Peak area precision ratio

Peak area precision is controlled by the sampling volume precision of the autosampler. In some specific instances, precision can be limited by the signal-to-noise ratio or the sampling rate as described by the equation below. 

Theoretical considerations shown in the above equation also indicate that peak area precision is inversely proportional to the peak sig-nal/noise ratio, and to the number of sampling points across the peak width. For very noisy peaks, the peak area precision is limited by random noise fluctuations (Figure 6). Figure 7 shows that the precision of the peak area degrades rapidly when the signal-to-noise ratio is less than 100. Statistical considerations also stipulate a minimum data sampling... 

FIGURE 6 Peak area precision study to delineate the effect of peak signal-to-noise ratio. 

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... 

For unbranched alkyl groups based on ratio of 30 ppm peak area to average of 14, 23 and 32 peak areas. Precision +10%... 

Figure 5.7. Diagram illustrating typical autosampler precision vs. peak signal/noise ratio (keeping injection volume constant at 10 pL). Note that the peak area precision worsens (increasing RSD) because precision was limited by the statistical variation of integration of noise peak when the signal-to-noise ratios (S/N) are less than 100. Reprinted with permission from reference 12. See the same reference for additional experimental details.

The relative instrumental sensitivity factors for cobalt and nitrogen were determined by measuring core level (Co 2p and N Is) XPS spectra for a series of pure cobalt amine complexes of established stoichiometry. To evaluate the core level photopeak intensities, peak areas, including shake-up satellite intensity were used. The precision for the measurements of the nitrogen to cobalt atomic ratio is 10% while the accuracy is approximately 15%. Additional details of the XPS measurements are contained in the literature (24,25). 

A highly versatile method for enantiomer analysis is based on the direct separation of enantiomeric mixtures on nonraceinic chiral stationary phases by gas chromatography (GC)6 123-12s. When a linearly responding achiral detection system is employed, comparison of the relative peak areas provides a precise measurement of the enantiomeric ratio from which the enantiomeric purity ee can be calculated. The enantiomeric ratio measured is independent of the enantiomeric purity of the chiral stationary phase. A low enantiomeric purity of the resolving agent, however, results in small separation factors a, while a racemic auxiliary will obviously not be able to distinguish enantiomers. 

To establish a sensitive and specific liquid chromatography-mass spectrometry (time-of-flight) [LC-MS (TOF)] method for the determination of donepezil in human plasma after an oral administration of 5 mg donepezil hydrochloride tablet [29]. Alkalized plasma was extracted with isopropa-nol-n-hexane (3 97) and loratadine was used as internal standard (IS). Solutes were separated on a Cis column with a mobile phase of metha-nokacetate buffer (pH 4.0) (80 20). Detection was performed on a TOF mass spectrometry equipped with an electrospray ionization interface and operated in positive-ionization mode. Donepezil quantitation was realized by computing the peak area ratio (donepezil-loratadine) (donepezil m/z 380 [M + H]+ and loratadine m/z 383[M + H]+) and comparing them with calibration curve (r = 0.9998). The linear calibration curve was obtained in the concentration range of 0.1-15 jUg/1. The detection limit of donepezil was 0.1 /zg/1. The average recovery was more than 90%. The intra- and inter-run precision was measured to be below 15% of RSD... 

To establish chiral separation method for donepezil hydrochloride enantiomers by capillary electrophoresis (CE) and to determine the two enantiomers in plasma [39], alkalized plasma was extracted by isopropa-nol-n-hexane (3 97) and L-butefeina was used as the IS. Enantioresolution was achieved using 2.5% sulfated-beta-cyclodextrin as chiral selector in 25 mmol/1 triethylammonium phosphate solution (pH 2.5) on the uncoated fused-silica capillary column (70 cm x 50 fim i.d.). The feasibility of the method to be used as quantitation of donepezil HC1 enantiomers in rabbit plasma was also investigated. Donepezil HC1 enantiomers were separated at a baseline level under the above condition. The linearity of the response was evaluated in the concentration range from 0.1 to 5 mg/1. The linear regression analysis obtained by plotting the peak area ratio (A(s)/A(i)) of the analyte to the IS versus the concentration (C) showed excellent correlation coefficient The low limit of detection was 0.05 mg/1. The inter- and intra-day precisions (RSD) were all less than 20%. Compared with chiral stationary phase by HPLC, the CE method is simple, reliable, inexpensive, and suitable for studying the stereoseletive pharmacokinetics in rabbit. 

Precision. The precision of multiple injections was evaluated using triplicate injections (at each nominal volume) of the same hGH/desamido hGH mixture that was used for the linearity study (Table I). The percent of desamido hGH in the mixture was calculated as the ratio of its peak area to the total peak area raw peak areas must be corrected for differences in migration velocity as previously described (15). 

The observed precision is comparable to the values we previously reported for biosynthetic human insulin (16). It also is similar to independent results obtained using a totally automated system (2.9% RSD) and much better than that reported for manual injection (11.8% RSD), both using a hydrodynamic injection technique (21). Finally, the observed precision for the percent desamido, which is really an area ratio similar to what would be obtained by comparison to an internal standard, is excellent for the 10-nL or larger injections. Although the data are insufficient to make a definitive conclusion, it suggests that the observed error is comparable to that obtained from many chromatographic techniques. It also suggests that one of the predominant sources of error is imprecision in the injection volume. The error in injection volume was recently characterized (19). They also reported approximately 1-3% RSD in peak areas for vacuum injection of various compounds. 

