Laboratories worldwide are now developing methods to test food products, including milk, for melamine.
Melamine is drawing international attention due to its recent discovery in certain food products in China. It is a substance commonly used to make plastics and fertilizers and it is normally found in plastic ware and industrial plastics such as Formica countertops and flooring. Recently, Chinese food producers have been found adding melamine to milk and dairy products to increase the amount of protein these products appeared to have. Proteins are usually the only source of nitrogen in food. By adding nitrogen-rich melamine to the infant formula and milk products, the formula would appear to contain more protein than it actually did.
To date, melamine has been found in Chinese infant formula, whole milk and dairy products like chocolates, and even in eggs laid by chickens believed to have been fed with melamine-tainted feed.
There are two main approaches to sample cleanup when analyzing melamine. One is the ‘dilute-and-shoot’ technique and the other uses a protein precipitation step.
The impact of melamine in food
Melamine was previously thought to be non-toxic until melaminespiked pet food exported from China to the US sickened and killed numerous dogs in 2006. Last year thousands of Chinese infants who had consumed melamine-tainted formula began to suffer from renal failure and kidney stones.
In September 2008, the FDA said more than 53,000 infants became ill from consuming the formula, and over 12,000 infants had to be hospitalized. At least four infants died as a result of these kidney problems and related complications.
According to a October 2008 report by the World Health Organization (WHO), melamine, when combined with cyanuric acid that was also found in the infant formula, forms crystals and eventually stones in the kidneys. However, WHO has not determined whether the cyanuric acid, which can also boost apparent protein content, was intentionally added to the formula.
Global pressure is now on China to improve on its food safety and to test their products for melamine contamination. However, the growing demand for more efficient food analysis methods and the increased emphasis on food safety is not limited to this country. Laboratories worldwide are now developing methods to test food products, including milk, for melamine.
The complex milk matrix
Milk is an essential component of the human diet and 100g of whole milk typically consists of 88% water, and the other 12% contains nutrients such as proteins (3.2g), fat (3.25g) and sugars (5.3g), all of which are completely solubilized.
Milk also contains minerals and vitamins such as calcium and potassium. From an analytical chemistry standpoint, milk is a complex matrix that can easily mask the presence of a contaminant.
We have used milk in our case study for the analysis of melamine. The brief summary of the analytical chemistry methods used in melamine analysis is seen in Figure 1.
Figure 1. Flow-diagram for melamine analysis
Sample cleanup employing solid phase extraction
The sample pre-treatment is critical in melamine analysis due to the complexity of milk. A cleanup step removes most of the non-essential components, leaving only the analyte of interest. Methods for melamine analysis typically use mass spectrometry detection, an analytical technique that has high specificity and selectivity on an analyte. However, it requires the sample to be ‘clean’, or free from other components.
Melamine can form supramolecular aggregates when it is bound to cyanuric acid that is present in the same sample. These aggregates are not soluble in water and most solvents.
The presence of melamine-cyanuric acid complex is the main cause for melamine toxicity. Analytical methods developed by the FDA suggested that exposing the samples to very high pH (basic or alkaline) or very low pH (acidic) and sample dilution is an effective method of disrupting the hydrogen bonds and breaking up the complex to separate melamine for analysis.
Approaches to sample cleanup
There are two main approaches to sample cleanup when analyzing melamine. One is the ‘dilute-and-shoot’ technique, which exposes the milk sample to low pH (such as 2.5% formic acid) followed by an exposure to an organic solvent (acetonitrile) for protein precipitation and centrifugation to remove the resulting precipitated components. This approach can be used for fast screening of samples for melamine.
The second cleanup approach uses a protein precipitation step (similar to the ‘dilute-and shoot’ technique) followed by solid phase extraction (SPE) step. This is used when a better cleanup protocol is required, such as when the amount of melamine needs to be quantified accurately.
The SPE step helps to further decrease the amount of co-extracted impurities and to achieve better selectivity for melamine from the sample matrix. Strong cation exchange SPE or a mixed-mode SPE that includes a strong cation exchanger is normally used in this procedure.
The SPE packing retains the basic melamine while the impurities are removed during the washing process. Melamine is subsequently eluted from the SPE cartridge using a strong base in methanol. Both polymer-based and silica-based SPE can be used. We used silica-based strong cation exchange SPE in this case. Figure 2 shows the SPE cartridges and the vacuum manifold used.
