Matrix effects problems

The diligent analyst would develop a robust method with rigorous matrix effect tests on multiple lots, including hemolyzed and lipidemic samples. An initial test would be a spike-recovery evaluation on at least six individual lots. Samples should be spiked at or near the LLOQ, and at a high level near the ULOQ. If matrix interference were indicated by unacceptable relative error (RE) percentage in certain lots, the spiked sample of the unacceptable lots should be diluted with the standard calibrator matrix to estimate the minimum dilution requirement (MDR) at and above which the spike-recovery is acceptable. The spike-recovery test should then be repeated with the test samples diluted at the MDR. Note that this approach will increase the LLOQ for a less sensitive assay. If sensitivity is an issue, then other venues will be required to address the matrix effect problem. For example, the method can be modified to include sample clean-up, antibodies and/or assay conditions may be changed, or the study purpose may be tolerable to acknowledge that the method may not be selective for a few patients (whose data may require special interpretation). 

While stable isotope-labeled ISs are generally considered safe in terms of matrix effects, Wang et al. [79] reported on an assay where matrix effects were an issue despite the use of a deuterium-labeled IS. In this case the reason that was given for the problem was that the chromatography was too good—there was some separation between the analyte and its IS and this led to the matrix-effect problem. 

Several strategies are generally incorporated into HPLC-MS method design and development to counter matrix effect problems. When the analytes are known, stable isotope-labeled internal standards (isotope dilution)... 

Another problem is that the Nernst equation is a function of activities, not concentrations. As a result, cell potentials may show significant matrix effects. This problem is compounded when the analyte participates in additional equilibria. For example, the standard-state potential for the Fe "/Fe " redox couple is +0.767 V in 1 M 1TC104, H-0.70 V in 1 M ITCl, and -H0.53 in 10 M ITCl. The shift toward more negative potentials with an increasing concentration of ITCl is due to chloride s ability to form stronger complexes with Fe " than with Fe ". This problem can be minimized by replacing the standard-state potential with a matrix-dependent formal potential. Most tables of standard-state potentials also include a list of selected formal potentials (see Appendix 3D). 

Sources of Error. pH electrodes are subject to fewer iaterfereaces and other types of error than most potentiometric ionic-activity sensors, ie, ion-selective electrodes (see Electro analytical techniques). However, pH electrodes must be used with an awareness of their particular response characteristics, as weU as the potential sources of error that may affect other components of the measurement system, especially the reference electrode. Several common causes of measurement problems are electrode iaterferences and/or fouling of the pH sensor, sample matrix effects, reference electrode iastabiHty, and improper caHbration of the measurement system (12). 

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. 

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. 

For APCI (if matrix effects become a problem in ESI), the mobile phase consisted of (A) 9 1 methanol-water containing 50 mM ammonium acetate and (B) water containing 50 mM ammonium acetate-methanol (9 1). The gradient was held at 50% A-50% B for 10 min and was then changed to 90% A-10% B in 22 min (held for 3 min). The HPLC column was a Zorbax RX-C8, 4.6-mm i.d. x 250 mm, 5 pm particle size, with a flow rate of l.OmLmin and a 50-pL injection. Table 8 shows the ion transitions (parent to product ions) that were monitored for HPLC/ESI-MS/MS. For single-stage HPLC/ESI-MS, Table 9 shows the ions that were monitored. 

APCI can help to reduce matrix effects when analyzing for carbamate insecticides. Eor example, when analyzing for methiocarb in citrus products, the apparent recoveries were in the region of 50% with ESI. However, on changing to APCI, the apparent recoveries were increased to 110%. This is an example where APCI can be an alternative API method if matrix effects are a problem with ESI. It is important to note that the analyte must show sufficient sensitivity to both API techniques. 

SFE has now been available long enough to allow an evaluation of its prospects for polymer/additive extraction. SFE is still around, but EPA and FDA approved SFE methods are still wanting. The main problem is strong matrix effects. SFE is not a cookbook method for one s matrix. Not unlike microwave extraction, SFE requires that a specific method be developed to optimise the recovery for each polymer/additive system. Therefore, the success of SFE depends on the polymer... 

Nondestructive radiation techniques can be used, whereby the sample is probed as it is being produced or delivered. However, the sample material is not always the appropriate shape or size, and therefore has to be cut, melted, pressed or milled. These handling procedures introduce similar problems to those mentioned before, including that of sample homogeneity. This problem arises from the fact that, in practice, only small portions of the material can be irradiated. Typical nondestructive analytical techniques are XRF, NAA and PIXE microdestructive methods are arc and spark source techniques, glow discharge and various laser ablation/desorption-based methods. On the other hand, direct solid sampling techniques are also not without problems. Most suffer from matrix effects. There are several methods in use to correct for or overcome matrix effects ... 

All available methods (TG-MS, PyGC-MS and LDI-MS) suffer from difficult quantitation, although for different reasons. In TG-MS, selective volatilisation may not reflect the composition in the solid the quantitation problem of PyGC-MS requires assessment of the importance of matrix effects. Laser ablation methods cannot easily be calibrated. Quantitation is simplified in case of dual detection (MS for identification, FID for quantitation). A general drawback of many direct methods, which allow only small sampling volumes, is granule-to-granule variations. 

False-positive results with bDNA have been observed with proficiency testing specimens for HTV-1 in the College of American Pathologists HIV-1 viral load survey and HCV in the viral quality control program administered by the Netherlands Red Cross. The reason for the false-positive results with these proficiency testing specimens is not known but may be sample matrix effects. The extent to which this problem occurs with clinical samples has not been determined. However, both the HIV-1 and HCV bDNA assays were designed to have a false-positive rate of 5%. 

Matrix effect is a phrase normally used to describe the effect of some portion of a sample matrix that causes erroneous assay results if care is not taken to avoid the problem or correct for it by some mechanism. The most common matrix effects are those that result in ion suppression and subsequent false negative results. Ion enhancement may lead to false positive results.126 127 Several reports about matrix effects include suggestions on what can cause them and how to avoid them.126-147 While various ways to detect matrix effects have been reported, Matuszewski et al.140 described a clear way to measure the matrix effect (ME) for an analyte, recovery (RE) from the extraction procedure, and overall process efficiency (PE) of a procedure. Their method is to prepare three sets of samples and assay them using the planned HPLC/MS/MS method. The first set is the neat solution standards diluted into the mobile phase before injection to obtain the A results. The second set is the analyte spiked into the blank plasma extract (after extraction) to obtain the B results. The third set is the analyte spiked into the blank plasma before the extraction step (C results) these samples are extracted and assayed along with the two other sets. The three data sets allow for the following calculations ... 

Total or partial ion suppression is a well-known LC-MS effect, which is induced by coeluting matrix components that can have a dramatic effect on the intensity of the analyte signal. As can be observed in Fig. 1, analyte suppression occurs as a consequence of the different matrix interferences present in waste-water samples, making the identification and/or quantification process difficult or unfeasible. Even when working under selection ion monitoring (SIM) conditions, these matrix effects can cause ion suppression in the detection of some analytes that are present at low levels of concentration, as seen in this figure. Several papers have reported this effect [30-32] and different alternatives to overcome these problems, such as the inclusion of a size-exclusion step [33] or sequential SPE [28], have been applied for the determination of pesticides in... 

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