Determination of 13C content in starch from paddy rice plants using stable isotope ratio mass spectrometry

Abstract

A new pretreatment method was developed to purify a starch hydrolysis mixture for isotope ratio mass spectrometry (IRMS) analysis. Ethanol-soluble (EtS) substances were removed from fine rice plant powder. Then, ethanol-insoluble solids were boiled with 10 mL of 2.5 mmol L−1 H2SO4 for 1 h and incubated with glucoamylase (from Aspergillus niger) at 52°C for 24 h to hydrolyze and convert to glucose. An acidified glucose–enzyme mixture was purified using serial cartridge solid-phase extraction (SPE), cation exchange resin and non-polar sorbent. This procedure aimed to purify glucose by removing considerable cationic and non-polar substances from the mixtures. The columns were rinsed with 2 mL of purified water. The eluate was regarded as the glucose fraction. The overall recovery examined using a rice sample with the addition of 13C glucose was 96.3%. This method was used to analyze 13C in plants that were fed with 13CO2 on the 14th day after heading time. At the end of 13CO2 feeding, 50% of newly fixed carbon was accumulated in superior leaves, mainly as EtS substances. At grain maturation, 95% of 13C was present in grains, 63% of which was in starch.