Identification and quantification of glucose degradation products

    CBS Articles

    Author: Sarah Leitzen

    Published in CBS 128

The subject of this article was investigated by Prof. Dr. Martin Brandl, Anette Engels, Dr. Matthias Vogel, Dr. Thomas Zapf and Sarah Leitzen [1]. Prof. Dr. Martin Brandl is a pharmacist and professor at the Department of Physics, Chemistry and Pharmacy at the University of Southern Denmark (SDU), Odense, Denmark and supervisor of Sarah Leitzen's doctoral thesis. Anette Engels, Dr. Matthias Vogel and Dr. Thomas Zapf work at the Federal Institute for Drugs and Medical Devices (BfArM) in Bonn. Sarah Leitzen is a pharmacist and PhD student working at both BfArM and SDU.


Sterile glucose solutions are commonly used as reconstitution solvents or diluents for injectable drugs and also for peritoneal dialysis solutions. In Germany, regulatory requirements for the different strengths of glucose solutions used for parenteral administration are regulated and published as standard marketing authorizations. During heat sterilization of glucose solutions for parenteral use, a variety of glucose degradation products (GDPs) may be formed. GDPs can cause cytotoxic effects after parenteral administration of these solutions. Therefore, the aim of the current study was to develop a simple and quick HPTLC method by which the major GDPs can be identified and (summarily) quantified in glucose solutions for parenteral administration. All GDPs were derivatized with o-phenylenediamine (OPD). The identity of the resulting GDP derivatives (quinoxalines) during method validation was confirmed via mass spectrometry

Standard solutions and pre-chromatographic derivatization

For each degradation product (glyoxal (GO), methylglyoxal (MGO) , glucosone (2-KDG) , 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), and the impurity 5-hydroxymethylfurfural (5- HMF)), individual 0.5 mg/mL solutions are prepared with fresh ultrapure water, as well as a mix of all seven GDPs. For quantification, calibration standards containing all seven GDPs at concentration levels of 1–75 μg/mL are prepared. All solutions also contain 50 mg/mL glucose and 0.75 mg/mL OPD. They are left to stand in the dark for 16 hours at room temperature.

Sample preparation and pre-chromatographic derivatization

An artificial mix is prepared to simulate expected concentrations of GDPs in an autoclaved 5% glucose solution containing GO and MGO (1.0 mg/mL, each), 2-KDG (7.0 mg/mL), 3-DG (45.0 mg/mL), 3-DGal (25.0 mg/mL), 3,4-DGE and 5-HMF (5.0 mg/mL, each). The mix also contains glucose in a concentration of 50 mg/mL and OPD as derivatization reagent in a concentration of 0.75 mg/mL. Samples are analyzed after a 16 h reaction time at room temperature.

Chromatogram layer

HPTLC glass plates silica gel 60 F254 (Merck), 20 x 20 cm are used.

Sample application

Samples and standard solutions are applied as bands with the Automatic TLC Sampler (ATS 4), band length 10.0 mm, distance between tracks of 19.0 mm, distance from lower edge 29.0 mm. 10 μL for sample and standard solutions are applied.


Plates are developed in the Twin Trough Chamber (TTC 20 x 20 cm) with chamber saturation (with filter paper) for 20 min, development with 25 mL of 1,4-dioxane – toluene – glacial acetic acid 49:49:2 (V/V) (each trough) to the migration distance of 160 mm (from the lower edge), followed by drying for 10 min.

Editor’s Note: in this special case, a migration distance of 160 mm leads to a significantly improved separation on HPTLC with the selected solvent system. Usually, a migration exceeding 80 mm on HPTLC plates does not improve resolution due to increasing diffusion

Post-chromatographic derivatization (second derivatization)

The plates are sprayed with the Derivatizer (yellow nozzle, level 4, 2 mL) with thymolsulfuric acid reagent (2 mL of a solution of 0.5 g of thymol in a mixture of 5 mL of sulfuric acid and 95 mL of ethanol 96%) and heated at 130˚C for 10 minutes on the TLC Plate Heater.

