Calibration internal standards used

Assays based on the use of an internal standard use response factors to compare the sample solution with the calibration solution. In this case a simple one-point calibration is used. The concentration of betamethasone can be ignored since it is the same in Solutions 1 and 3 it should usually be the case that the same concentration of internal standard is present in the calibration and sample solutions. If this is the case then for the assay described above ... 

Analysis Is carried out on a cream stated to contain 2% w/w of both miconazole and hydrocortisone. An ODS column is used with a mobile phase consisting of acetonitrile/ acetate buffer pH 4.0 (70 30) and the eluent is monitored at 220 nm. A narrow range calibration curve, within 20% of the expected concentration of each analyte in the sample extract was prepared for each analyte by plotting the ratio of the areas of the analyte peaks against fixed amounts of the internal standards for both analytes. The internal standards used were econazole and hydrocortisone 21-acetate for miconazole and hydrocortisone, respectively. 

ID MS involves the precise addition of an isotopically labeled form of the analyte to an accurately measured sample of the specimen, e.g., serum. After an appropriate equilibration time, the analyte and its labeled internal standard are isolated from the sample with a suitable extraction and purification step, and an aliquot is introduced, either directly or after (gas) chromatographic separation from remaining interferences, into the mass spectrometer. The latter accurately measures the ratio of analyte to internal standard using the intensities of an equivalent ion in the spectrum of each. From this ratio, the concentration of analyte is calculated by comparison with the ratios of the same ions in standard calibration mixtures. Critical points in this procedure are as follows ... 

The chromatogram given Fig. 1 illustrates the internal standard methodology. Here, methomyl was quantified using benzanilide as the internal standard. Using the calibration curve, the unknown methomyl assay in insecticides is deduced from the area ratio, after the addition to the sample of the same quantity of internal standard as in the calibration steps. 

Analysis. Multi-element calibration standards of 0.0, 5.0 and 10.0 ppm Fe, Ca, B, Cu and A1 are prepared by dissolving 0.5 and 1.0 ml of 1000 ppm multi-element standard control stock in tetralin solvent to 100 ml with the same concentration yttrium internal standard as for samples A and B. Both samples are stirred and nebulised to determine the metal content against a standard calibration curve and corrected with an internal standard using ICP-OES. 

There should be traceability to standards (e.g. national, regional or international standards) used in the calibration. 

Calibrant solutions should be made from high purity materials and dissolved in iso-octane. In general, it is recommended that an internal standard is used which is pure, thermally and photochemically stable and which is not present in the environment (e.g. DCBEs). This should be added to the calibrant solutions and to the samples prior to injection. If the internal standard used is a synthetised CB that is not present in industrial PCB mixtures it may be added at the commencement of the analysis (prior to the clean-up procedure). There was no significant difference in the results obtained using different internal standards, but some interferences of tetrachloronaphtalene (TCN) was detected in the chromatogram of some samples. 

The choice of internal standards was left to each individual laboratory. However, a series of pure dichlorobenzylalkyl eythers (DCBEs) was made available by the Scottish Office, Agriculture and Fisheries Department (United Kingdom) for use with the calibrants and samples. A list of internal standards used by the participating laboratories is given in the certification report [32]. 

A detailed description of the methods used is given in the certification report [33], including the internal standards used, GC conditions, calibration techniques etc. Clean-up procedures were based on well proven techniques to remove the bulk of the mineral oil and other organic compounds which would interfere with the chromatographic determination, e.g. decomposition with concentrated H2SO4, adsorption chromatography, separation on silica gel, or separation with Florisil or potassium silicate. For the separation, each CB was identified and confirmed using at least two capillary columns coated with different stationary phases to compare the relative retention times or... 

Figure 6 Structures of some Pd-PEPPSI complexes used in this study. Rate of formation of 67 by using catalysts 29 ( ), 63 (m), 64 (a.) and PdCI (y control). Conversion was determined by using GC/MS against a calibrated internal standard (undecane)...

Three primary methods are used for calibration external standard calibration, internal standard calibration, and standard addition. Figs. 1-3 show the typical response curves for each method. 

Quantitative analysis using external calibration, internal standard calibration, and the method of standard additions are all possible with SPME. Calibration is discussed in Section 1.5.2 and at greater length in Chapter 2. 

It is important to construct calibration curves by direct injection onto the column of standards of known concentrations (made with the same eluent as used for the tissue extracts) to determine the linearity range of the detector response. The results for such calibration curves are normally expressed as detector current (nA) vs. amount injected rather than peak height (cm). Similar calibration should be performed for any internal standard used. 

