Topical Area: Methods and Protocols
Thiamin plays an essential role during several metabolic reactions. Thiamin deficiency is a problem in developing countries, where a varied diet is lacking and people mainly rely on rice, which contains insufficient levels of thiamin, for their energy supply.
A cost-effective strategy to fight this deficiency is the improvement of the nutritional quality of staple crops via biofortification.
Although several methods for the determination of thiamin have been published, no method exists which allows a combined determination of thiamin, its phosphate derivatives and precursors.
LC-MS/MS was used to determine the low-level and labile thiamin vitamers. Volatile buffers are required for MS/MS detection, excluding commonly used phosphate buffers. Furthermore, determination of thiamin mono (TMP)- and diphosphate (TPP) is highly challenging due to their strong polar and ionogenic character, whereby regular C18 columns lack retention. HILIC columns are recommended for these types of compounds, however, the solubility of the phosphate derivatives is incompatible with the required organic injection solvent. Therefore, we tested numerous columns and mobile phases, in order to determine all five compounds in one chromatographic run from a single biological sample.
With the Gemini® C18 column (Phenomenex) we were able to separate all compounds of interest. However, TMP and TPP could only be detected under basic conditions (≥ pH 8.8), which resulted in secondary interactions between the charged silanol groups and the quaternary thiamin. We therefore transferred our method to a Gemini® NX C18 column, which offers a higher robustness at high pH and minimizes peak tailing since silanol groups are shielded. Finally, in an 8 minute run, we could achieve acceptable separation of all thiamin vitamers and precursors, with sufficient sensitivity for their determination in ≤200 mg of wild-type unpolished rice and Arabidopsis samples.
An LC-MS/MS method was successfully developed for the combined determination of all thiamin vitamers and precursors. Application of this method will allow to guide the effectiveness of biofortification strategies in order to fight thiamine deficiency in developing countries.
Funding Sources :
The research was supported by the Research Foundation – Flanders.