Using Internal Standardization

The classic way to compensate for a physical interference is to use internal standardization (IS). With this method of correction, a small group of elements (usually at the ppb level) are spiked into the samples, calibration standards, and blank to correct for any variations in the response of the elanents caused by the matrix. As the intensities of the internal standards change, the elanent responses are updated every time a sample is analyzed. The following criteria are typically used for selecting an internal standard  

The sample matrix or analyte elanents do not spectrally interfere with it. 

It does not spectrally interfere with the analyte masses. 

It should not be an eluent that is considered an environmental contaminant. It is usually grouped with analyte elanents of a similar mass range. For example, a low-mass internal standard is grouped with the low-mass analyte elements, and so on, up the mass range. 

It should be of a similar ionization potential to the group of analyte elements, so it behaves in a similar manner in the plasma. 

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Precision For samples and standards in which the concentration of analyte exceeds the detection limit by at least a factor of 50, the relative standard deviation for both flame and plasma emission is about 1-5%. Perhaps the most important factor affecting precision is the stability of the flame s or plasma s temperature. For example, in a 2500 K flame a temperature fluctuation of +2.5 K gives a relative standard deviation of 1% in emission intensity. Significant improvements in precision may be realized when using internal standards. 

Rice, G. W. Determination of Impurities in Whiskey Using Internal Standard Techniques, /. Chem. Educ. 1987, 64, 1055-1056. 

The proposed PAS-FTIR procedure allows the determination of 30 samples h does not require any sample pre-treatment and avoids waste generation. So, it can conclude that the PAS-FTIR methodology is a reliable alternative to long and tedious classical method of analysis of Mancozeb and improves the sample frequency achieved by the FTIR procedure based on the measurement of KBr disks using internal standard. 

Acetone and Ethanol (Grade AA only). Determine the acetone and ethanol content by the elution method of gas chromatography using internal standards... 

Table 3 Product Yield (czs-1,2 /1,4 of Scheme 5) as Weight Percent Using Internal Standard Method...

Compound Method of sample preparation Herman and Weidinger s method Using internal standard IR Solution calorimetry Stability at 50°C and 31% RH Reference... 

Holzbecker and Ryan [825] determined these elements in seawater by neutron activation analysis after coprecipitation with lead phosphate. Lead phosphate gives no intense activities on irradiation, so it is a suitable matrix for trace metal determinations by neutron activation analysis. Precipitation of lead phosphate also brings down quantitatively the insoluble phosphates of silver (I), cadmium (II), chromium (III), copper (II), manganese (II), thorium (IV), uranium (VI), and zirconium (IV). Detection limits for each of these are given, and thorium and uranium determinations are described in detail. Gamma activity from 204Pb makes a useful internal standard to correct for geometry differences between samples, which for the lowest detection limits are counted close to the detector. 

Internal standardization involves adding a chemical standard to the sample solution so that standard and sample are effectively measured at the same time. Internal chemical standards can be either the actual analyte, an isotopically labelled analyte or a related substance. The last one is usually chosen as something expected to be absent from the sample yet expected to behave towards the measurement process in a way similar to the analyte. There are a number of different ways of using internal standards and they sometimes serve a different purpose. 

Yields were determined by VPC using internal standard techniques, remaining material was starting halide. 

Yields were determined by VPC using internal standards. 

Use Internal Standards for bioanalytical samples to compensate for sample loss dnring sample preparation. The internal standards shonld have similar structures to the analytes and in most cases are isotopically labeled analytes. 

Sample-to-sample viscosity differences Use internal standards... 

Ohnesorge, J., Saenger-van de Griend, C., and Waetzig, H. (2005). Quantification in capillary electrophoresis-mass spectrometry long- and short-term variance components and their compensation using internal standards. Electrophoresis 26, 2360—2375. 

