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In this Interview, Tim Lumb (ALS Global, a leading global food testing organisation) and Dr. Kevin Nott (Oxford Instruments) discuss benchtop NMR techniques in food analysis, outlining some of the benefits that such a technique can bring to the laboratory environment.
Conventional high field NMR utilizes superconducting magnets, cryogens such as liquid helium and liquid nitrogen, and a wide range of specialized facilities, which includes a suitable air supply. These systems also require trained experts to maintain and supervise their use.
However, NMR instrumentation is not held back by these disadvantages because they leverage permanent magnets and are, therefore, cryogen-free. These instruments tend to be rather compact, needing only a main power supply, which means that they can be located in a standard laboratory setting.
Two types of benchtop NMR instrumentation are on offer. High-resolution NMR spectroscopy utilizes high magnetic field strength and homogeneity for the visualization of chemical information or spectra from an array of nuclei, such as hydrogen, carbon, sodium and phosphorus - all of which are of significant interest in food science. It is important to note that each of these samples should be either liquid or contained in a solution.
On the other hand, low-resolution Time Domain NMR (TD-NMR) leverages a reduced magnetic field strength and homogeneity for processing large sample sizes; therefore, it is impossible to obtain chemical information.
However, there are far fewer restrictions regarding the size and type of sample that can be analyzed due to the fact that radio frequency radiation can penetrate the entire sample, which may be solid, liquid, or a mixture of both.
Food companies generally employ Time Domain NMR analyzers in process and quality control, as well as in testing laboratories. The key requirement for both time-domain and spectroscopy is that the sample does not impede the magnetic field or stop the radiofrequency radiation from being able to penetrate it.
These already tend to be well resolved in some samples, and the signals can be read directly from the composite signal. This is standard in a number of time-domain NMR applications, but we may need to leverage fitting software to deconvolute the composite signal into a range of components in other samples.
This is typically known as ‘relaxometry.’ The free induction decay signal is comprised of various components, which can be categorized by their phase or state. The signal from solid and crystalline components decays rapidly, while the signal from solid-like components, such as residual and bound moisture (concentration below 10%), demonstrates slower decay rates. Comparatively, oils and fats show the slowest decay, making it possible to measure them when signals from all the other components have decayed.
For decades, TD-NMR has been applied as a solvent-free method to measure oil and fat in food. It is mostly used for quality control but can also be employed to help set up and monitor production lines for snack foods, such as nuts, chocolate and cocoa derivatives that are used in their manufacture.
Furthermore, it can also perform analysis of milk powder, infant and nutritional formula, food ingredients, and animal food without needing a drying step. Typically, foods with high moisture content, such as meat, fish, dairy and many processed foods, must be dried in advance. For instance, TD-NMR can be employed in accordance with the Nordic Committee on Food Analysis (NMKL) method for the measurement of dried fish products, by which simple calibration of the instrument is conducted using a cod liver oil sample.
There are other scenarios where it is possible to calibrate using pure oil, especially when working with processed foods and foods which contain high-fat contents. For instance, it is possible to calibrate certain snack foods using a sample of the pure oil used in the frying process. One of the leading advantages that TD-NMR offers over other secondary techniques is that it is generally insensitive to color, particle size and composition.
NMR calibrations are also linear, so results are generally valid even if the sample for analysis is outside of the existing concentration range. TD-NMR can clearly identify solids from liquids, making it possible to measure solid fat content at a range of temperatures, which refers to the melting properties of the edible oil or fat.
As well as pure oils, fats or blends, solid fat content is typically measured for both dairy and non-dairy spreads, including butter and margarine, as well as fats generally used across the bakery and confectionery industries.
The melting profile of fat usually confirms its sensory and physical properties, which is key for its eventual end-use. For instance, shortenings typically possess a greater content of solid fat across the temperature range to ensure the baked goods in which they are introduced remain solid but not greasy at ambient temperatures.
In contrast, the solid fat content of margarine is lower to ensure that it is soft and spreadable when removed from the fridge. TD-NMR is an internationally recognized methodology for measuring solid fat, and their official methods for the direct method include AOCS, ISO and IUPAC.
Benchtop NMR spectroscopy systems are generally compared to their high field counterparts. At just a fraction of the capital cost to purchase, these systems are less expensive to buy and are very cheap to run as cryogens are not required; the latter is a distinct advantage due to recent helium shortages.
