Reduction of Ion Suppression

Weaver, R., and Riley, R. J. (2006). Identification and reduction of ion suppression effects on pharmacokinetic parameters by polyethylene glycol 400. Rapid Commun. Mass Spectrom. 20 2559-2564. 

TheAPCImode may be preferable to E SI in terms of enhancing the sensitivity by reduction of ion suppression effects. 

There are several important reasons to include sample preparation in modem liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods. These include the elimination of matrix components from the sample reduction of ion suppression and, sometimes, improved sample utilization. 

The utility of UPLC has been demonstrated for both qualitative and quantitative analyses. In 2005, Castro-Perez et al. (2005) compared the performance of a HPLC with that of a UPLC and showed that improved chromatographic resolution and peak capacity attained with UPLC lead to reduction in ion suppression and increased MS sensitivity. Comparison of the mass spectrum obtained using HPLC with that from UPLC (Fig. 1.15) revealed that the higher resolving power of the UPLC-MS system resulted in a much cleaner mass spectrum than that obtained using the HPLC-MS system. The sensitivity improvement directly resulted in a higher ion count in the UPLC mass spectrum (855 vs. 176). 

A unique property of LC/API/MS is the extent to which the analyte signal is affected by the sample matrix or the existence of co-eluting analytes. This property can have a profound influence on sensitivity and assay reproducibility. Because of matrix-ion suppression, it is not possible to estimate extraction recovery by comparison of the signal from a neat sample to an extracted sample. This is because the reduction in signal represents the combined effects of recovery and ion suppression. As first shown by Buhrman et al., quantitative assessment of extraction efficiency is made by spiking the neat sample into an extracted blank and comparison of the result to a similar sample spiked before extraction [120]. Conversely, the extent of ion suppression is obtained by the comparison of the signals for a neat unextracted sample to the same neat solution spiked into an extracted matrix blank. 

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.)...

Over the last few years, due to improvements in the packing material of the chromatographic columns, the use of UHPLC technique has improved the reported HPLC methodologies in terms of peak efficiency, peak resolution, speed, sensitivity, and solvent consumption. However, this technique must operate at high backpressures [41-43]. Another advantage of the UHPLC technique is that, given this high peak resolution, when it is combined with MS as the detector system, it is less susceptible to matrix effects (MEs), one of the main problems when the MS is used to quantify minor components in food samples. This is because an efficient UHPLC separation may contribute to a reduction in ion suppression, when this is only produced by the coelution of two different compounds [44]. 

Sample Preparation Because large amounts of proteins are present in biological samples (except urine), conventional HPLC columns will not tolerate the direct introduction of these samples for quantitative analysis. Most bioanalytical assays have a sample preparation step to remove the bulk proteins from the samples [2], In addition, there are other important reasons for a sample preparation step when developing LC-MS/MS methods. These include the reduction of matrix components from the samples and minimization of ion suppression (also called matrix effects ) in the mass spectrometric detection [18]. Once a bioanalytical method has been developed, the method performance must remain consistent over the duration of the study. The results generated based on a validated method procedure should be free from systematic error and any other characterized errors and meet the predefined acceptance criteria. Sample preparation is used to... 

With suppressed migration, the only way to transport ions to and away from the electrode is dijfusion. For a reversible electrode reaction, the overall reaction rate is then said to be dijfusion controlled. The laws of diffusion are valid for ions as well as for neutral particles. Particles diffuse in the direction where a lack of substance exists, i.e. towards a negative concentration gradient. As soon as a substance is consumed at an electrode surface (e.g. by electrochemical reduction of ions), particles start to move in order to compensate the deficiency. 

The most favorable conditions for equation 9 are temperature from 60—75°C and pH 5.8—7.0. The optimum pH depends on temperature. This reaction is quite slow and takes place in the bulk electrolyte rather than at or near the anode surface (44—46). Usually 2—5 g/L of sodium dichromate is added to the electrolysis solution. The dichromate forms a protective Cr202 film or diaphragm on the cathode surface, creating an adverse potential gradient that prevents the reduction of OCU to CU ion (44). Dichromate also serves as a buffering agent, which tends to stabilize the pH of the solution (45,46). Chromate also suppresses corrosion of steel cathodes and inhibits O2 evolution at the anode (47—51). 

Any chemical (such as zinc hydroxide) that suppresses the reduction of oxygen to hydroxyl ion. A cathodic inhibitor suppresses that part of the electrolytic corrosion process at the cathodic sites on a metal surface. 

Ions at m/z 55, 60, 214 and 236 are observed but do some or all of these arise from the background and are present throughout the analysis, or are they present in only a few scans, i.e. are they from a component with insufficient overall intensity to appear as a discrete peak in the TIC trace An examination of reconstructed ion chromatograms (RICs) from these ions generated by the data system may enable the analyst to resolve this dilemma. The TIC shows the variation, with time, of the total number of ions being detected by the mass spectrometer, while an RIC shows the variation, with time, of a single ion with a chosen m/z value. The RICs for the four ions noted above are shown in Figure 3.15. These ions have similar profiles and show a reduction in intensity as analytes elute from the column. The reduction in intensity is a suppression effect. 

Several studies suggest that LA and DHLA form complexes with metals (Mn2+, Cu2+, Zn2+, Cd2+, and Fe2+/Fe3+) [215-218]. However, in detailed study of the interaction of LA and DHLA with iron ions no formation of iron LA complexes was found [217]. As vicinal dithiol, DHLA must undoubtedly form metal complexes. However, the high prooxidant activity of DHLA makes these complexes, especially with transition metals, highly unstable. Indeed, it was found that the Fe2+-DHLA complex is formed only under anerobic conditions and it is rapidly converted into Fe3+ DHLA complex, which in turn decomposed into Fe2+ and LA [217]. Because of this, the Fe3+/DHLA system may initiate the formation of hydroxyl radicals in the presence of hydrogen peroxide through the Fenton reaction. Lodge et al. [218] proposed that the formation of Cu2+ DHLA complex suppressed LDL oxidation. However, these authors also found that this complex is unstable and may be prooxidative due to the intracomplex reduction of Cu2+ ion. 

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