Authors’ Note: While establishing the method, two identical plates were run simultaneously (in separate twin trough chambers with chamber saturation), where one of the two plates was not treated with thymol-sulfuric acid reagent for substance confirmation by mass spectrometry.


Images of the plate are captured with the TLC Visualizer in UV 254 nm, UV 366 nm, and white light.


The HPTLC plate was scanned in absorbance mode at 330, 370, and 420 nm, and in fluorescencemode at 366>/400 nm.

Mass spectrometry

One of the duplicate plates (plate without 2nd derivatization step) is used for substance confirmation. The zones are localized with the help of the plate derivatized with thymol-sulfuric acid reagent. Zones of interest are scraped off at the expected position and extracted three times, using 200 μL of methanol each time, and centrifuged for 2 min at 16,000 rpm/21,130 rcf. The supernatants are evaporated with nitrogen and the residues are finally reconstituted in 200 μL of a 5 mM ammonium acetate buffer solution (pH = 3.5). The solutions are analyzed in positive electrospray ionization mode [1].

Editor’s Note: The zones can also directly be eluted to an MS for substance confirmation with the TLC-MS Interface 2. However, this equipment was not available at the BfArM.

Results and discussion

Glucose, as a highly polar molecule, does not react with OPD, or only to a lesser degree, and maintains its hydrophilic character without forming the quinoxaline system. Therefore, glucose is observed at the application position.

The presented method was successfully validated using the ICH Q2(R1) guideline. For 2-KDG, the linearity of the method was demonstrated in the range of 1–50 μg/mL, for 5-HMF and 3,4-DGE 1–75 μg/mL, for GO/MGO 2–150 μg/mL, and for 3- DG/3-DGal 10–150 μg/mL. All GDPs achieved a limit of detection (LOD) of 2 μg/mL or less and a limit of quantification (LOQ) of 10 μg/mL or less. R2 was 0.982 for 3.4-DGE, 0.997 for 5-HMF, and 0.999 for 2-KDG, 3-DG/3-DGal, and GO/MGO. The intraday precision was between 0.4 and 14.2% and the accuracy, reported as%recovery, between 86.4 and 112.7%. The proposed HPTLC method appears to be an inexpensive, fast, and sufficiently sensitive approach for routine quantitative analysis of GDPs in heat-sterilized glucose solutions.

Chromatograms of the derivatized seven GDPs and a mixture (= spiked glucose solution) in the solvent 1,4-dioxane – toluene – glacial acetic acid 49:49:2 (V/V)

Chromatograms of the derivatized seven GDPs and a mixture (= spiked glucose solution) in the solvent 1,4-dioxane – toluene – glacial acetic acid 49:49:2 (V/V). The chromatograms are shown in UV 366 nm without (left) and with (right) subsequent 2nd derivatization with thymolsulfuric acid reagent at concentration levels of 0.5 mg/mL [1]. (

Overview of the obtained RF values and signal responses of each analyte at different wavelengths *without and **with second derivatization.

Overview of the obtained RF values and signal responses of each analyte at different wavelengths *without and **with second derivatization.


HPTLC chromatograms in UV 366 nm after both derivatizations (ROI until RF 0.6)

HPTLC chromatograms in UV 366 nm after both derivatizations (ROI until RF 0.6). Track 1 shows the spiked 5% glucose solution spiked GDP-Mix-OPD); tracks 2–8 the calibration solutions in ascending concentrations (1.0 μg/mL–100.0 μg/mL GDPs) [1]. All solutions contain 50 mg/mL glucose. (

Calibration curve of GO/MGO at concentrations of 2–150 μg/mL (measured in absorbance mode at 330 nm)

Calibration curve of GO/MGO at concentrations of 2–150 μg/mL (measured in absorbance mode at 330 nm) [1]. (


[1] Leitzen et al. PLoS ONE (2021),

Further information is available on request from the authors.

Contact: Sarah Leitzen, Federal Institute for Drugs and Medical Devices, Bonn, Germany,

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CBS 128: Identification and quantification of glucose degradation products

Mentioned Products

The following instruments and devices were used in this work (discontinued products are replaced with current versions)