The First Comprehensive LC–MS/MS Method Allowing Dissection of the Thiamine Pathway in Plants

Abstract

Arabidopsis thaliana serves as a model plant for genetic research, including vitamin research. When aiming at engineering the thiamine (vitamin B1) pathway in plants, the 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 preset acceptance criteria for all analytes in both QC and real samples. All analytes were stable in extracted samples when stored for 48 h at 4 °C (autosampler stability) and when reanalyzed 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.

Metabolic engineering of rice endosperm towards higher vitamin B1 accumulation

“…The increasing DOP obtained by different polishing methodologies is depicted in Figure S2A, ranging from brown rice (unpolished, 0% DOP) to (thoroughly) polished white rice (60s M + 16h SP, ≈14% DOP), roughly spanning the range of DOP as applied in industrial polishing (Tran et al ., 2018). Thiamin and the biosynthesis intermediates were quantified by a validated LC‐MS/MS procedure (Verstraete et al ., 2020a; Verstraete et al ., 2020b). This analysis revealed the tremendous effect of polishing on the level of thiamin.…”

“…Quantification of metabolites (HMP, total HMP, HET, total HET, thiamin, TMP and TPP) was performed using a validated LC‐MS/MS method, as described by (Verstraete et al ., 2020a; Verstraete et al ., 2020b). The analysis requires 200 mg of freshly harvested rice material (polished, unpolished or cooked seeds (see below).…”

An optimized LC-MS/MS method as a pivotal tool to steer thiamine biofortification strategies in rice

“…Due to the complexity of the matrix, several parameters needed to be re-evaluated and optimized compared to our method in Arabidopsis thaliana [15]. Given that for the eventual application the availability of sample material would be limited, the minimal sample weight was assessed.…”

“…Similar to our method in Arabidopsis [15], a Shimadzu Prominence HPLC system (Shimadzu, Kyoto, Japan) coupled to a SCIEX API 5500™ triple quadrupole mass spectrometer (SCIEX, Framingham, MA, USA) was used for all analyses. The hardware system was controlled by the SCIEX Analyst® 1.6.2 software.…”

“…We previously reported on the chromatographic challenges we have overcome during the set-up of an LC-MS/MS method for the determination of thiamine, its intermediates and phosphate derivatives in Arabidopsis thaliana [15]. However, determination of these analytes in the starch-rich rice matrix implies a substantially different work-up, requiring extensive optimization of the sample preparation, taking into account factors such as matrix effect, recovery and stability.…”