HPLC–NMR Revisited: Using Time-Slice High-Performance Liquid Chromatography–Solid-Phase Extraction–Nuclear Magnetic Resonance with Database-Assisted Dereplication

Abstract

Time-based trapping of chromatographically separated compounds onto solid-phase extraction (SPE) cartridges and subsequent elution to NMR tubes was done to emulate the function of HPLC-NMR for dereplication purposes. Sufficient mass sensitivity was obtained by use of a state-of-the-art HPLC-SPE-NMR system with a cryogenically cooled probe head, designed for 1.7 mm NMR tubes. The resulting (1)H NMR spectra (600 MHz) were evaluated against a database of previously acquired and prepared spectra. The in-house-developed matching algorithm, based on partitioning of the spectra and allowing for changes in the chemical shifts, is described. Two mixtures of natural products were used to test the approach: an extract of Carthamus oxyacantha (wild safflower), containing an array of spiro compounds, and an extract of the endophytic fungus Penicillum namyslowski, containing griseofulvin and analogues. The database matching of the resulting spectra positively identified expected compounds, while the number of false positives was few and easily recognized.

The integration of LC‐MS and NMR for the analysis of low molecular weight trace analytes in complex matrices

“…The SPE cartridges were then eluted in 25 µL of solvent directly into 1.7 mm NMR tubes. The transfer of samples from the SPE to the NMR tube was all automated . Although limits of detection were not explicitly discussed, the data appear to show at least partial NMR spectra from compounds at the 1% level of a single 50 μg LC injection, in NMR acquisition times just under 10 min, consistent with expectations of the 1.7 mm micro‐cryoprobe …”

“…Most applications using microcryoprobes have been conducted using 3 mm microcryoprobes, however, a recent example from Johansen et al used a 1.7 mm microcryoprobe . They used SPE to collect NMR analytes from LC‐MS separations of a safflower and a Penicillum fungus.…”

Combined LC/UV/MS and NMR Strategies for the Dereplication of Marine Natural Products

“…Software tools such as ACD/Spectrus DB [139], MNova DB [140], or Bruker AMIX [141] allow searching for peaks, multiplets, spin systems, regions, or even whole spectra similarity. It has been shown that 1 H NMR spectra, obtained through analytical HPLC separation of extracts with time-sliced SPE trapping of eluting compounds and acquired with a microcryoprobe, can be used for dereplication purposes by matching an in-house database using an algorithm developed and operating under Matlab [142]. On the other hand, the spectra contained in the commercial SpecInfo database by Wiley can be conveniently searched for in installations under ACD Labs software [143] or the KnowItAll NMR Spectral Library by Bio-Rad [144], which also includes the 13 C NMR spectra collection of Wolfgang Robien [145].…”

Dereplication of microbial extracts and related analytical technologies

“…They connected the fraction collector to HPLC-UV or HPLC-UV-MS and used microtiter plates to collect fractions that were then subjected to HTS using robotic systems. This procedure has been used widely [42][43][44] and is called dereplication, in the narrow meaning of the word. Wagenaar 45 also reported a method of constructing a fraction library that was prepared before screening.…”

“…In order to avoid evaporation step, trap column (solid phase extraction cartridge) can be used to concentrate the fraction like LC-SPE-NMR technique. 44 The unstable compounds might be captured by this method. As mentioned in former chapter, the ion sources currently used in LC-MS instruments, ESI, atmospheric pressure chemical ionization and atmospheric pressure photo ionization, have been commercialized.…”