Validation calibration/standard curve

Working on the assumption that polystyrene standards are valid calibration standards for polyarylsulfones, the molecular weights (Mw) for resins IV and V were calculated from the molecular-weight distribution curves by the following equation. 

In order to validate sliding spark spectrometry results, plastic material was collected and the element concentration was determined via AAS after digestion. The samples were used as calibration standards. Additional standards were obtained by manufacturing known amounts of additives in the polymer matrix. Calibrations were made for Cd, Cr, Pb, Zn, Sb, Si and Ti in chlorine-free polymers Al, Ba, Ca, Cd, Pb, Sn, Ti, Zn in PVC chlorine (as PVC) and bromine in polyurethane (PUR). A calibration curve for Br as a flame retardant in PUR is shown in Figure 8.5. 

The Q-factor approach is based upon the weight-to-size ratios (Q-factors) of the calibration standard and the polymer to be analyzed. The Q-factors are employed to transform the calibration curve for the chemical type of the standards (e.g. polystyrene) into a calibration curve for the chemical type of polymer under study. The inherent assumption In such a calibration approach is that the weight-to-size ratio is not a function of molecular weight but a constant. The assumption is valid for some polymer types (e.g. polyvinylchloride) but not for many polymer types. Hence the Q-factor method is generally referred to as an approximation technique. 

A new calibration curve must be implemented every time a new stock of internal standard solution is prepared, and at least twice per year. New calibration curves are validated by the following criteria for acceptability point-to-point comparison (<10% difference from the previous calibration curve), coefficient of linear regression (>0.99), intercept and slope (<10% difference from previous calibration curve). Normal and abnormal control samples are calculated against the new and the old curve and compared to the current quality control (QC) mean as the final step in the validation of the new curve. The new calibration curve is then used with subsequent runs if the curve validation is acceptable. Curves are unique to each instrument and therefore must be established for each instrument prior to clinical use. 

Valid independent calibration curves can be established for each detector in a multidetector SEC system. In doing so the nature of the detector response and the MWDs of the calibration standards must be taken into... 

Procedurally, dilutional linearity should not be confused with the MRD of an assay. The MRD is a designed integral part of an analytical method and involves a predefined dilution of test samples, QC samples and, often, calibrators usually with a buffer-based matrix. In contrast, dilution linearity is used only to support analysis of study samples that exceed the assay s ULOQ and involves dilution(s) intended to result in an analyte concentration within the standard curve s validated range. Another notable difference is that, while MRD is usually performed in buffer, dilutional linearity is performed in matrix, often the same one used to prepare the standard curve. 

The acceptability of in-study batches/runs is based on the performance of standard calibrators and quality control samples run in an assay. As mentioned previously, it is desirable to have prestudy method acceptance criteria consistent with the in-study batch acceptance criteria. If not, a higher percentage of assay failures can be expected. This rationale was the genesis for the 4-6-30 rule as recommended by DeSilva et al. [3 5]. The standard curve acceptance criteria for macromolecule LB As are that at least 75% of the standard points should be within 20% of the nominal concentration (%RE of the back-calculated values), except at the LLOQ and ULOQ where the value should be within 25%. This requirement does not apply to anchor concentrations, which are typically outside the validation range of the assay and are used to facilitate and improve the nonlinear curve fitting. 

At a minimum, documentation of the characterization and stability of a standard, such as a certificate of analysis (Co A) and/or a certificate of stability (CoS), is typically available from the suppliers. The certificate should be obtained and recorded. The quantity of reference standard is typically limited in commercial kits designed for research use, and it is not uncommon that the reference material values may differ substantially between lots and manufacturers [16]. Novel biomarkers rarely have established gold standards against which their potency and abundance can be calibrated. A comparison of available sources can be useful, and when validating an assay for advanced applications it is desirable to plan ahead to obtain and reserve a sufficient supply of the same reference material. The example in Fig. 6.5 compares three reference standard curves, each prepared from a concentrated stock solution from a commercial supplier, an in-house reference standard, and a commercial kit, respectively. The instrument responses (optical density, OD) were highest with the standard from the commercial stock, the lowest with the kit, while the in-house reference standard response was intermediate. In this case, either the same commercial stock or the in-house reference standard can be used throughout the clinical study. 

