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Liquid phase mass spectrometry (LC-MS)-matrix effects in biological analysis

2021-03-24
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What is the matrix effect?

When analyzing drugs in biological samples based on liquid-phase-mass spectrometry (LC-MS) methods, some common extracts of the samples may have an impact on the ionization efficiency of the target compound. This effect can be observed from the response of the instrument, the signal of the compound It is enhanced or more commonly suppressed. This phenomenon is called the matrix effect.

What are the common causes of matrix effects?

During LC-MS/MS analysis, many substances in biological samples may have matrix effects. These matrix-effect substances must be present in the final extracted sample and eluted together with the compound or (and) the internal standard in the chromatographic system. However, even with these co-eluting substances, the matrix effect does not necessarily affect the analytical method, because the magnitude of the matrix effect depends on the type of atmospheric pressure ionization (API) used in the method (APCI is less susceptible to matrix effects than ESI The influence of interference), the relative concentration of interfering substances and the magnitude of the offsetting effect of the calibration standard or isotope internal standard.

The substances that cause the matrix effect can be divided into two categories: endogenous and exogenous according to their source.

Endogenous main disturber: usually some organic matter (protein, fat, phospholipid, etc.) and inorganic matter present in biological samples, and may also include a metabolite formed in the body. At present, plasma is the most common biological fluid used for sample analysis in the drug research and development industry. It generally contains high concentrations of salt, protein, fat and phospholipids, as well as a small amount of carbohydrates, peptides and other organic compounds. All of these components are possible. Lead to matrix effect.

For example, phospholipids, which may be one of the main causes of matrix effects, especially the general protein precipitation (PPT) method can not effectively remove phospholipids, you can consider using LLE to extract the test substance, which can eliminate the impact of phospholipids as much as possible . In addition, due to the hydrophobicity of long lipid chains, these compounds are generally eluted later under reversed-phase (RP) chromatographic conditions, so they are more likely to be eluted together with the compounds and become a source of interference. In contrast to this are highly polar salts, which cannot be retained in liquid chromatography (LC), and are generally eluted at the dead time before the elution of the compound, which may also cause matrix effects.

Exogenous main disturber: foreign substances introduced during the sample preparation process or sample processing. For example, anticoagulant lithium heparin, plasticizers used in sample collection and storage containers, buffers, ion pair reagents, and excipients added to pharmaceutical preparations, especially Tween-80 and PEG-400, may cause matrix effects.

What is the mechanism of matrix effect?

At present, it is generally believed that the matrix effect is caused by the endogenous or exogenous matrix material competing with the target compound for charge and neutralizing the ionization process during the sample ionization process in the mass spectrometry ion source. The following takes the most commonly used ESI (Electrospray Ion Source) as an example. Before the liquid sample separated in liquid phase enters the mass spectrometry analysis, the liquid sample must be desolventized in the front-end ion source of the mass spectrometer, and the target compound must be charged to form molecules The ions can be separated and captured by the quadrupole mass spectrometer, and the sample signal can be detected.

As shown in Figure 2-18, after the liquid sample is charged by the electric field at the capillary needle, the droplets form a Taylor cone under the action of the electric field force and form a series of tiny dotted droplets at the tip. These dotted droplets are at the ion source. It evaporates quickly under the action of auxiliary heating gas (nitrogen). Due to the large amount of solvent evaporation, a large amount of charged charges accumulate on the surface of the originally small charged droplets. The process of Coulomb explosion occurs, and finally the charged molecular ions in the gas phase enter the mass spectrum .

According to the principle of ionization process, there are five possible mechanisms for matrix effect.

① The most important one is the charge competition mechanism. When the analyte is in a high concentration range, when the ions are transferred to the gas phase, the ions are first required to reach the surface of the droplet, and the high concentration of matrix components can restrict the analyte from reaching the liquid. Drop the surface and compete for its charged position, such as some hydrophobic lipids or surfactants (Tween), mobile phase additives, ion pair reagents, etc., thereby inhibiting the ionization efficiency of the analyte.

②In addition, some matrix components can also affect the viscosity and surface tension of the droplets, reducing the generation of droplets and subsequent evaporation of the solvent, so that the ions reaching the gas phase are reduced and the signal is suppressed.

③At the same time, the matrix effect may also depend on the type and polarity of the compound to be tested. The compound with higher polarity will concentrate in the water phase of the droplet and will not stay on the surface. Therefore, the lower surface tension of the compound tends to produce more More ion suppression.

④ The non-volatile components in the matrix and the mobile phase may also be harmful. It will form solid particles of co-precipitation on the analyte during the volatilization of the dotted droplets, which will suppress the signal.

⑤In the gas phase, the analyte ions are still susceptible to the influence of the matrix components that enter the gas phase at the same time. The neutral matrix components may compete with the charged analyte through the proton transfer reaction in the gas phase, and those with higher The gas phase of alkalinity will remove the protons of the analyte, neutralize its charge and cause signal suppression.

