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A novel synthesis method of protocatechuic acid-(13COOH) was designed. 3,4-dimethoxy bromobenzene as raw material, Grignard reagent was prepared by Grignard reaction, then passed through the 13C labeled 13CO2 gas, the intermediate 3,4-dimethoxy benzoic acid-(13COOH) was prepared. The crude product was obtained viademethylation with BBr3, which was decolorizing by activated carbon. Protocatechuic acid-(13COOH) was obtained by cooling crystallization. Synthesis route had the advantages of simple operation, short process flow, less side product, the yield was more than 45%, 13C labeled isotope abundance was not diluted. The product was characterized by HPLC, MS, 1H NMR and 13C NMR, the chemical purity was more than 99%, 13C enrichment was more than 98%. As an important tracer, it can provide a basis for medical and chemical research.

A method for determination alanine, serine, glutamic acid, asparagine, theanine, glutamine in tea leaves by MSTFA derivatization has been established. And six amino acids were detected successfully. Isotope abundance of alanine, serine, glutamate, asparagine, theamine and glutamine are calculated accuracy, isotope abundance range from 10% to 98% (atom%). The relative standard deviations (RSD, n=3) were below 0.6%, and absolute deviation were below 0.6%. The method is quick, easy and accurate, which can meet the detection on isotope abundance of labeling amino acids in tea leaves. Besides, the method also can be applied to the determination of isotopic abundance of amino acids in other different crops, and provides technical support for the research of amino acids related transformation pathways and metabolic cycles in plants.

Stable isotope labelled p-methoxybenzoic acid is an important intermediate in the synthesis of stable isotope labelled preservative, anti-microbial, anti-fungal, spices, and other internal standard reagentsspices, drugs and other compounds. A new procedure for the synthesis of the ［13C］/D labelled p-methoxybenzoic acid has been developed in this paper. The stable isotope ［13C］/D labelled p-methoxybenzoic acid was synthesized from ethyl paraben and ［13C］/D labelled iodomethane (13CH3I or CD3I) under alkaline conditions via two steps. In this paper, the optimal reaction temperature, molar ratio, and reaction time were obtained by single factor experiment. The optimal reaction conditions were obtained as follows, the reaction temperature was 80 ℃, the molar ratio of ［13C］/D labelled iodomethane to ethyl paraben was 1.15∶1, the reaction time was 6 h. In the optimization conditions, the yield of ［13C］ labelled p-methoxybenzoic acid was 91.12%, and the utilization of 13CH3I was 79.32%. The melting point of product was 186.5~187.8 ℃. The product analyses were made with LC-MS, the chemical purity was 99.1%, and the isotopic abundance was 99.0 atom%13C. The qualitative analysis of product was confirmed by 1H NMR. By the same technological conditions, the stable isotope ［D］ labelled p-methoxybenzoic acid was synthesized from ethyl paraben and ［D］ labelled iodomethane (CD3I). The yield of ［D］ labelled p-methoxybenzoic acid was 90.49%, and the utilization of CD3I was 78.70%, and the melting point of product was 186.3~188.1 ℃. The product analyses were made with LC-MS, the chemical purity was 99.0%, and the isotopic abundance was 98.1 atom%D. The qualitative analysis of product was confirmed by 1H NMR.