Internal Standard Quantification

Perhaps no reagent is more important than the stable-isotope-labeled internal standard in any clinical assay utilizing mass spectrometry for quantification. Internal standards are important in many aspects of the analysis and are somewhat different than standards utilized in other clinical, non-mass-spectrometric assays. The ideal internal standard is an enriched isotopic version of the analyte being measure. For example, in the case of phenylalanine, a standard available may contain six 13C molecules rather than 12C in the aromatic ring. This has the net effect of shifting the mass of phenylalanine by six units while also maintaining nearly identical chemical... 

Chromatographic peak areas are calculated automatically by the data system by reference to the response obtained from certain specified, compound-dependent ions. From the peak areas of the target compounds, quantification is achieved by comparison with the internal standards, which are present in known concentration. The laboratory responsible for the analysis must report the target compounds and all tentatively identified (nontarget) compounds. Standard EPA forms must be completed and submitted. A laboratory is said to be in compliance when it has satisfied all aspects of its CLP contract. 

A further critical point are the intensities correlated to spectra of the pure elements. Calculated and experimentally determined values can diverge considerably, and the best data sets for 7 measured on pure reference samples still show a scatter of up to 10%. The use of an internal standard or a simultaneously measured external standard seems to be the most successful way to reducing the inaccuracy below 10%. (Eor a more detailed discussion of background subtraction and quantification see, e.g., Seah [2.9].)... 

In Raman spectroscopy the intensity of scattered radiation depends not only on the polarizability and concentration of the analyte molecules, but also on the optical properties of the sample and the adjustment of the instrument. Absolute Raman intensities are not, therefore, inherently a very accurate measure of concentration. These intensities are, of course, useful for quantification under well-defined experimental conditions and for well characterized samples otherwise relative intensities should be used instead. Raman bands of the major component, the solvent, or another component of known concentration can be used as internal standards. For isotropic phases, intensity ratios of Raman bands of the analyte and the reference compound depend linearly on the concentration ratio over a wide concentration range and are, therefore, very well-suited for quantification. Changes of temperature and the refractive index of the sample can, however, influence Raman intensities, and the band positions can be shifted by different solvation at higher concentrations or... 

Quantification External standard External and internal standard... 

Erbs, M., Hoerger, C.C., Hartmaim, N. and Bucheli, T.D. (2007). Quantification of six phytoestrogens at the nanogram per liter level in aqueous environmental samples using C-13(3)-labeled internal standards. Journal of Agricultural and Food Chemistry 55, 8339-8345. 

For HPLC, it is necessary to establish the relationship between the detector signal, of which the most used is peak area, and the concentrations of the pigments. Calibration curves for external quantification should be constructed for each carotenoid. Internal calibration is also used for quantification of carotenoids, using as internal standards all-trfln5 -p-apo-8-carotenal, ° Sudan 1, and decapreno-P-carotene. ... 

In both the GC/MS and the LC-FL analyses selected perdeuterated PAHs were added to the samples prior to extraction for use as internal standards for quantification. 

Alternatively, LC is used for the separation and quantification of PAHs using both UV and fluorescence detection. The analytes are identified based on their relative retention times and UV and/or fluorescence emission spectra. For UV detection an efficient cleanup is a prerequisite since this detection method is not very selective (almost universal for PAHs), and hence it also responds to many coeluting compounds. Due to the high specificity of fluorescence detection for most PAHs, this LC detection method is less susceptible to potential interferences. As in the case of GC the apphcation of internal standard(s) is mandatory since solvents have to be evaporated during the cleanup, which may result in partial losses of some of the more volatile analytes. 

Quantification is performed by comparing the sample response with an average response factor determined from the standard analyses. Internal standards are used... 

SFE and SFC require a high-purity feedstock of liquid C02 (electron capture impurities below 100 ppt, and mass responsiveness impurities below lOppb). Impurities can be detrimental to the use of SFE in trace analysis. Hinz and Wenclawiak [323] have investigated SFE/SFC grade C02 by means of GC with FID, ECD and MS detection. Quantification of the impurities, using FID or ECD, was achieved introducing an internal standard into the C02 flow. 

PP/Uvasil 299 fibres were analysed by 1II NMR after dissolution (toluene)/precipitation (methanol) and PP/Uvasil 299 pellets after reflux extraction with precipitation (methanol) of the oligomers [619]. The dried extracts were redissolved in CDCI3, containing the DMP internal standard. The method allows quantification of the high-MW HALS at very low levels (0.044 0.001 %). 

Headpace GC with electron-capture or flame-ionisation detection. Quantification by standard addition using 1-chloropropane internal standard Headspace GC of the polymer dissolved in N,N-dimethylacetamide with detection by electron-capture or flame-ionisation detection. Headpspace GC of the plastic if not soluble... 

Matrix effects were evaluated by Gago-Ferrero et al. [23]. Both signal suppression and signal enhancement were observed. The extent of these effects was found to be fairly dependent on the UV filter. Thus, quantification should be performed by standard addition or internal standard calibration. Since standard addition is a high time-consuming procedure, internal standard calibration with appropriate isotopi-cally labeled compounds is the best option. 

Quantification is usually achieved by a standard addition method, use of labeled internal standards, and/or external calibration curves. In order to allow for matrix interferences the most reliable method for a correct quantitation of the analytes is the isotope dilution method, which takes into account intrinsic matrix responses, using a deuterated internal standard or carbon-13-labeled internal standard with the same chemistry as the pesticide being analyzed (i.e., d-5 atrazine for atrazine analysis). Quality analytical parameters are usually achieved by participation in interlaboratory exercises and/or the analysis of certified reference materials [21]. 

In each of the profiles presented, several internal standards (20) are present and used for quantification. Increases or decreases in the relative concentration of these metabolites indicate disease and as such a positive screen. The complexity of the assay actually resides in the interpretation, which is illustrated in the flow chart shown in Fig. 14.3. 

The power of the system to overcome the problems associated with coeluting compounds is demonstrated in conjunction with the use of deuterated (or13C-labelled compounds) as internal standards. Such techniques could not be used in conventional gas chromatography as the deuterated compounds often co-elute, making quantification difficult if not impossible. With the ion-trap detector, however, it is easily possible to differentiate between the ions arising from the different compounds and the intensities of these ions could then be used for quantification of the compounds involved. The application of such techniques can be shown by... 

Hankin et al. [46] have used spacially residued time of flight mass spectrometry for quantification studies on polyaromatic hydrocarbons. Deuterated polyaromatic hydrocarbons were used as internal standards, chrysene-d being adopted in the final method. Theoretical values were obtained bj this procedure on standard reference soils. 

For the quantification of alkylphenol ethoxylates (APEOs), several related compounds have been used as internal standard, e.g. for recovery determination, decylphenol monoethoxylate [20]. Octylphenol nonaethoxylate (t-0PE09) was synthesised by reacting octylphenol (OP) with 1,2-6j.s(2-chloroethoxy)ethane to give the chloro derivative, followed by reaction with the sodium salt of hexaethylene glycol [21,22], Another approach used the synthetic standard l-(4,-methoxyphenyl)-hexan-l-ol as a surrogate to monitor the efficiency of the extraction for... 

For the quantification of ethoxylated non-ionic surfactants in LC-MS, the effect of changing response factors with changes in EO chain length requires particular attention. The urgent need for proper internal standards has recently been addressed with the 13C-labelled NP and NPEO compounds that have become available. These will help improve the quality of LC-MS quantification of ethoxylated non-ionic surfactants significantly. 

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