Figure 2. Example of vacuum manifold with solid phase extraction (SPE) tubes.
Detection using LC-MS
After the cleanup, the sample is injected into the liquid chromatography-mass spectrometry (LCMS) instrument for melamine identifi cation. LC is a popular technique used for melamine analysis. Early methods for melamine analysis also use gas chromatography (GC). In GC, however, melamine must be derivatized to enhance its gas chromatographic properties, or detection by that method would not be possible. Chromatography techniques further separate the melamine from the co-extracted impurities prior to mass spectrometry detection.
Ion exchange solid phase extraction (SPE) cartridges were used to extract melamine from milk, and good recoveries were seen for the melamine at concentrations between 100 ng/g and 500 ng/g.
Advantages with HILIC
Melamine (Figure 3) is a polar or water-soluble molecule, which makes it a good candidate for aqueous normal phase (ANP) liquid chromatography. In ANP, a polar hydrophilic analyte partitions between a relatively polar stationary phase and a relatively nonpolar mobile phase.
Figure 3. Chemical structure of melamine
ANP is commonly referred to as hydrophilic interaction chromatography or HILIC, but the term HILIC implies a mechanism that is one of several that may be operative under ANP conditions. Determining melamine content using the HILIC mode, results in better retention of this polar molecule than reversed-phase chromatography. In the HILIC mode, polar molecules are retained longer on the column. This provides greater flexibility in controlling the separation in the presence of interfering components.
If these interfering components elute at the start of the chromatogram, melamine retention can be changed to ensure that it elutes away from these interfering peaks.
In contrast to reversed-phase LC, if the melamine and interfering components elute close to the start of the chromatogram, there is minimal flexibility in separating the melamine from these interfering peaks, as there is no more time or space to move either the melamine or the interfering components to a longer or shorter retention period.
Mass spectrometry detection
Mass spectrometric detection of melamine was done in multiple reaction monitoring (MRM) mode when both parent and daughter ions for melamine were analyzed. MRM yields maximum sensitivity and selectivity for analytes of interest.
The two parent-daughter transitions for melamine were monitored in the positive ion mode: 127/85 and 127/68. The 127/85 MRM was used for quantitation. Figure 4 shows the specific chromatograms for melamine spiked into milk at a level of 100 parts per billion (ppb).
Figure 4. Specific chromatograms for melamine that was spiked into milk at a level of 100 parts per billion (ppb).
Evaluating techniques for food analysis
The performance of a food analysis technique is commonly evaluated by the recovery of the food contaminant from spiked samples. Noncontaminated food samples are used in the evaluation procedure.
The contaminant(s) under study are added to the food samples at known concentration levels and analytical methodology is applied to recover the contaminants with high efficiency.
The acceptable recovery range is usually between 70% and 120%. The milk samples we have used in this study were obtained locally and were found to be melamine-free. We then spiked the milk samples with 100 ppb (parts per billion) and 500 ppb of melamine and applied the cleanup and detection techniques as seen in Figure 1.
In summary, we precipitated the milk proteins and disrupted the melamine–cyanuric acid complex using pH 2.5 phosphate buffer and acetonitrile, performed sample cleanup using silica-based strong cation exchange SPE and used HILIC chromatography for further melamine separation prior to its detection by mass spectrometry. We achieved the recovery levels shown in Table 1. These recovery levels fall well within the acceptable range.
Table 1: Recoveries obtained for melamine from the spiked food samples.
Background melamine contamination was found in the reagents and plasticware used in this analysis. The background contamination levels were as high as 10–11 ppb (parts per billion) in some samples, when calculated as equivalent to 1g of the foods.
In a recent study of melamine in infant formula by the FDA, scientists have seen solvent background levels as high as 40 ppb. The presence of background melamine contamination shows the importance of using high-purity solvents and reagents during the analysis.
Given the background levels found here, we estimate the method limit of quantitation to be 50 ppb of melamine in milk. This value shows that the method can detect trace levels of melamine in milk. As a comparison, this analytical method is about 50 times more sensitive than the safety threshold for melamine in foods set by Hong Kong at the 2500 parts per billion (ppb), as reported in an October 2008 CNN News report.
