Quantitation by External Standardization

Note In practice, four or five runs at increasing concentration of the standard, preferably from well above the detection limit to close to saturation, should be mn in a sequence to obtain the data for the construction of a calibration curve. Extrapolation to concentrations outside the range covered has to be strictly avoided. One should also bear in mind that the limit of quantitation (LOQ) of a compound is found at higher concentration than its limit of detection (LOD). 

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Quantitation by External Standard. This quantitation technique is the most straightforward. It involves the preparation of one or a series of standard solutions that approximate the concentration of the analyte. Chromatograms of the standard solutions are obtained, and peak heights or areas are plotted as a function of concentration of the analyte. The plot of the data should normally yield a straight line. This is especially true for pharmaceuticals of synthetic origin. Other forms of mathematical treatment can be used but will need to be justified. 

Eatty acids from commercial fats and oils, such as peanut oil, are extracted with methanolic NaOH and made volatile by derivatizing with a solution of methanol/BE3. Separations are carried out using a capillary 5% phenylmethyl silicone column with MS detection. By searching the associated spectral library students are able to identify the fatty acids present in their sample. Quantitative analysis is by external standards. 

Quantitation by Internal Standard. Quantitation by internal standard provides the highest precision because uncertainties introduced by sample injection are avoided. In this quantitation technique, a known quantity of internal standard is introduced into each sample and standard solutions. As in the external standard quantitation, chromatograms of the standard and sample solutions are integrated to determine peak heights or peak areas. The ratio of the peak height or area of the analyte to an internal standard is determined. The ratios of the standards... 

A 25-g soil sample was extracted with methylene chloride and the extract was concentrated to a final volume of 2 mL. A 10-component alkane mixture at a concentration of each component as 50 pg/mL was used in quantitation performed by external standard method. 1 pL of the extract and the standard were injected onto the column for analysis. Determine the concentration of diesel range organics in the sample from the following data ... 

Identity is a general requirement for dosage forms. When determining specificity for identity, the assay and related substances or the content uniformity methods can be used. Assay and content uniformity methods are quantitated by external reference standard. This identity test confirms that the correct active ingredient (s) is present and is present in correct ratio if multiple variants are available. The method could also be used for post-packaging analysis. The general requirements are that the sample and standard chromatograms should correspond in retention time and normalized peak area within 10%. 

A quantitative Amp-RT assay is a useful laboratory tool for several applications, including the determination of HIV-1 levels in culture supernatant or biological fluids (e.g., plasma and serum), and the study of HIV-1 RT susceptibility to RT inhibitors. The authors describe a quantitative Amp-RT assay for measuring RT activity of HIV-1. Quantitation is based on a calibration curve made by external standards. The standards used can be known numbers of HI V-1 particles (from 10,000 to 1) or known numbers of HIV-1 RT molecules. Virion numbers of an HIV-1 stock can be easily determined from the concentration of p24 antigen on the basis of 10 4 pg of p24 antigen per virion as previously estimated (5). Alternatively, the virion numbers can be determined by RT-PCR analysis or by electron microscopy. HIV-1 RT with a well-characterized specific activity and known p66/p51 protein content can also be used to generate standard curves with known numbers of RT molecules. Commercially... 

Quantitation by external calibration The most common and straightforward method of calibration in atomic absorption spectrometry is the use of an external calibration with suitable standard solutions. It is based on the assumption that the standard solution matches the composition of the sample sufficiently well. This is an assumption that must always be examined with care, since, for example, samples of different viscosity may be aspirated at different rates in flame AAS,... 

A radioactive internal standard can be added to the sample to monitor analyte loss during the workup steps, and a nonradioactive internal reference standard can be added to all samples and standards to correct for variables such as spotting errors and layer variations and improve accuracy and precision (Aginsky, 1994). Relative standard deviations can reportedly be improved by a factor of 5-10 by use of an internal reference standard (Windhorst and de Kleijn, 1992), although other sources claim that internal standards are normally not required in quantitative HPTLC for good precision because samples and standards can be processed by external standardization on the same plate under essentially identical conditions. As an example, / -hydroxybenzaldehyde was used as the internal standard in the quantitative HPTLC determination of purine alkaloids in aqueous solutions such as daily foods and health drinks (Kunugi and Tabei, 1997). 

A quantitative determination using a single external standard was described at the beginning of this section, with k given by equation 5.3. Once standardized, the concentration of analyte, Ca, is given as... 

