The first comprehensive LC-MS/MS method allowing dissection of the thiamine pathway in plants.

Arabidopsis thaliana serves as a model plant for genetic research, including vitamin research. When aiming at engineering the thiamine (vitamin B1) pathway in plants, availability of tools that allow the quantitative determination of different intermediates in the biosynthesis pathway is of pivotal importance. This is a challenge, given the nature of the compounds and the minute quantities of genetically engineered material that may be available for analysis. Here, we report on the first LC-MS/MS method for the simultaneous quantification of thiamine, its mono- and diphosphate derivatives and its precursors 4-methyl-5-(2-hydroxyethyl) thiazole (HET) and 4-amino-2-methyl-5-hydroxymethylpyrimidine (HMP). This method was optimized and validated for the quantitative determination of these analytes in Arabidopsis thaliana. All analytes were chromatographically separated within less than 2.5 min during an 8 min run. No unacceptable interferences were found. The method was fully validated based on international guidelines. Accuracy (%bias) and total imprecision (%CV) were within pre-set acceptance criteria for all analytes in both QC and real samples. All analytes were stable in extracted samples when stored for 48h at 4°C (autosampler stability) and when re-analyzed after storage at -80°C and -20°C for 2 weeks (freeze/thaw stability). We demonstrated the start material should be stored at -80°C to ensure stability of all analytes during short- and long-term storage (up to 3 months). The validity and applicability of the developed procedure was demonstrated via its successful application on Arabidopsis lines, genetically engineered to enhance thiamine content.