We used ion exchange SPE cartridges to extract melamine from milk, and good recoveries were seen for the melamine at concentrations between 100 ng/g and 500 ng/g. Standards developed with highpurity melamine and cyanuric acid are needed to ensure valid results.
HILIC chromatography with a silica-based HPLC column gave good retention of the analyte, and the HILIC mode of chromatography gave enhanced response in the MS-MS analysis.
In October 2008, the US FDA says it plans to open more offices in China to inspect, in cooperation with Chinese officials and food safety groups, food and drug products to prevent the import of contaminated food and drugs from China into the US.
This means faster and more convenient methods for the analysis of melamine will emerge as food producers and importers continue to monitor for melamine contamination.
Unraveling the Melamine Maze
In order to ensure the safety of food for consumers, food testing laboratories will require effective analytical methods and instrumentation.
BY GUIPING LU, YOLANDA FINTSCHENKO, THERMO FISHER SCIENTIFIC
MIn September last year, the Hong Kong authority also ordered a withdrawal of a certain brand of food products after eight out of 30 samples tested positive for melamine. On 22 September 2008, Taiwan suspended imports of all milk products and plant-based proteins from China. The Japanese government has instructed importers to test dairy products from China for melamine or face huge fines and penalties. From 20 September to 19 October 2008, Japanese importers tested 2,540 products and 18 were found to be contaminated with melamine.
The Japanese government has instructed all importers to test dairy products from China for melamine or face huge fines and penalties.
The Korean government announced an official import ban on all Chinese dairy products on 25 September 2008 after trace amount of melamine was found in a popular custard snack made in China.
On 18 September 2008, China’s State Council announced the abolishment of inspection exemption policy for brand name products that was established in 1999. Due to the severe consequences of the melamine scandal, the head of General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (AQSIQ), Changjiang Li, resigned on 22 September 2008. In October 2008, the AQSIQ set up 1 ìg/kg and 2.5 ìg/kg maximum residue levels (MRL) for melamine in infant formula and milk products respectively.
Moving forward, the Chinese government now plans to impose controls over melamine production. In order to ensure the safety of food for consumers, food testing laboratories will require effective analytical methods and instrumentation.
Analysis of melamine in food
The toxicity of melamine is due to the formation of insoluble crystals between melamine and cyanuric acid (a by-product of melamine), and it can cause the development of kidney stones in pets and babies. Besides melamine and cyanuric acid, two melamine-related compounds, ammeline and ammelide (Figure 2), were also found in adulterated pet food in March 2007 in the US. Due to the severe consequences of melamine adulteration in milk products, government food safety agencies around the world such as China’s AQSIQ, US Food and Drug Administration (FDA) and the European Food Safety Authority (FAS) have established 1 mg/kg and 2.5 mg/kg MRL for melamine in baby formula and milk products respectively.
Figure 2. Structures of melamine, cyanuric acid, ammeline and ammelide.
Since melamine has been used as a fertilizer and was added to animal feed, other products face the possibility of melamine contamination. The testing list for melamine now extends beyond milk products or products with milk powder as an ingredient to meat, poultry, egg and vegetables.
Since the outbreak of melamine contamination in pet food, the US FDA has published six methods for melamine analysis, utilizing high performance liquid chromatography coupled with ultra-violet (UV) detector (HPLC-UV; US FDA Forensic Chemistry Center HPLC-UV method), gas chromatography coupled with mass spectrometer (GCMS or GC-MS/MS; US FDA Laboratory Information Bulletin 4423), and liquid chromatography coupled with mass spectrometer (LC-MS/ MS; US FDA Laboratory Information Bulletin 4396, 4421 and 4422). Manufacturers have also developed enzyme linked immuno-sorbent assay (ELISA) kits for detection of melamine using antigen-antibody reaction. Table 1 lists the major characteristics of these different methods.
Table 1: Comparison of methods for melamine analysis
The FDA has recently published six methods for the detection of melamine and cyanuric acid in milk and milk products using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LCMS/ MS method for melamine involves solid phase extraction (SPE) cleanup, which removes interfering compounds in dirty matrices such as seafood and meat.
In the selected reaction monitoring (SRM) mode, the LC-MS/MS method is able to reach low limit of quantifi cation (LOQ) of 10-50 ìg/kg for melamine. The GC-MS method requires a derivatization step to convert melamine and its related compounds cyanuric acid, ammeline and ammelide to GC amenable derivatives, which are analyzed by GC-MS.
