气相色谱-燃烧-同位素比值质谱法测定发酵液中尿素δ~(13)C和δ~(15)N

在酸性环境下,尿素与丙二醛-二甲基乙缩醛反应生成2-羟基嘧啶,随后由重氮甲烷甲基化形成2-甲氧基嘧啶。本研究以酵母发酵液为例,建立了气相色谱-燃烧-同位素比值质谱(GC-C-IRMS)法测定发酵液中尿素δ~(13)C和δ~(15)N,并对前处理方法和GC条件进行了优化。结果表明:尿素在不同13 C(0~5%)和15 N(0~10%)标记丰度下,标记量与GC-C-IRMS测定结果的相关性良好,线性相关系数R2分别为0.998和0.999,说明可在极低标记浓度下对尿素中δ~(13)C和δ~(15)N进行准确测定。采用该方法测定了酵母发酵液尿素中δ~(13)C和δ~(15)N,其重复性相对标准偏差分别为0.21‰和0.30‰,再现性相对标准偏差分别为0.23‰和0.29‰。该方法精度高、准确性好,可为低稳定同位素标记尿素示踪实验以及尿素循环代谢研究提供方法基础。

Determination ofδ13C andδ15N of Urea in Fermentation Broth by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry

A method for the determination ofδ¹³C andδ¹⁵N of urea in yeast fermentation broth by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was established. Urea was derived as 2-methoxypyrimidine by malonaldehydebis (dimethyl acetal) (MDBMA) and diazomethane in acidic condition, and then was analyzed by GC-C-IRMS. Sample pretreatment method and GC conditions were confirmed and optimized by GC-FID. The results show that the linear correlations of labeled level ofδ¹³C(0.5% labeled) andδ¹⁵N (0.10% labeled) and measured value of GC-C-IRMS are well. The correlation coefficients ofδ¹³C andδ¹⁵N are 0.998 and 0.999, respectively. The repeatability ofδ¹³C andδ¹⁵N of urea in yeast fermentation broth are 0.21‰ and 0.30‰, and the reproducibility are 0.23‰ and 0.29‰.δ¹⁵N of urea from three different sources was analyzed by EA-IRMS and GC-C-IRMS, and the correlation coefficient is 1. This method has high accuracy and good repeatability, which can provide foundation and technical support to low stable isotope labeling urea tracer experiment and analysis of urea cycle metabolism.