Standardizing the Method Equations 10.32 and 10.33 show that the intensity of fluorescent or phosphorescent emission is proportional to the concentration of the photoluminescent species, provided that the absorbance of radiation from the excitation source (A = ebC) is less than approximately 0.01. Quantitative methods are usually standardized using a set of external standards. Calibration curves are linear over as much as four to six orders of magnitude for fluorescence and two to four orders of magnitude for phosphorescence. Calibration curves become nonlinear for high concentrations of the photoluminescent species at which the intensity of emission is given by equation 10.31. Nonlinearity also may be observed at low concentrations due to the presence of fluorescent or phosphorescent contaminants. As discussed earlier, the quantum efficiency for emission is sensitive to temperature and sample matrix, both of which must be controlled if external standards are to be used. In addition, emission intensity depends on the molar absorptivity of the photoluminescent species, which is sensitive to the sample matrix. 

When possible, quantitative analyses are best conducted using external standards. Emission intensity, however, is affected significantly by many parameters, including the temperature of the excitation source and the efficiency of atomization. An increase in temperature of 10 K, for example, results in a 4% change in the fraction of Na atoms present in the 3p excited state. The method of internal standards can be used when variations in source parameters are difficult to control. In this case an internal standard is selected that has an emission line close to that of the analyte to compensate for changes in the temperature of the excitation source. In addition, the internal standard should be subject to the same chemical interferences to compensate for changes in atomization efficiency. To accurately compensate for these errors, the analyte and internal standard emission lines must be monitored simultaneously. The method of standard additions also can be used. 

Table 5.17 Quantitative results obtained for the determination of four diarrhetic shellfish poisons (DSPs) using external standards and the method of standard additions. Reprinted from J. Chromatogr., A, 943, Matrix effect and correction by standard addition in quantitative liquid chromatographic-mass spectrometric analysis of diarrhetic shellfish poisoning toxins , Ito, S. and Tsukada, K., 39-46, Copyright (2002), with permission from Elsevier Science...

Quantitation of anthocyanins has become simple and fast since many anthocy-anin standards became commercially available as external standards in the past decade. When the standards are not available, individual anthocyanins or total monomeric anthocyanins can be determined by the use of a generic external standard such as commercial cyanidin-3-glucoside or other compound structurally similar to the analytes of interest. Individual and total peak areas are measured at 520 nm or their and quantified using external standards by which values are typically slightly different from those via the pH differential method. ... 

The amounts of carfentrazone-ethyl, C-Cl-PAc, C-PAc, DM-C-Cl-PAc and HM-C-Cl-PAc were quantitated by the external standard calibration method. 

The amounts of sulfentrazone, SCA (analyzed as DMS), and HMS were quantitated by an external standard calibration method. A computer spreadsheet program (Microsoft Excel) was used for calculation and reporting. 

The determination of the relationship between detector response and the sample concentration is termed the calibration of the method. There are two types of methods in use for the quantitative analysis of a sample, i.e., the external standard and the internal standard method. An external standard method is a direct comparison of the detector response of a pure compound (standard) to a sample.2 The calibration of the method is performed by preparing standards of varying concentration and analyzing them by a developed method. Method 1 (below) was developed for toluene, and standards of varying concentration were prepared and analyzed. The results obtained are summarized in Table 2 see Figure 3. 

Each reaction step was monitored qualitatively by TLC using hex-ane ethyl acetate as the developing solvent and quantitatively by GC. Impurity peaks were identified by GC/MS. An HPLC external standard method (Method 2) was developed and used to determine the purity of the final isolated product (RWJ-26240). The following rugged HPLC method was developed to optimize scheme 1, step 6 ... 

Under some conditions, it is difficult to incorporate an internal standard into a method. If the chromatogram is very complex, an internal standard may interfere with quantitation of a peak of interest. The development of highly precise sample transfer techniques, including modem autoinjectors, reduces the dependence of the experimentalist on the use of an internal standard to correct for effects of dilution and transfer losses. In many cases, external standardization can be used effectively. The weight percent purity is determined by comparing the area of each peak in a chromatogram with those generated by separately injected pure standards of known concentration. 

Quantitative analysis using FAB is not straightforward, as with all ionisation techniques that use a direct insertion probe. While the goal of the exercise is to determine the bulk concentration of the analyte in the FAB matrix, FAB is instead measuring the concentration of the analyte in the surface of the matrix. The analyte surface concentration is not only a function of bulk analyte concentration, but is also affected by such factors as temperature, pressure, ionic strength, pH, FAB matrix, and sample matrix. With FAB and FTB/LSIMS the sample signal often dies away when the matrix, rather than the sample, is consumed therefore, one cannot be sure that the ion signal obtained represents the entire sample. External standard FAB quantitation methods are of questionable accuracy, and even simple internal standard methods can be trusted only where the analyte is found in a well-controlled sample matrix or is separated from its sample matrix prior to FAB analysis. Therefore, labelled internal standards and isotope dilution methods have become the norm for FAB quantitation. 

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