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Derivatization of sugars for GC-MS (Part 1): Analytical challenges and methods

8 Jun 2022

While evaluating the performance and selectivity of Restek’s Rtx-225, I came across a number of papers using 225 phase columns for separation of derivatized sugars, specifically, alditol acetate derivatives1–6. Sugars inform us about topics such as health, safety, and sustainability in fields like nutrition, medicine, and energy7–12. Considering the wide range of applications for sugar analyses, the popularity of derivatizations is noteworthy. Extra chemical reactions are usually unappealing for researchers, but in the case of sugars, are necessary. Sugars are highly polar, which complicates separation using LC methods, and they are non-volatile, which complicates separation by GC methods. Derivatization can be used to improve performance on both systems by adjusting polarity or volatility. The mass spectrometer detector is exceedingly helpful when analyzing unknown mixtures of sugar13,14, but derivatization can be avoided by using LC with a UV or RI detector9–11,15.

For GC analysis, there are a variety of derivatization methods, but the most common involve silylation or acetylation. In 1979, Knapp et al. suggested that trimethylsilyl (TMS) derivatizations were the preferred approach for analyzing sugars, despite their propensity to produce multiple isomers, and subsequent chromatographic peaks16. Work by Haas et al. (2018) now shows that adding an oximation step prior to TMS or trifluroacetyl (TFA) derivatization reduces the number of isomers (and chromatographic peaks) to just two, and improves separation of mixtures17. Lv et al. (2015), has also demonstrated that (in drug chemistry) alditol acetates are now the preferred derivative18. The TFA- or TMS- oximation approach is simple and effective for a wide range of sugars, but produces two isomers (Figure 1), which may complicate quantitation. The alditol acetatylation approach is effective for creating just one derivative peak (Figure 1), but multiple sugars may create the same derivative when carbonyl groups are reduced to hydroxyls (ex: arabinose and lyxose)16. Identification of sugars is a fascinating and complex field, but for the sake of this blog series, I will be focusing on the chromatographic performance of a few common, relatively simple, sugars (rhamnose, mannose, galactose, xylose, fucose, glucose, ribose, fructose, and arabinose) using three derivatization methods: TMS-oximation, TFA-oximation, and alditol acetylation.

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Figure 1: Chemical structures of various glucose derivatives.

In this series, I will provide details of my experience analyzing sugars by GC-MS. Methods for TMS- and TFA-oximation were based on those described by Haas et al, while my method for forming alditol acetates is a simplified combination of methods described by Reay et al., and Englyst et al17,19,20. All three methods involved two-step derivatizations. As expected, the alditol acetates produced one peak for each derivatized sugar, while the TMS- and TFA-oximes produced two peaks each.

Table 1: Methods for derivatizing sugars prior to GC-MS analysis

Alditol acetates TMS-oximes TFA-oximes
  1. Dissolve 2 mg sugar in 60 uL of 10 mg/L sodium borohydride in n-methylimidazole, and 250uL of water, then heat at 37oC for 90 minutes, then stop the reaction with 20 uL of glacial acetic acid.
  2. Allow sample to cool to room temperature for about five minutes, add 600 uL acetic anhydride, and heat again at 37oC for 45 minutes.
  3. Stop the reaction by freezing the samples (-15oC) for 15 minutes.
  4. Carefully quench with the dropwise addition of 2.5 mL water.
  5. Extract with 2 mL chloroform (bottom layer) three times, combine, evaporate to dryness, then re-constitute in 1.5 mL chloroform. The final concentration should be approximately 1.33 mg/mL alditol acetate derivative in chloroform.
  1. Dissolve 2 mg sugar in 200 uL in 40 mg/mL EtOx in pyridine and heat at 70oC for 30 min.
  2. Allow samples to cool to room temperature for about 5 minutes then add 120 uL BSTFA and heat at 70oC for 30 min.
  3. Dilute in 320uL ethyl acetate for an approximate final concentration of 3.125 mg/mL TMS-oxime in ethyl acetate.
  1. Dissolve 2 mg sugar in 200 uL of 40 mg/mL EtOx in pyridine and heat at 70oC for 30 min.
  2. Allow samples to cool to room temperature for about 5 minutes then add 120 uL MBTFA and heat at 70oC for 30 min.
  3. Dilute in 320uL ethyl acetate for an approximate final concentration of 3.125 mg/mL TFA-oxime in ethyl acetate.

