In various in-vitro and in-vivo studies, the naturally occurring amino acid found in fenugreek, 4-hydroxy-isoleucine (4-OH-Ile), has been shown to stimulate insulin secretion and promote insulin sensitivity in type-II diabetic models. In this study, ultrahigh-performance liquid chromatography and tandem mass spectrometry were used to separate and detect 4-OH-Ile in complex biological samples. The structure and purity of isolated standard 4-OH-Ile was determined using high field HNMR and CMR. Collison of the precursor 148 m/z with argon in Q2 resulted in three major SRM fragments with m/z ranges of: 74.28-74.33, 102.10-102.15, and 130.25-130.33. Increasing the collision energy in Q2 resulted in a much higher percentage of the 102 m/z peak, making it the most significant SRM for identifying and confirming 4-OH-Ile in complex mixtures. Samples of water, breastmilk, plasma, and urine were spiked with standard 4-OH-Ile at approximately 1 ppm, before and after methanol/chloroform extraction, and then analyzed. The precursor 148 m/z and the product 102 m/z were detected in all samples tested. GC-MS was also used to confirm the identity of the amino acid in the spiked breastmilk and plasma samples after derivatization.

Characterization of 4-hyroxy-isoleucine in complex mixtures by UHPLC-MS

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.