Although the real matrix effect inhibition mechanism may not be fully clarified, generally the matrix effect is caused by competing charges or neutralizing the ionization process. Conversely, when the matrix contains components that enhance ionization efficiency or gas phase transfer, it will Enhance the ionization process, resulting in increased signal.

Evaluation method of matrix effect

The most commonly used matrix effect evaluation method is to compare the response of the extracted sample and the pure solution in the standard analysis method. The absolute matrix effect of the compound is defined as the matrix effect factor (MF), which is calculated as follows: Without considering the recovery rate , Compare the response of the analyte in the presence and absence of matrix components, MF = the response of the extracted blank matrix added to the analyte/the response of the analyte in the pure solution. Since the chemical properties of the isotope internal standard are the same as the analyte, the influence of the matrix effect can be offset to the greatest extent during the extraction and ionization of the sample.

When MF=1, it indicates that there is no matrix effect.
When MF<1, it represents ion suppression. When MF>1, it represents ion enhancement.
When the tested samples come from different individual matrices, and there is an error in the test results caused by the uneven matrix effect, so the analysis needs to introduce the same concentration of the analog or isotope compound of the test compound as the internal standard anchor, so here is introduced The matrix effect factor MFi normalized by the internal standard,

According to the guiding principles, it is generally considered that the range of MFi normalized by the internal standard is acceptable when the range is 0.8-1.2. At the same time, the CV of MFi measured by different batches of matrix should be less than 15%. When the matrix effect is more serious, it is recommended Using isotope-labeled internal standards, because the chemical properties of the isotope internal standards are the same as the analyte, it can offset the influence of the matrix effect to the greatest extent during the extraction and ionization of the sample.

How to avoid and eliminate matrix effect?

After understanding the mechanism of matrix effect, we can consider the following five aspects.

① In the pretreatment stage of the sample, interferences can be selectively removed to reduce the matrix effect. The common pretreatment method is mainly PPT. Because of its simple process, wide range of applications to be tested, and low cost, it is the most commonly used, but for In terms of sample purification, PPT is easy to introduce substances that produce matrix effects, including many matrix components and phospholipids. When a more serious matrix effect occurs, the easiest way is to replace the appropriate extractant, such as changing from a methanol system. For the acetonitrile system, or adding an appropriate acid, changing the pH can help reduce the matrix effect. When PPT still cannot be improved, LLE or SPE sample extraction methods can be considered. LLE can largely eliminate the matrix effect caused by matrix components.

② The most common idea when selecting a suitable internal standard and using the internal standard to offset the influence of the matrix effect. If the internal standard and the analyte are suppressed and enhanced to the same degree, any change in ionization conditions will only affect the absolute peak. Area without affecting the ratio of the analyte to the internal standard, so it can offset the influence of the matrix effect. In order to offset and reduce the matrix effect to the greatest extent, the internal standard should have similar physical and chemical properties, ionization efficiency and chromatograms to the analyte Retention time, so it is recommended to use isotope-labeled internal standard (SIL-IS) when encountering serious matrix effects that cannot be resolved by ordinary internal standards.

③ Adjust the appropriate liquid phase separation conditions. Since the matrix effect is caused by the simultaneous elution of the matrix components and the analyte, the neutralization of the ion source is suppressed. Therefore, the chromatographic conditions can usually be modified to reduce the influence of the matrix effect. Gradient conditions, mobile phase elution strength and pH can effectively change the retention time of the analyte to keep it away from the ion suppression area. Generally, the most common matrix effect in the PPT method is phospholipids, and in high-throughput During the project, phospholipids tend to be retained in the conventional C18 column, making the matrix effect more and more obvious. At the same time, phospholipids have higher solubility in methanol systems than acetonitrile, so the methanol ratio in the mobile phase or washing solution can be increased to reduce the amount of phospholipids. The influence of the matrix effect.

④ Reducing the amount of sample entering the LC-MS system or diluting the sample before injection is a common method to reduce the matrix effect without drastically changing the method. Generally, the matrix effect increases with the incoming sample load or sample concentration. With gradual increase, as long as the sensitivity is within the allowable range, the matrix effect can be reduced simply by reducing the injection volume and diluting the sample before injection.

⑤ Choose the appropriate API ion source and ion polarity. Compared with the ESI source, the APCI source can be less affected by the matrix effect. If the thermal stability of the analyte is good and the sensitivity is not a problem, then switching from ESI to APCI is an effective reduction The matrix effect method, at the same time, it is generally believed that the negative ion mode has better selectivity and better ion suppression, but many analytes cannot be analyzed in the negative ion mode, and the selectivity of the ionization mode is also limited by the sensitivity of the method. When the ion source cannot be optimized, it can be improved in four aspects.

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