BSTFA and MBTFA are known for their sensitivity to water. Sugars are hydroscopic and readily absorb water from the atmosphere, so all samples were stored in a dehydrator until analysis. While my derivatizations with BSTFA were successful, I had trouble with the MBTFA. Assuming my difficulty with MBTFA was related to the presence of water in the samples or reagents, I added 30-60 mg of 5A, 8/12, crushed molecular sieve (activated at 300oC for 2 hours then cooled to room temperature). The sieve was effective in improving derivatization with MBTFA, but appeared to reduce peak heights, likely due to some retention of the derivatized sugars in addition to any water that may be present. More validation work would be necessary to use the sieve for quantitative methods, but for qualitative purposes, it was effective.

Performance on the Rtx-225 varied between the three derivatization methods. Part 2 of this series will explore how well the difference derivatives separated using the Rtx-225, and strategies for using EZGC to optimize separation for your research. Part 3 will discuss good practices for instrument maintenance, and Part 4 will explore automation of the TMS-oximation derivatization.

Further reading:

  1. Pawlaczyk-Graja, I. Polyphenolic-Polysaccharide Conjugates from Flowers and Fruits of Single-Seeded Hawthorn (Crataegus Monogyna Jacq.): Chemical Profiles and Mechanisms of Anticoagulant Activity. Int. J. Biol. Macromol. 2018, 116, 869–879. https://doi.org/10.1016/j.ijbiomac.2018.05.101.
  2. Fraś, A.; Gołębiewski, D.; Gołębiewska, K.; Mańkowski, D. R.; Gzowska, M.; Boros, D. Triticale-Oat Bread as a New Product Rich in Bioactive and Nutrient Components. J. Cereal Sci. 2018, 82, 146–154. https://doi.org/10.1016/j.jcs.2018.05.001.
  3. Tsirigotis-Maniecka, M.; Pawlaczyk-Graja, I.; Ziewiecki, R.; Balicki, S.; Matulová, M.; Capek, P.; Czechowski, F.; Gancarz, R. The Polyphenolic-Polysaccharide Complex of Agrimonia Eupatoria L. as an Indirect Thrombin Inhibitor - Isolation and Chemical Characterization. Int. J. Biol. Macromol. 2019, 125, 124–132. https://doi.org/10.1016/j.ijbiomac.2018.12.017.
  4. Pawlaczyk-Graja, I.; Balicki, S.; Wilk, K. A. Effect of Various Extraction Methods on the Structure of Polyphenolic-Polysaccharide Conjugates from Fragaria Vesca L. Leaf. Int. J. Biol. Macromol. 2019, 130, 664–674. https://doi.org/10.1016/j.ijbiomac.2019.03.013.
  5. Wu, J.; Elliston, A.; Le Gall, G.; Colquhoun, I. J.; Collins, S. R. A.; Wood, I. P.; Dicks, J.; Roberts, I. N.; Waldron, K. W. Optimising Conditions for Bioethanol Production from Rice Husk and Rice Straw: Effects of Pre-Treatment on Liquor Composition and Fermentation Inhibitors. Biotechnol. Biofuels 2018, 11 (1), 62. https://doi.org/10.1186/s13068-018-1062-7.
  6. Gołębiewska, K.; Fraś, A.; Gołębiewski, D.; Mańkowski, D. R.; Boros, D. Content of Nutrient and Bioactive Non-Nutrient Components in Different Oat Products. Qual. Assur. Saf. Crops Foods 2018, 10 (3), 307–313. https://doi.org/10.3920/QAS2018.1283.
