Simple Detection Methods for Antinutritive Factor β-ODAP Present in Lathyrus sativus L. by High Pressure Liquid Chromatography and Thin Layer Chromatography

Abstract

Lathyrus sativus L. (Grass pea) is the source for cheap and nutritious food choice in drought and famine susceptible zones in greater part of North India and Africa. The non-protein amino acid β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) has been known for decades for its potent neurotoxic effect, causing irreversible neurodegenerative disease “neurolathyrism”, present in both seed and leaf of Lathyrus sativus L. and other species in varying proportions. It is crucial to establish a rapid as well as reliable detection methodology for β-ODAP content in various Lathyrus plants. Currently available HPLC based methods involve multi-step derivatization of the sample. To overcome this, we have developed β-ODAP analysis method by HPLC without any prior derivatization. This method is statistically significant in the range of 2 to 100μg/ml and exhibited linear response with r 2 > 0.99. Limit of detection and quantitation of the later method was determined to be 5.56 μg/ml and 16.86 μg/ml, respectively. In addition to this, a TLC based method has also been developed. The limit of detection of β-ODAP is 0.6μg and for its substrate, L-1,2-diaminopropionic acid is 5μg. Both HPLC and TLC methods were validated by conducting in-vitro bioconversion test to detect the presence of biocatalyst in plant extract. This method is economical, rapid and simple.

An Improved HILIC HPLC-MS/MS Method for the Determination of β-ODAP and Its α Isomer in Lathyrus sativus

“…High-throughput accurate quantification methods for this specific compound are extremely important for these breeding programs, where a large number of samples need to be quantified in a simple and fast way. Several analytical methodologies have been suggested for β-ODAP quantification, but some were not able to distinguish the toxic β-ODAP from its non-toxic α isomer, such as colorimetric methods [12], GC-MS (gas chromatography-mass spectrometry) after sample derivatization [9] and TLC (thin layer chromatography) [13]. Some other methodologies have been successful in analyzing both α- and β-ODAP, namely by NMR (nuclear magnetic resonance) [14] and CZE (capillary zone electrophoresis).…”

“…Some other methodologies have been successful in analyzing both α- and β-ODAP, namely by NMR (nuclear magnetic resonance) [14] and CZE (capillary zone electrophoresis). However, the high operation pH (9.2) in CZE may result in β-ODAP hydrolysis to diaminopropionic acid (DAP) [13,15,16].…”

“…Although these methods have been employed, HPLC (high performance liquid chromatography) has been the most used for β-ODAP quantification [8,9,12,13,15,17,18,19,20,21]. However, the analysis of ODAP by HPLC is a challenge due to its high polarity and weak UV absorption [8].…”

“…The majority of the currently available HPLC based methods for β-ODAP quantification involve the use of a reversed phase C18 column and UV detection, after a multi-step derivatization of the sample [17,18,19,22]. However, poor derivative stability, side reactions, and reagent interferences may occur, which may lead to interference signals [8,13,20]. Furthermore, these methods are usually laborious, inconvenient, and time-consuming [8,13].…”

“…However, poor derivative stability, side reactions, and reagent interferences may occur, which may lead to interference signals [8,13,20]. Furthermore, these methods are usually laborious, inconvenient, and time-consuming [8,13]. More recently, a UHPLC-MS/MS method using a C18 column has been developed for the quantification of β-ODAP, L -homoarginine, and asparagine in Lathyrus sativus [21].…”