Quantitation response factors

It is crucial in quantitative GC to obtain a good separation of the components of interest. Although this is not critical when a mass spectrometer is used as the detector (because ions for identification can be mass selected), it is nevertheless good practice. If the GC effluent is split between the mass spectrometer and FID detector, either detector can be used for quantitation. Because the response for any individual compound will differ, it is necessary to obtain relative response factors for those compounds for which quantitation is needed. Care should be taken to prevent contamination of the sample with the reference standards. This is a major source of error in trace quantitative analysis. To prevent such contamination, a method blank should be run, following all steps in the method of preparation of a sample except the addition of the sample. To ensure that there is no contamination or carryover in the GC column or the ion source, the method blank should be run prior to each sample. 

It is seen that, individually, all the curves appear linear and any one of the five curves might be considered to give accurate quantitative results. However, the actual results that would be obtained from the analysis of a binary mixture containing 10% of one component and 90% of the other, employing detectors with each of the five response factors, is shown in Table 1. 

The refractive index detector, in general, is a choice of last resort and is used for those applications where, for one reason or another, all other detectors are inappropriate or impractical. However, the detector has one particular area of application for which it is unique and that is in the separation and analysis of polymers. In general, for those polymers that contain more than six monomer units, the refractive index is directly proportional to the concentration of the polymer and is practically independent of the molecular weight. Thus, a quantitative analysis of a polymer mixture can be obtained by the simple normalization of the peak areas in the chromatogram, there being no need for the use of individual response factors. Some typical specifications for the refractive index detector are as follows ... 

The use of an internal standard probably gives the most accurate quantitative results. However, the procedure depends upon finding an appropriate substance that will elute in a position on the chromatogram where it will not interfere or merge with any of the natural components of the mixture. If the sample contains numerous components, this may be difficult. Having identified a reference standard, the response factors for each component of interest in the mixture to be analyzed must be determined. A synthetic mixture is made up containing known concentrations of each of the components of interest and the standard. If there are (n) components, and the (r) component is present at concentration (Cr) and the standard at a concentration (Cst). 

The aim of all the foregoing methods of factor analysis is to decompose a data-set into physically meaningful factors, for instance pure spectra from a HPLC-DAD data-set. After those factors have been obtained, quantitation should be possible by calculating the contribution of each factor in the rows of the data matrix. By ITTFA (see Section 34.2.6) for example, one estimates the elution profiles of each individual compound. However, for quantitation the peak areas have to be correlated to the concentration by a calibration step. This is particularly important when using a diode array detector because the response factors (absorptivity) may considerably vary with the compound considered. Some methods of factor analysis require the presence of a pure variable for each factor. In that case quantitation becomes straightforward and does not need a multivariate approach because full selectivity is available. 

The method must be fast, rugged, and universal for the reaction products. The response factors of all components of interest should be equivalent to permit quantitation of all components without the use of extensive standardization. 

Much LC-MS work is carried out in a qualitative or semi-quantitative mode. Development of quantitative LC-MS procedures for polymer/additive analysis is gaining attention. When accurate quantitation is necessary, it is important to understand in depth the experimental factors which influence the quantitative response of the entire LC-MS system. These factors, which include solvent composition, solvent flow-rate, and the presence of co-eluting species, exert a major influence on analyte mass transport and ionisation efficiency. Analyte responses in MS procedures can be significantly affected by the nature of the organic modifier used in the RPLC... 

From the uv absorption spectra, a suitable wavelength is found for the simultaneous detection of aspirin, phenacetin and caffeine. Using phenacetin as internal standard, response factors are calculated for aspirin and caffeine and the results are used for the quantitative determination of aspirin and caffeine in an analgesic tablet. 

Surfactants are separated according to adsorption or partitioning differences with a polar stationary phase in NPLC. This retention of the polar surfactant moiety allows for the separation of the ethylene oxide distribution. Of all the NPLC packings that have been utilized to separate nonionic surfactants, the aminopropyl-bonded stationary phases have been shown to give the best resolution (Jandera et al., 1990). The separation of the octylphenol ethoxylate oligomers on an amino silica column is shown in Fig. 18.4. Similar to the capabilities of CE for ionic surfactants, the ethylene oxide distribution can be quantitatively determined by NPLC if identity and response factors for each oligomer are known. 

