Rothenhöfer, Martin and Scherübl, Rosmarie and Bernhardt, Günther and Heilmann, Jörg and Buschauer, Armin (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. Journal of chromatography. A 1248, pp. 169-77.
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Purified oligomers of hyalobiuronic acid are indispensable tools to elucidate the physiological and pathophysiological role of hyaluronan degradation by various hyaluronidase isoenzymes. Therefore, we established and validated a novel sensitive, convenient, rapid, and cost-effective high performance thin layer chromatography (HPTLC) method for the qualitative and quantitative analysis of small saturated hyaluronan oligosaccharides consisting of 2-4 hyalobiuronic acid moieties. The use of amino-modified silica as stationary phase allows a simple reagent-free in situ derivatization by heating, resulting in a very low limit of detection (7-19 pmol per band, depending on the analyzed saturated oligosaccharide). By this derivatization procedure for the first time densitometric quantification of the analytes could be performed by HPTLC. The validated method showed a quantification limit of 37-71 pmol per band and was proven to be superior in comparison to conventional detection of hyaluronan oligosaccharides. The analytes were identified by hyphenation of normal phase planar chromatography to mass spectrometry (TLC-MS) using electrospray ionization. As an alternative to sequential techniques such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), the validated HPTLC quantification method can easily be automated and is applicable to the analysis of multiple samples in parallel.
|Date:||27 July 2012|
|Institutions:|| Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer) |
Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical Biology (Prof. Heilmann)
|Keywords:||Hyalobiuronic acid; Hyaluronan; HPTLC; Amino-modified silica; Densitometric quantification|
|Subjects:||500 Science > 540 Chemistry & allied sciences|
500 Science > 570 Life sciences
600 Technology > 615 Pharmacy
|Refereed:||Yes, this version has been refereed|
|Created at the University of Regensburg:||Yes|
|Owner:||Prof. Armin Buschauer|
|Deposited On:||05 Nov 2012 06:02|
|Last Modified:||05 Nov 2012 06:02|