Matrix Effects and Sample Preparation

Bulk solid samples must be machined or polished into the correct shape, and this must be done without contaminating the surface from the grinding material or altering the surface composition by polishing out a soft phase, for example. The surface must be cleaned of any lubricant or oil used in the machining process before analysis. 

Advances in understanding of the spark and arc sources and improvement in electronics have led to redesign of the sources and spectrometers, so that in modem instruments, structure-induced interelement effects can be minimized by proper choice of excitation and measurement conditions. 

This has reduced the need for a common matrix and has permitted, in some cases, the use of one global calibration cnrve for multiple materials. However, if an interelement effect does exist, correction factors can be calculated to compensate for the effect. 

If there is no suitable analytical line free of spectral overlap, corrections can be made for the interfering element, but only if the overlapping element s intensity is less than 10% of the analyte intensity. Less than 1% is even better, if possible. Corrections cannot be made if the overlapping line is one of the major elements in the sample (e.g., overlap from an iron line on Cr in a steel sample cannot be corrected mathematically). 

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Overall Accuracy. In addition to the matrix effect and sample preparation error, method accuracy can also be affected by calculation error for example, difference in relative response, significant -intercept, and nonlinearity. Therefore, a more vigorous approach is to determine the overall accuracy, which incorporates the effect from all aspects of the method ... 

Recovery of an analyte spike added to a sample prior to sample preparation. Determination of spike recovery is based on results provided by spiked and unspiked sample. Used to estimate matrix effects and sample preparation losses surrogate standard. 

Apart from automating the matrix application process, it is critical to evaluate the resulting matrix crystals and coating for analyte extraction, localization, and effect on tissue architecture. Several reports have shown that even for standard analytes mixed with matrices, there is an uneven distribution of the analyte within the MALDI crystals and that sample preparation influences the resulting distribution [18-20], Thus, other active areas of research are focused on optimizing matrix application and sample preparation protocols. 

Standards used to constmct a cahbration curve must be prepared such that the matrix of the standard is identical to the sample s matrix because the values of the parameters k and b associated with a linear cahbration curve are matrix dependent. Many areas of chemical analysis are plagued by matrix effects, and it is often difficult to duphcate the sample matrix when preparing external standards. Because it is desirable to eliminate matrix effects, cahbration in the sample matrix itself can be performed. This approach is called the standard addition method (SAM) (14). In this method, the standards are added to the sample matrix and the response of the analyte plus the standard is monitored as a function of the added amount of the standard. The initial response is assumed to be Rq, and the relationship between the response and the concentration of the analyte is... 

Analyte dilution sacrifices sensitivity. Matrix matching can only be applied for simple matrices, but is clearly not applicable for complex matrices of varying composition. Accurate correction for matrix effect is possible only if the IS is chosen with a mass number as close as possible to that of the analyte elements). Standard addition of a known amount of the element(s) of interest is a safe method for samples of unknown composition and thus unknown matrix effect. Chemical separations avoid spectral interference and allow preconcentration of the analyte elements. Sampling and sample preparation have recently been reviewed [4]. 

It has to be stressed that selectivity of both separation methods depends on the matrix effect and the additives used, which can strongly influence chromatographic or electrophoretic equilibria. For that reason proper sample preparation plays a crucial role in the analytical process. 

One alternative is to compare the results of the method with results from an established reference method. This approach assumes that the uncertainty of the reference method is known. Second, accuracy can be assessed by analyzing a sample with known concentrations (e.g., a certified reference material) and comparing the measured value with the true value as supplied with the material. If such certified reference material is not available, a blank sample matrix of interest can be spiked with a known concentration by weight or volume. After extraction of the analyte from the matrix and injection into the analytical instrument, its recovery can be determined by comparing the response of the extract with the response of the reference material dissolved in a pure solvent. Because this accuracy assessment measures the effectiveness of sample preparation, care should be taken to mimic the actual sample preparation as closely as possible. 

Posyniak, A., Zmudzki, J., and Semeniuk, S. (2001). Effects of the matrix and sample preparation on the determination of fluoroquinolone residues in animal tissues. J. Chromatogr. A 914 89-94. 

The analyte binding efficiency is matrix dependent. Some matrices, such as urine and tissue extracts, can be directly loaded onto the column, other matrices such as milk may need sample processing prior to loading onto an immunoaffinity column. The simplest sample preparation method is dilution this method has been applied to serum, liver, and kidney extracts after removal of particulates. Sometimes dilution alone is not sufficient to eliminate the matrix effect and classical sample preparation techniques (solvent/solvent extraction, solid phase extraction, etc.) will be necessary prior to immunoaffinity chromatography. We found milk often needs this type of treatment. 

Matrix effects play an important role in the accuracy and precision of a measurement. Sample preparation steps are often sensitive to the matrix. Matrix spikes are used to determine their effect on sample preparation and analysis. Matrix spiking is done by adding a known quantity of a component that is similar to the analyte but not present in the sample originally. The sample is then analyzed for the presence of the spiked material to evaluate the matrix effects. It is important to be certain that the extraction recovers most of the analytes, and spike recovery is usually required to be at least 70%. The matrix spike can be used to accept or reject a method. 

Internal standard calibration can be used to compensate for variation in analyte recovery and absolute peak areas due to matrix effects and GC injection variability. Prior to the extraction, a known quantity of a known additional analyte is added to each sample and standard. This compound is called an internal standard. To prepare a calibration curve, shown in Figure 4.6b, the standards containing the internal standard are chromatographed. The peak areas of the analyte and internal standard are recorded. The ratio of areas of analyte to internal standard is plotted versus the concentrations of the known standards. For the analytes, this ratio is calculated and the actual analyte concentration is determined from the calibration graph. 

The recovery experiment carried out for this assay resembles somehow the sum of two potential effects The loss of compound during the sample preparation process (in this case on-line extraction) and a potential matrix effect during sample analysis. However, since an online sample clean up procedure is used here, these two potential effects cannot be separated in the usual way (One experiment would be the comparison of a processed spiked plasma sample with a blank plasma sample spiked post sample processing in order to determine the recovery of the sample preparation step. In a second experiment, again a processed blank plasma sample would be spiked post processing and the result would be compared with the response of a spiked solvent sample in order to reveal a potential matrix effect during analysis). 

A spiked sample is a sample prepared by adding a known quantity of analyte to a matrix which is close to or identical to that of the sample of interest. Spiked samples may be used in method validation experiments to help identify matrix effects and determine the recovery (see Section 2.3) of an analyte or the selectivity of the method. 

The quality of analytical results obtained in the analysis of biological and environmental materials substantially depends on the sample preparation procedures applied, particularly when low concentrations of the metals and species of low stability are determined in complex matrices. Concentrations below the detection limits (DLs) of the available instrumental techniques and interfering effects from the matrix components require separation and preconcentration procedures. Instrumental techniques applicable to direct examination of solid materials are valuable for minimizing the effect of sample preparation steps on the quality of obtained results. In the analysis of noble metal samples, in particular those of complex environmental origin, use of such techniques is strongly limited because of interference from matrix components and the heterogeneity of examined materials. 

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