Post-Detection Signal Enhancement

We found it quite simple instead to write and deploy a Gaussian convolution program that was applied to every data point in turn and took only a fraction of a second to run with our limited number of 512 data points. Although in its simplest form this is mathematically equivalent to the Fourier transformation described earlier, we modified it so that the convolution was taken with the first-or second derivative of a Gaussian profile of arbitrarily chosen width (Sections 4.2 and 4.3). With this we were able to discriminate against pedestal, linear and 

MMW spectra possess a number of features that make direct readout of sample concentration from a measurement less than straightforward. Although the peak absorption coefficient of the sample may be determined in several ways itemised below. Equations 1.24 and 1.48 show that additional information about linewidth, temperature, occupation of vibrational states and even Doppler broadening is required to obtain the analyte concentration. Such calculations can be incorporated into any anal5dic program, but the parameters required are often not readily available in the literature and may need to be determined directly. 

This applies most particularly to the linewidth broadening parameter, which can vary with the sample composition by up to an order of magnitude. The values for concomitant gases on the NH3 3,3 line range typically from 10 (He) to 205 (NH3) kHz Pa (ref. 11, p. 364). This parameter reflects the different cross-sections experienced by the two molecules in collision. Its effect on the analytical 

175843 MHz and N N 0 at 175855 MHz, at 1.6 Pa (Reprinted from Baker et al. with permission from Elsevier Science) 

Although some correction must still be made for changes in the linewidth broadening parameter, the calculations required are much less detailed. In particular, practical measurements are much simplified by exploiting the insensitivity of the peak absorption coefficient to pressure (Section 1.2). This technique becomes especially effective if combined with the line profile integration described in the next section. 

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In a certain sense, the detector provides us with more details than that bargained for. The goal of the primary post-detection signal handling is to get rid of those features which are irrelevant in a given context and enhancing those which are essential. In many cases, for example, it makes... 

The two constraints that make direct observation of weak absorption signals impracticable are the presence of pink noise, which contains a preponderance of low-frequency power compared with white noise, and the enhancement of this and other sources of noise by the rectification process through which MMW signals are detected. Both predicate the use of some kind of modulation at a frequency greater than the noise comer frequency and the use of a phase-coherent detector to convert the resulting modulated signal to a DC level suitable for display or for post-detection computer processing. 

Measurement of samples at low concentration may be expected to yield signals badly contaminated by random and systematic noise. Techniques for noise reduction may be applied both in the design and the operation of the spectrometer and in post-detection data processing (Section 4.2). The utility and flexibility of these techniques have been greatly enhanced by the computing power now available from desk-top computers that can be attached on-line to a spectrometer in order simultaneously to process its output and optimise its working parameters. Some of the techniques that have been used are described in Section 4.3 and their development for measurements up to atmospheric pressure is discussed in Section 4.4. 

A protein-binding assay (BA) coupled with hplc provided a highly sensitive post-column reaction detection system for the biologically important molecule biotin and its derivative biocytin, biotin ethylenediamine, 6-(biotinoylamino) caproic acid, and 6-(biotinoylamino)caproic acid hydrazide (71). This detection system is selective for the biotin moiety and responds only to the class of compounds that contain biotin in their molecules. In this assay a conjugate of streptavidin with fluorescamine isothiocyanate (streptavidin—FITC) was employed. Upon binding of the analyte (biotin or biotin derivative) to streptavidin—FITC, an enhancement in fluorescence intensity results. This enhancement in fluorescence intensity can be directly related to the concentration of the analyte and thus serves as the analytical signal. The hplc/BA system is more sensitive and selective than either the BA or hplc alone. With the described system, the detection limits for biotin and biocytin were found to be 97 and 149 pg, respectively. 

Of course, coincidence of molecular masses will always result in direct interferences in an SIM channel used for the SIS and thus directly affect the quantitation of the analyte, but not necessarily in an MRM channel for the SIS depending on the nature of the product ions (a good example of the increase in detection selectivity provided by MRM). Another approach to facilitating detection of metabolites is via a similar comparison for pre-dose and post-dose samples from the same test subject. It is also emphasized that aU of the preceding discussion refers to direct interferences with the analytical signals for analyte and SIS, not to indirect interferences arising from suppression or enhancement of ionization efficiency. These matrix effects are almost always the result of endogenous components of the matrix rather than of metabolites of the analyte. 

Matrix Effect and Recovery For LC-MS/ MS-based methods, the signal suppression or enhancement of the analyte due to the presence of the matrix interferences (matrix effects) in MS/MS detection should be evaluated by comparing the response (peak area) of the analyte and the IS from the extracted blank samples post-fortified with the analyte and the IS with the response of neat solutions with both the analyte and the IS at the same concentrations as above. Matrix effects should be evaluated in one pooled batch of animal matrix or in at least three different batches of human matrix, using three replicates at a minimum of three QC concentrations (e.g., low quality control [LQC], medium quality control [MQC], high quality control [HQC]) with IS at working concentration. The coefficient of variation (CV%) of the matrix effect variability should be <15% at each concentration level and between the three (LQC, MQC, and HQC) concentration levels. 

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