Testing for Toxins: How Can NIR Be Used to Identify Toxins?

In the detective’s office, Eggcule tells the detectives about his time learning about traditional port production methods in the Douro Valley. Suddenly, a distressed Shallot Holmes bursts through the door. “What’s the matter, Holmes?” asks Nancy Beef. “I’ve been… contacted… by a… government agency,” says Holmes out of breath. “This isn’t anything to do with what happened in the forest in Dorset, is it?” asks Eggcule, looking nervous. “No, why, what did happen in the forest?” asks Holmes. “Umm… nothing really… anyway, what did the agency want?” asks Eggcule trying to change the conversation.

Shallot Holmes explains the details to the detectives. A government agency has been informed of a fraud case involving powdered baby milk that has caused a severe health risk. “The milk in question was found to contain melamine, which has resulted in kidney stones and other kidney damage in infants,” says Holmes. “Melamine, isn’t that used for making plastic? Why would there be melamine in milk?” asks Miss Mapple. “Melamine is rich in nitrogen and relatively cheap,” says Holmes. “So, someone has been trying to make the protein content appear higher,” says Miss Mapple. “Protein content… how does adding melamine increase the protein content?” asks Cornlumbo. “Standard quality tests to estimate protein levels measure the nitrogen content in a sample using a conversion factor,” explains Miss Mapple. “How does that work?” asks Cornlumbo.

What is the Kjeldahl Method?

This most common method is the Kjeldahl method, which is based on the fact that proteins are composed of amino acids containing nitrogen. The Kjeldahl method involves:

• Digestion: The sample is digested with sulfuric acid to convert the nitrogen into ammonium sulfate. 
• Alkalization and Distillation: The digest is neutralized with a strong base (usually sodium hydroxide), which converts ammonium sulfate into ammonia gas that gets distilled and absorbed in a known volume of standard acid. 
• Titration: Titration with a standard acid solution determines the amount of ammonia.
• Nitrogen Content Calculation: The nitrogen content is calculated based on the amount of ammonia produced.
• Protein Content Calculation: The nitrogen content is multiplied by a conversion factor to estimate protein content. Proteins, on average, contain 16% nitrogen, so a conversion factor of 6.25 is used (100/16 = 6.25).

How can you quantify melamine?

“Melamine is rich in nitrogen and relatively cheap; therefore, adding it to sub-standard milk raises the milk’s perceived protein levels. As Miss Mapple pointed out, the Kjeldahl method would not have identified the melamine, only the nitrogen content,” explains Holmes. “So how do you quantify the melamine?” asks Cornlumbo. “Analytical methods such as high-performance liquid chromatography (prep HPLC), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS) are excellent at determining such things,” says Miss Mapple. “You’re right, but the agency has a problem. The milk containing the melamine was donated, and the agency is unsure where it came from. The methods you suggested are expensive and time-consuming. We need to find the agency a simple, accurate, and inexpensive technique to detect melamine in milk powder,” says Holmes. 

“What about NIR spectroscopy?” suggests Nancy Beef. “That is fast and cheap, but not typically used for food safety testing as toxins are generally contained in such low amounts that they cannot accurately be quantified using NIR, which is a method for the percentage range,” says Miss Mapple. “Perhaps we don’t need to quantify the amount,” says Holmes, pacing back and forth. “I don’t understand, how can we know how much melamine is in the milk without quantification?” asks Miss Mapple. “The agency's first problem is finding out where the milk containing melamine originated. This will involve testing milk from potentially hundreds of producers, which would take ages and be very expensive using pHPLC or other such methods. Although NIR could not accurately quantify the levels of toxins or adulterants, it could be used to identify their presence,” says Holmes. 

How can you identify melamine using NIR?

Holmes gets the detectives to adulterate some milk powder samples with melamine and begins to analyze the spectral data obtained using NIR. After a short period of analysis, Holmes notices some unique spectral features of the melamine-contaminated samples. To increase the pattern recognition rate and reduce the computation time, Holmes performs another analysis using only the wavelength where melamine was detected. “And there it is, in the 5300-4900cm-1 region. By focusing just on this area using NIR, the agency will be able to detect the presence of melamine in milk. Once they have found the contaminated batch, they can then use traditional methods, such as HPLC, to accurately quantify the precise amount of melamine present. Can anyone tell me what the limits are for the amount of melamine present in milk?” Holmes asks. “Let me see,” says Miss Mapple. “It seems there is global agreement among the various international agencies. The FDA, the EU, and the Codex Alimentarius that provides international standards all agree that the levels of melamine should not exceed 2.5 ppm for foods and animal feed, and no more than 1 ppm for infant formula,” says Miss Mapple. “What’s ppm?” asks Cornlumbo. “Parts Per Million, it is equivalent to one part of a substance to one million parts of the total mixture,” explains Miss Mapple. 

“Great work,” says Holmes as he dashes off to inform the client of their findings. “Great work indeed, detectives; how about we celebrate with some of the port I was given for solving my last case?” asks Eggcule. “Excellent idea,” says Cornlumbo. “Just remember Cornlumbo, only 1 PPD,” says Eggcule. “PPD?” says Cornlumbo, looking confused. “Yes, only 1 PPD, 1 Port Per Detective!” says Eggcule to the amusement of his colleagues.