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, 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.

Vitamin B1 in critically ill patients: needs and challenges.

BACKGROUND: Thiamine has a crucial role in energy production, and consequently thiamine deficiency (TD) has been associated with cardiac failure, neurological disorders, oxidative stress (lactic acidosis and sepsis) and refeeding syndrome (RFS). This review aims to explore analytical methodologies of thiamine compound quantification and highlight similarities, variances and limitations of current techniques and how they may be relevant to patients. CONTENT: An electronic search of Medline, PubMed and Embase databases for original articles published in peer-reviewed journals was conducted. MethodsNow was used to search for published analytical methods of thiamine compounds. Keywords for all databases included "thiamine and its phosphate esters", "thiamine methodology" and terms related to critical illness. Enquiries were also made to six external quality assurance (EQA) programme organisations for the inclusion of thiamine measurement. SUMMARY: A total of 777 published articles were identified; 122 were included in this review. The most common published method is HPLC with florescence detection. Two of the six EQA organisations include a thiamine measurement programme, both measuring only whole-blood thiamine pyrophosphate (TPP). No standard measurement procedure for thiamine compound quantification was identified. OUTLOOK: Overall, there is an absence of standardisation in measurement methodologies for thiamine in clinical care. Consequently, multiple variations in method practises are prohibiting the comparison of study results as they are not traceable to any higher order reference. Traceability of certified reference materials and reference measurement procedures is needed to provide an anchor to create the link between studies and help bring consensus on the clinical importance of thiamine.

[Thiamine Hydrochloride Solution Reference Standard (Control 021) of National Institute of Health Sciences].

The raw material of thiamine hydrochloride was examined for preparation of the "Thiamine Hydrochloride Reference Standard". The analytical data obtained were: assay by HPLC, 100.5%; spectrophotometric assay 101.2%. Based on the above results, the candidate material was authorized as the Thiamine Hydrochloride Reference Standard (Control 021) of the National Institute of Health Sciences.

[Thiamine Hydrochloride Solution Reference Standard (Control 991) of National Institute of Health Sciences].

The raw material of thiamine hydrochloride solution was examined for preparation of the "Thiamine Hydrochloride Solution Reference Standard (Control 991)". The analytical data obtained were: assay by HPLC, 101.0%; spectrophotometric assay, 100.4%. Based on the above results, the raw material was authorized as the Thiamine Hydrochloride Solution Reference Standard (Control 991) of the National Institute of Health Sciences.

[Thiamine Hydrochloride Solution Reference Standard (Control 971) of National Institute of Health Sciences].

The raw material of thiamine hydrochloride solution was examined for the preparation of the "Thiamine Hydrochloride Solution Reference Standard (Control 971)". Analytical data obtained were as follows: assay by HPLC, 100.8%; spectrophotometric assay, 99.8%. Based on the above results, the raw material was authorized to be the Thiamine Hydrochloride Solution Reference Standard of the National Institute of Health Sciences.

[The Thiamine Hydrochloride Reference Standard (Control 931) of the National Institute of Health Sciences].

Raw thiamine hydrochloride material was tested for preparation of the "Thiamine Hydrochloride Reference Standard (Control 931)". Analytical data obtained were as follows: melting point, 242.7 degrees C (decomposition); infrared spectrum, the same as that of the JP Thiamine Hydrochloride Reference Standard; thin-layer chromatography, one impurity was detected; high-performance liquid chromatography (HPLC), a trace amount of one impurity was detected; assay results, 100.4% by UV spectrophotometry and 100.0% by HPLC, respectively. Based on the above findings, the raw material was authorized as the JP Thiamine Hydrochloride Reference Standard (Control 931).

[Thiamine Hydrochloride Reference Standard (Control 891) of National Institute of Hygienic Sciences].

The raw material of thiamine hydrochloride was examined for preparation of the "Thiamine Hydrochloride Reference Standard". Analytical results were as follows: melting point 242.7 degrees; pH 3.2 (1 g, water, 100 ml); IR spectrum the same as that of JP Reference Standard (Control: 841); one and two impurities detected by TLC and by HPLC analyses, respectively; assay by thiochrome method 100.3% and by HPLC 100.1% against the JP Reference Standard. Based on the results, the present raw material was authorized to be the Reference Standard of the National Institute of Hygienic Sciences.