For research applications, where maximal accuracy is desirable even at the expense of time and effort, two maneuvers are possible. The first involves derivation of the initial dissociation curve using the standard calibration curve. A second computation follows, using the derived curve for calibration. This process is repeated until the derived and calibrating dissociation curves match each other. This method requires considerable calculations, but it is theoretically valid even for abnormal hemoglobins. The other technique for accurate dissociation curve analysis is simply a variation on the traditional method of carefully equilibrating aliquots... 

The standard curve should be monitored during in-study validation with at least one set of calibrators per patch run. As for prestudy validation, the curve should be constructed from six concentrations in duplicate. Anchor points may be used. The final number of points used for curve lit must be either 75% of the total number or a minimum of six calibrator samples not including the anchor points. The relative error of the back-calculated samples should be <20% (<25% at the LLOQ). If either the high or low calibrator standards have to be deleted, the range for this particular run must be limited to the next standard point. Samples out of range must be repeated. 

As for immunoassays for pharmaceutical proteins, in-study validation of biomarker assays should include one set of calibrators to monitor the standard curve as well as a set of QC samples at three concentrations analyzed in duplicate for the decision to accept or reject a specific run. Recommended acceptance criterion is the 6-4-30 rule, but even more lenient acceptance criteria may be justified based on statistical rationale developed from experimental data [14]. 

Batch A set of standard curve calibrators, validation samples or QC samples, or study samples that are analyzed in a single group of measurements it is synonymous with run. 

The MSA is often used if no suitable external calibration curve has been prepared. There may be no time to prepare calibration standards— for example, in an emergency situation in a hospital it may be necessary to measure sodium rapidly in a patient s serum. It may not be possible to prepare a valid set of calibration standards because of the complexity of the sample matrix or due to lack of sufficient information about the sample—for example, industries often require the analysis of mystery samples when something goes wrong in a process. MSA calibration is very useful when certain types of interferences are present in the sample matrix. MSA permits us to obtain accurate results without removing the interferences by performing the calibration in the presence of the interferences. It is often used when only one sample must be analyzed, and the preparation of external standards would be inefficient. 

This is only valid for areas and concentrations within the limits of the standard curve calibration levels. While in principle one may calibrate against a standard curve which has a substantial y-axis intercept, this will yield valid results only if the the background signal level causing the large intercept is the same in both the standards and unknown sample matrices. This is a dangerous and often unverifiable assumption to make, and the use of such calibration curves should be avoided. 

Quantitative methods are usually based on a comparison of the response from an analyte in a sample with the response from standards of the analyte in solution at known concentrations. In method development and validation, the calibration curve should first be determined to assess the detector response to standards over a range of concentration. These concentrations should cover the full range of analytical interest, and, although it is usually recommended practice to include a suitable blank with the calibration samples, this does not imply that it is acceptable to extrapolate into the region of the curve below the lowest calibration standard or to force the curve through the origin. 

When preparing the calibration curve experiments in a method characterization or validation experiment, there are some key things to remember. It is important to first examine and plot the detector response to analyte in pure or defined solvent (calibration function) and also at various concentrations in the matrix (i.e., in the presence of interferants, or analytical function). Remember that more standard solutions and more calibration points are required when a non-linear response is observed. Extrapolation above or below the analytical range may be used to approximate behavior at zero if a blank is not included in the calibration standards, but the curve should not be forced through the origin. Typically, a curve prepared by... 

Using the 500-ppm BTEX stock reference solution, prepare a series of calibration standards in which the BTEX is present in 10 mL DDI which is contained in a 22-mL HS vial with PTFE/silicone septa and aluminum crimp caps. Refer to the above calibration for reference as you prepare a series of working calibration standards for HS-GC analysis. Following the development of a calibration curve, inject the ICV (only one injection per vial is acceptable in HS GC), then inject the headspace above the aqueous samples. Following the development of a calibration curve, inject the ICV (only one injection per HS vial is valid) and the contaminated aqueous samples. 

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