HPLC–SPE–NMR in pharmaceutical development: capabilities and applications

Abstract

High-performance liquid chromatography-solid phase extraction-NMR spectroscopy (HPLC-SPE-NMR) has recently become commercially available and has been evaluated with regard to its applicability in a pharmaceutical environment. The addition of an automated SPE unit to an HPLC-NMR system for peak trapping results in an improved NMR signal-to-noise ratio (S/N) and also has other practical advantages. The trapping efficiency is shown to depend on compound polarity and is highest for compounds eluting late on reversed-phase HPLC systems. Multiple peak trapping further increases the S/N, again with the best results for less polar compounds. For polar compounds, multiple peak trapping resulted in no S/N gain as the amount of material retained on the SPE cartridge was equivalent to that from a single injection. When compared with conventional HPLC-NMR, a S/N gain of up to five-fold could be achieved for some compounds in a single trapping step. A major advantage of the technique is the independence of the chromatographic step from the NMR step, resulting in greater versatility than conventional HPLC-NMR in the HPLC solvents and NMR solvents that can be used. Practical applications from both drug metabolite and drug impurity identification are presented.

Metabolism of Dicentrine: Identification of the Phase I and Phase II Metabolites in Miniature Pig Urine

“…Hence, the HPLC-NMR technique has been applied to study the metabolic profile of some drugs (Burton et al, 1997;Spraul et al, 2003). The HPLC-SPE-NMR technique has also been successfully applied in analysis of natural products (Wang and Lee, 2005;Lam et al, 2007;Lee et al, 2007) as well as in identification of certain metabolites (Godejohann et al, 2004;Sandvoss et al, 2005). In the present study, HPLC-SPE-NMR, along with HPLC-HRESIMS, was applied to the characterization of the dicentrine metabolites in the urine of miniature pigs.…”

Metabolism of (2S)-Pterosin A: Identification of the Phase I and Phase II Metabolites in Rat Urine

“…Prominent among these is the technique of HPLC-SPE-NMR, for which a SPE cartridge is used to trap multiple injections of a metabolite to enable sufficient accumulation for NMR measurement and thereby eliminating the eluent effect on 1 H NMR signals. Various studies successfully applied such a technique to identify the metabolites of drugs or bioactive compounds (Burton et al, 1997;Spraul et al, 2003;Godejohann et al, 2004;Sandvoss et al, 2005;Lai et al, 2010) and the constituents in complex natural products (Wang and Lee, 2005;Lam et al, 2007;Lee et al, 2007). In the present study, HPLC-SPE-TT-NMR, which equipped a liquid handler between SPE and NMR to transfer compound from SPE to a 2-mm NMR tube for NMR measurement, and HPLC-HRESIMS were applied to characterize the metabolites of (2S)-pterosin A (1) in rat urine, collected in 24 h after intragastrical oral administration (Brant and Remon, 1991).…”

Which Hydroxy? Evidence for Species Differences in the Regioselectivity of Glucuronidation in Rat, Dog, and Human in Vitro Systems and Dog in Vivo

“…Recent advances in NMR technology (Bernstein and Lewis, 2004), such as capillary NMR (Lewis et al, 2005) and HPLC-solid-phase extraction-NMR (Sandvoss et al, 2005), have brought improvements to sensitivity in metabolite identification. Despite this, much metabolite identification work is performed close to the sensitivity limits, and it is often only feasible to obtain proton NMR spectra or simple proton-proton correlations within a reasonable time frame.…”