Mass spectrometry sample pretreatment methods and steps are key to ensuring the accuracy and reliability of mass spectrometry analysis. Before mass spectrometry analysis, proper pretreatment of the sample is crucial, as it can remove interfering substances in the sample, enrich the target analytes, and adjust the sample to a state suitable for mass spectrometry analysis. The following will introduce in detail several commonly used mass spectrometry sample preparation methods and their operating steps. These methods cover a variety of techniques from simple dilution to complex solid-phase extraction, aiming to meet different sample types and analysis needs.

1. Sample pretreatment method 1. Dilution method (1) Method description: Dilute the sample with an LC-MS compatible solvent so that the target concentration in the sample reaches the detection range of the instrument. (2) Advantages: simple operation, strong compatibility, and high recovery rates for almost all analytes. (3) Disadvantages: There is no selectivity, and all components in the sample will enter the LC-MS system.

2. Protein precipitation method (PPT) (1) Method description: The protein in the sample is precipitated by adding organic solvents (such as methanol, ethanol, acetonitrile, etc.) to remove macromolecular impurities such as proteins. (2) Advantages: simple operation, low cost, and easy to implement. (3) Disadvantages: Only proteins are removed, endogenous impurities are not completely removed, and long-term detection may have an impact on the life of the chromatographic column and instrument; the concentration of the processed sample will be diluted, which is not suitable for targets with low concentrations and The mass spectrum response is low. (4) Step: Take an appropriate amount of sample and add it to the centrifuge tube. Add precipitation reagent (such as acetonitrile or methanol) in proportion and vortex to mix. Centrifuge and take the supernatant for subsequent analysis.

3. Liquid-liquid extraction (LLE) (1) Method description: Based on the different distribution coefficients of the target analyte between mutually immiscible solvents, the target analyte is transferred from one solvent to another solvent. (2) Advantages: Strong selectivity, suitable for non-polar to moderately polar analytes, can achieve enrichment and concentration effects, reduce matrix effects, low cost, easy method transfer, and short development time. (3) Disadvantages: Not suitable for hydrophilic polar substances, easy to contaminate during operation, and time-consuming. (4) Step: Take an appropriate amount of sample and add it to the separatory funnel. Add an appropriate amount of extraction solvent and shake to mix. Let stand and separate, and collect the organic phase or aqueous phase for subsequent analysis.

4. Solid-phase extraction (SPE) (1) Method description: Load the sample onto the solid-phase extraction column. The target analyte is retained on the column through adsorption, while the impurities are eluted. (2) Advantages: It is suitable for various types of compounds from polar to non-polar, can be automated, and can enrich and analyze trace samples. (3) Disadvantages: The operation is cumbersome, time-consuming and costly, and requires rich operating skills and separation scientific knowledge. (4) Step: Activate the solid-phase extraction column. Load the sample onto the extraction column. Wash away impurities with eluent. The target analytes are eluted with the eluent for subsequent analysis.

5. Immunoaffinity extraction (IA-SPE) (1) Method description: Extraction is performed using the principle of binding of specific antibodies to target analytes. (2) Advantages: high selectivity and specificity, high sensitivity and good reproducibility. (3) Disadvantages: The operation is cumbersome, time-consuming and costly. (4) Step: Activating immunosorbent. Load the sample onto the immunosorbent. Elution removes non-specifically adsorbed impurities. The target analytes are eluted for subsequent analysis.

2. Notes 1. Sample type: The pretreatment methods for different sample types (such as cells, tissues, body fluids, etc.) may be different, and the appropriate method needs to be selected according to the characteristics of the sample. 2. Properties of the target analyte: Understanding the physical and chemical properties of the target analyte (such as lipid-water partition coefficient, pH, solubility, etc.) is crucial to selecting an appropriate pretreatment method. 3. Instrument compatibility: Ensure that the solvents and reagents used in the pretreatment process are compatible with the mass spectrometry instrument. 4. Operating specifications: Carry out pre-treatment operations strictly in accordance with operating procedures to avoid introducing contamination and errors.

Mass spectrometry sample preparation involves multiple steps, and each step is crucial. Only by doing good sample pre-processing can the accuracy and reliability of subsequent mass spectrometry analysis be ensured. We hope that the introduction in this article can help you better understand the methods and steps of mass spectrometry sample preprocessing.