Evaluation of a liquid chromatography method for compound-specific δ13C analysis of plant carbohydrates in alkaline media

RATIONALE Isotope analysis of carbohydrates is important for improved understanding of plant carbon metabolism and plant physiological response to the environment. High-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS) for direct compound-specific d13C measurements of soluble carbohydrates has recently been developed, but the still challenging sample preparation and the fact that no single method is capable of separating all compounds of interest hinder its wide-spread application. Here we tested in detail a chromatography method in alkaline media. METHODS We examined the most suitable chromatographic conditions for HPLC/IRMS analysis of carbohydrates in aqueous conifer needle extracts using a CarboPac PA20 anion-exchange column with NaOH eluent, paying specific attention to compound yields, carbon isotope fractionation processes and the reproducibility of the method. Furthermore, we adapted and calibrated sample preparation methods for HPLC/IRMS analysis. OnGuard II cartridges were used for sample purification. RESULTS Good peak separation and highly linear and reproducible concentration and d13C measurements were obtained. The alkaline eluent was observed to induce isomerization of hexoses, detected as reduced yields and 13C fractionation of the affected compounds. A reproducible pre-purification method providing similar to 100% yield for the carbohydrate compounds of interest was calibrated. CONCLUSIONS The good level of peak separation obtained in this study is reflected in the good precision and linearity of concentration and d13C results. The data provided crucial information on the behaviour of sugars in LC analysis with alkaline media. The observations highlight the importance for the application of compound-matched standard solution for the detection and correction of instrumental biases in concentration and d13C analysis performed under identical chromatographic conditions. The calibrated pre-purification method is well suited for studies with complex matrices that disable the use of a spiked internal standard for the detection of procedural losses. Copyright (c) 2012 John Wiley & Sons, Ltd.