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Sample Preparation for NMR by Solid Phase Extraction

SPE-NMR - Sample preparation for NMR by Solid Phase Extraction

SPE-NMR is a process that uses solid phase extraction (SPE) to prepare solutions for NMR. It is applied to complex aqueous samples like food matrices or body fluids. It simplifies the mixture by removal of disturbing compounds like inorganic salts and highly concentrated polar matrix components. Medium to non-polar compounds are retain and concentrated. And later eluted in a well-defined organic solvent (i.e. methanol). The SamplePro SPE is a robotic system which performs this process in a completely automatic way and supplies the solutions in NMR tubes ready for NMR measurement.

Screening complex aqueous samples with NMR

While NMR spectroscopy can analyze, classify mixtures and quantify for compounds in the mixtures, it often suffers from the fact that the spectrum is dominated by the intense signals of the matrix components of the mixture. Signals from characteristic minor compounds can be covered completely or at least may be difficult to detect. In addition matrix effects from salts and acids may lead to variations of signal positions and shape.

In contrast to SPE-NMR, LC-SPE-NMR is used for the identification or structure elucidation of single components the combination of chromatography and NMR with post-column-SPE. A single compound is isolated by a chromatographic separation. The SPE is here used to for enrichment of this isolated, pure component that elutes from the chromatography. The SPE collection enriches the sample, separates it from the complex chromatography solvent and provides it a concentrated solution in a deuterated NMR solvent.

For untargeted screening applications the isolation into individual pure components is not required and too time consuming. A coarse separation into fractions based on the polarity of the compounds is sufficient. SPE-NMR is the method of choice to achieve this goal.

SPE-NMR with SamplePro SPE

The SamplePro SPE performs the SPE process with the original sample mixture and generates typically one or two fractions per incoming sample. The major components (typically polar like sugars in beverages or endogenous metabolites from bodyfluids) are not retained and are collected in the aqueous load fraction.
The compounds of interest (typically unpolar) are retained on the cartridge, enriched, dried and eluted as concentrated solution with an organic, NMR friendly solvent.
For ion exchange SPE applications, a third fraction is generated by flushing the cartridge with a different SPE solvent containing an acid or base according to the ions to be released from the sorbent.

The complete process of preparation, loading, drying, elution and transfer of the fractions into NMR tubes is fully automated and can process up to 48 samples yielding 96 fractions in an unattended process.
A standard method is provided that is optimized and validated by BRUKER for NMR screening. Quality and reproducibility is essential as small changes in water content, pH value or salt content may considerably affect position and shape of NMR signals and make the evaluation of the data difficult or impossible.

Solid Phase Extraction (SPE)

The principle of solid phase extraction: an aqueous sample (e.g. fruit juice, wine or body fluid) is passed through a conditioned and equilibrated SPE cartridge, washed, dried and finally eluted with a small volume of organic solvent for further measurement.

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The classical SPE follows six steps:

  1. Condition of the sorbent bed with organic solvent
  2. Equilibration of the cartridges with water
  3. Loading of the sample
  4. Washing of the cartridges
  5. Drying in order to remove residual water
  6. Elution with a small volume of organic solven

Two versions of the Sample SPE are available. For the Foodscreener™, SPE extracts are transferred into 7" tubes while for the IVDr™ SampleJet™ racks are employed.

Liquid Handling Properties

The SamplePro SPE uses 25ml vials for the sample input and typically 10ml of sample is subjected to the SPE process.
For the SPE step 3ml SPE cartridges are used (200mg sorbent material).
After the drying step, extracts of 2-3ml volume are produced. A fraction of it is transferred into NMR tubes as part of the automated process.

Reproducibility and removal of polar matrix constituents

SPE-NMR removes polar matrix components from the sample while moderately polar metabolites are quantitatively and reproducibly retained and eluted from the SPE cartridge after applying a dying step:


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Statistical analysis of wine extracts


NMR and LC-MS data obtained from the combined use of the SamplePro SPE™, UPLC-TOF-MS and FoodScreener™ can be used for subsequent chemometric approaches as demonstrated below after processing NMR data of corvina and primitivo wines clearly showing differences in the aromatic region:

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Structure elucidation of unknown marker

LC-SPE-NMR-MS can be used for the isolation and structure elucidation of marker differentiating between both grape varieties. After cleaning of the pre-isolated peak from a LC-time slice-SPE-NMR run, acquisition of all necessary NMR data and determination of the molecular formula, the structure can be generated with the use of the CMC-se software.

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