Ion-Moderated Partitioning for Carbohydrate Analysis

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Carbohydrate analysis using HPLC separation techniques includes two options: normal-phase and reversed-phase. Normal-phase HPLC employs a polar stationary and a nonpolar mobile phase. Reversed-phase HPLC utilizes a nonpolar hydrophobic stationary phase and a polar mobile phase for separation. This article discusses ion-moderated partitioning, a form of normal-phase HPLC for carbohydrate analysis that is often referred to as ion chromatography (IC) and is commonly used in the food and biopharmaceutical industries.

Multiple uses of IC in carbohydrate analysis

Ion chromatography has been in use for a wide variety of applications, including carbohydrate analysis in the pharmaceutical and food industries and for environmental, agricultural, and biotechnology applications. The technique was first used to reduce the hardness of water during the Industrial Revolution in Europe. Its adoption for a wide range of other applications has led to the inclusion of chapters on ion chromatography in the United States Pharmacopeia-National Formulary (USP-NF) in the past decade. Many USP-NF test procedures and assays are now available for the identification and quantification of carbohydrates and other analytes that are important factors in many industries.

USP classification

The USP has classified different IC resin-packed columns into USP numbers L1 through L116. Carbohydrate analysis columns include L17, L19, L22, L34, and L58 (Table 1).¹ These designations are referred to by the USP-NF as well as other international agencies such as the Food and Agriculture Organization (FAO), Food Chemicals Codex (FCC), and European Pharmacopeia (EP).

Table 1 – Carbohydrate analysis columns

Carbohydrate analysis in the food industry

The food industry is required by the FDA and compelled by consumers to ensure food quality and safety. Ion chromatography has been used to demonstrate food safety to meet mandatory and voluntary government standards, to ensure quality control of raw materials and final products, and to provide nutritional values for labeling and identification of ingredients.² Its use in carbohydrate analysis is also growing in food allergen testing. Foods such as dairy, gluten, shellfish, and nuts are routinely tested in order to meet regulatory guidelines for food labeling and ensure consumer health.

For such purposes, the dairy industry uses USP L34 IC columns to assess and quantify different sweeteners added to or naturally occurring in yogurt, milk, and lactose-reduced milk. Figure 1 shows the carbohydrate profile for three different yogurt flavors with different sugar contents and quantities.³ This information provides valuable nutritional information that allows consumers, including diabetics, to make betterinformed decisions.

 Figure 1 – Carbohydrate profiles of plain and flavored yogurts on a USP L34 column.

Sugar profile determination can also help identify food spoilage or degradation by microbes, by detecting sugar byproducts released due to microbial metabolism. This allows quick quality control of raw materials and thereby helps food manufacturers save considerable costs and protect consumer health. For example, sorghum, an important gluten-free grain crop, is subject to fungal stalk rot, which makes it unfit for human consumption.⁴ Carbohydrate analysis can be used to identify crops affected by this disease as sorghum stalk carbohydrate profiles change with disease (Figure 2).³

 Figure 2 – Carbohydrate profile of healthy sorghum using a USP L19 column.

Carbohydrate analysis for biomedical and pharmaceutical samples

Ion chromatography has also grown in popularity in the pharmaceutical industry, where it is used for drug authentication and detection of impurities. Impurities are defined as anything that is not the active pharmaceutical ingredient (API). These impurities can lead to loss of drug efficacy and can be mutagenic, teratogenic, or carcinogenic. This jeopardizes the safety and health of patients taking these drugs. For these reasons, impurities must be controlled and monitored. Limits and regulations have been set by international pharmacopeias including the British, Indian, United States, and European Pharmacopeias, as well as the International Conference on Harmonization (ICH). Limits are set to parts per millions (ppm) for a host of impurities identified in drug development processes.⁵

For example, IC has been used to monitor impurities in aminoglycoside antibiotics, an important class of antibiotics used in oral, topical, and injectable forms, that contain many carbohydrate moieties. Aminoglycosides do not contain chromophores for UV detection, which is one of the commonly used detection methods on an HPLC system. Aminoglycosides, however, are oxidizable, and high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) is used for its separation and detection. HPAEC-PAD is another technique for analysis of carbohydrates which are weak acids with pKa values in the 12–14 range.⁶

Excipients are another class of molecules commonly quantified by IC. Excipients are inactive ingredients used in pharmaceutical formulations as antimicrobial preservatives, stabilizers, anti-foaming agents, and tablet binders. Deaths have resulted from adverse reactions to inappropriate or poor-quality excipients in drug formulations.⁷ Excipients, like aminoglycosides, can also lack UV-absorbing chromophores but are easily oxidizable and thus detectable by HPAEC-PAD and IC. Ion chromatography can therefore be used to easily identify, resolve, and separate the different sugars and sugar alcohol isomers used in excipient formulations to ensure their quality (Figure 3). USP columns L17, L19, L34, and L58 have been used in various methods that not only allow detection and quantitation of excipients, but also analysis of their impurities.

 Figure 3 – Sugar alcohol separation using a USP L19 column.

Conclusion

Ion chromatography carbohydrate analysis is used for a wide range of applications. It is not only used in the food industry to ensure food safety and quality, but has become increasingly popular in the pharmaceutical industry, where it is used to confirm the integrity and safety of drugs, thus making IC an important contributor to human health.

References

1. www.uspnf.com/pharmacopeial-forum/pf-table-contents
2. Nielsen, S., Ed. Food Analysis, 5th ed., Springer, 2017.
3. Overview and strategies for Bio-Organic Molecule Purification, Bulletin 1928, Rev. B, 2012, Bio-Rad Laboratories.
4. Undersander, D. Sorghums, Sudangrasses, and sorghum-Sudan hybrids, 2003. Univ. of Wisconsin Focus on Forage 5:5, Madison; http:// www.uwex.edu/CES/crops/uwforage/SorghumsFOF.htm (accessed July 1, 2012).
5. Rama Rao, N.; Mani Kiran, S.S. et al. Pharmaceutical impurities: an overview. Ind. J. Pharm. Educ. Res. 2010, 44(3); https://www.researchgate. net/publication/260172609.
6. Corrandini, C.; Cavazza, A. et al. High-performance anion-exchange chromatography coupled with pulsed electrochemical detection as a powerful tool to evaluate carbohydrates of food interest: principles and applications. Int. J. Carbohyd. Chem. 2012, Article ID 487564, 13 pages; http://dx.doi.org/10.1155/2012/487564.
7. Wong, Y.L. Adverse effects of pharmaceutical excipients in drug therapy. Ann. Acad. Med. Singapore 1993 Jan, 22(1), 99–102.

Mona Chin and Payal Khandelwal, Ph.D., are global product managers for Protein Purification, Life Science Group, and Anna Quinlan, Ph.D., is editorial manager, Bio-Rad Laboratories, Inc., 6000 James Watson Dr., Hercules, CA 94547, U.S.A.; tel.: 510-741-5831; e-mail: [email protected]; www.bio-rad.com