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Summary of LC-MS Experience

2021-03-24
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Liquid chromatography-mass spectrometry technology is also called liquid chromatography-mass spectrometry (LC-MS), which uses liquid chromatography as the separation system and mass spectrometry as the detection system. LC/MS embodies the complementarity of the advantages of chromatography and mass spectrometry. It combines the high separation ability of chromatography for complex samples with the advantages of MS with high selectivity, high sensitivity, and the ability to provide relative molecular mass and structural information. Many fields such as food analysis and environmental analysis have been widely used.

The liquid-mass spectrometry interface technology is mainly developed along three branches:

1) The mobile phase enters the mass spectrometer for direct ionization, forming continuous-flowfast atom bombarment (CFFAB) technology, etc.;

2) After the mobile phase is atomized, the solvent is removed, and the analyte is evaporated and then ionized, forming a “moving-belt interface” and an ion-beam interface (particle-beam interface), etc.;

3) The small droplets formed after the mobile phase atomization are desolvated, gas-phase ionized or ionized after ion evaporation, forming a thermospray interface (thermospray interface), atmospheric pressure chemical ionization (APCI) and electrical Spray ionization (electrosprayionization, ESI) technology, etc.

*The development of liquid-mass spectrometry analysis technology depends on the joint development of liquid-mass spectrometry interface technology and mass spectrometer technology. Connecting the liquid chromatography with the mass spectrometer through a suitable interface will obtain a liquid-mass spectrometer with special analytical performance. In addition, connecting the mass spectrometer and mass spectrometer in series through the interface can make up for the shortcomings of various mass spectrometers, and achieve synergy. Improved effect.

In order to achieve the purpose of connecting the liquid phase and the mass spectrometer, a device acting as an “interface” is needed to connect the liquid phase and the mass spectrometer. This interface has to solve three main problems:

1) The flow rate used in liquid chromatography is relatively large, while mass spectrometry requires a high vacuum environment to work;

2) To provide enough ions from the mobile phase for mass spectrometry analysis;

3) Remove the possible contamination of the mass spectrometer caused by impurities in the mobile phase.

Analytical characteristics HPLC-MS can analyze strongly polar, hardly volatile, and thermally unstable compounds that cannot be analyzed by gas chromatography-mass spectrometry (GC-MS), but also has the following advantages:

① The analysis range is wide, and MS can detect almost all compounds, which can easily solve the problem of analyzing thermally unstable compounds;

② Strong separation ability. Even if the analyzed mixture is not completely separated on the chromatogram, the characteristic ion mass chromatogram of MS can also give their respective chromatograms for qualitative and quantitative;

③The qualitative analysis results are reliable, and the molecular weight and rich structure information of each component can be given at the same time;

④ The detection limit is low and MS has high sensitivity. Through the selective ion detection (SIM) method, its detection capability can be improved by more than an order of magnitude;

⑤ The analysis time is fast. The liquid chromatography column used by HPLC-MS is a narrow-diameter column, which shortens the analysis time and improves the separation effect;

⑥ High degree of automation, HPLC-MS has a high degree of automation.

Matrix effect in LC/MS

1) The influence of components other than the analyte in the specimen on the measured value of the analyte.

2) The matrix interferes with the analytical method’s ability to accurately determine the analyte. Broadly speaking, matrix effects should also include known interfering substances (for example, bilirubin, hemoglobin, ascorbic acid, etc. are all interfering substances in the Chol determination), but the matrix effect is currently limited to unknown or undetermined substances in biological materials. Or the influence of factors (such as viscosity, pH, etc.).

Matrix bias

The deviation of the analysis result caused by the matrix effect is called the matrix deviation. For the processed mixed serum used as a calibration material or quality control material, due to the changes in the physical and chemical properties of the serum matrix during the processing, matrix deviations often occur in routine determinations. The occurrence of matrix deviation is also related to the analysis system (including: methods, reagents and equipment used), so some people characterize the matrix effect as a specific reaction of the method, material and matrix.