Additionally, maintaining and running the instrument requires minimal expertise. Even though it has only been in use for the past ten years, benchtop NMR spectroscopy is now considered a mature field. The powerful combination of enhanced magnet design, state-of-the-art electronics and computing power has led to the development of numerous industrial applications, especially in the food sector.
Oxford Instruments has been working closely with Dr. Kate Kemsley and her group at the Quadram Institute in the UK to evaluate whether benchtop NMR spectroscopy of hydrogen nuclei could offer compositional information appropriate for labelling.
Edible oils are primarily comprised of triglycerides. These are made up of a glycerol backbone bound to three fatty acids, which vary in length and numerous unsaturated bonds between and within oils. With regard to food composition, the primary interest is establishing the proportions of monounsaturated, polyunsaturated and saturated fatty acids.
Spectra will typically display clear differences and similarities between oil types, most distinctly in terms of the band height or areas. The glyceride region is analogous across all oils and can serve as an internal standard and chemical shift reference.
Olefinic bands emerge from any hydrogen atom affixed to carbon that is involved in a double bond. Bis-allylic bands are produced by hydrogen atoms attached to carbons situated in between pairs of double bonds. Kemsley was able to calculate the number of monounsaturated, polyunsaturated and saturated fatty acids in oils directly by unpicking these other signals.
This meant that the team was able to find a decent correlation between the results from the NMR method and those from gas chromatography for a set of test oils. They also discovered that the relationships between predicted and actual values are greater for monounsaturated and polyunsaturated fatty acids. The correlation for saturated fatty acids was slightly lower but still acceptable. This covered a narrower concentration range in contrast to the others.
The result of this work was the development of a five-minute test on the purity of oils. This analysis method has since been inserted into a simple software tool, which sends the monounsaturated, polyunsaturated and saturated fatty acid values back for each spectrum. The team was able to determine and quantify the concentration of omega-3 fatty acids. In addition to the compositional data, benchtop NMR can also offer a fingerprint that can be used for potential authentication.
Kate Kemsley’s group then examined benchtop NMR spectroscopy to see whether it could offer a solution for fraud detection in the coffee sector. Nearly all commercial production derives from just two different species of coffee. Arabica is the most in-demand, but it is harder to grow, while Robusta delivers higher yields but has limited sensory properties meaning it is used in lower-grade coffee products.
These differences are reflected in the trading prices. The limit of detection for Robusta in Arabica coffee is between 1% and 2%, which is at the boundary between what could be considered accidental contamination and that which is fraudulent. Kemsley’s group was able to identify Robusta coffee by the occurrence of a chemical shift which was shown as a 16-Omethylcafestol peak at 3.16 ppm.
Previously, it was believed that this cafestol derivative was present in Arabica, but the team soon discovered this was not the case by analyzing a range of beans known to be authentic. They also developed a software tool to perform this type of analysis.
Across most of Europe, we are now seeing TD-NMR being accepted as the standard method. Popularity is also growing in Asia, Australia and Latin America, and we are witnessing the acceptance of NMR for total fat analysis in the USA and for products such as meat and dairy. Many standard methods now refer to TD-NMR methods.
ALS Global started to work with Oxford Instruments on TD-NMR in 2008. Before that, we employed a conventional acid hydrolysis technique for the analysis of total fat – which is a long process with a series of steps.
Firstly, we took a chunk of sample and weighed this out into a vessel. We then introduced concentrated hydrochloric acid and heated this to boiling point for several hours before washing and drying this to eliminate the remaining aqueous acid. We then boiled petroleum ether and let this condensate and drip back through the sample, pulling out the fat with it.
After finishing this part of the process, the solvents were then extracted by evaporation or collecting the solvent for reuse in later extractions. Finally, the remaining oil was weighed. The way that TD-NMR functions in this process is precisely why many laboratories covet this technique.
Water presents a real challenge to TD-NMR, especially if levels greater than 10% water are present, which must be dried away. There are two major techniques for doing this. The first involves drying samples in an oven for approximately 16 hours. This method can be employed to dry numerous samples at once, and while there are rapid drying methods, these tend to only be appropriate for one sample at a time.
As well as knowing how much fat is in a product, a number of manufacturers want to know how much water is present. Weighing the samples once drying is complete allows manufacturers to calculate the moisture content of samples. Subject to how the NMR is set up, analysis usually involves reporting an average result by taking between eight and 16 specific measurements over a 20-second period.
The impact of TD-NMRs on the food industry has revolved around cost control. Since these methods were introduced, we have seen a huge increase in labor and consumable costs. The cost to the laboratories only goes one way. Yet, when we focus on total fat analysis, a major parameter for all food manufacturers and retailers, a significant decrease in the cost of that analysis can be seen over that same period.
