Food adulteration, whether intentional or accidental, poses a risk to consumer health and defames food manufacturers. In addition to maintaining best manufacturing practices, it is crucial for food scientists to develop reliable methods to test food quality, detect traces of unauthorized adulterants, and remain compliant with regulatory requirements. For a variety of food products, carbohydrate components serve as authenticity markers and are often used to validate food quality.
Despite their widespread use, analytical techniques such as liquid chromatography (LC) or gas chromatography (GC) often present challenges when it comes to obtaining accurate carbohydrate measurements, compromising important information at the expense of public health. Here, we explain why it’s necessary to choose sensitive and robust methods for carbohydrate analysis within the food industry. We also discuss how using high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAE-PAD) can identify food adulterants with increased confidence.
Carbohydrate Detection: The Need for Sensitive and Robust Methods
Carbohydrate profiles in certain foods, such as honey, agave syrups, fruit juices, and coffee, act as markers for authenticity and can be used to detect food fraud. Adulteration of honey or agave syrup can involve their dilution with cheaper, often unhealthy alternatives, such as high fructose corn syrup or saccharose syrup, produced from beets or canes. In these instances, analytical methods that can accurately measure sucrose levels in honey or perform oligosaccharide profiling in agave syrup help distinguish the genuine food products from their fraudulent counterparts.
The familiarity and widespread use of LC and GC prompt scientists to use these techniques as a default approach for carbohydrate analysis; however, these methods aren’t the best choice to detect, measure, and study carbohydrates. The high polarity of carbohydrates makes them difficult to reliably retain and separate using reverse phase chromatography. As carbohydrates are weakly acidic, dissolving high concentrations of them imparts higher acidity to the samples. At extreme pH levels, metals from the column’s surface strip away and adhere to the packing materials, tampering with the column’s integrity. Moreover, the inherent viscosity of these samples will also require optimized column heating to ensure a consistent flow through the column. Any fluctuations to lower temperatures can result in changes in viscosity of the sugar samples, causing them to stick to the column surface, generating backpressure and making the method irreproducible.
Additionally, carbohydrates tend to have very few chromophore groups and cannot, therefore, be detected with adequate sensitivity using ultraviolet (UV)-based detectors. Switching to refractive index or low-wavelength UV detection methods prevents the use of gradients due to their sensitivity to the eluent and sample matrix components. Gradients can also increase the baseline noise, thereby reducing the signal-to-noise ratio and decreasing the sensitivity of measurements.
Relying on discrete elution times is also challenging as the monosaccharide components, glucose and fructose, both have the same molecular weights of 180.16 g/mol. When in solution, they also both exist in ring forms, making them indistinguishable, especially given the lack of chromophores. Using a strong base can push the equilibrium to one side and stop the interconversion between ring and chain structures, providing a slight difference in retention time. However, at higher base concentrations, the monosaccharides are not retained for too long and will elute out very quickly.
One option to retain carbohydrates and boost sensitivity for measurement is to derivatize the samples. Though several isomers and chains of a carbohydrate molecule can be derivatized, requiring a summation to yield the total result for one carbohydrate can make method validation complicated, laborious, and time-consuming. Furthermore, due to the diversity of sample matrices used in the food industry, a thorough sample cleanup prior to injection is often necessary to prevent any assay carryovers, making the sample preparation process more tedious.
HPAE, on the other hand, is a chromatographic technique better suited to separate, detect, and measure carbohydrates as food authenticity markers. It takes advantage of the weakly acidic nature of carbohydrates for highly selective separations at high pH using strong anion exchange stationary phases. At higher pH, carbohydrates are partially ionized and can, therefore, be separated by anion exchange mechanisms.
Coupling HPAE with PAD allows direct quantification of nonderivatized carbohydrates at even low-picomole levels with minimal sample preparation and cleanup. The direct form of analysis precludes any biased selectivity toward certain carbohydrate structures, as may be seen in other analytical methods measuring derivatized sugars. This simplifies method validation and brings much-needed reproducibility to these techniques, enabling intra- and inter-batch testing for quality control.
There are two key reasons why HPAE-PAD is more selective and specific for carbohydrate analysis compared to LC or GC approaches. First, the specific detection voltages used in the pulsed amperometry ensure that it only measures analytes that are oxidizable at those particular voltages. In the case of carbohydrate analysis, the settings provide a sensitivity that is several orders of magnitude greater than other classes of analytes. Second, due to the anion exchange separation, neutral or cationic sample components that may be oxidizable at the same voltages elute into or closer to the void volume of the column, thereby removing any analyte that may otherwise interfere with the carbohydrate analysis.
Food Safety Testing with HPAE-PAD
When it comes to food safety, the data obtained are only as good as the method used. HPAE-PAD methods are commonly used to detect and quantify unauthorized additives in food products that have carbohydrates as their quality markers. Additionally, the method is regularly used to characterize the carbohydrate components present in the food sample to gain deeper insights into their composition, serving as another testing parameter for future measurements. Below, we have listed how HPAE-PAD can be used to perform safety testing and combat food fraud in popular food items.
