Introduction to UPLC-MS Sample Preparation

Sample Purity

The use of high-purity solvents is critical to reducing the risk of contamination. Such contamination could affect the quality of results. Larger biomolecules may require an additional digestion phase to ensure complete dispersion into solution prior to analysis.

Concentration 

Optimizing sample concentration is crucial for accurate analysis. Some techniques allow for concentrating analytes to enhance sensitivity, while others may require dilution to bring samples within the optimal range for the instrument.

Solvent Selection

Choosing appropriate solvents is critical for effective sample preparation. The selected solvents should be compatible with both the sample and the UPLC-MS system. Common solvents may include water, ACN, MeOH, acetic acid, or ammonium acetate depending on the different analytes utilized. 

Sample Stability

Proper storage and handling of samples are essential to prevent degradation during preparation. Consider the stability of analytes and use appropriate preservation methods. Possible sources of analyte loss to consider are thermal degradation, oxidation, or analyte volatility.

Liquid-Liquid Extraction (LLE)

LLE is the transfers of solutions and analytes from a liquid phase to another immiscible liquid phase based on differences in solubility. Benefits of this include:

- Concentration of analytes, enhancing sensitivity
- Depletion of matrix components, increasing selectivity

Common solvents used in liquid-liquid extraction include n-hexane, heptane, dichloromethane, ethyl acetate, and methyl tert-butyl ether. The aqueous matrix pH level should be set two pH units above the pKa of a basic analyte, or two pH units below the pKa of an acidic analyte. This is to ensure 99% of the analyte is uncharged (1).

Solid-Phase Extraction (SPE)

Solid Phase Extraction is a more selective sample preparation technique that can effectively remove matrix components and concentrate analytes. Key considerations for SPE include:

- Selection of appropriate SPE cartridges
- Optimization of loading, washing, and elution steps
- Ensuring consistent flow rates across multiple samples

Protein Precipitation

Protein precipitation is a simple and cost-effective method, particularly useful for biological samples. Common precipitants include:

- Water-miscible organic solvents (acetonitrile, acetone, ethanol, methanol)
- Acids (trichloroacetic acid, perchloric acid)
- Metal ions and salts

Acetonitrile, trichloroacetic acid, and zinc sulfate are optimal at removing protein in their categories (>96%, 92%, and 91% protein precipitation efficiency at a 2:1 ratio of precipitant to plasma, respectively).

Filtration 

Sample filtration is crucial for removing particulates that could clog the UPLC system. Various filter types and pore sizes are available, depending on the sample properties.

Derivatization

Derivatization can be used to improve the chromatographic behavior or detection sensitivity of certain analytes. Common derivatization reactions include sialylation, acylation, and alkylation.

Biological Samples

Samples of blood, plasma and serum typically utilize methods of protein precipitation or SPE. Urine samples may need dilution or SPE, while tissue samples typically require homogenization followed by extraction.

Environmental Samples 

Water and soil samples may require filtration, SPE, or LLE, depending on the target analytes. Extraction techniques for environmental contaminants often involve multi-step processes to isolate compounds of interest.

Food and Beverage Samples

QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) is a popular method for food sample preparation. It combines salt-out extraction with dispersive SPE cleanup and is adaptable for various analyte classes.

Pharmaceutical Samples

Drug formulation samples may require dissolution, extraction, or dilution, depending on the dosage form. Consideration must be given to potential excipient interferences.

Vial Selection

Choose autosampler vials compatible with your UPLC-MS system. Consider factors such as sample volume, solvent compatibility, and potential adsorption issues. For samples containing chloroform or acetonitrile, use glass vials and glass inserts to prevent plasticizer contamination.

Sample Storage

Proper storage conditions are crucial to maintain sample integrity. Consider temperature, light exposure, and potential degradation pathways when storing prepared samples.

Matrix effects are a major constraint in UPLC-MS methods. Complex sample matrices, especially biological ones, can interfere with the analysis by causing ion suppression or enhancement. Strategies to minimize these effects include:

- Optimizing chromatographic separation
- Using appropriate internal standards
- Implementing matrix-matched calibration
- Enhancing sample cleanup procedures

Implement quality control measures such as:

- Use of internal standards
- Analysis of blank samples
- Regular validation of sample preparation methods

These steps ensure consistency and reliability in your UPLC-MS analyses.

Automation of sample preparation processes can improve reproducibility, precision, and throughput. Examples include:

- Automated liquid handling systems
- Robotic sample preparation platforms
- Online coupling of sample preparation to UPLC-MS systems

1. Tailor sample preparation methods to specific sample types and analysis goals
2. Use high-purity solvents and reagents to minimize contamination
3. Optimize chromatographic separation to complement sample preparation
4. Regularly maintain and clean sample preparation equipment and UPLC-MS instruments
5. Consider miniaturization and online coupling of sample preparation techniques for improved efficiency and sensitivity

Effective sample preparation is crucial for successful UPLC-MS analysis. By carefully considering sample characteristics, selecting appropriate techniques, and optimizing methods, analysts can significantly improve the accuracy, sensitivity, and reliability of their results. As UPLC-MS technology continues to advance, ongoing research into innovative sample preparation strategies will further enhance the capabilities of this powerful analytical technique.

Be sure to check out our interview with Mark Dennis Chico Retrato, a PhD candidate at Uppsala University in Sweden, who has developed a novel method for analyzing phospholipids in total parenteral nutrition (TPN) products using Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS).

References:

(1) Israel D. Souza, Maria Eugenia C. Queiroz. LCGC Supplements. Volume 39. Issue 11 (2021).