Determination of glucosamine and monitoring of its mutarotation by hydrophilic interaction liquid chromatography with evaporative light scattering detector
Abstract:Saccharides and their derivatives are typical polar analytes without a suitable UV-chromophore that are nowadays analyzed by HPLC (high-performance liquid chromatography) under HILIC (hydrophilic interaction liquid chromatography) mode. Usually an evaporative light scattering detector (ELSD) is utilized which, however, gives a nonlinear response. A procedure to overcome the problem of mutarotating (time-varying) analytes recorded with such a nonlinear response detector is described. The procedure was applied f… Show more
“…As such, it was concluded that the two peaks corresponded to two anomeric forms of glucosamine. The ratio of the peak areas was calculated as 25.8/74.2 which was in good agreement with previously published results (38,39), meaning that the obtained peaks corresponded to the β-anomer (tR = 0.87 min) and α-anomer (tR = 1.03 min) of glucosamine, respectively. The incomplete baseline separation can be attributed to the interconversion between the two anomers through mutarotation, as reported previously (15,(38)(39)(40).…”
Section: Chromatographic Conditionssupporting
confidence: 91%
“…The ratio of the peak areas was calculated as 25.8/74.2 which was in good agreement with previously published results (38,39), meaning that the obtained peaks corresponded to the β-anomer (tR = 0.87 min) and α-anomer (tR = 1.03 min) of glucosamine, respectively. The incomplete baseline separation can be attributed to the interconversion between the two anomers through mutarotation, as reported previously (15,(38)(39)(40). For calibration and quantification, both peaks as well as the plateau were integrated and the total area was used.…”
As chitin is gaining an increased attention as feedstock for industry, quantification thereof is becoming increasingly important. While gravimetric procedures are long, not specific, and highly labour-intensive; acidic hydrolysis of chitin into glucosamine followed by quantification of the latter is more performant. Even though several quantification procedures for the determination of chitin can be found in the literature, they give inconsistent results and their accuracy was not assessed due to the lack of certified analytical standards. Therefore, in the present study commercially available chitin from practical grade was characterised in detail, allowing the assessment of method accuracy. The procedure for the hydrolysis of chitin into glucosamine and subsequent quantification via UPLC-MS was investigated in detail as well.Using 9-fluorenylmethyl chloroformate (FMOC-Cl) as derivatisation reagent, glucosamine was quantified using reversed-phase chromatography. For the chitin hydrolysis, the highest glucosamine recovery was obtained with 8.0 M HCl for 2 hours at 100 °C. The entire procedure for chitin quantification, including the hydrolysis, was characterised by a high inter-and intraday precision and accuracy. The specificity of the procedure was assessed as well by analysing different mixtures of cellulose and chitin. Chitin recoveries from these analyses ranged from 98.8 to 105.8 % while no signal was observed for 100 % cellulose, indicating the high specificity of the procedure. It was also concluded that the procedure is much faster and less labour-intensive compared to the gravimetric procedure.
“…As such, it was concluded that the two peaks corresponded to two anomeric forms of glucosamine. The ratio of the peak areas was calculated as 25.8/74.2 which was in good agreement with previously published results (38,39), meaning that the obtained peaks corresponded to the β-anomer (tR = 0.87 min) and α-anomer (tR = 1.03 min) of glucosamine, respectively. The incomplete baseline separation can be attributed to the interconversion between the two anomers through mutarotation, as reported previously (15,(38)(39)(40).…”
Section: Chromatographic Conditionssupporting
confidence: 91%
“…The ratio of the peak areas was calculated as 25.8/74.2 which was in good agreement with previously published results (38,39), meaning that the obtained peaks corresponded to the β-anomer (tR = 0.87 min) and α-anomer (tR = 1.03 min) of glucosamine, respectively. The incomplete baseline separation can be attributed to the interconversion between the two anomers through mutarotation, as reported previously (15,(38)(39)(40). For calibration and quantification, both peaks as well as the plateau were integrated and the total area was used.…”
As chitin is gaining an increased attention as feedstock for industry, quantification thereof is becoming increasingly important. While gravimetric procedures are long, not specific, and highly labour-intensive; acidic hydrolysis of chitin into glucosamine followed by quantification of the latter is more performant. Even though several quantification procedures for the determination of chitin can be found in the literature, they give inconsistent results and their accuracy was not assessed due to the lack of certified analytical standards. Therefore, in the present study commercially available chitin from practical grade was characterised in detail, allowing the assessment of method accuracy. The procedure for the hydrolysis of chitin into glucosamine and subsequent quantification via UPLC-MS was investigated in detail as well.Using 9-fluorenylmethyl chloroformate (FMOC-Cl) as derivatisation reagent, glucosamine was quantified using reversed-phase chromatography. For the chitin hydrolysis, the highest glucosamine recovery was obtained with 8.0 M HCl for 2 hours at 100 °C. The entire procedure for chitin quantification, including the hydrolysis, was characterised by a high inter-and intraday precision and accuracy. The specificity of the procedure was assessed as well by analysing different mixtures of cellulose and chitin. Chitin recoveries from these analyses ranged from 98.8 to 105.8 % while no signal was observed for 100 % cellulose, indicating the high specificity of the procedure. It was also concluded that the procedure is much faster and less labour-intensive compared to the gravimetric procedure.
“…Initial experimental conditions for the method optimization were based on recently published papers on study of monosaccharides and disaccharides retention on a polyol stationary phase [ 25 , 26 , 27 , 55 ], where a diol column (Lichrosphere100 DIOL, Merck, Darmstadt, Germany) [ 25 , 26 ] was mostly applied, but in this paper we used a HALO PentaHILIC column that showed a better performance [ 27 ]. Due to the high affinity towards the stationary phase, a flow rate of 2.0 mL/min could be maintained in all experiments.…”
Section: Resultsmentioning
confidence: 99%
“…Therefore, a compromise between high retention (high resolution) and sufficient peak area must be chosen. To find better experimental conditions we followed our own experience [ 27 , 55 ]; in the following experiments several ammonium buffers (formate, acetate, bicarbonate) in a mixture with acetonitrile were tested.…”
Section: Resultsmentioning
confidence: 99%
“…A possible approach is to find an exponent m < 1 that the peak area is powered to. Typically, the value lies in the range 0.65–0.80 [ 27 , 55 ]. The exponent can be easily found as a slope of a graph log (concentration) vs. log (peak area).…”
This paper presents a rapid HPLC method for the separation of isomaltulose (also known as Palatinose) from other common edible carbohydrates such as sucrose, glucose, and maltodextrins, which are commonly present in food and dietary supplements. This method was applied to determine isomaltulose in selected food supplements for special diets and athletic performance. Due to the selectivity of the separation system, this method can also be used for rapid profiling analysis of mono-, di-, and oligosaccharides in food.
Saccharides and biocompounds as saccharide (sugar) complexes have various roles and biological functions in living organisms due to modifications via nucleophilic substitution, polymerization, and complex formation reactions. Mostly, mono‐, di‐, oligo‐, and polysaccharides are stabilized to inactive glycosides, which are formed in metabolic pathways. Natural saccharides are important in food and environmental monitoring. Glycosides with various functionalities are significant in clinical and medical research. Saccharides are often studied with the chromatographic methods of hydrophilic interaction liquid chromatography and anion exchange chromatograpy, but also with capillary electrophoresis and mass spectrometry with their on‐line coupling systems. Sample preparation is important in the identification of saccharide compounds. The cases discussed here focus on bioscience, clinical, and food applications.
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