HiFSA Fingerprinting Applied to Isomers with Near-Identical NMR Spectra: The Silybin/Isosilybin Case

Abstract

This study demonstrates how regio- and diastereo-isomers with near-identical NMR spectra can be distinguished and unambiguously assigned using quantum mechanical driven, 1H iterative Full Spin Analysis (HiFSA). The method is illustrated with four natural products, the flavonolignans silybin A, silybin B, isosilybin A, and isosilybin B, which exhibit extremely similar coupling patterns and chemical shift differences well below the commonly reported level of accuracy of 0.01 ppm. The HiFSA approach generated highly reproducible 1H NMR fingerprints that enable distinction of all four isomers at 1H frequencies from 300 to 900 MHz. Furthermore, it is demonstrated that the underlying numeric 1H NMR profiles, combined with iterative computational analysis, allow parallel quantification of all four isomers, even in difficult to characterize reference materials and mixtures. The results shed new light on the historical challenges to the qualitative and quantitative analysis of these therapeutically relevant flavonolignans and open new opportunities to explore hidden diversity in the chemical space of organic molecules.

Identification of Diet-Derived Constituents as Potent Inhibitors of Intestinal Glucuronidation

“…Silymarin was obtained from Euromed S.A. (Barcelona, Spain) and consisted of the flavonolignans silybin A (16%), silybin B (24%), isosilybin A (6.4%), isosilybin B (4.4%), silydianin (17%), silychristin (12%), and isosilychristin (2.2%); the remainder consisted of the flavonoid taxifolin (1.6%) and uncharacterized polyphenols and aliphatic fatty acids (Davis-Searles et al, 2005). The individual flavonolignans were purified as described previously (Graf et al, 2007) and were .97% pure as determined by ultra-high-performance liquid chromatography (UHPLC) (Napolitano et al, 2013). Methanol (liquid chromatography-mass spectrometry grade), ethanol, Tris-HCl, Tris base, and formic acid were purchased from Fisher Scientific (Waltham, MA).…”

Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product–Drug Interaction

“…Silymarin was obtained from Euromed S.A. (Barcelona, Spain) and consisted of silybin A (16%), silybin B (24%), isosilybin A (6.4%), isosilybin B (4.4%), silydianin (17%), silychristin (12%), and isosilychristin (2.2%); the remainder consisted of the flavonoid taxifolin (1.6%) and uncharacterized polyphenols and aliphatic fatty acids (Davis-Searles et al, 2005). Individual flavonolignans were purified as described previously (Graf et al, 2007) and were .97% pure as determined by ultra high-performance liquid chromatography (UHPLC) (Napolitano et al, 2013). Dimethylsulfoxide, methanol (liquid chromatography/mass spectrometry grade), ethanol, Tris-HCl, Tris base, and formic acid were purchased from Fisher Scientific (Waltham, MA).…”

Chemoenzymatic Synthesis, Characterization, and Scale-Up of Milk Thistle Flavonolignan Glucuronides

“…Alamethicin was purchased from Cayman Chemical Company (Ann Arbor, MI). The individual milk thistle flavonolignans were purified as described in detail previously (Graf et al, 2007) and were .97% pure as determined by ultra-performance liquid chromatography (UPLC) (Napolitano et al, 2013). Methanol [liquid chromatography/mass spectrometry (MS) grade], ethanol, Tris-HCl, Tris base, and formic acid were purchased from Fisher Scientific (Waltham, MA).…”

“…S3-S6; Table 3). With respect to the characterization of flavonolignans by NMR, in general (Napolitano et al, 2013), this study varied in one major aspect. Based on earlier research that derivatized the flavonolignans Althagafy et al, 2013), the phenol moieties were hypothesized to be glucuronidated readily; indeed, other researchers have reported this observation (Kren et al, 2000;Jan cová et al, 2011;K ren et al, 2013;Charrier et al, 2014).…”