Quantification methods can be based on standard additions or external calibration. Internal standards may be used to correct for some losses of material but for many analyses suitable compounds are unavailable. In LC-MS and LC-MS/MS the ideal internal standards are stable isotope analogs of the target compounds as these behave exactly as the native compound used but can be distinguished by their different masses. For LSD-related compounds with slightly different structures such as methysergide have been used, but trideuterated LSD is available commercially. 

Reference values for the acylcarnitines are derived from >500 patients, mostly pediatric (0.2-16 yrs), evaluated for metabolic disorders in the author s laboratory but with no manifest biochemical evidence of disease. Individuals with any markedly abnormal values were discounted. The analytical method used was tandem mass spectrometry with electrospray ionization. Internal standards used were stable isotope-labeled analogs of acetyl, propionyl, butyryl, octanoyl and palmitoyl carnitine. The values for straight-chain C2, C3, C4, C5, C6, C8, CIO, C14, C16 and Cl8 1 species are in pmol/1 and are derived from calibration curves using analytical standards all other values are ratios of the signal for the compound to an appropriate internal standard. All values are mean + 2 std. dev. except where a range is given... 

The internal standard used was 2-(hydroxyethyl)pyridine (I), dissolved in methanol (2 mg/mL) and diluted appropriately. The calibration standard was 4-methyl-5-(2-hydroxyethyl)thiazole (II), which was obtained commercially or by cleavage of thiamine with NaHSOs, as described above. The calibration was done by use of aliquots of the standard solution of compound n, which were mixed with an aliquot of compound 1 and then chromatographed by GC at an appropriate temperature. The difference in the retention times of compounds I and II is quite clear and can vary from less < 1 up to 5 min, depending on the temperatures. The concentration of a given standard obtained by the cleavage of thiamine agreed within 5% with a calibration standard of the same concentration prepared from compound II. 

Wang et al. (47) developed a rapid and sensitive method to determine lovastatin in human plasma with sinvastatin as an internal standard using UHPLC-MS/MS. The analysis was carried out on an ACQUITY UPLC BEH Cig column. The calibration curve of this method was found to be linear over the concentration range of 0.025 to 50 ng/mL. The lower limit of quantification for lovastatin was 0.025 ng/mL. The interday and intraday precision (relative standard deviation) were less than 11%, and the accuracy (relative error) was within 6.0%. The analysis time was shorter than 1.7 min per sample, which met a high-throughput determination of biological samples. 

With a microsyringe, add 50 pL of internal standard to a 100-mL volumetric flask about three-fourths full of the calibration mixture. Mix well. Add calibration mixture to mark and again mix well. If n-heptane is used as the internal standard, using a density of 0.684 for n-heptane and 0.906 for styrene, this solution will contain 0.0377 weight % n-heptane. 

After the analyses of the calibration standards are complete, integrate the peak area of each calibration component and internal standards using the reconstructed ion chromatogram (RIC) of the characteristic calibration ion listed in Table 2. Obtain the area under the extracted ion at the retention time of the expected aromatic component (or internal standard). 

After the compounds in Table 1 have been properly identified, measure the areas of each peak at the specified primary quantitation mass and that of the internal standards using the same procedure used for the calibration solutions. Using the sample RIC plots in Fig. 2, follow the steps below for quantitation. 

Flow measurements using tracers are performed in all piping systems carrying oil, gas or water including separators, compressors, injector systems, and flares. Calibration of elsewhere difficult accessible flow meters is regularly performed by the tracer methods, which are based on international standards. Tracer flow measurements are also well suited for special purposes... 

Analytical chemists make a distinction between calibration and standardization. Calibration ensures that the equipment or instrument used to measure the signal is operating correctly by using a standard known to produce an exact signal. Balances, for example, are calibrated using a standard weight whose mass can be traced to the internationally accepted platinum-iridium prototype kilogram. 

Using the Regression Equation Once the regression equation is known, we can use it to determine the concentration of analyte in a sample. When using a normal calibration curve with external standards or an internal standards calibration curve, we measure an average signal for our sample, Yx, and use it to calculate the value of X... 

What is the %w/w Pb in a sample of brass that gives an emission intensity of 9.25 X lO" The analysis for Ni uses an internal standard. Results for a typical calibration are shown in the following table. ... 

