Internal standards quantitative calculations

Quantitative results were produced for each compound on the basis of internal standard method calculations. A three-point calibration curve was generated for each compound by using peak areas of a quantitation ion extracted from the mass spectrum of the compound. The ion was selected on the basis of it being a uniquely characteristic mass of the compound. The use of extracted ion quantitation produces more accurate results than total ion-current quantitation in cases in which two or more components are not completely resolved chromatographically. This situation is generally the case in complex mixture analysis. The quantitation ions selected for each of the compounds in the mix are listed in the box. 

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. 

It is assumed in the course of the calculation procedure that for the same molar quantities of the two compounds the intensities of both molecular ions are identical. This is obviously not true for most compounds. To establish the quantitative relationship between the intensity of the molecular ion of an internal standard and that of the compound in question, a calibration procedure based on measuring standard samples is performed. These standard samples contain both the internal standard and the compound in question in known quantities (two standard samples for each of about 5 different amounts within the expected operating range). Assuming the above quantitative relationship, the content of the compound in question is calculated for each standard sample giving a calculated and an observed (actual) value. A regression line is calculated for the whole calibration set of pairs of expected and observed quantities. From the equation for the regression line a corrected amount of the internal standard is calculated. The corrected internal standard is subsequently used in all experimental samples to calculate the amount of the compound in question. 

Quantitative mass spectrometry, also used for pharmaceutical appHcations, involves the use of isotopicaHy labeled internal standards for method calibration and the calculation of percent recoveries (9). Maximum sensitivity is obtained when the mass spectrometer is set to monitor only a few ions, which are characteristic of the target compounds to be quantified, a procedure known as the selected ion monitoring mode (sim). When chlorinated species are to be detected, then two ions from the isotopic envelope can be monitored, and confirmation of the target compound can be based not only on the gc retention time and the mass, but on the ratio of the two ion abundances being close to the theoretically expected value. The spectrometer cycles through the ions in the shortest possible time. This avoids compromising the chromatographic resolution of the gc, because even after extraction the sample contains many compounds in addition to the analyte. To increase sensitivity, some methods use sample concentration techniques. 

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. 

Both vibrational spectroscopies are valuable tools in the characterization of crystalline polymers. The degree of crystallinity is calculated from the ratio of isolated vibrational modes, specific to the crystalline regions, and a mode whose intensity is not influenced by degree of crystallinity and serves as internal standard. A significant number of studies have used both types of spectroscopy for quantitative crystallinity determination in the polyethylenes [38,74-82] and other semi-crystalline polymers such as polyfethylene terephthalate) [83-85], isotactic poly(propylene) [86,87], polyfaryl ether ether ketone) [88], polyftetra-fluoroethylene) [89,90] and bisphenol A polycarbonate [91]. 

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). 

These are the retention times and/or mass spectra or other spectra for qualitative analysis and peak areas, including that of the internal standard, for quantitative analysis. For quantitative analysis, the peak area ratio is calculated as with the standards. 

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. 

A limit test is a semi-quantitative test that allows the determination of the presence of a specific compound above a well-defined concentration level. The determinations are done according to an external calibration calculation procedure, either with or without internal standardization. The calibration is perfotmed against a reference solution at threshold level (e.g., 0.10% w/w compared to the nominal concentration of the sample solution at 100% w/w). 

Drifts of migration times can partly be compensated by calculating the mobility for analyte identification and using corrected PAs or internal standards for quantitation. 

For quantitative estimation of identified monomers, response factors were calculated, using 4-ethyl-resorcinol as an internal standard. The relative error in the determination of all the compounds was 4%. 

Calculation of the molecular weights of the -butyl esters of acylcarnitines, expressed as (M+H)+ molecular ions, allows the designation of each peak (Table 3.2.1). Quantitation software provided by the instrument vendor (i.e., Chemoview for SCIEX instruments) allows calculation of quantitative and semiquantitative concentrations for these acylcarnitines by comparison of the detected abundance of each acylcarnitine versus that of a designated internal standard with a known concentration. The results for each sample are further compared to age-appropriate reference ranges (Table 3.2.2). 

Amounts of injected compounds are proportional to the peak area. BA concentrations are calculated by comparison to standard calibration curves. This method does not need an internal standard for quantitative analysis. 

In clinical chemistry, interpretation of the data can be quite simple or complex. In the case of MS/MS applications pertaining to a single analyte, all that is needed is the intensity value from the mass of a peak of interest and its internal standard. Viewing of a spectrum is not necessary. For profile methods such as full-scan acylcarni-tines, amino acids, or other compound families, the interpretation is more complex. With multiple related components, calculation of the concentration of many key metabolites is required. The system generally has multiple internal standards, external standards, or both. In addition to the concentration calculations, examination of a profile is often best achieved by viewing the spectra together with the quantitative information. 

Quantitative analysis by isotope dilution. In isotope dilution, a known amount of an unusual isotope (called the spike) is added to an unknown as an internal standard for quantitative analysis. After the mixture has been homogenized, some of the element of interest must be isolated. The ratio of the isotopes is then measured. From this ratio, the quantity of the element in the original unknown can be calculated. 

Since trace analysis also includes air or gas samples, it is appropriate to point out that proper addition of an internal standard to this type of sample is difficult. This difficulty lies, not in the mechanical problem of transfer, but in the difficulty of knowing that the precisely intended volume has properly been transferred. However, the internal standard technique is still not widely used here for the same reason it is not generally used in trace analysis. This reason again is because the analyst normally has no prior knowledge of the variation in composition from sample to sample. The continual risk exists that any given sample in a series will have a component, not present in others, which elutes with the internal standard. This occurrence would introduce significant error into the quantitative calculations which result. 

