Dilutional Linearity and Parallelism

Parallelism, or linearity of dilution in biological samples, is a practical approach in a fit-for-purpose validation for new biomarkers. Preparing and analyzing dilutions of individual samples containing measurable amounts of endogenous 

BEST PRACTICES IN NOVEL BIOMARKER ASSAY FIT-FOR-PURPOSE TESTING 

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For PK assays, it may be the case that a human-derived molecule is being dosed to a different species. In this case, an assessment should be made of whether structurally similar molecules in that species are likely to cross-react. If the molecule does not appear naturally in the species, for example, a humanized monoclonal antibody, then the assay can be used for that species, but potential matrix differences have to be investigated and solved, that is, parallelism, dilution linearity, and so on. 

Preliminary analyte stability in biological matrix Parallelism/dilutional linearity Robustness and ruggedness... 

Parallelism and Dilutional Linearity to Evaluate Matrix Effects... 

Linearity has been described by some workers in a way which, by the current authors, would be interpreted as matrix parallelism, whereas others will use the term to describe the extent to which a calibration curve is linear in nonligand-binding assays. For the purpose of this chapter, the term linearity or dilution linearity is used to describe the results of experiments conducted using spiked samples to demonstrate the potential for high-concentration samples to be able to be diluted into the analytical range and read with acceptable accuracy and precision. It is often used to give an indication that matrix effects will not cause a problem upon sample dilution in circumstances where incurred or volunteer samples are not available with concentrations of analyte sufficiently high to conduct parallelism experiments. 

Neat PE was also studied in the melt. The 2930 cm"l and 2850 cm"l bands behave as expected from the alkane study. A plot of the ratio of their intensities versus temperature is shown in Fig. 6. The function is also a straight line and parallel to the lines for neat n-C and diluted n-C2 5 in COCl3(Fig. 5). Therefore, the same procedure can be used to calibrate the mentioned Raman intensities for PE as was used for li this way, a quantitative measurement of the gauche bond content of a linear polymer can be obtained. 

The adsorption transition also shows up in the behavior of the chain linear dimension. Fig. 6(a) shows the mean-square gyration radii parallel, i gl, and perpendicular, to the adsorbing plate. While these components do not differ from each other for e for e > ej i g strongly increases whereas Rh decreases. In the first case (non-adsorbed chain) oc oc N as a dilute solution in a good solvent in the bulk. For adsorbed chains R /N 0 for oo because the thickness is finite it is controlled by the distance e- e from the adsorption threshold, but does not diverge as N oo. The adsorbed chain follows in fact a... 

Grant et al. (2002) designed a parallel system employing two HTLC columns (Cyclone, 50 x 1 mm, Cohesive Technologies) connected to one analytical column (Zorbax SB-C18, 50 x 2 mm, Hewlett Packard) on a 2300 HTLC. A polyarylethyl ketone (PAEK) six-port Valeo (Valeo Instruments, Texas) was used to increase switching speed and reduce carry-over. Peak focusing was used when the analyte was flushed from the TFC column into the analytical column by aqueous dilution. Compared to the dual column method, the overall time reduction was 1.5 to 4 min per sample with comparable data quality at the linear range of 0.1 to 100 ng/mL. 

The order and timing of the addition of reagents in the KA-process is varied but in a typical procedure three reagents, namely, acetic anhydride, a solution of ammonium nitrate in nitric acid, and solid hexamine dinitrate, are added slowly, in small portions and in parallel, into the reaction vessel which is preheated to 60-80 °C. On completion the reaction mixture is often cooled to 50-60 °C and the RDX filtered and sometimes washed with acetic acid. This process produces a product which melts over a 2 °C range but the RDX still contains up to 10 % HMX as a by-product. Dilution of the reaction mixture with water before removing the RDX produces a very impure product containing numerous unstable linear nitramine-nitrates. Based on the assumption that one mole of hexamine dinitrate produces two mole of RDX the KA-process commonly yields 75-80 % of RDX. 

The most widely used type of simultaneous assay is the one in which the response has a homoscedastic linear regression on a logarithmic scale. Homoscedasticity means that the variance of all experimental groups is the same. For such an assay, the condition of similarity requires that the straight lines of the standard and the samples should be parallel. Otherwise, the condition of similarity between the sample and standard is not established, that is, it would not be valid to assume that dilutions of one behave the same as dilutions of the other, which is the assay s underlying principle. 

This type of test is called parallel-line assay and is based on the comparison of a sample response with that of a reference standard (Finney, 1978). In general, it determines the response - at least by duplicates - of a series of dilutions of each preparation (sample and standard) while plotting the means of their corresponding doses on a logarithmic scale. As this test requires analysis of a linear portion of the curves, at least three points of each curve belonging to such a portion should be selected. The more selected points, the better the comparison. 

Interfacing the TEA to both a gas and a HPLC has been shown to be selective to nitro-based explosives (NG, PETN, EGDN, 2,4-DNT, TNT, RDX and HMX) determined in real world samples, such as pieces of explosives, post-blast debris, post-blast air samples, hand swabs and human blood, at picogram level sensitivity [14], The minimum detectable amount for most explosives reported was 4-5 pg injected into column. A pyrolyser temperature of 550°C for HPLC-TEA and 900°C for GC/TEA was selected. As the authors pointed out, GC uses differences in vapour pressure and solubility in the liquid phase of the column to separate compounds, whereas in HPLC polarity, physical size and shape characteristics determine the chromatographic selectivity. So, the authors reported that the use of parallel HPLC-TEA and GC-TEA techniques provides a novel self-confirmatory capability, and because of the selectivity of the technique, there was no need for sample clean-up before analysis. The detector proved to be linear over six orders of magnitude. In the determination of explosives dissolved in acetone and diluted in methanol to obtain a 10-ppm (weight/volume) solution, the authors reported that no extraneous peaks were observed even when the samples were not previously cleaned up. Neither were they observed in the analysis of post-blast debris. Controlled experiments with handswabs spiked with known amounts of explosives indicated a lower detection limit of about 10 pg injected into column. 

The same model can be applied to methane- and trifluoromethanesulphonic acids.93 Also in these two cases, there is a linear dependence of 33S Ti on concentration in very large ranges (approximately 0.1-8 M for methanesulphonic acid and 0.1-5 M for trifluoromethanesulphonic acid). Moreover, Ti in methane-sulfonic acid increases linearly with the degree of ionization of the acid. In both cases, 170 Ti has a parallel behaviour. For methanesulphonic acid, T] at infinite dilution is 11.6 ms at 293 K and 13.5 ms at 313 K. For trifluoromethanesulphonic... 

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