Retention time windows

MS detection does not necessarily require as highly resolved GC separations as in the case of selective detectors because the likelihood of an overlapping mass spectral peak among pesticides with the same retention time is less than the likelihood of an overlapping peak from the same element. Unfortunately, this advantage cannot always be optimized because SIM and current gas chromatography/tandem mass spectrometry (GC/MS/MS) methods, it is difficult to devise sequential SIM or MS/MS retention time windows to achieve fast GC separations for approximately > 50 analytes in a single method. 

Peaks are identified from absolute or relative retention times by comparison with data from previously run standards stored in RAM or in libraries on disk. To take account of the variability of retention times from successive runs, retention time windows are used. These are defined as being /R x% for a standard, the unknown being positively identified if its retention time falls within the specified range. The size of the window can be varied by the user to conform with the degree of certainty required. Reference peaks can be selected for the calculation of relative retention times or as internal standards in quantitative analysis (pp. 9, 114). 

Interfering compounds will vary considerably from source to source, and samples may require a variety of cleanup steps to reach required method detection limits. The emission wavelengths used (EPA 8310) are not optimal for sensitivity of the small ring compounds. With modem electronically controlled monochromator, wavelength programs can be used which tune excitation and emission wavelengths to maximize sensitivity and/or selectivity for a specific analyte in its retention time window. 

Thus, the presence of the methoxyphenols in pyrolysis products indicates that lignin-like components have survived for approximately 3(X) million years. That the specific peak was indeed methoxyphenol was determined by retention time and by examination of the fragment ions at m/z 109 and 81. Similarly, the verification of the presence of methy thoxyphenol was made by retention time and by examination of fragment ions at nVz 123 and 95. Dir t comparison of mass spectra to library spectra of audientic methoxyphenols was not possible due to the trace quantities and the complexity of die pyrolysis mixture in the retention-time windows for elution of the methoxyphenols. 

Reference vector. After calibration a reference vector is created. This vector is used to match all the same fractions together in a data matrix and is created by ordering of all existing calibrated retention time values. The number of the values is reduced by replacing each value that falls into a small retention time "window" with its mean value. The "window" was set wide enough to reduce the number of values to 250. 

Chemical Analysis of Extracts. The extracts were analyzed by capillary column GC-MS for OCs, TAAPs, and PAHs (see the list on page 313). The GC-MS parameters used at the two laboratories are shown in Table II. The identification and quantitation were all done by using automatic routines based on a mass spectra library created from authentic standards of the selected compounds. Compounds were located by searching the reconstructed ion chromatogram for each library entry within a narrow retention time window relative to the internal standard (anthracene-dio or phenanthrene-dio). Quantitation was achieved by comparison of characteristic ion areas in the field samples with ion areas of the internal standard. These ion areas were normalized by response factors established by comparison of ion ratios of a standard mixture of all 66 analytes at a concentration of 2.5 ng//zL. 

Recognizing the fact that shifts in retention time of individual compounds may cause false negative results, laboratories use retention time windows for target analyte identification. Retention time windows are experimentally determined retention time ranges for each target analyte. To minimize the risk of false positive results, EPA methods require that chromatography analysis be performed on two columns with dissimilar polarities. This technique, called second column confirmation, is described in Chapter 4.4.3. It reduces the risk of false positive results, but does not eliminate them completely. 

Qualitative compound confirmation may be performed either with a second column or with a second detector. Second column confirmation technique consists of analyzing the sample on two columns with dissimilar polarities. Each column is calibrated with the same standards, and the same calibration acceptance criteria are applied. For the presence of a compound to be confirmed, its retention time values obtained from each column must fall into respective retention time windows. If a peak falls within the retention time window on one column, but not on the second column, the compound is not considered confirmed and should not be reported. 

Sample cleanup is particularly important for analytical separations such as GC, HPLC, and electrophoresis. Many solid matrices, such as soil, can contain hundreds of compounds. These produce complex chromatograms, where the identification of analytes of interest becomes difficult. This is especially true if the analyte is present at a much lower concentration than the interfering species. So a cleanup step is necessary prior to the analytical measurements. Another important issue is the removal of high-boiling materials that can cause a variety of problems. These include analyte adsorption in the injection port or in front of a GC-HPLC column, false positives from interferences that fall within the retention window of the analyte, and false negatives because of a shift in the retention time window. 

In most data systems, a given peak in a sample mixture is identified if its retention time falls within a user-defined retention time window. The retention time window is typically centered about the retention time of that peak in a standard solution (i.e., 5%). However, the size of the window may be altered depending on the number of other components in the sample that may fall within a given window and the reproducibility of the method. The more reproducible the retention times, the smaller are the windows that may be defined. 

Packed GC columns afforded moderately well-resolved, reproducible profiles of the hydrocarbon fraction. Each gas chromatogram contained 40 peaks corresponding to a set of standardized retention time windows. Further details about the collection of this data can be found in the literature [90],... 

