Linearity assays, calibration curves

The assay was carried out using a Varian gas chromatograph (model 5000 LC) under the following experimental condition. The oven injector and flame ionization detector temperatures were 125°C and 225°C respectively. A Porapak column was used, the eluent was N2 at a flow rate of 30 ml/min and the injected volume 2 pi. Various concentrations of purified methylene chloride in purified methanol were injected (both solvents were distilled to discard any impurity which might interfere with the sensitive assay). Calibration curves were linear in the range 50-500 ppm (the limit of detection was 10 ppm). Methylene chloride detection in the microspheres was performed by dissolving various amounts (20-200 mg) of microspheres in 220 ml of purified methanol prior to the injection. 

Following a 10-fold dilution of the sample with 0.6 M perchloric acid, the product of this reaction absorbs maximally at 420 nm. The original assay, developed by Kissane and Robins,12 uses DABA dihydrochloride for depurination (no perchloric acid was used) and employs fluorescence measurements at 520 nm after excitation at 420 nm. More recent work shows that the assay with perchloric acid can be conducted with 10-pL DNA sample volumes and provides a linear fluorescence calibration curve over the 10-500-ng total DNA range (1-50 pg/mL).13 The DABA method has since been modified for colorimetric measurements at 420 nm, and has a detection limit of 25 pg.14 The DABA reaction is specific for DNA, but... 

The concentration of analyte in the unknown sample is extrapolated from the calibration curve. To obtain an accurate and precise quantitative value, the optical density (OD) for the sample solutions must fall on the linear portion of the calibration curve. If the sample OD is too high, the sample solution must be diluted until the OD falls within the quantitative range of the assay. The concentration of the analyte in the original sample is calculated by correcting for any dilution factor that was introduced in preparing the sample for application to the microplate. 

Because of the possibility that the herbicide alachlor could adulterate food if either poultry or livestock consumed contaminated materials, Lehotay and Miller evaluated three commercial immunoassays in milk and urine samples from a cow dosed with alachlor. They found that milk samples needed to be diluted with appropriate solvents (1 2, v/v) to eliminate the matrix effect. One assay kit (selected based on cost) was also evaluated for use with eggs and liver samples from chickens. Egg and liver samples were blended with acetonitrile, filtered, and diluted with water. Linear calibration curves prepared from fortified egg and liver samples were identical... 

For those scientists who had to perform quantitation, the linearity of the A/D was also critical. Linearity is the condition in which the detector s response is directly proportional to the concentration or amount of a component over a specified range of component concentrations or amounts. It is imperative that the A/D not add any additional error or variability to the performance of the detector. The resulting calibration curve now becomes dependent on the combined linearity of the detector and the /VD. Accurate quantitation requires that the system is linear over the range of actual sample concentrations or amounts. Many pharmaceutical assays, like degradation and stability studies, require that the system be able to identify and quantitate very disparate levels of peaks. In many cases, this translates into a 3 to 4 order of magnitude difference between the main active component and the impurities that need to be quantitated. 

The rules for level I (screening) assays are shown in Table 13.1. An example of the type of samples where a level I assay could be used is the CARRS samples [85] that can be used for screening NCEs using a rat PK model [vide supra). The concept behind this assay is that it should use a small number of standards and a simple linear extrapolation. For level II assays (see Table 13.2) that might be used for discovery PK studies in preclinical species, a complete standard curve is required. In this case a complete standard curve is defined as 10-15 standards in duplicate assayed with at least five standards used in the final calibration curve. Neither level I nor level II assays require the use of quality control (QC) standards. When a compound is in the lead qualification stage, then a level III assay would be required. As shown in Table 13.3, the main distinction for level III assays is that they are required to include at least six QC standards. As described in Tables 13.1-13.3, these rules show the requirements for how an assay should be set up before the samples are assayed and then these rules describe the acceptance criteria for the assays after they have been performed. 

