Calibration matrix matching

Internal standards could be used in external calibration, matrix-matched external calibration, and standard addition calibration [2], However, the use of internal standards in LC-MS quantitative methods should not be confused with internal calibration in which an internal standard is employed as a calibrant and the concentration of a unknown sample is calculated from the concentration of this internal standard and its analyte/IS signal ratio, i.e., the concentration of the unknown sample is calculated without the need for a calibration curve [3], The use of internal standards in most LC-MS quantitative methods belongs to signal-ratio calibration or internal standardization [2,4], In fact, the majority of bioanalytical LC-MS methods use matrix-matched signal-ratio external calibration. 

Calibrators (Matrix Matched Calibration Solutions) Samples of blank matrix to which known amounts of the analytical standard (and of SIS if available) have been added before extraction and clean-up used to determine the concentration range of the complete assay and to calibrate the instrument response for analyte concentration while correcting for variations in extraction... 

The method of standard additions can be used to check the validity of an external standardization when matrix matching is not feasible. To do this, a normal calibration curve of Sjtand versus Cs is constructed, and the value of k is determined from its slope. A standard additions calibration curve is then constructed using equation 5.6, plotting the data as shown in Figure 5.7(b). The slope of this standard additions calibration curve gives an independent determination of k. If the two values of k are identical, then any difference between the sample s matrix and that of the external standards can be ignored. When the values of k are different, a proportional determinate error is introduced if the normal calibration curve is used. 

In most quantitative analyses we are interested in determining the concentration, not the activity, of the analyte. As noted earlier, however, the electrode s response is a function of the analyte s activity. In the absence of interferents, a calibration curve of potential versus activity is a straight line. A plot of potential versus concentration, however, may be curved at higher concentrations of analyte due to changes in the analyte s activity coefficient. A curved calibration curve may still be used to determine the analyte s concentration if the standard s matrix matches that of the sample. When the exact composition of the sample matrix is unknown, which often is the case, matrix matching becomes impossible. 

Requirements for standards used In macro- and microspectrofluorometry differ, depending on whether they are used for Instrument calibration, standardization, or assessment of method accuracy. Specific examples are given of standards for quantum yield, number of quanta, and decay time, and for calibration of Instrument parameters. Including wavelength, spectral responslvlty (determining correction factors for luminescence spectra), stability, and linearity. Differences In requirements for macro- and micro-standards are considered, and specific materials used for each are compared. Pure compounds and matrix-matched standards are listed for standardization and assessment of method accuracy, and existing Standard Reference Materials are discussed. 

Only a direct matrix match of sample and CRM, and the CRM s use as a direct calibrant will allow the user to demonstrate accuracy and subsequently traceability close to the uncertainties established during the CRM certification ( note matrixmatching may not be necessary with matrix-independent techniques). This reality places a significant burden on the CRM producers, since large uncertainties in the certified values may degrade the perceived value of the CRM. 

The effect of co-extracted matrix components on the analyte response in the final determination step should be assessed. Normally, this is done by comparing the response of standards in solvent with matrix-matched standards, i.e., standards prepared in the extract of a control sample without residues. Because matrix effects tend to be inconsistent, the guidelines propose the general use of matrix-matched calibration unless it is demonstrated to be unnecessary. 

It is often difficult to define where sample extraction ends and cleanup procedures begin. Sample extracts may be injected directly into a gas or liquid chromatograph in certain cases, but this will be dependent on the analyte, sample matrix, injection, separation and detection system, and the limit of determination (LOD) which is required. It is also more likely that matrix-matched calibration standards will be needed in order to obtain robust quantitative data if no cleanup steps are employed. 

Pd removal was determined as follows. An aliquot of a representative liquid or solid sample was accurately weighed and subsequently digested by refluxing in nitric and/or hydrochloric acid using a closed vessel microwave procedure (CEM MARS5 Xpress or Milestone Ethos EZ). Cooled, digested samples were diluted, matrix matched to standards, and referenced to a linear calibration curve for quantitation an internal standard was employed to improve quantitation. All samples were analyzed by an Inductively Coupled Plasma Mass Spectrometer or ICP/MS (Perkin Elmer SCIEX Elan DRCII) operated in the standard mode. 

As XRF is not an absolute but a comparative method, sensitivity factors are needed, which differ for each spectrometer geometry. For quantification, matrix-matched standards or matrix-correction calculations are necessary. Quantitative XRF makes ample use of calibration standards (now available with the calibrating power of some 200 international reference materials). Table 8.41 shows the quantitative procedures commonly employed in XRF analysis. Quantitation is more difficult for the determination of a single element in an unknown than in a known matrix, and is most complex for all elements in an unknown matrix. In the latter case, full qualitative analysis is required before any attempt is made to quantitate the matrix elements. 

In addition to statistical peculiarities, special features may also result from certain properties of samples and standards which make it necessary to apply special calibration techniques. In cases when matrix effects appear and matrix-matched calibration standards are not available, the standard addition method (SAM, see Sect. 6.2.6) can be used. 

Brenner et al. [ 169] applied inductively coupled plasma atomic emission spectrometry to the determination of calcium (and sulfate) in brines. The principal advantage of the technique was that it avoided tedious matrix matching of calibration standards when sulfate was determined indirectly by flame techniques. It also avoided time-consuming sample handling when the samples were processed by the gravimetric method. The detection limit was 70 ig/l and a linear dynamic range of 1 g/1 was obtained for sulfate. 

