Unlocking the Secrets of Carbohydrates: A Comprehensive Analysis

Due to their diversity, natural carbohydrates are increasingly being used in a variety of scientific fields, such as medicine, food industry, agriculture, etc. Enantiomers, diastereoisomers and epimers a can all be created from carbohydrate molecules. As a result, there is a growing need for reliable analysis of carbohydrates.

Sample Pre-Treatment

One of the important steps in the analysis of carbohydrates is proper sample pre-treatment. Lipids, proteins, carboxylic acids, or polyphenols in complex matrices may affect the quantitative or qualitative analysis of major components. Common sample pre-treatment methods include solvent extraction, enzyme digestion and precipitation. Before the analysis of target carbohydrates, it is necessary to select appropriate sample pre-treatment methods to reduce the interference of impurities to the analysis.

Colorimetric Methods

Among colorimetric methods, the phenol-sulfuric acid is the most reliable method to measure the content of simple sugars and oligosaccharides and their derivatives (including methylated pentose groups in polysaccharide proteoglycans, glycoproteins, and glycolipids). This method is suitable for the determination of carbohydrates composed of the same repeating unit and homopolymer, but it is not effective for complex carbohydrates. MBTH (3-methyl-2-benzothiazoline hydrazine hydrochloride) method is a suitable method for sugar detection and quantification. The detection limit of this method is low (420-500nM), and the accuracy is less than 10% at μM level. Its disadvantage is that it is time-consuming and laborious, because it involves three chemical reactions. The method that can be used to determine the content of reducing sugars is Somogyi and Nelson's (SN) method. The SN method depends on the oxidation ability of reducing sugar and the measurement of copper reagent reduction in alkali solution.

Fig. 1. Schematic illustration of monosaccharide detection based on phenol-sulphuric acid, anthrone and dinitrosalicylic acid (DNS) colorimetric methods. (Kurzyna-Szklarek M, et al.,2022)

HPLC

High performance liquid chromatography (HPLC) is considered as the most effective and innovative method for quantitative and qualitative identification of sugars because of its ability of high sensitivity, accuracy to analyze complex mixtures. The analysis speed is relatively fast and can be used in a wide concentration range and produce accurate results. Many different types of HPLC are considered useful tools for carbohydrate analysis, including ion chromatography (after bringing charges to monosaccharides), reversed-phase chromatography and hydrophilic interaction chromatography.

Table 1. Application of chromatographic methods and capillary electrophoresis for monosaccharide composition. (Kurzyna-Szklarek M, et al.,2022)

Sample	Analyte	Column	Method details	Detection
Leaves and fruits of strawberry and blueberry	glucose, fructose, sucrose, sorbitol, trehalose, arabinose, turanose, galactose, ribose, isomaltose, isomaltotriose, maltose, maltotriose, xylose, panose, rhamnose, raffinose	CarboPac PA100	Gradient 600 mM sodium hydroxide:500 mM sodium acetate: water	HPAEC-PAD
Himalayan crab apple fruits	inositol, arabinose, fructose, glucose, sucrose	Lichrospher RP-18	Gradient 0.14 M sodium acetate: mix of acetonitrile and water (60%:40%)	HPAEC-PAD
Wolfberry fruits	xylitol, fructose, glucose, sucrose, maltose	Acquity UPLCTM BEH Amide	Gradient water and acetonitrille with 0.2% triethylamine Flow rate = 0.15 mL/min	UPLC-ELSD
Food samples including fruits	fructose, glucose, sucrose	Phenomenex Luna 5u NH2 100 Å	Isocratic acetonitrile–water (78%:22%) Flow rate = 1.5 mL /min	HPLC-ELSD
Goji Berry	fructose, glucose, sucrose, maltose	Prevail Carbohydrate ES	Gradient acetonitrile–water Flow rate = 1.0 mL /min	HPLC-ELSD
Cashew apple, soursop	sucrose, fructose, glucose	Supelcogel Ca	Isocratic water Flow rate = 0.5 mL/min	HPLC-RID
Raw cocoa beans	glucose, fructose, sucrose, maltose, mannitol	Shodex Asahipak NH2 P-50 4E	Isocratic acetonitrile/water (75%:25%) Flow rate = 1.0 mL/min	HPLC-CAD
Schisandra chinensis fruits	fructose, glucose, sucrose, arabinose, galactose	Phenomenex Gemini C18	Gradient 5 mM formic acid in water; 25 mM formic acid in acetonitrile Flow rate = 0.4 mL/min	LC-MS-ESI
Apple's leaf and fruit peel	glucose, fructose, sorbitol, sucrose	Carbosep Corgel 87H3	Isocratic 5 mM sulfuric acid Flow rate = 0.3-0.7 mL/min	HPAEC-RID
Strawberry, potato	fructose, glucose, sucrose	Accucore 150 Amide HILIC	Isocratic acetonitrile: water containing 10 mM ammonium formate (84%:16%) Flow rate = 0.8 mL/min	LC-ESI-MS
Nopal	rhamnose, arabinose, xylose, galactose, glucose, galacturonic acid	Agilent Poroshell 120 EC-C18 (4.6 × 100 mm, 2.7 µm)	Isocratic acetonitrile: phosphate buffer 0.1 mol/L (84.3:15.7) pH 6.7 Flow rate = 1.8 mL/min	UHPLC-DAD (PMP derivatives)
Pears	arabinose, fucose, galactose, glucose, mannose, rhamnose, xylose	Capillary column of 30 m × 0.25 mm id coated with DB225 MS, 0.25 µm film thickness	Hydrogen Flow rate = 1.3 mL/min	GC-FID
Carob	sucrose, glucose, fructose, myo-inositol	Agilent DB-5 ms	Helium Flow rate = 1.1 mL/min	GC–MS
Blackberry	fructose, glucose, sucrose	60 cm (effective length – 8.5 cm) of 50 μm I.D.) Uncoated fused‐silica	Voltage: 25 kV BGE (optimal background electrolyte): 20 mmol/ L sorbic acid, 0.2 mmol/L CTAB and 40 mmol/L NaOH at pH 12.2	CE-DAD
Juçara fruit	sucrose, glucose, fructose.	60 cm (effective length – 8.5 cm) of 50 μm I.D.) Uncoated fused‐silica	Voltage: 25 kV BGE: 20 mmol/ L sorbic acid, 0.2 mmol/L CTAB and 40 mmol/L NaOH at pH 12.2	CE-DAD

Gas Chromatography

Gas Chromatography (GC) is another powerful tool in the carbohydrate analysis arsenal. Unlike HPLC, GC employs a gaseous mobile phase and a capillary column, making it well-suited for analyzing volatile and thermally stable carbohydrates. GC is particularly valuable for the analysis of sugar alcohols and complex carbohydrates, as well as for determining the structural configuration of monosaccharides. It offers high sensitivity and is capable of detecting trace amounts of carbohydrates in various samples.

In conclusion, the analysis of carbohydrates is a dynamic field with significant implications for various scientific disciplines. As technology continues to advance, our understanding of the chemical composition and functions of carbohydrates in nature will undoubtedly expand, offering exciting opportunities for research and application in the future.

Reference

Kurzyna-Szklarek M, Cybulska J, Zdunek A. Analysis of the chemical composition of natural carbohydrates - An overview of methods. Food Chem. 2022, 394:133466.