The detection limit for individual conjugated BA is 1 ng and the assay is linear over the range of analysis. In Table 5.4.13 are shown equations of calibration curves for all analysed BAs. 

In some instances, calibration test data may need to be subjected to some kind of mathematical transformation, prior to the regression analysis, in order to obtain linear calibration plots. In some cases, however, such as in immunochemical assays, linearity cannot be demonstrated even after any transformation. The use of nonlinear calibration curves for analysis has been discussed (27). 

Another criterion for linearity is that the y-intercept of the calibration curve (after the response of the blank has been subtracted from each standard) should be close to 0. An acceptable degree of closeness to 0 might be 2% of the response for the target value of analyte. For the assay of impurities, which are present at concentrations lower than that of the major component, an acceptable value of R2 might be 0.98 for the range 0.1 to 2 wt% and the y-intercept should be 10% of the response for the 2 wt% standard. 

The cholesterol assay as used in this experiment is linear up to 500 mg/100 mL serum. A calibration curve is not essential and a single standard can be used. Use Equation El 1.8 to calculate the cholesterol concentration. If the serum sample was turbid, you should have determined A5W of serum in saline water, Ac. If so, subtract this from A5U)(x)... 

The reducing sugar-based assays are predicated on the correct measurement of enzymegenerated product. A typical calibration curve for the BCA assay, using galacturonic acid as the calibration standard, is presented in Figure Cl.2.5. Note that the assay is linear for the... 

Fig. 18.6. Calibration curve obtained by the immuno-assay procedure in the detection of anti-E2 using the ITO-Poly (pyrrole-benzophenone) coated optical fibers. The linear range of the calibration curve was obtained for titer 1 64,000 and lower. The curve was fitted according to the equation y = A+B(x), where x is the human sera (anti-E2 antibodies) dilution value and y is the chemiluminescence response. The obtained correlation coefficient was R2 = 0.988.

A selective, sensitive, and rapid hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry was developed for the determination of donepezil in human plasma [32], Donepezil was twice extracted from human plasma using methyl-ferf-butyl ether at basic pH. The analytes were separated on an Atlantis HILIC Silica column with the mobile phase of acetonitrile ammonium formate (50 mM, pH 4.0) (85 15, v/v) and detected by tandem mass spectrometry in the selective reaction monitoring mode. The calibration curve was linear (r = 0.9994) over the concentration range of 0.10-50.0 ng/ ml and the lower limit of quantification was 0.1 ng/ml using 200 /d plasma sample. The CV and relative error for intra- and inter-assay at four quality control levels were 2.7% to 10.5% and —10.0% to 0.0%, respectively. There was no matrix effect for donepezil and cisapride. The present method was successfully applied to the pharmacokinetic study of donepezil after oral dose of donepezil hydrochloride (10 mg tablet) to male healthy volunteers. 

Zarghi et al. [76] developed an HPLC method, using a monolithic column, for quantification of omeprazole in plasma. The method is specific and sensitive with a quantification limit of 10 ng/ml. Sample preparation involves simple, one-step extraction procedure, and analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 x 4.6 mm) column with an isocratic mobile phase consisting of 0.01 mol/1 disodium hydrogen phosphate buffer-acetonitrile (73 27) adjusted to pH 7.1. The wavelength was set at 302 nm. The calibration curve was linear over the concentration range 20-1500 ng/ml. The coefficients of variation for intra- and interday assay were found to be less than 7%. 

Sultana et al. [88] developed a reversed-phase HPLC method for the simultaneous determination of omeprazole in Risek capsules. Omeprazole and the internal standard, diazepam, were separated by Shim-pack CLC-ODS (0.4 x 25 cm, 5 m) column. The mobile phase was methanol-water (80 20), pumped isocratically at ambient temperature. Analysis was run at a flow-rate of 1 ml/min at a detection wavelength of 302 nm. The method was specific and sensitive with a detection limit of 3.5 ng/ml at a signal-to-noise ratio of 4 1. The limit of quantification was set at 6.25 ng/ml. The calibration curve was linear over a concentration range of 6.25—1280 ng/ml. Precision and accuracy, demonstrated by within-day, between-day assay, and interoperator assays were lower than 10%. 

