"Just Enough" Sample Preparation: A Proven Trend in Sample Analysis - - Chromatography Online
"Just Enough" Sample Preparation: A Proven Trend in Sample Analysis


LCGC Europe
Volume 25, Issue 12, pp. 670-674

Ion Suppression in LC–MS and LC–MS-MS

An area of potential problem in the just enough sample preparation approach is unique to LC–MS and LC–MS-MS. The impact of un-extracted matrix compounds that may coelute with the analytes of interest may end up in the ionization chamber of the mass spectrometer. Ion suppression in MS is one form of a matrix effect that impacts analyte ionization in the MS source. Most often a loss in response occurs; hence the term ion suppression is generally used. Ion suppression effects impact reproducibility and signal strength. They are most noticeable when trace analytes are in the presence of complex matrices such as biological fluids. In some cases, an increasing response of the desired analyte may occur; ion enhancement or a strongerthan-expected signal results.

Ion suppression results from the presence of less volatile compounds that can change the efficiency of droplet formation or droplet evaporation, which in turn affects the amount of charged ion in the gas phase that ultimately reaches the detector. Materials shown to cause ion suppression include salts, ion pairing reagents, endogenous compounds, drugs, metabolites and proteins. The electrospray ionization detector (EID) is strongly affected by the presence of certain coeluted compounds. Atmospheric pressure chemical ionization detectors are also affected by ion suppression but to a lesser extent than the electrospray detector. The presence of ion suppression can be determined by the use of infusion. The infusion experiment involves the continuous introduction of the standard solution containing the analyte of interest and its internal standard by means of the syringe pump connected to the column of fluid. A drop in constant baseline after a blank sample extract is injected into the LC system indicates suppression in ionization of the analyte because of the presence of the interfering material.

Although beyond the scope of this article, there are a number of strategies for reducing ion suppression. Among them are changing the ionization mode (such as switching to negative ionization), sample dilution or volume reduction, reducing the flow rate, improving chromatographic selectivity or performing better sample preparation. In the latter case, just enough sample preparation to meet the analytical needs may be the use of SPE, liquid–liquid extraction or even additional techniques. The use of formic acid rather than trifluoroacetic acid in the HPLC mobile phase can also help. For more information, a simple discussion of ion suppression effects and their elimination was published earlier (2).

Examples of Just-Enough Sample Preparation


Figure 4: Steps in protein precipitation.
Many sample preparation methodologies have already been published in earlier instalments of "Sample Preparation Perspectives". Figure 2 provides a number of sample preparation protocols that could qualify as just enough procedures. As mentioned earlier, the fewer sample preparation steps in an analytical method the less chance of errors, better analyte recovery and less time spent handling samples. However, as one proceeds down Figure 2, just enough may require more sophisticated sample preparation processes.


Figure 5: LC–MS analysis of fluticasone proprionate in plasma. Shown is an ion chromatogram (MRM transition 501.2→293.1) for 2.5 fg injected on-column with a 1-fg limit of detection. The standard curve was linear over the range of 5 pg/mL to 50 ng/mL. The plasma sample was precipitated with acetonitrile and then diluted four-fold with water. Adapted from reference 3.
Let's look at a few examples of sample preparation procedures that may qualify as just enough and see if they provide acceptable results. In recent years, for the determination of drugs and their metabolites in biological fluids such as plasma, many pharmaceutical companies have switched their sample preparation to protein precipitation (Figure 4) and reversed-phase HPLC analysis but using a more selective, sensitive LC-triple-quadrupole MS-MS detector with multiple reaction monitoring (MRM) at defined transitions. The first example shows the direct analysis of fluticasone proprionate in human plasma using an LC–triple quadrupole MS system. This compound is a synthetic steroid of the glucocorticoid family of drugs for treating allergic conditions. When used as a nasal inhaler or spray, medication goes directly to the epithelial lining of the nose, and very little is absorbed into the rest of the body. Because of its low systemic levels, a high sensitivity LC–MS assay is required to determine its concentration in human plasma. Figure 5 shows the LC–MS results from a plasma protein precipitation followed by dilute and shoot using the MRM transition shown in the figure caption. In this case, the dilute and shoot method has more than adequate sensitivity at the lowest calibration level of 5 pg/mL. Thus, dilute and shoot sample preparation has an assay performance well within the accepted regulatory guidelines and was just enough to meet the analytical needs.


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