sampled and directly analysed, as opposed to mixing with water as per the preparation instructions. Thus, the MEL concentrations provided in this paper are for the retail products, rather than for prepared food items. However, solid food items were homogenised using a food processor or a coffee grinder to obtain a homogenous sample prior to analysis. All samples were processed using liquid and solid phase extraction and analysed by combined liquid chromatography–tandem mass spectrometry (Xu et al. 2009). Sample preparation, sample analysis and data analysis were all performed using the methods described by Tittlemier et al. (2009). Briefly, each sample was fortified with 13C3-MEL internal recovery standard (Wellington Laboratories, Guelph, ON, Canada), left to equilibrate for a specified period of time, and extracted with a 1.0 N HCl solution. Solid samples were mixed using a hand held homogeniser. Samples were then centrifuged, aliquotted and mixed with dichloromethane. The aqueous top layer was
Downloaded by [Pennsylvania State University] at 11:09 10 May 2013
Food Additives and Contaminants: Part B cleaned using mixed mode cation exchange/reversed phase solid phase extraction (Oasis MCX, 150 mg, 30 mm, 6 cc; Waters Corp., Milford, MA, USA). Sample eluate was evaporated to dryness and reconstituted in 90:10 (v/v) acetonitrile/H2O and mixed well. The reconstituted extract was filtered, diluted by a factor of five and spiked with 15N3,13C3-MEL internal performance standard (Cambridge Isotope Laboratories, Andover, MA, USA). Samples were mixed well and stored at room temperature until instrumental analysis. Samples were chromatographed on a hydrophilic interaction (HILIC) column using a binary mobile phase of aqueous ammonium formate and formic acid, and formic acid in acetonitrile. The analysis of MEL and stable isotope labelled internal standards was performed using multiple reaction monitoring (MRM) in the positive ion electrospray mode. The transition of m/z 127 ! 85 was used for quantitation of MEL; m/z 127 ! 68 and m/z 127 ! 43 were used for confirmation. The analogous transitions of m/z 130 ! 87 and 130 ! 44, and m/z 133 ! 89 and 133 ! 45 were monitored for the internal standards 13C3-MEL and 15 N3,13C3-MEL, respectively.
Data analysis Analyte peaks were positively identified as MEL if the retention time of analytes in samples and blanks were within 0.1 min of the retention times of 13C3-MEL and 15 N3,13C3-MEL, the peak had a signal to noise ratio greater than 9:1, and the ratio of the peak height of the quantitation transition to the peak height of the primary confirmation transition (m/z 127 ! 68) was within 20% of the average ratio in calibration standards. MEL was quantitated using a seven-point calibration curve (ranging from 0.10 to 100 ng/ml; r2 4 0.99) of standards prepared in 90:10 (v/v) ACN/H2O. A 1/X weighted linear curve was used to plot the MEL response factor versus the MEL concentration in the calibration standards. The MEL response factor was calculated as the ratio of the peak height of the MEL quantitation MRM transition to the peak height of the 15 N3,13C3-MEL quantitation MRM transition. Concentrations of MEL in samples were blank and recovery corrected by subtracting the concentration of MEL in the associated reagent blank and dividing by the percentage of 13C3-MEL internal recovery standard present in each sample.
Quality control procedures Reagent blanks consisting of 5 g H2O were processed along with every 10 samples to monitor the background concentration of MEL. Solvent blanks of 90:10
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(v/v) ACN/H2O were run throughout the instrumental analysis to monitor for carryover. Two in-house reference materials, one liquid and one powdered infant formula each known to contain approximately 25 ng/g MEL, were processed as QC samples along with every 40 samples. Duplicate analyses were performed on a sample randomly selected from every group of 20 extracted. In addition, a solution of MEL (50 ng/ml) prepared using a source independent of the calibration standards was also ran during instrumental analysis to monitor stability of the standard calibration on a day-to-day basis. This method used to analyse MEL in dairy products and soy-based dairy replacement products was also used to analyse two samples in an international proficiency test. A milk powder and a powdered baking mix were analysed as part of the test organised by the Institute for Reference Materials and Measurements {Breidbach, 2010 3190/id}, and the results reported had associated satisfactory z-scores of 0.4 and 0.0, respectively.
Results and discussion MEL was infrequently detected in the dairy products and soy-based dairy substitutes analysed in this study. Only 14% of the individual food items (Table 3) and 11% of the TDS dairy composites (Table 2) contained quantifiable levels of MEL (method quantitation limit (MQL) ¼ 0.004 mg/kg). MEL was more often found in individual food items that contained milk (21%), as opposed to the soy-based dairy product substitutes (2%). Aside from one candy sample, all concentrations of MEL were at least 10 times lower than the 2.5 mg/kg interim standard for melamine in products containing milk and milk-derived ingredients (Health Canada 2009). The one sample that contained MEL above Health Canada’s interim standard was a candy that was a product of China; it contained 7.29 mg/kg of MEL. This was not considered to be a new violative result, because a recall of this particular brand of candy was initiated by the Canadian Food Inspection Agency just prior to the September 2008 time period, in which this sample was collected. Of the 32 samples that contained MEL above the MQL, 22 were self-identified as being a product of a particular country on their packaging. The median MEL concentration in the 13 products that indicated they were a product of China or Taiwan (0.0513 mg/kg) was significant greater than in those nine that indicated they were product of Canada or the USA (0.00825 mg/ kg) (Mann–Whitney rank sum test; p ¼ 0.045). There are few data in the peer reviewed literature regarding baseline levels of melamine in dairy products or soy-based dairy replacement products. Most of the
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S.A. Tittlemier et al.
