Abstract
Thin-layer chromatography (TLC) is a simple, fast and inexpensive separation method which can be applied to virtually all natural products including oligosaccharides. Unfortunately, however, the unequivocal identification of a TLC spot is normally difficult. Fortunately, this problem can be minimized when mass spectrometry (MS) such as matrix-assisted laser desorption and ionization time-of-flight is used to identify the TLC spots. This work is dedicated to the TLC/MS analysis of oligosaccharides derived from native chondroitin sulfate and hyaluronan. We will show that the thickness of the silica gel layer (200 versus 100 µm) has a tremendous influence on the quality of the mass spectra: a reduced silica gel thickness enhances the spectral quality and, in particular, improves the achievable signal-to-noise ratio. Additionally, unwanted formylation of the GAG oligosaccharides (which occurs due to the high moiety of formic acid in the mobile phase) can also be minimized if MS-grade HPTLC plates are used.
This is a preview of subscription content, log in via an institution to check access.
References
Fuchs B, Süß R, Nimptsch A, Schiller J (2009) Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) directly combined with thin-layer chromatography (TLC)—a review of the current state. Chromatographia 69:95–105
Scherz H, Bonn G (1998) Analytical chemistry of carbohydrates. Georg Thieme Verlag, Stuttgart
Patzsch K, Netz S, Funk W (1988) Quantitative HPTLC of sugars—part 2: Determination in different matrices. J Planar Chromatogr 1:177–179
Schnabelrauch M, Scharnweber D, Schiller J (2013) Sulfated glycosaminoglycans as promising artificial extracellular matrix components to improve the regeneration of tissues. Curr Med Chem 20:2501–2523
Zhang Z, Xie J, Zhang F, Linhardt RJ (2007) Thin-layer chromatography for the analysis of glycosaminoglycan oligosaccharides. Anal Biochem 371:118–120
Nimptsch K, Süss R, Riemer T, Nimptsch A, Schnabelrauch M, Schiller J (2010) Differently complex oligosaccharides can be easily identified by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry directly from a standard thin-layer chromatography plate. J Chromatogr A 1217:3711–3715
Rothenhöfer M, Scherübl R, Bernhardt G, Heilmann J, Buschauer A (2012) Qualitative and quantitative analysis of hyaluronan oligosaccharides with high performance thin layer chromatography using reagent-free derivatization on amino-modified silica and electrospray ionization-quadrupole time-of-flight mass spectrometry coupling on normal phase. J Chromatogr A 1248:169–177
Hillenkamp F, Peter-Katalinić J (2014) MALDI MS—a practical guide to instrumentation, methods, and applications. Wiley-Blackwell, Weinheim
Griesinger H, Fuchs B, Süß R, Matheis K, Schulz M, Schiller J (2014) Stationary phase thickness determines the quality of thin-layer chromatography/matrix-assisted laser desorption and ionization mass spectra of lipids. Anal Biochem 451:45–47
Fuchs B, Bischoff A, Süss R, Teuber K, Schürenberg M, Suckau D, Schiller J (2009) Phosphatidylcholines and -ethanolamines can be easily mistaken in phospholipid mixtures: a negative ion MALDI-TOF MS study with 9-aminoacridine as matrix and egg yolk as selected example. Anal Bioanal Chem 395:2479–2487
Schiller J, Arnhold J, Benard S, Reichl S, Arnold K (1999) Cartilage degradation by hyaluronate lyase and chondroitin ABC lyase: a MALDI-TOF mass spectrometric study. Carbohydr Res 318:116–122
Nimptsch K, Süß R, Schnabelrauch M, Nimptsch A, Schiller J (2012) Positive ion MALDI-TOF mass spectra are more suitable than negative ion spectra to characterize sulphated glycosaminoglycans. Int J Mass Spectrom 310:72–76
Nimptsch A, Schibur S, Schnabelrauch M, Fuchs B, Huster D, Schiller J (2009) Characterization of the quantitative relationship between signal-to-noise (S/N) ratio and sample amount on-target by MALDI-TOF MS: determination of chondroitin sulfate subsequent to enzymatic digestion. Anal Chim Acta 635:175–182
Laremore TN, Murugesan S, Park TJ, Avci FY, Zagorevski DV, Linhardt RJ (2006) Matrix-assisted laser desorption/ionization mass spectrometric analysis of uncomplexed highly sulfated oligosaccharides using ionic liquid matrices. Anal Chem 78:1774–1779
Lemmnitzer K, Schiller J, Becher J, Möller S, Schnabelrauch M (2014) Improvement of the digestibility of sulfated hyaluronans by bovine testicular hyaluronidase: a UV spectroscopic and mass spectrometric study. Biomed Res Int 2014:986594
Nurok D (2000) Forced flow techniques in planar chromatography. Anal Chem 72:634A–641A
Acknowledgments
This work was supported by the German Research Council (DFG Schi 476/16-1 as well as TR 67, project A2 and A8). We would like to thank Dr. Detlev Suckau and Dr. Martin Schürenberg (Bruker Daltonics, Bremen, Germany) for many helpful discussions.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lemmnitzer, K., Griesinger, H., Süß, R. et al. Analysis of Glycosaminoglycan Oligosaccharides by Combined HPTLC/MALDI-TOF MS: Reduced Silica Gel Thickness Leads to Improved Spectral Qualities and Reduced Side Reactions. Chromatographia 78, 1409–1413 (2015). https://doi.org/10.1007/s10337-015-2971-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10337-015-2971-2