An ideal method for the preconcentration of trace metals from natural waters should have the following characteristics it should simultaneously allow isolation of the analyte from the matrix and yield an appropriate enrichment factor it should be a simple process, requiring the introduction of few reagents in order to minimise contamination, hence producing a low sample blank and a correspondingly lower detection limit and it should produce a final solution that is readily matrix-matched with solutions of the analytical calibration method. 

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). 

The matrix is the major component of the solution (or of the solid if using laser ablation or SEM). Matrix differences between standards and samples or between samples may result in differences of elemental sensitivity. Therefore, it is desirable that all the blanks, calibration standards, and samples have the same matrix (i.e., are matrix matched). This is relatively easy to achieve in solution, but can be a major problem with the analysis of solid samples. 

Two types of blanks need to be prepared with each batch of samples. To subtract background levels of contamination occurring during instrumental analysis, an instrument blank, which consists of a matrix-matched solution with no internal standard, is made. Generally, an instrument blank is run at the start of the analysis and will be included in the calibration with assigned concentrations of zero for all elements to be measured, so, in effect, this is subtracted from all the subsequent samples. 

Water samples are amongst the simplest to prepare, simply requiring acidification to keep the sample elements in solution and to matrix-match with the calibration solutions, and the addition of an internal standard. The procedure is as follows. 

Empirical Analysis uses reference standards to establish an empirical calibration line (Piorek Rhodes 1988) and can be useful for samples where all major elements present cannot be analyzed. The standards should be matrix-matched and contain a range of element concentrations bracketing the desired level of quantification. 

Use as a matrix matched calibrant (direct or via working standards) to ensure traceabiiity of results to an external reference (the CRM)... 

Systematic effects are estimated by repeated measurements of a CRM, suitably matrix matched. Any difference between the CRM and a routine sample for which the measurement uncertainty is being estimated should be considered and an appropriate uncertainty component added. Suppose a concentration measurement is routinely made in a laboratory that includes measurement of a CRM in the same run as calibration standards and unknowns. The bias (6) is given by... 

Primary calibrators, indeed all calibrators, must be commutable that is, they must behave during measurement in an identical manner to the native analyte material being measured. Matrix reference materials made by mixing a pure reference material with the components of the matrix are unlikely to be entirely commutable, and for this reason some authorities (EURACHEM for one [EURACHEM and CITAC 2002]) advise against using matrix-matched CRMs for calibration, recommending instead their use to establish recoveries, after calibration by a pure reference standard. 

Another calibration technique - standard addition - minimizes matrix effects because analytes with well defined increasing concentrations are added to a set of sample solutions to be analyzed. The measured calibration curve in the standard addition mode plots the measured ion intensities of analytes versus the concentration added to the sample solution. The concentration of analytes in the undoped sample is then determined by extrapolation of the calibration curve with the x-axis. Matrix matching is subsequently performed and the matrix effects (signal depression or interference problems) are considered. An example of the standard addition technique is described in Section 6.2.6 using solution based calibration in LA-ICP-MS. 

Three different calibration strategies for solution based calibration in LA-ICP-MS have been developed in our laboratory. These are similar to the solution calibration in solution analysis by ICP-MS external calibration if a high purity matrix-matched blank target is available,29,71 the standard addition technique (e.g., for high purity platinum)76 or the isotope dilution technique.43... 

Figure 9.38 Concentration profile of copper and zinc in the scanned area of interest measured by LA-ICP-MS on brain samples (hippocampus). Calibration was performed via synthetic matrix matched laboratory standards for 1, 5 and 10 fxgg-1 of analyte (see inserted figures on left). Bottom histologically processed brain tissue in which cell bodies were stained (cresyl violet staining) in order to demonstrate the layered structure of the analyzed region. (]. S. Becker, M. Zoriy, C. Pickhardt, N. Palomero-Gallagher and K. Zilles, Anal. Chem., 77, 3208 (2005). Reproduced by permission of American Chemical Society.)...

Inorganic mass spectrometry requires the development of suitable reference materials, such as matrix matched standard reference materials for trace, surface (including depth profiling and microlocal) analysis and/or the creation of matrix independent calibration procedures. The development of species specific standards will be intensified for speciation studies in the future. 

Conventional external calibration uses pure standard solutions (single- or multielement) and is therefore unable to compensate for matrix effects, fluctuations or drifts in sensitivity. Matrix effects can be compensated for by using matrix-matched calibration solutions. In this case, the degree of compensation depends on the proper matrix adjustment. 

Calibration using simple solutions of well-characterised pure substance standards or matrix matched standard solutions. Calibration solutions are prepared from materials whose identity and purity have been established to an appropriate level of uncertainty and where the effects of any impurities have been evaluated. Where appropriate and where available, standards provided by metrology institutes with demonstrated capability are used. In other cases, materials from other reputable suppliers or prepared in-house are used after appropriate characterisation. Where necessary, professional judgement is used to estimate the uncertainty associated with chemical standards. The target uncertainty of the identity is for practical purposes zero and for purity less than one-fifth of the desired overall uncertainty. 

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