Liquid Mass Spectrometry (LC-MS) – Matrix Effects in Bioanalysis


LCMS Analysis

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

2. What are the common causes of matrix effects?
Many substances in biological samples can have matrix effects during LC-MS/MS analysis. These matrix-effect substances must be present in the final extracted sample and eluted with the compound or/and the internal standard in the chromatographic system. However, even in the presence of these co-eluting species, matrix effects do not necessarily affect the analytical method because the magnitude of matrix effects depends on the type of atmospheric pressure ionization (API) used in the method (APCI is less susceptible to matrix effects than ESI). effect), relative concentrations of interfering substances, and the magnitude of the offsetting effect of the calibration standard or the isotopic internal standard.

Substances that cause matrix effects can be divided into two categories: endogenous and exogenous according to their origin.
Endogenous main disruptors: usually some organic substances (proteins, fats, phospholipids, etc.) and inorganic substances present in biological samples, and may also include other metabolites formed in the body. Currently, plasma is the most common biological fluid used for sample analysis in the drug development industry, and generally contains high concentrations of salts, proteins, fats, and phospholipids, as well as small amounts of carbohydrates, peptides, and other organic compounds, all of which may be lead to matrix effects.

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

Exogenous Primary Contaminants: Foreign substances introduced during sample preparation or sample processing. For example, the anticoagulant lithium heparin, plasticizers in containers used for sample collection and storage, buffers, ion-pairing reagents, and excipients added to pharmaceutical formulations, especially Tween-80 and PEG-400, may cause matrix effects

3. What is the mechanism of the matrix effect?
It is generally believed that the generation of matrix effects is caused by the competition between endogenous or exogenous matrix substances and target compounds during the ionization process of the sample in the mass spectrometry ion source and the neutralization ionization process. The most commonly used ESI (electrospray ion source) is used as an example to illustrate that before the liquid sample separated by liquid phase enters the mass spectrometry analysis, the liquid sample must be desolvated at 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.

After the liquid sample is charged by the electric field at the capillary nozzle, the droplet forms a Taylor cone under the action of the electric field force and forms a series of tiny dotted droplets at the tip. These dotted droplets are heated by auxiliary heating gas at the ion source ( Under the action of nitrogen), it evaporates rapidly. Due to the evaporation of a large amount of solvent, a large amount of charged charges accumulate on the surface of the originally small charged droplet, and a process of Coulomb explosion occurs, and finally charged molecular ions in the gas phase are formed into the mass spectrometer.

According to the principle of the ionization process, there are five possible mechanisms for the generation of the matrix effect:
① The most important one is the charge competition mechanism. When the analyte is in the high concentration range, when the ions are transferred into the gas phase, the ions are first required to reach the surface of the droplet, and the high concentration of matrix components can limit the analyte from reaching the liquid. Drop surface and compete for its charged sites, such as some hydrophobic lipids or surfactants (Tweens), mobile phase additives, ion-pairing reagents, etc., thus inhibiting the ionization efficiency of the analyte.

② In addition, some matrix components can also affect the viscosity and surface tension of the droplets, reducing the generation of droplets and the 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 compounds to be tested. Compounds with higher polarity will be concentrated in the water phase of the droplet and will not stay on the surface, so compounds with lower surface tension tend to produce more Multiple ion suppression.

④ Non-volatile components in the matrix and the mobile phase may also be harmful, which will form solid particles of co-precipitate with the analyte during the volatilization of the droplet, which will suppress the signal.

⑤ In the gas phase, the analyte ions are still susceptible to the influence of the matrix components entering 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 protons from the analyte, neutralizing its charge and causing signal suppression.

Although the true matrix effect inhibition mechanism may not be fully understood, in general matrix effects are caused by competing charges or neutralizing ionization processes. Conversely, when the matrix contains components that enhance ionization efficiency or gas phase transfer, it will Enhances the ionization process, resulting in increased signal.

4. Evaluation method of matrix effect
The most common method for evaluating matrix effects is to compare the responses of extracted samples and pure solutions in standard analytical methods, where the absolute matrix effect of a compound is defined as the matrix effect factor (MF), which is calculated as: , compare the response of the analyte in the presence and absence of the matrix component, MF=the response of the blank matrix after extraction with the analyte added/the response of the analyte in the pure solution. Since the chemical properties of the isotope internal standard are the same as that of the analyte, the influence of the matrix effect can be counteracted to the greatest extent during the extraction and ionization of the sample.

When MF=1, there is no matrix effect.
When MF<1, it represents ion suppression.
When MF>1, it represents ion enhancement.

When the samples to be tested come from different individual matrices, and the detection result error caused by the uneven matrix effect occurs, it is necessary to introduce the analog or isotopic compound of the test compound at the same concentration as the internal standard anchor in the analysis, so here is introduced. Internal standard normalized matrix effect factor MFi,

According to the guidelines, it is generally considered that the range of MFi normalized by the internal standard is acceptable when the range of 0.8-1.2 is acceptable. Meanwhile, the CV of MFi measured by different batches of matrices should be less than 15%. When the matrix effect is serious, it is recommended to Using an isotope-labeled internal standard, since the chemical properties of the isotopic internal standard are the same as the analyte, the influence of matrix effects can be counteracted to the greatest extent during sample extraction and ionization.

5. How to avoid and eliminate matrix effects?
After understanding the mechanism of matrix effect, the following five aspects can be considered.

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

② The most common idea is to select a suitable internal standard and use 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 chromatographic properties of the analyte. Therefore, when the matrix effect is serious and the common internal standard cannot be resolved, it is recommended to use the isotope-labeled internal standard (SIL-IS).

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, and the neutralization of the ion source is inhibited, the chromatographic conditions can usually be modified to reduce the influence of the matrix effect. Gradient conditions, elution strength of the mobile phase and pH can effectively change the retention time of the analyte and keep it away from the ion suppression region. Usually, phospholipids are the most common cause of matrix effects in the PPT method. During the project, phospholipids are easily retained in the conventional C18 column, which makes the matrix effect more and more obvious. At the same time, compared with acetonitrile, the solubility of phospholipids in methanol system is higher, so the proportion of methanol in the mobile phase or washing solution can be increased to reduce the phospholipids. matrix effect.

④ Reducing the amount of sample entering the LC-MS system or injecting the sample after dilution is a common method to reduce the matrix effect without significantly changing the method. Usually, the matrix effect increases with the amount of incoming sample or the concentration of the sample. Gradually increasing, as long as the sensitivity allows, matrix effects can be reduced simply by reducing the injection volume and diluting the sample before injection.

⑤ Select 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. 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 When the ion source cannot be optimized, the above four aspects can be improved.