Summary of experience (lessons) exchange:

1. Maintain the instrument well

First, the flow rate should not be too high, and the liquid quality cannot carry too much flow rate; secondly, the voltage should not be increased to the limit, especially when the positive and negative ions are converted properly; finally, the pipeline must be flushed and purged in time after the sample is completed.

2. Condition optimization of LC and MS is the key to success

1. Due to the small flow rate of the liquid (ESI is generally 0.2mL/min), the solvent strength of the configured sample should not be too large, and should be less than the initial ratio as much as possible, otherwise, problems such as retention time shift will occur.

2. If the conditions are good on the liquid phase, pay attention especially to the composition of the mobile phase to be converted into suitable LC-MS analysis.

3. Phosphate and other non-volatile buffer salts will precipitate in the ion source and block the capillary, etc. They should be replaced with volatile organic buffer salts.

4. Buffer salt will cause ion suppression, so the strength of the buffer should be controlled;

5. Detergents and surfactants will cause ion suppression. The adducts and ion clusters produced by surfactants will interfere with mass spectrometry data. Therefore, do not use detergents to clean glassware when used as an LC/MS instrument. If the container must be used, it is recommended to ultrasonically clean it several times.

3. start small

For example, the analyzed sample must be clean, which can protect the chromatographic column and prevent contamination of the mass spectrometer. After analyzing a large number of biological samples, when flushing the system, first rinse with a high proportion of water to rinse the source. Then switch to a high-proportion organic phase. At this time, remove the post-column tubing from the source to prevent impurities in the column from rushing into the mass spectrometer.

4. chromatographic column maintenance

1. Pre-treatment: The sample must be clean, whether it is for mass spectrometry or for column protection, biological samples are better extracted. If you directly precipitate, you must pay attention to the high speed above 12000rpm, low temperature centrifugation, and it is best to separate twice (be safe ), transfer the sample carefully, slowly suck from the middle, sometimes there will be floating objects.

2. Sample concentration: Mass spectrometry is a highly sensitive instrument. The injection concentration must not be too high. 1~2μg/mL is already ok. Too high a concentration is more likely to cause residues for the instrument, and the quantification will be inaccurate. .

3. Mobile phase: try to add volatile salt to the mobile phase, try not to add surfactants and the like, it is easy to ion suppression, if you encounter ion suppression, you can try to push your sample peak back or change the extraction Method, you can also try the APCI source. If your liquid phase is mixed at low pressure, try not to run the gradient as it takes a lot of time. If there is no alternative, if one needle takes a long time, you can consider switching and only measure the time before and after the peak of the sample. Can protect the mass spectrum. But if you use thick columns, you can also consider running gradients with higher flow rates, such as API4000, but try to minimize the dead volume.

4. Washing: Needless to say, washing the column, the low organic phase and the high organic phase are washed for a certain period of time (each for at least half an hour), and the column is stored in the high organic phase. After the experiment, the impulse source is also very important. It is also low organic phase and high organic phase impulse, but the time can not be so long. You can charge the source first and then the column, or separate the two. I personally think it is better to wash separately. The dirt on the pillar will not enter the source. The qi can be lowered when rushing to the source.

5. matters needing attention

1. The sample must be filtered through a 0.22μm filter membrane without particulates;

2. The solvent used for loading must be chromatographically pure, preferably consistent with your mobile phase ratio;

3. In reversed phase system, it is not allowed to dissolve the sample and load the sample with a weakly polar solvent such as n-hexane;

4. The sample is not allowed to contain metal ions, surfactants (do not use detergent to wash the bottle), phosphate, borate and other non-volatile salts;

5. The buffer solvent of the eluent must be volatile, such as acetic acid, ammonium acetate, tetrabutylammonium hydroxide, etc., chromatographically pure grade.

6. It is strictly forbidden for samples pH5-7 to contain strong inorganic or organic acids and alkalis;

7. The six-way valve is changed to three-way, and it is best to switch the area with no signal to waste liquid, so as to reduce source pollution;

8. Regularly vibrate the gas, clean the ion source, and change the oil;

Related Articles:

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LC-MS/MS Method Development

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