As scientists have shortened assay times by using shorter columns or higher mobile phase flow rates (vide supra), a new problem has become more apparent. This problem is often referred to as ''matrix effects''. Matrix effects can be described as a component in the sample that is injected into the HPLC-MS/MS system that results in a reduction of the analyte signal (aka ion suppression) or an increase in analyte signal. There have been multiple papers written on various aspects of matrix effects in recent years [97, 114-124]. The problem that can be caused by matrix effects (if undetected) is that they can lead to erroneous results in a bio-analytical assay. One of the best ways to test a bioanalytical method for matrix effects is to use the post-column analyte infusion method as shown schematically in Fig. 13.6. The post-column infusion method was described by King et al.  in their report on ionization suppression and the possible causes and solutions of this problem. Briefly, in the post-column infusion method, one has a constant infusion of the analyte into the post-column eluant, this provides a steady signal of the analyte; the comparison is made by injecting either a mobile phase aliquot or a sample from control matrix that has been prepared using the sample preparation procedure [6, 97, 121, 124]. The resulting two chromatograms can be prepared and the difference is attributed to matrix effects. For example, as shown in Fig. 13.7, Dunn-Meynell et al.  used the post-column infusion technique to ensure that a generic ballistic gradient would be free from matrix effects in the part of the chromatogram where the analytes would be expected to elute; in this example, the generic HPLC-MS/MS method should be unaffected by matrix effects.
While matrix effects are generally attributed to sample constituents, sample preparation can also lead to matrix effects. Mei et al. [115, 124] demonstrated that matrix effects could be caused by the brand of sample tubes that are used in the sample storage step of the assay. In this example, the solution was to switch to a different supplier for the tubes. In addition, while it is generally reported that
Divert Jo Waste Valve
Divert Jo Waste Valve
Post-column Infusion Experiment:
1. Inject mobile phase or solvent blank while infusing anaJyte into the column eluant at a flow rate of 5 - 10 jiL/min ¡mil follow path A,
2. Inject control sample extract while infusing analvte intn the column eluant at a flow rate of 5 - 10 pL/mi n and follow path A.
3. Compare the mass chromatograms—diffcrences arc due to matrix effects.
Analysts of samples;
L inject sample (syringe pump off) and divert the flow to waste (path B) for the first 10-25% of the assay run time, then sw itch the divert valve to send the eluant into the MS/MS system (path A) for the analysis of the sample.
Fig. 13.6 Post-column infusion technique as part of the HPLC-MS/MS system. Adapted from , with permission from Elsevier.
ESI is more likely to exhibit matrix effects than is atmospheric pressure chemical ionization (APCI), Mei et al. [115, 124] reported that matrix effects could be seen in both APCI and ESI assays. In general, these matrix effects can be detected by using the post-column infusion technique as described above.
More recently, there have been reports of matrix effects that were caused by the formulation that was used in a preclinical PK study [6, 9, 119, 125-127]. For example, Shou and Naidong  used the post-column infusion technique to show that Tween 80 and PEG 400 (both commonly used as part of preclinical dosing formulations) could lead to significant matrix effects. As an example, they showed that if they used a fast HPLC-MS/MS method in a dog study where PEG 400 was used in the dosing formulation then a significant matrix ion suppression resulted and the analytical results were significantly in error for the plasma samples collected at timepoints up to 2 h. The authors reported that correct results could be obtained by extending the HPLC gradient so that the analyte and its internal standard eluted later and were outside of the matrix ion suppression "window".
Xu et al.  discussed the problem of matrix effects caused by dosing formulation components in PK studies in a recent publication. Xu et al.  showed that using either Tween 80 or PEG 400 could lead to matrix effects for analytes that are very polar and elute early in a fast HPLC reversed phase method. In addition, Xu et al.  showed that these matrix effects were time-dependent; generally, early PK timepoints were more susceptible to these effects than were later PK timepoints. For example, as shown in Fig. 13.8, there was a time-dependent nature to the matrix effects caused by the Tween 80; the reason for this is that the amount of the Tween 80 in the plasma samples changed over the 24-h sampling time . As shown in Fig. 13.8, the matrix effects were also dependent on the instrument vendor and the route of dosing the formulation. As discussed by Xu et al. , this problem could be avoided by not using Tween 80 or PEG 400 in formulations for PK studies. If these compounds have to be used in the formulation, then one should be careful when performing the HPLC-MS/MS assay on the samples that are obtained from the PK study. Generally, the matrix effect problem can be avoided by using a longer HPLC gradient or by doing a more extensive sample cleanup before performing the HPLC-MS/MS assay.
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