Identification, Characterization, and Quantification of an Antipyridoxine Factor from Flaxseed Using Ultrahigh-Performance Liquid Chromatography−Mass Spectrometry

In the present study, the anti pyridoxine compounds linatine (1-[(n-gamma-L glutamyl) amino]-D proline) and 1 amino D proline (1ADP) were quantified following extraction from defatted flaxseed using aqueous isopropanol as a solvent, with extraction variables including time, temperature, and the solid/solvent ratio. Both linatine and 1ADP were identified,
characterized, and quantified via UPLC/ESI MS using authentic standards. To optimize the extraction conditions for these antipyridoxine compounds, a response surface methodology was applied using a second order polynomial to describe the experimental data. The predicted model for the optimal extraction was significant  with a R2 of 0.82. A varietal analysis
showed that the amount of anti pyridoxine present in flaxseed ranged from 177 to 437 μg 1ADPE per g of whole seed. The current study establishes the content of specific anti pyridoxine factors in flaxseed and positions the data for use in subsequent risk assessment modeling. (Authors abstract)

Flaxseed contains several antinutritive compounds, including phytic acid,  cyanogenic glycosides, and the dipeptide linatine.  Linatine (1-[(n-γ-L-glutamyl)amino]-D-proline), is a polar compound first identified  to possess antipyridoxine activity and it acts as an asymmetrically substituted secondary hydrazine that is thus responsible for the in vivo hydrazone toxicity of the active pyridoxyl 5 phosphate (PLP), ultimately leading to the vitamin B6 deficiency symptoms. 
The present study was designed to develop an ultrahigh-performance liquid chromatography mass spectrometry (UPLC-MS) technique for the measurement of linatine and 1ADP. The objectives included the optimization of extraction conditions using response surface methodology (RSM) with four factors (solvent concentration, time, and temperature, and the liquid/solid ratio), each tested at three distinct levels. The final objective of this study was the determination of the content of the anti-pyridoxine components in selected varieties of commercial flaxseed.

This study established a new method for assessing the anti pyridoxine providing an improved extraction of 1ADP and linatine from flaxseed by optimizing the extraction parameters using a RSM design. The UPLC MS/MS method, which has higher specificity and sensitivity for those analytes, revealed that the amount of antipyridoxine content in flaxseed was up to 437 μg/g of whole seed, much higher than previously reported. The presence of free 1ADP in flaxseed, which has never been reported before, is reported. Although varietal differences in 1ADP equivalents were apparent, further work is required to ascertain the impact of genotype by environment effects on the anti-pyridoxine content of flaxseed. Future work will assess the oral toxicity of anti-pyridoxines, especially 1ADP and linatine.(Editros comments)