For quantitative analysis, the cell constant k is determined with a weighed internal standard and then used in calculating the amount of other compounds present. Starting with Equation 2, Paul and Umbreit (1) derived the following equation for molecular weight determination in a dual chromatographic system ... 

In this procedure, the instrument is set up and standards are prepared, and then the sample is prepared as described elsewhere (see Support Protocol). Addition of an internal standard to the sample is also important for the analysis of organic acids. This provides a means not only for determining whether the analysis is working, but also for quantitating the percent recovery of the method. The sample is then run and concentrations are calculated. 

Once again, a literature review must be done to determine which acid to use as an internal standard. The internal standard will be used to calculate the percent recovery, which quantitates organic acid losses during sample preparation. 

A prerequisite for the calculation of OAVs are exact quantitative data. Aroma compounds, which are relatively stable and are present in food extracts in higher concentrations (>100 pg/kg food) are often quantified by using an internal standard containing a similar pattern of functional groups as the analyte. In a quantitative study on cherry odorants [63] it has been shown, that the results are significantly influenced by the isolation technique used and by the structure of the odorant. However, under appropriate conditions the values differed only between 7 % (benzaldehyde) and 26 % ((E,Z)-2,6-... 

If standard mixtures of known weight ratios of components do not give the same ratio of areas, the detector is not responding equivalently to each component. In this case a quantitative analysis of the mixture requires preliminary experimentation. One method is the use of an internal standard (for a more detailed discussion of other procedures, specialist texts should be consulted65,66). Thus for a two-component mixture (A + B), a third component C is selected as the internal standard. Mixtures of A + C and B -I- C are prepared in which the weights of each component in each mixture are known. The relevant chromatogram is recorded and the detector response factors calculated from the following relationships ... 

The Raman effect has also been broadly applied to online and bench-top quantitative applications, such as determination of pharmaceutical materials and process monitoring [4-6], in vivo clinical measurements [7], biological materials [8, 9], to name only a few. Because the absolute Raman response is difficult to measure accurately (sample presentation and delivered laser power can vary), these measurements are almost always calculated as a percentage with respect to the response from an internal standard. This standard is typically part of the sample matrix in a drug product, the standard may be an excipient in a biological sample, it is commonly water. 

In the determination of steady state reaction kinetic constants of enzyme-substrate reactions, FABMS also provides some very unique capabilities. Since these studies are best performed in the absence of glycerol in the reaction mixture, the preferred method is that which analyzes aliquots which are removed from a batch reaction at timed intervals. Quantitation of the reactants and products of interest is essential. When using internal standards, generally, the closer in mass the ion of interest is to that of the internal standard, the better is the quantitative accuracy. Using these techniques in the determination of kinetic constants of trypsin with several peptide substrates, it was found that these constants could be easily measured (8). FABMS was used to follow the decrease in the reactant substrate and/or the increase in the products with time and with varying concentrations of substrate. Rates of reactions were calculated from these data for each of the several substrate concentrations used and from the Lineweaver-Burk plot, the values of Km and Vmax are obtained. 

The methyl esters can be also determined by GC-FID. Using a 30 m x 0.32 mm ID x 0.25 pm (film thickness) capillary column, such as DB-1701 or equivalent, the compounds can be adequately separated and detected by FID. The recommended carrier gas (helium) flow rate is 35 cm/s, while that of the makeup gas (nitrogen) is 30 cm/min. All of the listed herbicides may be analyzed within 25 min. The oven temperature is programmed between 50 and 260°C, while the detector and injector temperatures should be 300 and 250°C, respectively. The herbicides may alternatively converted into their trimethylsilyl esters and analyzed by GC-FID under the same conditions. FID, however, gives a lower response as compared with ECD. The detection level ranges from 50 to 100 ng. For quantitation, either the external standard or the internal standard method may be applied. Any chlorinated compound stable under the above analytical conditions, which produces a sharp peak in the same RT range without coeluting with any analyte, may be used as an internal standard for GC-ECD analysis. U.S. EPA Method 8151 refers the use of 4,4,-dibromooctafluorobiphenyl and 1,4-dichlorobenzene as internal standards. The quantitation results are expressed as acid equivalent of esters. If pure chlorophenoxy acid neat compounds are esterified and used for calibration, the results would determine the actual concentrations of herbicides in the sample. Alternatively, if required, the herbicide acids can be stoichiometrically calculated as follows from the concentration of their methyl esters determined in the analysis ... 

Quantitation. The organic species identified by the GC-MS analyses were quantitated by GC analysis using internal and external standardization methods. Pure compounds representative of the various compound classes identified by GC-MS were selected as standards and methylated. A specific amount of each standard was co-injected with each sample to confirm the GC-MS identifications. For quantitation purposes, each standard was injected onto the gas chromatograph prior to and following sample analyses. The response factor of each standard was calculated under analytical conditions identical to those of the sample analyses. 

Internal standards could be used in external calibration, matrix-matched external calibration, and standard addition calibration [2], However, the use of internal standards in LC-MS quantitative methods should not be confused with internal calibration in which an internal standard is employed as a calibrant and the concentration of a unknown sample is calculated from the concentration of this internal standard and its analyte/IS signal ratio, i.e., the concentration of the unknown sample is calculated without the need for a calibration curve [3], The use of internal standards in most LC-MS quantitative methods belongs to signal-ratio calibration or internal standardization [2,4], In fact, the majority of bioanalytical LC-MS methods use matrix-matched signal-ratio external calibration. 

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