Analytical performance parameters Recovery

The purpose of an analytical method is the deliverance of a qualitative and/or quantitative result with an acceptable uncertainty level. Therefore, theoretically, validation boils down to measuring uncertainty . In practice, method validation is done by evaluating a series of method performance characteristics, such as precision, trueness, selectivity/specificity, linearity, operating range, recovery, LOD, limit of quantification (LOQ), sensitivity, ruggedness/robustness, and applicability. Calibration and traceability have been mentioned also as performance characteristics of a method [2, 4]. To these performance parameters, MU can be added, although MU is a key indicator for both fitness for purpose of a method and constant reliability of analytical results achieved in a laboratory (IQC). MU is a comprehensive parameter covering all sources of error and thus more than method validation alone. 

Ensuring high-quality analytical performance in trace analysis, if separation of sample components by extraction is indispensable, requires implementation of the appropriate extraction method and establishment of suitable operational parameters to ensure a high efficiency of extraction. Selection of extraction conditions is crucial for quantitative recovery of analyte, or at least for sufficient effectiveness. If an aqueous solution is one of the extraction phases, problems such as complex-ation, hydrolysis, and solvation can play an important role. Extraction of elements from aqueous to organic phase often requires selection of appropriate ligands and control of pH. 

Knowing the most influential parameters of a specific biosensor architecture is the basis to understand and fine tune the performance of these devices in a rational manner. Figure 1.8 summarizes the key features of typical biosensors and lists several that are of additional importance for commercial devices. Among these, selectivity, sensitivity, accuracy, response, and recovery time as well as operating lifetime are some of the most important key factors. Keeping in mind the needs of the specific analytical task of interest, it seems to be necessary to characterize at least the key parameters mentioned in Figure 1.8 in order to specify the analytical performance of a biosensor design. 

The performance of the new analytical method was adequate to ensure that measured GB concentrations in the hydrolysate were low enough for secondary treatment using supercritical water oxidation (SCWO). Method detection limit (MDL) values in hydrolysate were 2.2 pg/L (2.2 ppb) with 68 percent recovery and were calculated in accordance with standard U.S. Environmental Protection Agency (EPA) methods (40 CFR Part 136, Appendix B). This value is well below the release criteria of 75 ppb for GB in the hydrolysate. A more significant performance parameter is the target action limit (TAL), which is the concentration for which 95 percent of the measurements will be below the release criteria (Malloy et al., 2007). In an analysis of GB performed on hydrolysate generated from two batch reactor studies conducted at Battelle, the TAL values were calculated at 57 ppb and 52 ppb. 

Once the analytical scale method conditions are optimized, the next step is to choose a column and scale up the analytical HPLC parameters so that preparative chromatography can be performed and the unknown compound(s) can be isolated for identification by MS and NMR. For ease of transition, a preparative column consisting of the same packing material and particle size should be chosen. The column is the most important component of the process because it determines the amount of material that can be loaded for the desired purity and recovery. An important step in the scale-up procedure is determining the maximum load on the analytical column. The maximum analytical load is essential in determining the loading capacity of the preparative column. When an appropriate column is chosen, the analytical isolation can be scaled up using Eq. (5) 2 ... 

In 2002, Indyk and co-workers evaluated vitamin B12 in a range of foods such as milks, infant formulas, meat and liver using SPR technology with performance parameters including a quantitation range of 0.08-2.4 ng/mL with recoveries of 89-106% (Indyk et al. 2002). The analytical technique was biosensor based utilizing bimolecular interaction. 

Development of SPE methods requires a sound knowledge of liquid chromatography. The most important parameters are the SPE cartridge (type of the sorbent) and the type of solvents used. The size of the cartridge (which determines the sample amount) is also important Too small sorbent size is easily overloaded, while too large sorbent size may bind the analyte, thereby decreasing recovery. Like in chromatography, various additives, especially buffers, may be used to improve performance. 

The most economic way of using CRMs for calibration purposes is to validate a procedure for routine analysis. The analytical procedure is carried out with the CRMs analysed as samples. "IMth the results achieved, all relevant analytical parameters can be determined, e.g. uncertainty, recovery, reproducibility, selectivity, linearity, etc. The procedure is then well known for the specific sample type and the specific analytes for which it is validated and can be applied routinely for this analytical problem, with a few regular reviews of the analytical performance. CRMs in this case are not used for calibration but rather for validation of the procedure and regular review of the method performance. 

If analytical methods are validated in inter-laboratory validation studies, documentation should follow the requirements of the harmonized protocol of lUPAC. " However, multi-matrix/multi-residue methods are applicable to hundreds of pesticides in dozens of commodities and have to be validated at several concentration levels. Any complete documentation of validation results is impossible in that case. Some performance characteristics, e.g., the specificity of analyte detection, an appropriate calibration range and sufficient detection sensitivity, are prerequisites for the determination of acceptable trueness and precision and their publication is less important. The LOD and LOQ depend on special instmmentation, analysts involved, time, batches of chemicals, etc., and cannot easily be reproduced. Therefore, these characteristics are less important. A practical, frequently applied alternative is the publication only of trueness (most often in terms of recovery) and precision for each analyte at each level. No consensus seems to exist as to whether these analyte-parameter sets should be documented, e.g., separately for each commodity or accumulated for all experiments done with the same analyte. In the latter case, the applicability of methods with regard to commodities can be documented in separate tables without performance characteristics. 

Method performance study All laboratories follow the same written protocol and use the same test method to measure a quantity (usually concentration of an analyte) in sets of identical test samples. The results are used to estimate the performance characteristics of the method, which are usually within-laboratory- and between-laboratory precision and - if relevant - additional parameters such as sensitivity, limit of detection, recovery, and internal quality control parameters (IUPAC Orange Book [1997, 2000]). 

Acceptability is determined for each parameter by comparison of the standard deviation (s) and the average recovery (X) with the corresponding acceptance criteria for precision and accuracy as published in the method for the analytes of interest. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples may begin. If any individual s exceeds the precision limit, or if any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter. The analyst must locate and correct the source of the problem and repeat the test for all parameters that failed. 

The performance characteristics of any analytical method involve the evaluation of the following parameters calibration range, limit of detection, precision, trueness, specificity, recovery, and robustness [40]. 

The recovery of an analyte in an assay is defined by the FDA in a strictly operational way as the detector response obtained Ifom an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the pure authentic standard. Recovery pertains to the extraction efficiency of an analytical method within the limits of variability. Recovery of the analyte need not be 100 %, but the extent of recovery of an analyte and of the internal standard should be consistent, precise, and reproducible. Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (low, medium, and high) with unextracted standards that represent 100 % recovery (FDA 2001). In terms of the symbols used in Section 8.4, the recovery is thus defined as the ratio (R /R"), and is equivalent to determination of F provided diat no suppression or enhancement effects give rise to differences between R and R" and that the proportional systematic errors and 1 are negligible. The FDA definition of recovery also corresponds to that of the PE ( process efficiency ) parameter (Matuszewski 2003) discussed in Section 5.3.6a, since the former (FDA 2001) measures a combination of extraction efficiency and matrix effects (if any). 

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