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Application of high-field NMR spectroscopy for characterization and quantitation of submilligram quantities of isolated natural products.

ABSTRACT: We have investigated and compared a number of sample conditions on different NMR platforms in the search of maximum SNR and optimal experiment time efficiency for structure elucidation and quantitation of natural products. Using restricted volume 3?mm Shigemi microcell assembly in conjunction with a 900?MHz NMR spectrometer equipped with a 5?mm carbon-sensitive inverse cryoprobe, it was possible to achieve a substantial increase in SNR (46-fold) as compared with a conventional room temperature 400?MHz instrument. Switching from standard 5?mm NMR tube to 3?mm Shigemi microcell assembly typically improved SNR by threefold on either 600 or 900?MHz cryoplatform. A quantitation method that relies on a calibrated residual protonated NMR solvent signal as internal standard was developed using the same hardware setup and restricted sample volume tubes. Linearity of the method spans over 3 orders of magnitude, from low microgram to milligram quantities. We successfully applied this method to quantify a low micrgram sample of paclitaxel, verified by a UV/VIS quantitation measurement.

SUBMITTER: Krunic A

PROVIDER: S-EPMC4752180 | biostudies-other | 2015 Dec

REPOSITORIES: biostudies-other

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Application of high-field NMR spectroscopy for characterization and quantitation of submilligram quantities of isolated natural products.

Krunić Aleksej A Orjala Jimmy J

Magnetic resonance in chemistry : MRC 20150820 12

We have investigated and compared a number of sample conditions on different NMR platforms in the search of maximum SNR and optimal experiment time efficiency for structure elucidation and quantitation of natural products. Using restricted volume 3 mm Shigemi microcell assembly in conjunction with a 900 MHz NMR spectrometer equipped with a 5 mm carbon-sensitive inverse cryoprobe, it was possible to achieve a substantial increase in SNR (46-fold) as compared with a conventional room temperature 4 ...[more]

PMID: 26289113

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Project description:A current challenge in metabolomics is the reliable quantitation of many metabolites. Limited resolution and sensitivity combined with the challenges associated with unknown metabolite identification have restricted both the number and the quantitative accuracy of blood metabolites. Focused on alleviating this bottleneck in NMR-based metabolomics, investigations of pooled human serum combining an array of 1D/2D NMR experiments at 800 MHz, database searches, and spiking with authentic compounds enabled the identification of 67 blood metabolites. Many of these (?1/3) are new compared with those reported previously as a part of the Human Serum Metabolome Database. In addition, considering both the high reproducibility and quantitative nature of NMR as well as the sensitivity of NMR chemical shifts to altered sample conditions, experimental protocols and comprehensive peak annotations are provided here as a guide for identification and quantitation of the new pool of blood metabolites for routine applications. Further, investigations focused on the evaluation of quantitation using organic solvents revealed a surprisingly poor performance for protein precipitation using acetonitrile. One-third of the detected metabolites were attenuated by 10-67% compared with methanol precipitation at the same solvent-to-serum ratio of 2:1 (v/v). Nearly 2/3 of the metabolites were further attenuated by up to 65% upon increasing the acetonitrile-to-serum ratio to 4:1 (v/v). These results, combined with the newly established identity for many unknown metabolites in the NMR spectrum, offer new avenues for human serum/plasma-based metabolomics. Further, the ability to quantitatively evaluate nearly 70 blood metabolites that represent numerous classes, including amino acids, organic acids, carbohydrates, and heterocyclic compounds, using a simple and highly reproducible analytical method such as NMR may potentially guide the evaluation of samples for analysis using mass spectrometry.

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Dataset Information

Application of high-field NMR spectroscopy for characterization and quantitation of submilligram quantities of isolated natural products.

Publications

Application of high-field NMR spectroscopy for characterization and quantitation of submilligram quantities of isolated natural products.

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