Quality Assurance/Quality Control. QA/QC measures included field blanks, solvent blanks, method blanks, matrix spikes, and surrogates. Percent recovery was determined using three surrogate compounds (nitrobenzene-d5, 2-fluorobiphenyl, d-terphenyl-diQ and matrix spikes (naphthalene, pyrene, benzo[ghi]perylene) the recoveries ranged from 80 to 102%. Separate calibration models were built for each of the 16 PAHs using internal standards (naphthalene-dg, phenanthrene-dio, perylene-di2). Validation was performed using a contaminated river sediment (SRM 1944) obtained from NIST (Gaithersburg, MD) accuracy was <20% for each of the 16 analytes. 

Q. Why must care be exercised when using internal standardization ... 

Recovery of extraction. The recovery of extraction can be monitored using internal standard and spiking methods. For meat products, 8-tocopherol is used as an internal standard because it is usually not detectable in these samples. Ueda and Igarashi (1987) also introduced 2,2,5,7,8-pentamethyl-6-chromanol as an internal standard for the recovery examination. As uneven recoveries occurred in the different forms of tocopherols and tocotrienols during extraction, spiking, which is adding the standards into the sample prior to saponification and extraction, is a reliable method to obtain the recoveries of the tocopherols and tocotrienols. 

The main advantage of quantification using internal standards and GC-FID is the simple, inexpensive instrumental set-up as well as the availability of standards compared to IDA. 

Normal-phase/reversed-phase chromatography is the ideal combination for semipreparative and quantitative separations in two-dimensional HPLC. Vitamins D2 and D3 coelute during the semipreparative stage, allowing a narrow retention window to be collected for analysis using internal standardization. By this means, Johnsson et al. (201) obtained a vitamin D3 detection limit of 0.1 yug/kg for milk and milk products. 

Methyl esters were identified by comparison with authentic compounds on both the Carbowax 20M column and a XE-60 (a General Electric cyanosilicone gum) column. Some of the earlier identifications were made with a Carbowax 20M + H3P04 column and confirmed on l,2,3-tris(2-cyanoethoxy) propane. Also, some of the analyses of free acids were run on a Carbowax 20M-TPA(terephthalic acid terminated) column rather than the FFAP. Quantitative analysis of free acids and methyl esters was carried out using internal standards. Acetophenone was used with the acids, and veratrole was used with the methyl esters. Formic acid in the nitrobenzene and nitric acid liquors was analyzed quantitatively by direct calibration. 

In the determination of steady state reaction kinetic constants of enzyme-substrate reactions, FABMS also provides some very unique capabilities. Since these studies are best performed in the absence of glycerol in the reaction mixture, the preferred method is that which analyzes aliquots which are removed from a batch reaction at timed intervals. Quantitation of the reactants and products of interest is essential. When using internal standards, generally, the closer in mass the ion of interest is to that of the internal standard, the better is the quantitative accuracy. Using these techniques in the determination of kinetic constants of trypsin with several peptide substrates, it was found that these constants could be easily measured (8). FABMS was used to follow the decrease in the reactant substrate and/or the increase in the products with time and with varying concentrations of substrate. Rates of reactions were calculated from these data for each of the several substrate concentrations used and from the Lineweaver-Burk plot, the values of Km and Vmax are obtained. 

As individual sample QC checks, internal standards are important in compound quantitation. They should be monitored with the same care as other QC checks. The deterioration of internal standard area counts (the area under a chromatographic peak) reflects the changes in the analytical system that may eventually degrade the quality of analysis to an unacceptable level. EPA Methods 8260 and 8270 require that the area for each internal standard be within the range of —50 to +100 percent of the area of the internal standards in the most recent CCV (EPA, 1996a). This requirement may be used as a general rule for all other methods that use internal standard calibration. 

Fish, birds, seals (CDDs) No methods details extraction and clean-up on silica, modified silica, and alumina columns used internal standards added HRGC/LRMS (EI/SIM) HRGC/LRMS (NCI/SIM) 1-50 pg 0.01-0.1 pg No data Buser et al. 1985... 

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