Determination of sugars in food provides important nutritional information but can be challenging to execute. Current methods suffer from laborious sample preparation steps, lack of specificity, and interference from complex food matrices. A high-resolution, direct detection approach can overcome these obstacles and provide a cost-effective and reliable solution.
The determination challenge
Carbohydrates are present in virtually all foods and their accurate quantification is essential for quality control in food production. Yet, determination of sugars is notoriously challenging. Not only does their lack of a chromophore make them difficult to detect, but similarity in sugar structures complicates separation.
Nevertheless, methods have been developed to overcome these shortcomings and allow determination of sugars. Enzyme-based techniques, for example, are quick, indirect methods that convert saccharides to easily measured compounds[i]. While these techniques save time, they lack specificity and can’t determine all sugars simultaneously.
Another option is high-performance liquid chromatography (HPLC). This method doesn’t require a UV or fluorescent active functional group in the sugars, as nonselective detection techniques, such as refractive index (RI), are available. With RI detection, though, mobile phase gradients cannot be used, as they induce large changes in the baseline, limiting the number of sugars that can be separated.
Gas chromatography (GC) offers another solution for sugar profile analysis. While effective, it requires costly and time-consuming derivatization of sugars, and suffers from inadequate separation[ii].
Finding the analytical sweet spot
High-performance anion-exchange chromatography (HPAE) with pulsed amperometric detection (PAD) is a great method for carbohydrate analysis that doesn’t require a choice between time or separation quality. With no derivatization required, HPAE-PAD can directly detect carbohydrates with excellent sensitivity. The technique also gives high-resolution separation, meaning many sugars can be resolved in a single injection.
Determination of nutritionally relevant carbohydrates (including galactose, glucose and sucrose) using HPAE-PAD with sodium hydroxide eluents was approved as an AOAC 2018.16 First Action Method[iii]. Now, the method has been modified to utilize dual eluent generation cartridge (EGC) mode conditions using a Thermo Scientific™ Dionex™ CarboPac™ PA20-1 mm column (Figure 1).
Figure 1: Thermo Scientific™ Dionex™ ICS-6000 HPIC system in Dual EGC mode.
We recently reported the development of the Dual EGC-mode modified approach in an application note. The Dual EGC method avoids the need to manually prepare the sodium hydroxide/sodium acetate eluents, thereby saving set-up time. Instead, the mode uses a methanesulfonic acid (MSA) EGC and a potassium hydroxide (KOH) EGC in series, which creates a highly reproducible and accurate KOH/KMSA eluent gradient needed for separating complex carbohydrates.
Dual EGC mode: accurate and precise
We evaluated the Dual EGC mode HPAE-PAD method for the determination of nutritionally relevant carbohydrates in three food samples: infant formula, whey flour and chocolate powder. As shown in Figure 2, the Dionex CarboPac PA20-1 mm column gave excellent separation of target sugars (galactose, glucose, sucrose, fructose, lactose, and maltose) from potentially interfering sugars.
Figure 2: Separation of target sugars from interference sugars using a Dionex CarboPac PA20-1 mm column set.
Our results showed the method was accurate, with sugar recovery in three spiked diluted sugar extract samples ranging from 91 to 110 percent. Results also demonstrated it was precise: the calculated area precision varied from 0.42 to 1.6 percent, with retention time precision <0.2 percent for all target carbohydrates.
A simpler and improved choice
When compared to the traditional HPAE-PAD separation of sugars with manually prepared sodium hydroxide eluent, the Dual EGC mode method showed:
- Similar resolution of sugars
- Simplified operation
- Improved retention time precision
The Dual EGC mode method modification to the AOAC 2018.16 First Action Method is therefore an optimal choice for sugar determination in food analysis, with reproducible eluent generation and excellent separation, precision and accuracy.
For more details on determining sugars with HPAE-PAD, read our application note, and keep an eye out for our next blog to learn more about determination of tromethamine in pharmaceutical formulations using an IC method.
[i] Vennard, T.R.; Ruosch, A.J.; Wejrowski, S.M.; Ellingson, D.J. Sugar Profile Method by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection in Food, Dietary Supplements, Pet Food, and Animal Feeds: First Action, 2018.16, Journal of AOAC INTERNATIONAL 2020, 103(1), 89–102.
Dr. Jeffrey Rohrer, Director of Applications Development, contributed to this blog post.
[i] Official Methods of Analysis (2012) 19th Ed., AOAC INTERNATIONAL, Rockville, MD, Method 996.11.
[ii] Li, K.; Lui, S.; Tan, Y./ Chao, N./ Tian, X./ Qi, L./ Powell, W.A.; Jiang, X.; Gai, Y. Optimized GC-MS Method to Simultaneously Quantify Acetylated Aldose, Ketose, and Alditol for Plant Tissues Based on Derivatization in a Methyl Sulfoxide/1-Methylimidazole System, J. Agric. Food Chem. 2013, 61, 4011–4018.
[iii] Vennard, T.R.; Ruosch, A.J.; Wejrowski, S.M.; Ellingson, D.J. Sugar Profile Method by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection in Food, Dietary Supplements, Pet Food, and Animal Feeds: First Action, 2018.16, Journal of AOAC INTERNATIONAL 2020, 103(1), 89–102.