Today, prices for total fat analysis are now 25% of the price that it was in 2008 due to this change of method. Utilizing acid hydrolysis for most samples would not allow effective cost control across the industry, leading to increased costs throughout the supply chain to the final consumer.
The precision of TD-NMR has also had a significant impact on the industry. This precision is crucial because the trueness or bias of the method may see considerable variability based on how well the calibration is set up. Irrespective of how that calibration is assembled, the precision of NMR will always surpass that of the manual method.
We have seen improvements in the quality of proficiency tests, and over time, there has been an overall reduction in RSD (Relative Standard Deviation) as an increasing number of laboratories have adopted NMR. Laboratories have benefitted significantly from TD-NMR over the past ten years, but there is still more that can be done with this technology, especially as the industry starts utilizing benchtop NMR spectrometers operating at 60 MHz.
For instance, TD-NMR can reveal how much total oil and fat is in a material, but benchtop NMR spectroscopy can be used to characterize the oil and determine what fat-soluble compounds and other components are present. These may include groups of or individual fatty acids within foodstuffs, as well as sterols, stanols and cholesterols. It is already possible to see and characterize these components, but as the technology progresses, we will be able to quantify those using innovative hydrogen and carbon-13 NMR techniques.
The real potential and scope of benchtop NMR are held in its characterization capabilities, and that is where it is a key tool in the authentication of food products and detecting fraud. One such study employed benchtop NMR to authenticate argan oil. The study started with an examination of the spectra for pure oil and assessed how oil with just a small amount of adulteration influenced a change in the spectra’s peaks.
These peaks may vary depending on the nature and degree of contamination, enabling us to identify the likelihood of contamination of a certain substance. This is extremely practical when conducting root cause analysis into an adulteration issue to establish if it stemmed from deliberate adulteration or accidental contamination within the factory itself.
Besides oils, benchtop NMR can also be a useful tool for validating the authenticity of coffee, as we have previously mentioned as well as high-value herbs and spices. Many herbs and spices are comprised of essential fatty compounds in part; thus, benchtop NMR can be employed to confirm their authenticity. For instance, herbs such as Oregano include aromatics and phenolics that are unique to that particular spice. If this was cut with olive leaves, then one would expect to observe a reduction in some of those aromatics or see the appearance of other properties from another source.
This approach was applied to other spices, such as cumin and paprika. One of the key applications of NMR and high-field NMR is its ability to identify if honey has undergone adulteration.
Watch the webinar "Benchtop NMR analysis and the future for the food industry" here
Honey and urine are related in terms of their potential for solvent suppression on NMR. Substantial efforts have been made across clinical settings to identify specific compounds in urine to diagnose diseases by investigating specific metabolites that should or should not be present in urine.
One of benchtop NMR’s most challenging historical issues is associated with the size of the water peak. In some matrices, it is difficult to detect anything else when the water peak is excessively large. However, modern solvent suppression on NMR enables all of these analytes of interest to be revealed, including organic acids, proteins and sugars.
These components can also be found in a number of foodstuffs, and one of the most common uses for high-field NMR is looking at these specific components. When investigating a standard honey spectrum on a high field spectrometer, the acids, proteins and sugars can be observed.
If we are able to evaluate this level of detail using benchtop NMR, this could revolutionize the means of validating and authenticating foodstuffs’ using several other parameters beyond those fatty components.
Contact our food analysis experts
Tim Lumb - ALS Global : Food Chemistry Technical Manager (UK), Food Chemistry Technical Co-ordinator (Europe) and Life Sciences Innovation Co-ordinator (Europe). Tim has been working in the food industry for 15 years, starting as a Laboratory Technician studying nutrients in a range of foodstuffs. After holding various management and technical roles both in the UK and Europe, Tim now holds the position of Food Chemistry Technical Manager (UK), Food Chemistry Technical Co-ordinator (Europe) and Life Sciences Innovation Co-ordinator (Europe) at ALS. In these roles Tim is responsible for all technical development for the food business in the UK – advising producers, manufacturers and retailers of the most appropriate means to verify that the food they produce and sell is safe, nutritious and authentic. Tim has been working with NMR instruments from Oxford Instruments since 2008.
Dr Kevin Nott - Oxford Instruments: Product Manager. Kevin has worked at Oxford Instruments since May 2005. Kevin was previously at the University of Cambridge where he researched into non-medical applications of TD-NMR and MRI.
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