Lin and Wu [137] established a simple capillary zone electrophoresis method for the simultaneous analysis of omeprazole and lansoprazole. Untreated fused-silica capillary was operated using a phosphate buffer (50 mM, pH 9) under 20 kV and detection at 200 nm. Baseline separation was attained within 6 min. In the method validation, calibration curves were linear over a concentration range of 5-100 /iM, with correlation coefficients 0.9990. RSD and relative error were all less than 5% for the intra- and interday analysis, and all recoveries were greater than 95%. The limits of detection for omeprazole and lansoprazole were 2 fiM (S/N = 3, hydroxynamic injection 5 s). The method was applied to determine the quality of commercial capsules. Assay result fell within 94—106%. 

Assay calibration involves the use of human serum samples fortified with CP-80,794 at 11 concentrations (six replicates per concentration) ranging from 0.05 to lOng/mL. In this particular case, due to a narrow linear dynamic range, two standard curves, ranging from 0.05 to lOng/mL, are constructed to provide the best accuracy. Serum blanks and an 11-point standard curve (two samples per concentration) are analyzed with each set of unknown samples. 

EDTA has been assayed in foods through its reaction with Fe(III) [28]. Excess Fe(III) is removed by chloroform and N-benzoyl-iV-phenylhydroxylamine. The calibration graph was linear over the range of 0.5 - 40 pg/mL [28], A similar method for EDTA in food involving reaction with Cu(II) in Tris-HCl buffer at pH 8.5 has been reported [29]. The excess Cu(II) was been determined at 477 nm using 2,9-dimethyl-4,7-diphenyl-l,9-phenanthroline. This calibration curve was found to be linear over the range of 4-12 pM EDTA. 

A colorimetric method based on the inhibitory effect of EDTA on the Mn(II) catalyzed oxidation of malachite green by periodate was reported [32]. An alternative method based on using Fe(III) instead of Mn(II) was proposed [33]. The reduction of the absorbance of Bi(III) bromo-pyrogallol red tenside ternary complex upon the addition of EDTA has been exploited for its determination. Calibration curves obtained at 650 nm were linear over the range of 0.2-6 pg/mL of EDTA [34]. EDTA in ophthalmic solutions could be assayed by spectrophotometric titration using Mg(II) as the titrant and Arsenazo I as the indicator. The working range was 0.05-2 pg/mL [35]. 

A typical chromatogram obtained using this method is shown in Figure 12, and the retention times (Rt) and the relative retention time (Rrt) for pantoprazole sodium and some of its related compounds are shown in Table 6. The calibration curve for the assay determination, obtained over a concentration range of228-670 pg/mL, was found to be linear with a correlation coefficient of0.999. The recovery and relative standard deviation for various assays were 97.3-101.5 and 1.1, respectively. A calibration curve was also developed for pantoprazole sodium related compounds, covering a concentration range of 1 to 3 pg/mL, and which was found to be linear with a correlation coefficient of more than 0.999. The limits of detection and limits of quantitation were calculated as 0.15 pg/mL and 0.49 pg/mL, respectively. 

Determine the concentration of host-cell proteins from the calibration curve. If the samples do not show linear dilution then the capture antibody was not in excess and the assay results are invalid. 

Before any column is used for assay purposes, a calibration curve should be constructed to verify that the instrumental response is linear over the required range and that the curve passes through the origin. If the compound to be analyzed is adsorbed within the system, the calibration curve will intersect the abscissa at a nonzero value. This may result in error, particularly for compounds at low concentrations determined by a procedure based on a single reference point. At high concentrations, the liquid phase may be overloaded, leading to loss of peak height and symmetry. 

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