  7. Staš, M.; Auersvald, M.; Kejla, L.; Vrtiška, D.; Kroufek, J.; Kubička, D. Quantitative Analysis of Pyrolysis Bio-Oils: A Review. TrAC Trends Anal. Chem. 2020, 126, 115857. https://doi.org/10.1016/j.trac.2020.115857.
  8. Tarasov, D.; Leitch, M.; Fatehi, P. Lignin–Carbohydrate Complexes: Properties, Applications, Analyses, and Methods of Extraction: A Review. Biotechnol. Biofuels 2018, 11 (1), 269. https://doi.org/10.1186/s13068-018-1262-1.
  9. Ren, Y.; Bai, Y.; Zhang, Z.; Cai, W.; Del Rio Flores, A. The Preparation and Structure Analysis Methods of Natural Polysaccharides of Plants and Fungi: A Review of Recent Development. Molecules 2019, 24 (17), 3122. https://doi.org/10.3390/molecules24173122.
  10. Al-Sanea, M. M.; Gamal, M. Critical Analytical Review: Rare and Recent Applications of Refractive Index Detector in HPLC Chromatographic Drug Analysis. Microchem. J. 2022, 178, 107339. https://doi.org/10.1016/j.microc.2022.107339.
  11. Cortés-Herrera, C.; Artavia, G.; Leiva, A.; Granados-Chinchilla, F. Liquid Chromatography Analysis of Common Nutritional Components, in Feed and Food. Foods 2018, 8 (1), 1. https://doi.org/10.3390/foods8010001.
  12. Alyassin, M.; Campbell, G. M. Chapter 15 Challenges and Constraints in Analysis of Oligosaccharides and Other Fibre Components. In The value of fibre; González-Ortiz, G., Bedford, M. R., Bach Knudsen, K. E., Courtin, C. M., Classen, H. L., Eds.; Wageningen Academic Publishers: The Netherlands, 2019; pp 257–277. https://doi.org/10.3920/978-90-8686-893-3_15.
  13. Grabarics, M.; Lettow, M.; Kirschbaum, C.; Greis, K.; Manz, C.; Pagel, K. Mass Spectrometry-Based Techniques to Elucidate the Sugar Code. Chem. Rev. 2021, acs.chemrev.1c00380. https://doi.org/10.1021/acs.chemrev.1c00380.
  14. Wang, J.; Zhao, J.; Nie, S.; Xie, M.; Li, S. Mass Spectrometry for Structural Elucidation and Sequencing of Carbohydrates. TrAC Trends Anal. Chem. 2021, 144, 116436. https://doi.org/10.1016/j.trac.2021.116436.
  15. Pokrzywnicka, M.; Koncki, R. Disaccharides Determination: A Review of Analytical Methods. Crit. Rev. Anal. Chem. 2018, 48 (3), 186–213. https://doi.org/10.1080/10408347.2017.1391683.
  16. Daniel R. Knapp. Handbook of Analytical Derivatization Reactions; John Wiley & Sons, 1979.
  17. Haas, M.; Lamour, S.; Trapp, O. Development of an Advanced Derivatization Protocol for the Unambiguous Identification of Monosaccharides in Complex Mixtures by Gas and Liquid Chromatography. J. Chromatogr. A 2018, 1568, 160–167. https://doi.org/10.1016/j.chroma.2018.07.015.
  18. Lv, G.; Hu, D.; Zhao, J.; Li, S. Quality Control of Sweet Medicines Based on Gas Chromatography -Mass Spectrometry. 14.
  19. Reay, M. K.; Knowles, T. D. J.; Jones, D. L.; Evershed, R. P. Development of Alditol Acetate Derivatives for the Determination of 15 N-Enriched Amino Sugars by Gas Chromatography–Combustion–Isotope Ratio Mass Spectrometry. Anal. Chem. 2019, 91 (5), 3397–3404. https://doi.org/10.1021/acs.analchem.8b04838.
  20. Englyst, H. N.; Cummings, J. H. Simplified Method for the Measurement of Total Non-Starch Polysaccharides by Gas-Liquid Chromatography of Constituent Sugars as Alditol Acetates. The Analyst 1984, 109 (7), 937. https://doi.org/10.1039/an9840900937.