Matrix effects method development

An understanding of the mechanisms of solid-phase extraction (SPE) are crucial for effective methods development. The four mechanisms outlined in Chapter 2 are sufficient for the majority of SPE and are an effective set of tools for methods development. The molecule s structure and the sample matrix are the main features used to choose a mechanism of isolation and separation. This chapter will discuss a six-step approach to methods development, how to execute the SPE recovery experiment, troubleshooting and optimizing conditions for the SPE recovery experiment, and how to critically evaluate previously published methods. 

All LC-MS techniques tend to be subject to matrix effects, especially suppression, although enhancement effects may also be observed. A procedure has been suggested to systematically investigate matrix effects when developing and validating methods using LC-MS or LC-MS/MS for detection. First, run pure standards to determine the analyte response in the absence of matrices. Next, either prepare standards in a matrix extract or infuse standards in the presence of matrix extract into the mass spectrometer and determine whether the response differs from that observed for pure standards. Differences in response may be attributed to matrix suppression (or enhancement) effects. Finally, fortify blank tissue with standards, perform the extraction and clean-up steps of the method, and then determine the detector response. The difference between the response observed for fortification into matrix extract and fortification into matrix prior to extraction and clean-up is attributed to method recovery. The evaluation of matrix effects is discussed in detail in Chapter 6. 

The issue of selectivity is one that is often difficult to address. Initial method development is invariably carried out by using standards made up with pure solvents, i.e. free from any matrix effects. It is often only when real samples are analysed that the true extent of interference becomes apparent and the value of the method can be properly assessed. An added complication is that interferences , by their very nature, are not constant and a number of samples may have a combination of interferences that defy analysis by a method that is otherwise successful on a routine basis (another example of Murphy s law ). 

The fact that APCl and electrospray are soft ionization techniques is often advantageous because the molecular ion alone, in conjunction with HPLC separation, often provides adequate selectivity and sensitivity to allow an analytical method to be developed. Again, method development is important, particularly when more than one analyte is to be determined, when the effect of experimental parameters, such as pH, flow rate, etc., is not likely to be the same for each. Electrospray, in particular, is susceptible to matrix effects and the method of standard additions is often required to provide adequate accuracy and precision. 

A fully automated instrumental procedure has been developed for analyzing residual corrosion inhibitors in production waters in the field. The method uses ultraviolet (UV) and fluorescence spectrophotometric techniques to characterize different types of corrosion inhibitors. Laboratory evaluations showed that fluorescence is more suitable for field application because errors from high salinity, contamination, and matrix effect are minimized in fluorescence analysis. Comparison of the automated fluorescence technique with the classic extraction-dye transfer technique showed definite advantages of the former with respect to ease, speed, accuracy, and precision [1658],... 

Using a simple solvent extraction procedure to minimize matrix effects, a diclofop-methyl immunoassay was developed for milk, a number of edible plant products, and other matrices. Gas chromatography (GC) and liquid scintillation counting (LSC) of a C-labeled analyte were used as reference methods to compare with enzyme immunoassay (EIA) results. The methods were well correlated, with comparison of EIA... 

Although SPME was applied initially for the analysis of relatively volatile environmental pollutants in waters, rapid developments have enabled SPME to be successfully applied for the analysis of pesticides in water, wine and more complex food samples such as honey, fruit juice and pears, vegetables and strawberries. With food samples, most analysts recognize the need for some sample pretreatment in order to minimize matrix effects. The matrix can affect the SPME efficiency, resulting in a reduced recovery of pesticides. The most common method is simply to dilute the sample or sample extract with water. Simpltcio and Boas comminuted pears in water prior to the determination of pesticides. Volante et al. extracted over 100 pesticides... 

The development of new fiber coatings in the near future should further improve the specificity of SPME and overcome some of the observed matrix effects. Quantification by stable isotope dilution gas chromatography/mass spectrometry (GC/MS) may assist in improving analytical performance. Along with the possible application of micro LC and capillary LC columns to in-tube SPME, the development of novel derivatization methods and the potential for the analysis of fumigant pesticides, SPME appears to be a technique with a future in the analysis of pesticide residues in food. 

Dougherty and co-workers have conducted an especially detailed treatment of matrix effects in inhomogeneous solids, and have developed several valuable tools for modeling the kinetics of such systems. The details and applications of these methods on specific systems will be described later in this chapter. 

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... 

Quantitative XRF analysis has developed from specific to universal methods. At the time of poor computational facilities, methods were limited to the determination of few elements in well-defined concentration ranges by statistical treatment of experimental data from reference material (linear or second order curves), or by compensation methods (dilution, internal standards, etc.). Later, semi-empirical influence coefficient methods were introduced. Universality came about by the development of fundamental parameter approaches for the correction of total matrix effects... 

Maximum power heating, the L vov platform, gas stop, the smallest possible temperature step between thermal pretreatment and atomisation, peak area integration, and matrix modification have been applied in order to eliminate or at least reduce interferences in graphite furnace AAS. With Zeeman effect background correction, much better correction is achieved, making method development and trace metal determinations in samples containing high salt concentrations much simpler or even possible at all. 

A method of analysis frequently requires the separation of the analyte from interfering materials present within the sample matrix. Many different procedures are used to effect such separations (Chapter 4), and the establishment of quantitative separations is a singularly important step in method development. Careful study and control of experimental conditions are needed and a large number of experiments may be required to establish the most suitable. 

The method development process with the multisorbent plate consists of three steps. In step 1, the sorbent chemistry and the pH for loading, washing, and elution are optimized. In step 2, optimization of the percentage organic for wash and elution and the pH of the buffer needed is carried out. Step 3 is validation the method developed from the results of the previous two steps is tested for linearity, limits of detection, quantitation of recovery, and matrix effects using a stable isotope-labeled analyte as an IS. 

For PK assays, it is generally believed that most matrix effects are due to the sample matrix (typically plasma). While this is correct in many cases, this assumption has some exceptions (vide infra). One of the most useful tools for avoiding matrix effects is studying the sample matrix and proposed assay by using the post-column infusion technique described by Bonfiglio et al.14S This technique allows visualization of the portion of the chromatographic step affected by ion suppression.161721 Xu et al.101 recommended inclusion of this step in the method development process for drug discovery PK assays. 

The development of multiclass methods for the detection of antibacterials and coccidiostats in food samples has shown a growing interest during the last years since the regulations concerning the presence of such chemicals in animal-derived foodstuffs is becoming more and more stringent. The challenges that these types of analyses pose to the analysts mainly have to do with the complexity of the matrix and the different physicochemical properties of the antibacterial families. Therefore, very often, a purification and preconcentration step is required prior to analysis in order to minimize matrix effects and reach the desired sensitivities [192, 193]. 

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