Table 3. Summary results of the analysis of soy- and milk-containing dairy and dairy substitute food items marketed in Canada for melamine (MEL).
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Food item type
n analysed
n 4 MQL
11 2 1 1 1 47 12 2 35 1 12 4 1 38 1 6 1 23 9
3 0 0 1 0 1 4 2 11 0 3 0 0 1 0 0 0 0 2
Candy Cheese, soft Cookies Custard Ice cream Milk Milk, condensed and evaporated Milk, powder Milk-containing beverage Milk-containing cereal mix Milk-containing drink mix Soy-based cheese substitute Soy-based sour cream substitute Soy-containing beverage Soy-containing cereal mix Soy-containing drink mix Soy-containing yogurt Yogurt Yogurt beverage
Max MEL (mg/kg)
Min MEL (mg/kg)
7.29
Geometric mean MEL (mg/kg)
0.638
0.0792
0.00742 0.0307 0.0122 0.282
0.0175 0.00528 0.00435
0.0254 0.00802 0.0489
0.0213
0.00765
0.00920
0.00696
0.00712
0.0160
0.00663
0.00728
Table 4. Concentrations of melamine (MEL) quantitated in Chinese dairy products.
Food item Liquid milk Powdered infant milk Powdered soy milk Liquid milk Milk tea Raw liquid milk Semi-finished liquid milk Flavoured liquid milk Ice cream, liquid milk, powdered milk Japanese bun with cream Cream bun Creamed corn crepe
n analysed
–a –
n4 MQL
Max MEL (mg/kg)
1 3 1
33.8 mg/L 23.63 31.73 0.28b 11.07b 2.19 1.89 1.74 6175 4.0 37.0 13.6
1 6 1 – – 2 3 5 105 1 4 1
2 3 5 4 1 4 1
Min MEL (mg/kg)
Geometric mean MEL (mg/kg)
1.32
8.5
2.09 1.67 0.08 0.01
2.14 1.81 0.36
0.8
– 8
Reference (Chen and Yan 2009) (Chen and Yan 2009) (Chen and Yan 2009) (Miao et al. 2009) (Miao et al. 2009) (Sun et al. 2010) (Sun et al. 2010) (Sun et al. 2010) (Xu et al. 2009) (Fujita et al. 2009) (Fujita et al. 2009) (Fujita et al. 2009)
Notes: aNot provided in reference. b Not specified in reference whether this value is a mean, maximum, or minimum.
reported data is associated with adulterated food items produced in China, since that is where the deliberate addition of MEL to milk occurred. All concentrations observed in products examined in this study, apart from the recalled candy, were lower than those concentrations reported in Chinese domestic dairy products (Table 4). The majority of concentrations reported in Chinese dairy and milk-containing products in other studies were greater than 1 mg/kg. Aside from the recalled candy, the highest concentration of MEL detected in this study was 0.282 mg/kg. It should be kept in mind that the data on MEL in Chinese domestic dairy products and milk-containing
items in Table 4 are not necessarily representative of baseline levels of MEL in these foods. Samples in these studies may have been selected for analysis based on the fact that they were likely to contain adulterated milk (Fujita et al. 2009). However, a comparison of MEL concentrations in Canadian and Chinese domestic milk products strongly suggests that for the few instances in which sub-ppm levels of MEL were found in products sold in Canada that these sub-ppm levels were not due to misuse or adulteration. Over 90% of the Chinese dairy products sampled in the fall of 2008, such as liquid milk and yogurt and powdered milk products
Food Additives and Contaminants: Part B contained greater than 1 mg/kg MEL (Gossner et al. 2009). The relatively low levels of MEL concentrations found in products sold in Canada are orders of magnitude lower than those reported in Chinese products, and are consistent with the designation as baseline levels (World Health Organization 2009).
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Summary Canadians are infrequently exposed to low levels of MEL in dairy products and soy-based dairy replacement products. The concentrations observed in products examined in this survey, aside from one recalled sample of candy, were at least 10 times lower than the 2.5 mg/kg interim standard for melamine in products containing milk and milk-derived ingredients (Health Canada 2009). Therefore, it is considered that the consumption of foods containing these low levels of MEL does not constitute a health risk for consumers.
Acknowledgement Karen Pepper and John Moisey assisted with sampling. ST organised and managed the study and wrote the manuscript. BPYL performed the LC–MS/MS analyses. CM, CC, DG and MS prepared the samples for instrumental analysis. XLC and BD provided samples from the Canadian Total Diet Study.
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