As this example clearly shows, the variation in individual peak areas between injections is substantial. The use of an internal standard, however, corrects for these variations, providing a means for accurate and precise calibration. 

Continuing calibration for a Series Method is performed using calibration check compounds. Surrogate compounds are added to the matrix before sample preparation to evaluate recovery levels. To check GC retention times, internal standards are added to a sample after its preparation for analysis. 

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. 

Calibration Cahbration entails the adjustment of a measurement device so that the value from the measurement device agrees with the value from a standard. The International Standards Organization (ISO) has developed a number of standards specifically directed to cahbration of measurement devices. Furthermore, compliance with the ISO 9000 standards requires that the working standard used to cahbrate a measurement device must be traceable to an internationally recognized standard such as those maintained by the National Institute of Standards and Technology (NIST). 

HR-ICP-MS EEEMENT-2 (Pinnigan MAT, Germany) equipped with a standard introduction system (quartz water-cooled spray chamber, concentric nebulizer, torch with 1.5 mm i.d. injector and nickel cones) was used for measurements. The following operating conditions were used RP power 1150 W, coolant gas flow rate 16 1 min k auxiliary gas flow rate 0.85 1 min nebulizer gas flow rate 1.2 1 min k Sample uptake rate was 0.8-1 ml min k Measurements were performed with low and middle resolutions. Rh was used as an internal standard. Por calibration working standard solutions were prepared by diluting the multielemental stock solutions CPMS (SPEX, USA) with water to concentration range from 5 ng to 5 p.g I k... 

Calibration curves must be made using a series of standards to relate emission intensities to the concentration of each element of interest. Because ICP-OES is relatively insensitive to matrix effects, pure solutions containing the element of interest often are used for calibration. For thin films the amount of sample ablated by spark discharges or laser sources is often a strong function of the sample s composition. Therefore, either standards with a composition similar to the sample s must be used or an internal standard (a known concentration of one element) is needed. 

Because of the complex nature of the discharge conditions, GD-OES is a comparative analytical method and standard reference materials must be used to establish a unique relationship between the measured line intensities and the elemental concentration. In quantitative bulk analysis, which has been developed to very high standards, calibration is performed with a set of calibration samples of composition similar to the unknown samples. Normally, a major element is used as reference and the internal standard method is applied. This approach is not generally applicable in depth-profile analysis, because the different layers encountered in a depth profile of ten comprise widely different types of material which means that a common reference element is not available. 

The procedure comprises the addition of a constant amount of internal standard to a fixed volume of several synthetic mixtures which contain varying known amounts of the component to be determined. The resulting mixtures are chromatographed and a calibration curve is constructed of the percentage of component in the mixtures against the ratio of component peak area/standard peak area. The analysis of the unknown mixture is carried out by addition of the same amount of internal standard to the specified volume of the mixture from the observed ratio of peak areas the solute concentration is read off using the calibration curve. 

To use KBr discs for quantitative measurements it is best to employ an internal standard procedure in which a substance possessing a prominent isolated infrared absorption band is mixed with the potassium bromide. The substance most commonly used is potassium thiocyanate, KSCN, which is intimately mixed and ground to give a uniform concentration, usually 0.1-0.2 per cent, in the potassium bromide. A KBr/KSCN disc will give a characteristic absorption band at 2125 cm 1. Before quantitative measurements can be carried out it is necessary to prepare a calibration curve from a series of standards made using different amounts of the pure organic compound with the KBr/KSCN. A practical application of this is given in Section 19.9. 

Because an increase in resolution causes a decrease in sensitivity, it is best to operate at the lowest resolution commensurate with good results. Some instrument data systems will allow calibration with an external reference material such as perfluorokerosene and then use of a secondary reference material for the internal mass reference. Tetraiodothiophene, vaporized using the solids probe inlet, is recommended as the secondary reference. The accurate masses are 79.9721, 127.9045, 162.9045, 206.8765, 253.8090, 293.7950, 333.7810, 460.6855, and 587.5900. For a higher mass standard, use hexaiodobenzene. Because the mass defect for these internal reference ions are so large, a resolution of 2000 is ample to separate these ions from almost any sample ions encountered in GC/MS. 

Because the two analytical lines differ in wavelength, an internal standard can never compensate absorption and enhancement effects completely. If Cases II and IV %re avoided in selecting ah internal standard, the use of such a standard will usually prove satisfactory. Special cases may require special calibration curves run with the disturbing elements present. 

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