Target compound chromatograms

Fig. 5.19. Ion chromatograms from a DIP measurement. The TIC (upper trace) shows a bump during early scans that is also reflected in the RICs of m/z 179.0 and 194.1. Spectrum (a) belongs to volatile impurities, whereas the RICs of m/z 382.2 and 453.3 are related to the target compound (b) that evaporates upon heating (cf. Fig. 5.20). By courtesy of R. Gleiter, Organisch-Chemisches Institut, Universitat Heidelberg.

On the other hand, the SIM analysis of a predefined set of analytes (Fig. 3.1.4a) require visual inspection, but only to exclude unexpected interferences in the signal of a target compound. For the purpose of detecting unexpected interferences, it is necessary, and is also a CAP accreditation requirement, to monitor at least two ion species per compound (Fig. 3.1.4b and c), so as to recognize artificial variations in the intensity ratios between two ion chromatograms. Figure 3.1.5 shows examples of characteristic organic acid profiles in patients with selected conditions [32]. 

The effect on chromatography is to complicate the separation greatly. If we consider a reverse-phase separation, the first thing we notice is an almost irreversible binding of protein to the column. Even after protein removal, we find polar peaks, which overload the early part of the chromatogram and tail into the compounds of interest. The components that are more nonpolar than our compounds of interest adhere to the column and must be washed off before the next injection. To ensure polar elution before our target compounds and nonpolar removal afterwards, we are almost forced to run solvent gradients. 

Finally, these relative heights or areas are compared with equivalent values obtained from standards curves prepared from known amounts of target compounds to yield values for the amount of each target compound present in the chromatographic injection. Unknowns in the chromatogram can be identified with relative retention times, areas, and heights, but the amount of each present cannot be determined until they are identified, standard curves run, and response factors calculated for each compound. 

Calibration standards can be of two types external standards and internal standards. With external standards, multiple concentrations of the standards are injected, areas are measured, and a calibration curve is platted. Unknown samples are then injected, chromatograms run, and areas are calculated and compared with the calibration curves to determine amounts of each compound present. With internal standards, known amounts of an internal standard are added to each known concentration of standard compound and areas or peak height response factors relative to those of the internal standard are calculated. When unknowns are run, a known amount of internal standard is added to the unknown sample, response factors are calculated relative to the internal standards, and amounts of each unknown present are calculated from the standards calibration factors. Internal standards are usually used to correct for variations in injection size due to different operators and injection techniques. Internal standards can also be used to correct for extraction variation in GC/MS target compound quantitation, this standard is referred to as a surrogate standard. Generally, an internal standard is used for one purpose or the other, not both at the same time. 

Target compound identification (from spectra, chromatograms)... 

To test this hypothesis, the pLC performance was evaluated as a function of Vinj for analyte-spiked plasma samples. Typical chromatograms are shown in Fig. 5 [4]. The retention times of target compounds increased only slightly as Vinj was increased,... 

Figure 11.7 Typical results of a post-column infusion experiment. In the top chromatogram the separation of the parent drag, its metabolite, and an ANIS is shown. The bottom chromatogram shows the matrix effect on the response of the parent drag. The ion suppression and ion enhancement effects prevent the rehable determination of the target compounds. Reprinted from W.M.A. Niessen, J. Chromatogr. A, 1000 (2003) 413 with permissiom 2003, Elsevier Science BV.

After analysis, target compounds (those compounds that the analysis is aiming to detect) are recorded by the data processing facilities of the mass spectrometer (Figure 41.3). The correct location of target peaks in a gas chromatogram is verified by the use of target-compound databases,... 

PB/LC/MS Detection and Quantitation of Chloronhenoxv Acid Herbicides in Soil. Detection and quantitation of these target compounds is via PB/LC/MS with SIM and El ionization (using 4-ions each). Figure 4 shows the PB/LC/MS chromatogram for the actual soil extract using EI/SIM. Table II compares the reference values from the interlaboratory check to the LC/MS values. 

The quantification is based on multi level response factors covering both differences in extraction behaviour, volatility and GC/MS response of the compounds and of the deuterated internal standards. 1 Utre pentane washed water is spiked with 50 pg deuterated internal standards and normally 1,5,25 and 100 pg of the different target compounds. Calibration curves are automatically calculated and stored on the disk, and quantification is based on area of ion chromatogram of one specific mass for each compoimd. 

Figure 6.3. Example chromatograms of two target compounds isolated by the high-throughput purification system shown in Figure 6.2. Chromatograms illustrate the performance of fast gradient analysis (ELSD and UV detector trace before purification). Reprinted with permission from reference 12.

Quantitative data of selected target compounds were obtained by integration of specific ion chromatograms extracted from the TIC. Injection volume and sample volume inaccuracies were corrected for by using internal standard compounds as a surrogate standard. An external four-point-calibration generated from reference compounds was used for quantification. 

Deprotonated molecule peaks for BPA (and NP) were predominant in the ESI spectra, while APCI spectra indicated slight thermal fragmentation. " " Signal intensities and signal-to-noise (S/N) values, based on mass chromatograms for [M-H] of each analyte, were 50 to 100 times larger in the ESI mode than those obtained in the APCI mode. This indicates that ESI is preferred to APCI for accurate quantification and sensitive detection of the target compounds. " ... 

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