Figure 13. Chromatogram of silylated ether extract (pH 5.5) of human urine prior to enzyme hydrolysis. (Chromatographic conditions same as Figure 4). Shaded area denotes retention time window during which the Olfax searched for the 9-THC-ll-oic acid/TMS contracted spectrum. Inset Auto-Assay analysis for THC panel of 8 compounds in which 240 ng (2.40 X 10 1 fig) of A9-THC-ll-oic acid was found with a Confidence Index of 62.

NOTE Parameters for Selected Ion Monitoring (SIM)—Set up two separate retention time windows and monitor four ions within each window using 150 msec dwell time per ion. 

Retention Time Windows and Calibration of Target Analytes... 

The identification of all other PCDD/PCDF isomers is based on their retention times falling within their respective PCDD/PCDF retention time windows as established by a window defining mix. Confirmation of all PCDDs/PCDFs is based on a comparison of the ratio of the integrated ion abundance of the molecular ion species to the theoretical ion abundance ratio. 

Any compound that yields ions listed in Table 5 and also elutes within the retention time window of the corresponding homologue is a potential interference. PCDDs/PCDFs are often associated with other chlorinated compounds such as polychlorinated biphenyls (PCBs) and polychlorinated diphenyl ethers (PCDPEs). These compounds may be found at concentrations several orders of magnitude higher than that of the analytes of interest and may otherwise interfere with the analysis of PCDDs/PCDFs. Therefore the retention time of the target analytes must be verified using reference standards and compared to retention time... 

For all calibration solutions, the retention times of the isomers must fall within the appropriate retention time windows established by the window defining mix analysis. In addition, the absolute retention times of the recovery standards, 13Ci2-1,2,3,4-TCDD and 13Ci2-1,2,3,6,7,8-HxCDD, shall not change by more than 10 seconds between the initial CC3 analysis and die analysis of any other standard. 

For non-2,3,7,8-substituted compounds (tetra through hepta), the retention time must be within the retention time windows established by the window defining mix for the corresponding homologue (see Section 7.1). 

The time advantage of MS/MS also is exemplified by the ability to select from a mixture of ions in the source any parent ion, in any order, and to return as necessary to that parent ion for precise measurements. This independence of access persists for the duration of the sample residence time in the source. This is in marked contrast to the situation in GC/MS, where each sample is available for examination by the mass spectrometer only during the retention time window. To repeat the measurement, the entire sample must be reinjected. The source residence time for most samples introduced via the direct insertion pjrbbe is on the order of a minute or two, depending on the temperature of the source and the rate of heating of the prcbe tip itself. 

Six 50-mg blank hair samples were extracted by SFE and the noise was integrated for the ion used for quantification (m/z = 355 for codeine, 369 for ethylmorphine, 383 for 6-MAM, and 397 for morphine) in a retention time window of tj + 0.5 min. The LOD and LOQ were determined (n = 6) using lUPAC methods. For each substance the standard deviation of the blank value (Sg) was determined. The mean area converted from the noise was calculated as concentration equivalent based on a calibration graph. The LOD is defined as 3Sg and the LOQ as lOSg. 

Adjust the column oven temperature, if required, to obtain retention time windows for etofenprox (approximately 19 minutes) and dicyclohexyl phthalate (approximately 9.5 minutes), but do not exceed 300°C. 

Retention time windows and identification of the analytes and internal standard, and should allocate the method to calculate the analyte amount or concentration... 

This method was dependent on product scans of m/z 157. a characteristic fragment ion for the dansyl moiety. The added specificity and chromatographic resolution was shown to remove ambiguity in cases where spurious peaks in the HPLC fluorescence masked the retention time window for a code. Fig. 5.21 shows a comparison of the HPLC fluorescence trace and the CEC/MS/MS result for such a case. 

In quantitative bioanalysis, the application of SRM in a triple-quadrupole instrument is the method-of-choice. SRM provides ultimate selectivity, because only compounds are detected, which after sample pretreatment of the matrix are detected within a selected retention time window in the chromatogram as the stracture-specific fragment at a particular m/z in MSj, generated by collision-induced dissociation (CID) from a precursor ion with a particular m/z of the compound of interest, selected in MS . The ultimate selectivity results in good sensitivity and low detection limits. Software procedures for the automatic optimization of instrament... 

However, spectral identity is a necessary but not a sufficient condition for compound identification. If the instrument parameters that determine how spectra are acquired are matched suitably, the spectral match of an unknown to a known spectram can be used as strong indication of compound similarity or confirmation of identity. In a chromatogram, the retention time of a given peak offers an obvious means of preselection of candidate spectra through the application of a retention time window. All spectra from the set of possible candidates that fall in the window are compared to the unknown spec-tram. The candidate with the best match factor above a certain threshold level is then used to identify the compound for the peak. Another possibility is to weigh both the match factor and the similarity in retention times and use the combined information for identification. 

While glucuronide and sulfate molecular ion species are not seen in the PB mass spectra, the SAX-LC separation groups the compounds by conjugate class. Phenols elute first, followed by glucuronides, and then sulfates. Thus, tentative identification of unknown metabolites is possible based upon identification of the aglycone and the retention time window. 

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