Select one of the quantitation procedures we have discussed (response factor method, internal standard method, or standard addition method) and describe ... 

This method assumes that the response factor is constant over a range of concentrations and it is often more acceptable to determine the response factor for a range of test concentrations. In this method, a calibration curve is produced by incorporating a fixed amount of the internal standard in samples that contain known amounts of the test compound. For each concentration the ratio of peak heights is determined and plotted against concentration (Procedure 3.2). For quantitation of a test sample, the same amount of the internal standard is introduced in its usual way and the ratio of peak heights for the standard and unknown is used to determine the concentration of the unknown from the calibration curve. 

The colour or fluorescence produced per mole of amino acid varies slightly for different amino acids and this must be determined for each one to be quantitated. This is done by loading a mixture of amino acids containing the same concentration of each amino acid including the chosen internal standard and from the areas of the peaks on the recorder trace calculating each response factor in the usual way (Figure 10.19). These values are noted and used in subsequent calculations of sample concentrations. 

The truncated peptide analogs were used to demonstrate the specificity of the method and to evaluate the limit of quantitation of potential impurities. Potential impurities were spiked into a solution of IB-367 at 0.05%, 0.1%, 0.2%, 0.5%, and 1% to assay the linearity of potential impurities at low concentrations. The method exhibited acceptable linearity for impurities from 0.05 to 1%. The relative response factors of these analogs were assessed to determine area normalization feasibility. 

It is critical when performing quantitative GC/MS procedures that appropriate internal standards are employed to account for variations in extraction efficiency, derivatization, injection volume, and matrix effects. For isotope dilution (ID) GC/MS analyses, it is crucial to select an appropriate internal standard. Ideally, the internal standard should have the same physical and chemical properties as the analyte of interest, but will be separated by mass. The best internal standards are nonradioactive stable isotopic analogs of the compounds of interest, differing by at least 3, and preferably by 4 or 5, atomic mass units. The only property that distinguishes the analyte from the internal standard in ID is a very small difference in mass, which is readily discerned by the mass spectrometer. Isotopic dilution procedures are among the most accurate and precise quantitative methods available to analytical chemists. It cannot be emphasized too strongly that internal standards of the same basic structure compensate for matrix effects in MS. Therefore, in the ID method, there is an absolute reference (i.e., the response factors of the analyte and the internal standard are considered to be identical Pickup and McPherson, 1976). 

Inappropriate response factors may have been used for quantitation of impurities/degradation products (i.e., molar extinction coefficients of degradation products are less than those of the API). 

Further discussion of method validation can be found in Chapter 7. However, it should be noted from Table 11 that it is frequently desirable to perform validation experiments beyond ICH requirements. While ICH addresses specificity, accuracy, precision, detection limit, quantitation limit, linearity, and range, we have found it useful to additionally examine stability of solutions, reporting threshold, robustness (as detailed above), filtration, relative response factors (RRF), system suitability tests, and where applicable method comparison tests. 

Most frequently, the design results, or more specifically the factor effects, are analyzed graphically and/or statistically, to decide on method robustness. A method is considered robust when no significant effects are found on responses describing the quantitative aspects. When significant effects are found on quantitative responses, non-significance intervals for the significant quantitative factors can be defined, to obtain a robust response. However, no case studies were found in CE where such intervals actually were determined. 

In 1981, two eminent British cancer experts. Sir Richard Doll and Richard Peto published a paper in the Journal of the National Cancer Institute entitled The causes of cancer Quantitative estimates of avoidable risks of cancer in the United States today. The authors drew upon a vast body of literature of the type mentioned above, and attempted to allocate the deaths caused by cancers among various responsible factors. The authors concluded that a certain percentage of human cancer deaths could be avoided if exposure to the responsible factors could be eliminated or controlled in some way, although the appropriate degree and nature of control for some of the lifestyle factors, especially diet, is still highly uncertain. The Doll and Peto estimates are presented in Table 5.2. The factors are listed in a somewhat different order from how they were listed by the original authors, because of our interest in clearly separating lifestyle factors (the first... 

Quantitation method System number Manual injector Total i nj ect i ons Sarriple rate Sample amount Scale factor Response factors Channel to calibrate... 

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