Quantification of a Botanical Negative Marker without an Identical Standard: Ginkgotoxin in Ginkgo biloba

Abstract

A new strategy for the analysis of natural products uses a combination of quantitative (1)H NMR (qHNMR) and adsorbent-free countercurrent separation (CS) methodology to establish a quantification method for ginkgotoxin (4'-O-methylpyridoxine) in Ginkgo biloba preparations. The target analyte was concentrated in a one-step CS process using the ChMWat +2 solvent system (CHCl3-MeOH-H2O, 10:5:5) and subsequently assayed by qHNMR. While commercial G. biloba seeds contained 59 μg of ginkgotoxin per seed, the compound was below the limit of detection (9 ppm) in a typical leaf extract. Due to the enrichment potential and loss-free operation of CS, the combination of CS and qHNMR is a generally suitable approach for threshold assays aimed at quantifying target compounds such as botanical negative markers at the low ppm level. As the proof of principle is demonstrated for relatively small CS capacities (20 mL, 1:40 loading) and modest NMR sensitivity (n = 16, 400 MHz, 5 mm RT probe), the approach can be adapted to quantification at the ppb level. The procedure enables the quantification of a botanical negative marker in the absence of identical reference material, which otherwise is a prerequisite for LC-based assays.

Countercurrent assisted quantitative recovery of metabolites from plant-associated natural deep eutectic solvents

“…Briefly, if the organic phase of a solvent system or an equivalent organic-only solvent system reported in [19] can develop a given analyte to a R f close to 0.5 on a normal phase TLC plate, the parent solvent system will likely deliver the analyte into the CCS K value sweet spot. This approach has been validated [20,23]. Although only H exane- E tOAc- M eOH- Wat er (HEMWat) and CH Cl 3 - Me OH- Wat er (ChMWat) solvent system families were fully detailed in the original GUESS method publication [19], in practice, this consideration can be applied to most available biphasic solvent systems.…”

“…As a nondestructive and near universal detection method, qHNMR is ideal for quantitative analysis. Because it is a mole-based determination, qHNMR does not need an identical, high purity, calibrant for the establishment of a standard curve [20]. Additionally, based on the UHPLC results, there was a small peak with a close retention time and a major absorption (Fig.…”

“…CCS was performed as previously described [20] in a TBE-20 A HSCCC instrument (16 mL, Tauto Biotech, China). The NADES-analyte solution (100 μL) was diluted with both upper and lower phase (200 μL of each), then loaded into the sample loop (2 mL).…”

The Generally Useful Estimate of Solvent Systems (GUESS) method enables the rapid purification of methylpyridoxine regioisomers by countercurrent chromatography

“…3- O -methylpyridoxine (1) [11], 4′- O -methylpyridoxine (2) [9], and 5′- O -methylpyridoxine (3) [12,13] were synthesized as shown in Fig. 1.…”

“…Thus, the present study was performed as an extension of the original GUESS method, and demonstrates that TLC-based solvent system selection may be applied in a single step to different set of analytes than what was proposed in the original article. The present study was also motivated by a recent study on 4′- O -methylpyridoxine or ginkgotoxin (2), which established a CCC- and qHNMR-based threshold assay for this botanical negative marker [9]. The synthesis and purification of the ginkgotoxin regioisomers, 3- O- methylpyridoxine (1) and 5- O -methylpyridoxine (3), enables their targeted CCC-based detection in complex (natural) mixtures with the aim of providing a better understanding of these alleged anti-vitamins [10].…”