Modified fast UHPLC method for quantification of soil amino sugars – improved sensitivity compared to the GC method

Soil microbial necromass analysis through measurement of soil amino sugars is a common technique in soil science. Traditionally, the measurement is performed by aldononitrile acetate derivatization and gas chromatographic (GC) analysis. Long pretreatment times and high limits of quantification (LOQ) have led to the development of faster and more sensitive high performance liquid chromatography (HPLC) methods. In this study, we enhanced a previously discovered ortho-phthalaldehyde derivatization and HPLC separation method for soil samples by converting it to work with ultra-high performance liquid chromatography (UHPLC) column and equipment. We also added an internal standard to control for pretreatment variation. In addition, we explored the factors that must be optimized to establish the method with alternative equipment, and the differences in the results between the UHPLC and the GC methods. We found that the UHPLC method produced similar results to the GC methods with glucosamine and galactosamine, while the performance of the UHPLC method was significantly better in determining muramic acid, especially at low concentrations. Mannosamine results were not correlated between the methods. The speed of the UHPLC analysis was much higher and LOQ much lower compared to the GC method. In the majority of soil samples (13 out of 18), muramic acid concentrations were found to be below LOQ for the GC method, but clearly detectable with the developed UHPLC method. It was found that the UHPLC method is at least twice as fast as the GC method and requires only few of the hazardous chemicals traditionally used in amino sugar analysis. The UHPLC method also improved on the HPLC method by consuming only 1/5^th of the total solvent and by reducing analysis time from 30 to 18 minutes.