In selected ion monitoring (SIM) mode, the GC-MS method can obtain LOQ of about 100 ìg/kg in melamine. Using SRM of GC-MS/MS, this method can reach a lower LOQ of 10 ìg/kg.
The LC-UV method is fast and does not need sample cleanup for melamine in milk products. If upgraded to ultra high performance liquid chromatography (UHPLC), this method can detect melamine within two minutes in a LC run.
The ELISA method is fast and easy to use and up to 96 samples can be run within two hours in a microplate. The results from ELISA have a possibility of cross-reaction with related compounds. If there is a nondetect, there is no need for further analysis. Other tests may have to be run if it is detected in order to meet regulatory requirements and ensure that a cross-reacting compound did not cause the response.
In any laboratory analysis, the workflow begins and ends with data. The suspect product needs to be sampled correctly, the sample data needs to be entered into a tracking system and the sample needs to be correctly tracked through preparation, analysis and reporting.
Once the report is prepared, it needs to be in an actionable format. If the product is perishable or is an ingredient in a process, paper reports that require extensive analysis will not provide the timely data required to make a decision. A modern laboratory information management system (LIMS) can be used to automate workflow, increase productivity and centralize lab data.
The testing list for melamine now extends beyond milk products or products with milk powder as an ingredient to meat, poultry, egg and vegetables.
Food ingredients distributors can ensure the safety of their products before they reach consumers.
BY PETER KAM, REGIONAL DIRECTOR-FOOD INGREDIENTS, CONNELL BROS. COMPANY
The food chain, which consists of production, processing, distribution and preparation, has generated positive interest in handling the world’s food due to new innovations and technologies. However, it also presents new concerns of global food contamination outbreak.
When food contamination is suspected in a product, stores would typically take at-risk products off their shelves until the authorities had given a green light on the safety of the product and results from investigations were reported to the industry. In the process, the issue is publicized, consumers begin to distrust the brands involved and the product categories; and product sales would drop – even after the issue is resolved.
Avoid such a scenario by having a safe, accountable and traceable supply process. There are also ways for food ingredients distributors to ensure the safety of their products before they reach consumers. Here are some of them.
Food ingredients should ideally be stored in a dedicated warehouse whenever possible, and they should be stored separately in order to prevent cross-contamination.
Food ingredients distributors need to ensure that their manufacturers are held accountable for food safety standards and are using quality ingredients with safe production, storage and transportation. Food ingredients distributors need to evaluate their personnel, techniques and procedures while tracking and monitoring products as they are distributed in the market.
Train food technologists
Ensure products are imported and delivered to customers in compliance with the highest food safety standards. Have an intensive training program that educates food technologists about the latest food safety trends in order to equip them to develop solutions and capabilities in case of a food contamination issue.
Monitor the delivery of goods constantly. Food ingredients should be transported in designated food delivery trucks or containers.
Store and transport food ingredients properly
Food ingredients should ideally be stored in a dedicated warehouse whenever possible, and they should be stored separately in order to prevent cross-contamination. Conduct routine audits on the warehouses in areas such as hygiene, pest control, cleaning/ sanitation, contamination control and food traceability. Put on record damaged food products and have a licensed disposal company to dispose them. Damaged products should not be repacked or reworked.
Monitor the delivery of goods constantly. Food ingredients should be transported in designated food delivery trucks or containers. Food contamination may arise if a truck or container carries non-food items or hazardous items with food. If it is not possible to obtain a designated food delivery truck, food materials should be shrinkwrapped and stored in separate sections of the truck.
Monitor supply chain
Certification and auditing can help food ingredients distributors to determine if manufacturers are compliant with food safety standards. Manufacturers that are certified by the International Organisation for Standardisation (ISO) or Hazard Analysis and Critical Control Point (HACCP) are committed to follow common stringent quality standards.
A ‘site audit’, where food ingredients distributors visit a manufacturing site to understand the manufacturer’s compliance to standards, also enables distributors to make sound decisions before agreeing to a business partnership.
When a food contamination incident occurs despite having precautions, food ingredients distributors need to act quickly and effectively in order minimize the impact it has on consumers. Have a system to track food deliveries and the product codes delivered. This ensures that all items are easily and efficiently accounted for in case of a product recall.
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