LC-MS/MS, Sample prep, SPE, Solid phase extraction, Microelution, detection limit

Nigel Tooke

How to meet demands for detection limits with the right sample prep

October 15, 2020 at 9:04 AM / by Nigel Tooke

Highly sensitive techniques such as LC-MS/MS are enabling analytical laboratories to generate more data on more analytes in more samples, in less time, at a lower cost, and using smaller sample volumes. Achieving this performance involves pushing the limits of detection and quantitation with high levels of sensitivity and reproducibility that are only possible with high quality samples.

Sample prep must remove matrix interferences and also concentrate the sample to give high performance analytical instrumentation the best possible chance of consistently delivering accurate results with high sensitivity. The question is, how can this be done in an efficient way?

The need for sample concentration

For most biological matrices, sample prep focuses on sample clean up and analyte concentration. Solid-phase extraction (SPE) has become especially popular due to the availability of a large range of sorbents and affinity phases that can be incorporated into SPE columns and plates. These deliver high enrichment with good recovery combined with low consumption of organic solvents.

Sample volumes used in biofluid analysis are typically limited to 100–500 µL and extracted using SPE columns or 96-well plates containing 10–100 mg sorbent. With these formats, the minimum elution volume is typically 150 µL, which can mean that the analyte concentration may be below detection or quantitation limits. The standard method of reducing sample volume and therefore increasing analyte concentration is to add an evaporation step followed by reconstitution in a smaller volume of solvent, but there are several drawbacks with evaporation.

Evaporation isn’t just solvent removal

The evaporation step takes time and increases the complexity of the overall workflow. Even when using the most efficient systems, evaporation can add 30–60 mins to the overall extraction procedure. Some analytes are also prone to losses during evaporation, which can reduce sensitivity, and make it difficult to reach the required detection limits. This is particularly problematic for:

  • some ‘sticky’ peptides, and other hydrophobic molecules, which can bind irreversibly to the walls of the evaporation vial/plate, or be difficult to solubilize after evaporation
  • highly volatile analytes such as amphetamines
  • analytes that are sensitive to thermal degradation

A better way forward is to improve the SPE step by reducing the elution volume.

Miniaturizing elution is the sample prep solution

As in many developments in the analytical laboratory, miniaturization brings the possibility of doing more with less. In the case of sample prep, an attractive alternative to concentration by evaporation is miniaturization of the SPE device. Microelution SPE devices are essentially miniature versions of the traditional 96-well SPE plate that contain tiny amounts of sorbent. They are ideal for analysis that involves small sample volumes, or for analysts that need to improve assay sensitivity and save time or eliminate sample losses by avoiding the evaporation step. We will look at microelution SPE for sample preparation in more detail in the next article in this series.

Want to know more about the benefits of miniaturization of SPE?

Learn More

Topics: Sample preparation, Solid-phase extraction, detection limit, Microelution

Nigel Tooke

Written by Nigel Tooke