Honey. Composed of several sugars based on its floral source, honey is tested for adulteration using sucrose as its quality indicator. Adding cheap sweeteners, such as cane sugar or refined beet sugar, can artificially increase the levels of sucrose in honey. The Codex Alimentarius Committee on Sugars has, therefore, specified the maximum value of sucrose as 5 g in 100 g of honey. Carbohydrate analysis with HPAE-PAD can be used to measure these parameters as well as determine the floral origins of honey, using a few minor sugars as a “fingerprint.” Using the Thermo Scientific Dionex CarboPac PA210-4μm column in an HPAE-PAD protocol allows for the separation of 15 sugars in honey with minimal sample preparation, 80-120% precision and accuracy, and a detection limit of as low as 10% adulteration with added syrups.
Agave syrup. Another food product that has recently become a target for food fraud due to its growing popularity is agave syrup. An alternative to traditional sweeteners, such as table sugar and honey, agave syrup has a low glycemic index, causing a slower rise in blood glucose and insulin levels. As most of its sugars are in fructose form with very little glucose, adulteration with high fructose corn syrup is common. The main producer of agave, Mexico, has recently created a governmentally approved guideline for the characterization of pure agave syrup. In the method prescribed by the Norma Oficial Mexicana, HPAE-PAD is used to determine levels of the main sugars (fructose, glucose, and sucrose), polyols (sorbitol, mannitol), and 5-hydroxymethyfural. After the agave syrup is diluted with water, the carbohydrate profiles are analyzed before and after enzymatic hydrolysis with amyloglucosidase and fructanase to measure the content of sugars as well as fructan.
Fruit juices. The billion-dollar fruit juice industry often encounters dilution and blending with inexpensive and synthetic sweeteners, a ploy designed to achieve higher margins and larger economic gains. A common adulterant known as medium invert sugar, in which one half of the sucrose has been hydrolyzed to glucose and fructose, closely matches the composition ratio of approximately 1:1:2 (glucose: fructose: sucrose) found in orange juice. When cane sugar is the source of the invert sugar, stable isotope ratio analysis (SIRA) can be used to detect adulteration due to the differing ratios of 13C:12C in orange juice and cane sugar; however, if beets are used to produce the invert sugar, the 13C:12C ratio between orange juice and beet sugar do not differ much as the sugars are produced using similar metabolic pathways. In this case, SIRA can no longer detect adulteration by beet sugar, providing a convenient loophole in food fraud.
Scientists have resorted to HPAE-PAD to characterize beet invert sugar and discover several sugar components that are not present in orange juice. One such sugar not found in pure orange juice is raffinose, a trisaccharide of D-glucose, D-fructose, and D-galactose, which has been used as an adulteration marker for orange juice. Additionally, the signature pattern of late-eluting components appearing at about 60 minutes during the HPAE-PAD run can also be used to identify adulteration.
Coffee. Carbohydrates also serve as tracers to assess the authenticity of instant coffee. Although an unlikely candidate for sugar analysis due to its characteristic bitter taste, at least 50 percent of the dry weight of raw coffee beans comprises coffee carbohydrates. As these undergo Maillard reaction during the roasting process, they contribute to the flavor, aroma, and viscosity of coffee. An HPAE-PAD-based method to determine the free and total carbohydrates in instant coffee has been prescribed by the Association of Analytical Chemists (AOAC) Official Method 995.136 and is currently used by the British Standards Institution. In a recent application study, the AOAC method was tested using the Thermo Scientific Dionex CarboPac SA10 column. The former method, which typically has a run time of 80 minutes, was made significantly faster by using the column. The quicker method had a run time of 10 minutes, only needed deionized water for continuous operation, and offered the same level of accuracy and sensitivity, differing only in its total analysis time and number of resolved peaks for coffee carbohydrate analysis.
All the food testing examples mentioned above justify the argument that, with the increasing demand for reproducible, fast, and simple methods to profile a wide variety of analytes in the food industry, HPAE-PAD has steadily emerged as a reliable method of choice to analyze carbohydrates.
Better Methods, Safer Food
As traditional methods used in the food industry start becoming outdated, new and problematic adulterants that are similar in structure to the genuine components can sneak into the food industry by taking advantage of either inadequate sensitivity or lack of specificity. Food testing laboratories will need to continually evaluate, test, and validate new methods to stay ahead of food fraud, while keeping up to date with regulations. Similarly, upgrading conventional methods with the latest technology makes them more robust and productive. Choosing the most appropriate method to accurately detect carbohydrate-based authenticity markers, such as HPAE-PAD, will result in safer food for the community and sustain consumer trust with the manufacturer.
Man is product marketing manager, IC/SP, chromatography and mass spectrometry for Thermo Fisher Scientific. Reach her at firstname.lastname@example.org.