Matrix Effects in LC-MS

In many cases when methods involve internal or external standards, the solutions used to construct the calibration graph are made up in pure solvents and the signal intensities obtained will not reflect any interaction of the analyte and internal standard with the matrix found in unknown samples or the effect that the matrix may have on the performance of the mass spectrometer. One way of overcoming this is to make up the calibration standards in solutions thought to reflect the matrix in which the samples are found. The major limitation of this is that the composition of the matrix may well vary widely and there can be no guarantee that the matrix effects found in the sample to be determined are identical to those in the calibration standards. 

It is well known that electrospray ionization (El) suffers from suppression effects when polar/ionic compounds other than the analyte(s) of interest, such as those originating from the sample matrix, are present, with this phenomenon being attributed to competitive ionization of all of the appropriate species present [33], Matrix effects can, therefore, be considerable and these have two distinct implications for quantitative procedures, as follows  

One approach to the problem of matrix effects is to prevent the matrix materials reaching the electrospray source by carrying out some form of clean-up prior to analysis and/or to employ chromatographic separation. It is not always possible, however, to develop a simple procedure for sample clean-up and since this approach involves further work-up with the associated increase in analysis time and potential for sample loss it is therefore not ideal. 

Chromatogr., A, 907, Choi, B. K., Hercules, D. M. and Gusev, A. I., Effect of liquid chromatography separation of complex matrices on liquid chromatography-tandem mass spectrometry signal suppression , 337-342, Copyright (2001), with permission from Elsevier Science. 

This work [34, 35] reinforces the view that chromatographic separation itself is not always sufficient to effect adequate clean-up to remove all matrix effects but that these will be reduced if the minimum possible injection volume is used. 

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From reviewing the LC-MS literature, it first appears that matrix effects are only observed with ESI-MS. However, already in 1988, Gelpi et al. [71] discussed sample effects observed in the thermospray LC-MS analysis of labelled serotonin. More recently, Hajslova and Zrostlikova [72] reviewed matrix effect in the ultratrace GC-MS and LC-MS analysis of pesticide residues in food and biotic matrices. The discussion in this section is focussed on matrix effects in LC-MS, especially with respect to quantitative bioanalysis. A minireview was written by Annesley [73]. 

Chambers, E. et al., Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 852(1-2), 22, 2007. 

Figure 5.33 Top schematic diagram of the post-column infusion apparatus introduced to establish the time dependence of ionization suppression and other matrix effects in LC/MS analyses. Extracts of blank matrix or of pure mobile phase are injected into the HPLC and a standard solution of pure analyte is infused post-column while the mass spectrometer is set to continuously monitor the signal specific for the analyte.

Separation of tertiary PAs and of PANOs has been carried out on a C8 HPLC column with the aid of ion pairing with hexanesulfonic acid and detection by UV [7], but this approach has not been widely adopted, partly because the ion pan-reagents are non-volatile and cannot be used with LC-MS. Some extracts have been analyzed with minimal clean-up simplified and less elaborate protocols [45]. Simple dilution and filtration of extracts and filtraticMi can reduce matrix effects in LC-MS methods and the lack of coeluting compounds improves the background signal and the signal to noise ratio, therefore the sensitivity. 

J. L. Matrix effect in LC-ESI-MS and LC-APCI-MS with off-line and on-line extraction procedures. J Chromatogr A 2004, 1058, 61-66. 

Although PPE is the most efficient and inexpensive extraction technique, it is also the most nonspecific extraction procedure which is known to be susceptible to matrix effect for LC-MS/MS assay. In contrast, LLE provides a much cleaner extract. LLE, also known as solvent extraction and partitioning, separates analytes based on their relative solubility in two different immiscible solvents, usually water and an organic solvent. The most commonly used solvents for LLE are ethyl acetate (EtOAc), methyl ferf-butyl-ether (MtBE), methylene chloride (CH2C12), and hexane or the combination of the above solvents. In order to manipulate the polarity of the analytes, often a volatile acid or base such as FA or NH4OH respectively at 5-10 %... 

Figure 6.4 Schematic overview of the two commonly used methods to assess matrix effects in LC/ESI-MS/MS. (a) The post-column infusion method. The dashed line represents the signal of the analyte. The full line is obtained when injecting blank matrix. The arrow indicates the region of ion suppression, (b) The post-extraction spike method. The dashed peak represents the standard in neat solution. The full-line peak is obtained with standard spiked in matrix post-extraction. A clear reduction of the peak area is observed, which indicates ion suppression. (Reproduced from Van Eeckhaut et al. with permission from Elsevier copyright 2009.)...

Kiuve A, Leito I, Herodes K, Combating matrix effects in LC/ESI/MS The extrapolative dilntion approach. Anal. Chim. Acta 2009 651(l) 75-80. 

With the application of FIA in the mixture analytical mode for the analysis of environmental samples and after a marginal sample pretreatment by SPE, matrix effects are a high probability. But, as cited previously [27—31], matrix effects were not only observed with FIA but also in LC-MS and MS—MS modes. Advice to overcome these problems by, e.g. an improved sample preparation, dilution of the analyte solution, application of stable isotopic modification of LC conditions [29] or even application of two-dimensional LC separations [27], postcolumn standard addition [29], addition of additives into the mobile phase (e.g. propionic acid, ammonium formate) [34,35] or even matrix compounds [32] were proposed and discussed. 

Dams, R., Huestis, M. A., Lambert, W. E., and Murphy, C. M. (2003). Matrix effect in bio-analysis of illicit drugs with LC-MS/MS influence of ionization type, sample preparation, and biofluid. Am. Soc. Mass Spectrom. 14, 1290—1294. 

Matuszewski, B. K. Constanzer, M. L. Chavez-Fng, C. M. Matrix effect in quantitative LC/MS/MS analyses of biological fluids a method for determination of finasteride in human plasma at picogram per milliliter concentrations. Anal Chem 1998, 70, 882-889. 

Due to the complexity of the food samples, it is possible that the presence of some compounds in the matrix interferes with analyte determination even when working in LC-MS/MS, certain compounds present in the sample can affect the initial ionization of the analyte through what is often called ion suppression/ enhancement or matrix effects. 

APCI source should be used in LC-MS/MS. An APCI source has less matrix effect than an ESI source or no matrix effect. 

The decision tree and infusion tuning experiments used in the systematic method development for choosing the mobile phase to achieve the best sensitivity in LC-MS/MS and reducing the matrix effect are shown in Figures 8.1 to 8.3. The run time was 3.5 min using 96.5 3.5 acetonitrile/1 mM ammonium acetate as mobile phase. Only the peaks of interest were collected all the rest were... 

Matuszewski BK, Constanzer ML, Chaver-Eng CM Matrix effect in quantitative LC/MS/MS analyses of biological fluids ... 

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