Range calibration

The linear dynamic range of this method is 0.1 p.gl to 100 J.gl i The method detection limit (MDE) depends on selected operating conditions and a calibration range. It is important to use high purity reagents in all determinations. 

Instrument makers nonetheless provide this less-than-useful information, but hardly anybody recognizes as the outflow of the wide calibration range, the linear concentration-to-signal transfer function, and the excellent repeatability. 

Figure 2.2. Examples of correlations with high and low coefficients of determination. Data were simulated for combinations of various levels of noise (a = 1,5, 25, top to bottom) and sample size (n - 10, 20, 40, left to right). The residual standard deviation follows the noise level (for example, 0.9, 5.7, 24.7, from top to bottom). Note that the coefficient 0.9990 in the top left panel is on the low side for many analytical calibrations where the points so exactly fit the theoretical line that > 0.999 even for low n and small calibration ranges.

Choose the endpoints of the calibration range the calibration concentrations are now displayed in the green field. 

In the author s experience, such confirmation is not appropriate when the calibration range is greater than one order of magniffide or calibration points are not chosen carefully. The reason is that lower concentration levels of a calibration graph influence the correlation coefficient to a much smaller extent than higher concentrations. The hypothetical example of calibration results presented in Table 3 demonstrates this very simply. If the amount injected is correlated with the observed peak area in the second column in Table 3, the calibration graph in Figure 2 is obtained. 

Calibration range Not specified usually > 4 Not specified but at least one calibration in each sequence Linearity not required any vahd calibration is accepted Calibration with standards in matrix strongly recommended... 

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. 

Each commodity required a specifically customized workbook, containing a worksheet for each analyte determined in the commodity. Each laboratory received electronic copies of either three or four workbooks, which served as templates for the three or four commodities assigned to the laboratory. Each set of up to 10 commodity samples scheduled for colleetion and analysis required the creation of a copy of the appropriate template. Each workbook template contained one primary worksheet for each analyte, in which analytical data were recorded and residue levels were calculated, as described below. Eor example, the template for green beans contained 17 primary worksheets, one for each of the 17 analytes determined in each green bean sample. Additional worksheets were inserted into copies of the template as needed, to describe results of further analyses, such as confirmation of analytes present above the limit of quantitation (LOQ) or dilutions to bring the concentration of the analyte into the calibration range. 

The calibration curve is generated by plotting the peak area of each analyte in a calibration standard against its concentration. Least-squares estimates of the data points are used to define the calibration curve. Linear, exponential, or quadratic calibration curves may be used, but the analyte levels for all the samples from the same protocol must be analyzed with the same curve fit. In the event that analyte responses exceed the upper range of the standard calibration curve by more than 20%, the samples must be reanalyzed with extended standards or diluted into the existing calibration range. 

Fig. 4. Securing the lower limit of the calibration curve. The calibration range is from X (lower limit concentration) to X2 (upper limit concentration). The calculated value of Xp (p = 0.05) must be

Nano-structuring also results in a decreased detection limit. Since the latter has different explanations in analytical literature, we define it as the lower limit of the linear calibration range. For nano-structured Prussian blue the detection limit was found to be of 1 X 10 9 mol L 1 (Fig. 13.6). 

The resulting Prussian blue-based nano-electrode arrays in FIA demonstrate a sub-ppb detection limit (1 X 10 9 mol I. ) and a linear calibration range starting from the detection limit and extending over seven orders of magnitude of H202 concentrations (1 X 10 9 1 X 10 2 mol L ), which is the most advantageous analytical performance in electroanalysis. As a conclusion from the evidence in this chapter, Prussian... 

Sample preconcentration was performed by means of an automated on-line SPE sample processor Prospekt-2 (Spark Holland, Emmen, The Netherlands). Oasis HLB cartridges (Waters, Barcelona, Spain) were used to preconcentrate cannabi-noids present in the water samples whereas isolation of the rest of the compounds was done in PLRPs cartridges (Spark Holland). Before extraction, influent samples were diluted with HPLC water (1 9, v/v) to reduce matrix interferences and to fit some analyte concentrations, e.g., cocaine (CO) and benzoylecgonine (BE), within the linear calibration range. A sample volume of 5 mL was spiked with the internal standard mixture (at 20 ng/L) in order to correct for potential losses during the analytical procedure, as well as for matrix effects. Elution of the analytes to the LC system was done with the chromatographic mobile phase. 

After overcoming the instability of the thermospray ionization source, a sensitivity of 2 ng/mL was achieved with a calibration range of 10 to 103 ng/mL for human plasma samples. In recent years, the online SPE LC/MS/MS technology has matured and is now easy to build and use. It is used widely in the pharmaceutical industry (Jemal et al. 2000 Hsieh 2004 Hennion 1999). 

These systems have been used in many bioanalytical applications. A Prospekt system coupled with MS quantitated eserine N-oxide, a cholinesterase inhibitor, in human plasma for low level (4.5 mg) oral administration pharmacokinetic studies (Pruvost et al. 2000). After conditioning of the SPE cartridge (PLRP-S, Spark) with methanol (5 mL/min, 0.5 min) and water (5 mL/min, 0.5 min), a volume of 250 jj.L plasma plus internal standard was injected and washed (water, 1 mL/min, 3 min). The analytes were flushed out with 80 20 ammonium acetate (20 mM, pH 3.5 adjusted with formic acid) and acetonitrile (0.3 mL/min) and separated on a Zobax SB-CN column (150 x 2.1 mm inner diameter, 5 jim). A calibration range of 25 pg/mL to 12.5 ng/mL was achieved with a run time of 10.5 min. 

---