Signal response matrix effect

Table 5.15 Relative signal responses from various injection volumes for the LC-MS-MS analysis of a wheat forage matrix sample. Reprinted from J. Chromatogr., A, 907, Choi, B. K., Hercules, D. M. and Gusev, A. L, 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...

Table 5.16 LC-MS-MS signal responses" obtained from wheat forage matrix samples using various mobile-phase additives (injection volumes of 50 p,l). From Choi, B. K., Hercules, D. M. and Gusev, A. I., LC-MS/MS signal suppression effects in the analysis of pesticides in complex environmental matrices , Fresenius J. Anal. Chem., 369, 370-377, Table 2, 2001. Springer-Verlag GmbH Co. KG. Reproduced with permission...

The analytical response generated by an immunoassay is caused by the interaction of the analyte with the antibody. Although immunoassays have greater specificity than many other analytical procedures, they are also subject to significant interference problems. Interference is defined as any alteration in the assay signal different from the signal produced by the assay under standard conditions. Specific (cross-reactivity) and nonspecific (matrix) interferences may be major sources of immunoassay error and should be controlled to the greatest extent possible. Because of their different impacts on analyses, different approaches to minimize matrix effects and antibody cross-reactivity will be discussed separately. 

HPLC/MS and HPLC/MS/MS analyses are susceptible to matrix effects, either signal enhancement or suppression, and are often encountered when the cleanup process is not sufficient. To assess whether matrix effects influence the recovery of analytes, a post-extraction fortified sample (fortified extract of control sample that is purified and prepared in the same manner as with the other samples) should be included in each analytical set. The response of the post-extraction fortified sample is assessed against that of standards and samples. Matrix effects can be reduced or corrected for by dilution of samples, additional cleanup, or using calibration standards in the sample matrix for quantitation. 

A particular issue that must be considered for all calibration procedures is the possibility of matrix effects on the analyte signal. If such effects are present they may be allowed for in many cases by matrix matching of the standard to the sample. This of course requires an accurate knowledge of the sample matrix. Where this is not available, the method of standard addition is often effective. This involves spiking at least three equal aliquots of the sample with different amounts of the analyte, and then measuring the response for both spiked and unspiked aliquots. A plot of response vs analyte, extrapolated back, will give abscissae intercepts from which the amount of analyte in the sample may be deduced (Figure 2.8). 

Luminescence is often much more sensitive to molecular dynamics than other optical techniques where temperature, viscosity, pH and solvent effects can have a significant influence on the emission response. Analyte degradation for light sensitive fluors and photobleaching for static measurements also influence the emission signal. Because of the wide variety of potential matrix effects, a thorough investigation should be conducted or the sample matrix well understood in terms of its potential impact on emission response. A complete discussion on the fate of the excited states and other measurement risk considerations can be found elsewhere. ... 

Because so many factors determine the response obtained for a chemical substance in a sample, it is usually not possible to derive directly the concentration from the measured response. The relationship between signal, or response and concentration has to be determined experimentally, a step which is called calibration. The complexity of the calibration depends upon the type of expected problems. These are roughly divided into three categories interferences, matrix effects or interactions and a combination of both, a so-called interacting interference. 

Figure 5-3 shows a strong matrix effect in the analysis of perchlorate (CIO4 ) by mass spectrometry. Perchlorate at a level above 18 p,g/L in drinking water is of concern because it can reduce thyroid hormone production. Standard solutions of C104 in pure water gave the upper calibration curve in Figure 5-3. The response to standard solutions with the same concentrations of CIO4 in groundwater was 15 times less, as shown in the lower curve. Reduction of the ClOj" signal is a matrix effect attributed to other anions present in the groundwater.

In principle, methods should be validated for each type of matrix and for the extraction agent applied. Matrix effects may affect strongly the calibration (e.g. loss of signal, interferences). Standard addition techniques are, therefore, the only way to control the validity of the detection, but only if the addition is performed with the proper identical form of the compound to be determined. As stressed above, it is of primary importance to evaluate this linear range before starting the analysis so that the spiked levels remain in the linear range of the detector response (Quevauviller et al., 1996a). 

The method of standard addition is the least widely used method of quantitation, but it is used to ensure that the calibration standards experience the same matrix effects as the sample constituents. In this method, the sample is analyzed first in order to estimate the concentration of the solute(s) of interest. Several different, known concentrations of the solute(s) of interest are then added to portions of the sample, to provide approximate incremental increases in detector response. Each portion of the sample is then reanalyzed. The principle of the method is that the extra signal produced by the addition of the standards is proportional to the original signal. The method is applicable only when a known straight-line relationship has been established between the instrument response and the analyte concentration.18... 

Thrombin is added to plasma diluted 1 100 in a concentration range 0-200 nM. The response is linear in the tested concentration range (R2 = 0.990) and reproducibility was good (CV% = 13%). The matrix effect with a blank signal of-21 Hz is present but, despite the high complexity of the matrix, the increase in thrombin concentration could be detected (Fig. 6). 

Chemical matrix effects were encountered in the HVAA determination. When a constant amount of chromium was added to different petroleum samples, the atomization response varied considerably from that obtained in THF alone (Table 8.II). Since almost parallel effects were noted after ashing, the effect of Fe, Ni, and V was studied. Excesses of vanadium had a significant effect at levels which often occur in crude oils (10 to > 100 ppm). Once vanadium was introduced in the atomizer, the chromium signal was consistently suppressed until the atomization furnace was baked at maximum temperature to remove residual vanadium. 

From the data in Table 8.II it is apparent that components other than vanadium affect the chromium signal. No explanation has been found for the enhancement observed in some cases. Since a linear response is obtained after the initial injection of a given sample solution, the method of standard additions can be applied to compensate for these chemical matrix effects. 

Matrix effect and matrix suppression/enhancement are terms in bioanalysis that are often used interchangeably. However, the matrix effect is a more specific terminology in bioanalysis and is primarily used to describe whether an assay s performance can be reproduced for individual subject samples, regardless of the difference in matrices. If the response ratio of an analyte to its ISTD can be kept consistent under the same concentration level in different matrices, then no adverse matrix effect can be claimed for the bioanalytical assay. On the other hand, matrix suppression/enhancement is more explicit to evaluate whether an analyte signal is affected by coeluted matrix components even though they are not directly measured by mass spectrometry. The suppression/ enhancement can be ascribed to an analyte s ionization efficiency, which is impacted, either negatively or positively, by the underlying matrix components. 

---