For example, little is known about the isotopic composition of formaldehyde in the atmosphere. Formaldehyde is a chemical intermediate in hydrocarbon oxidation. The carbon (8 C) and hydrogen (8D) isotopic composition of atmospheric formaldehyde is analyzed using continuous flow gas chromatography isotope ratio mass spectrometry." Isotope ratios were measured using GC-IRMS (Finnigan MAT 253 stable isotope ratio mass spectrometer, single-sector field with electron impact ion source and multiple ion collection) with a precision of 1.1 and 50%(lo ) for 8 C and 8D, respectively. The accuracy of the online continuous flow isotope technique was verified by calibrating three aliquots of the gas phase standard via the offline dual inlet IRMS technique. The concentration of formaldehyde in ambient air was determined on IRMS major ion peak areas (i.e., mass 44 for 8 C and mass 2 for 8D)." ... 

Certain evaluation methods were examined based on the quantification processes. These can be divided into three groups, employing only the values of peak normalization (PN). The effect of the use of IS and certain evaluation methods, such as correction of peak area (normalization), was calculated by dividing the related peak area into on which the precision was examined. These can be divided into three groups a) employing only the area values of PN (PN no IS), b) computing the ratio values (IS no PN), and c) using the area values of peak normalized IS and ENX (IS and PN). The precision of the peak areas was calculated as shown in Table 1. 

The internal standard (I.S.) method is a more accurate method. The I.S. technique can compensate for both instrumental and sample preparation errors and variations (e.g., dilution and extraction) [45, 46], Sample pretreatment steps such as extraction often result in sample losses, and a proper I.S. standard should be chosen to mimic the variations in these steps. Thus, both the accuracy and precision of quantitative data increase if an I.S. is included in the procedure. The I.S. should be similar but not identical to the analyte, and the two should be well resolved in the chromatographic step. The standard curves are obtained from standards of blank samples spiked with different known concentrations of the analyte of interest and addition of an I.S. at constant concentration. Also to the unknown samples the same constant concentration of the I.S. is added. The standard samples are processed in parallel with the unknown samples. In the calibration curve, the ratios of analyte to I.S. peak area (or height) are plotted versus the concentration of the analyte. A proper I.S. in a bioanalytical chromatographic method should fulfill the following requirements [44] ... 

The RATIO method table (Table I) includes provision for specifying upper and lower limits of integration for both primary and reference bands with the peak area evaluation procedure. The practical limits of the integration can be determined empirically by evaluating a set of spectra stored on microfloppy disks with varying limits set in the appropriate locations in the method table. Optimum limits can be determined from the calibration plots and related error parameters. The calibration plots shown in Figures 4 and 5 indicate that both evaluation procedures, peak height and peak area provide essentially the same level of precision for the linear least squares fit of the data. The error index and correlation coefficients listed on each table are both indicators of the relative scatter in the data from the least squares fit line. The correlation coefficient is calculated as traditionally defined in statistics. 

Fig. 3 The relationship between signal-to-noise ratio and precision of reported peak area...

The highest precision for quantitative GC is obtained using internal standards because the uncertainties introduced by sample injection, flow rate, and variations in column conditions are minimized. In this procedure, a carefully measured quantity of an internal standard is introduced into each standard and sample (see Section 8C-3), and the ratio of analyte peak area (or height) to internal-standard peak area (or height) is used as the analytical parameter (see Example 31 -1). For this method to be successful, it is necessary that the internal standard peak be well separated from the peaks of all other components in the sample. It must appear close to the analyte peak, however. Of course, the internal standard should be absent in the sample to be analyzed. With a suitable internal standard, precisions of 0.5% to 1 % relative are reported. 

The area of each peak is obtained from a series of replicate (5+) injections of a mixture containing equal (or known) amounts of all the components. Acceptable precision is essential to obtain satisfactory data. One component is chosen as the reference and the relative responses of the other components are determined by dividing the peak areas by that of the reference component. The detector response factors (Z>rf) may then be used to calculate corrected peak areas (.<4correct) for other analyses involving these components and hence their percentage ratios in the mixture may be... 

Thus, by substitution in this equation for the peak areas from the chromatogram, the relative response factors, derived from the calibration analysis, and the concentration of the internal standard added to the sample, the concentration of the components in the sample can be calculated. Since this method involves ratios of peak areas rather than absolute values, it should be noted that the precision of analysis is not dependent on the injection of an accurately known amount of sample. However, the accuracy does depend on the accurate measurement of peak area. Assay and quantitation by the internal standard is often the preferred method as it takes account of variable compound response and removes potential errors due to variation in sample injection. The 80 and 200 mg% (mg per 100 ml) standard solutions are used to confirm the linearity of response over this concentration range. 

For a given spectrometer, a set t>f relative values of. V can be developed for the elements of interest. Note that the ratio US is directly proportional to the concentration n on the surface. The quantity / is usually taken as the peak area, although peak heights are also used. Often, for quantitative work, internal standards are used. Relative precisions of about 5% are typical. For the analysis of solids and liquids, it is necessary to assume that ihc surface composition of the sample is the same as its bulk composition, l- or many applications this assumption can lead to signiticani errors. Detection of an element hy XPS requires that it be pres-cnl at a level of at least 0.1 %. Quantitative analysis can usually he performed if 5 m of the element is present,... 

The crystal phases in the glass-ceramics were determined by XRD analysis. All instruments were precisely and identically set to ensure a high precision to obtain the integral peak area. The microstructure of the fresh fractured cross section of the glass-ceramics was observed by SEM. The thermal expansion coefficient (TEC) was calculated from room temperature to 500 °C at a heating rate of 5°C/min in the dilatometry analyser (NETZSCH, DIL402PC). The flexural strength was determined in a 3-point bend test at a constant strain ratio of 0.5mm/min. 

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