Interferences internal standard method

To measure serotonin (5-hydroxytryptamine), by the internal standard method, a 1 ml aliquot of the unknown solution is added to 1 ml of a solution containing 30 ng of N-methyl-serotonin. This mixture is then treated to remove all other compounds which could interfere with the experiment. The operation performed was an extraction in the solid phase to isolate the serotonin and its methyl derivative, diluted in a suitable medium. 

With attention to the purity of the standards and to the lack of interference of any solvent impurities, the precision of the internal standard method is controlled by the ability to quantitate peak size. That certainly qualifies this technique as the most precise method of quantitative analysis by GC, and where precision is paramount, the internal standard technique should be applied. Its advantages far outweigh the slight increase in effort required for standard and sample preparation. An excellent, detailed, how-to approach for the internal standardization technique as applied to a practical problem has been detailed by Barbato, Umbreit, and Leibrand (35). 

If the internal standard method is applied properly, it may be assumed that the ratio of the weight of component to the weight of internal standard is exactly proportional to the peak area ratio, and under these conditions no correction factor is needed. The sample is first run by itself to determine whether the internal standard would mask any component by peak superposition. If there is no interference, a mixture is prepared of the sample and of the internal standard in the specified weight ratio, and the percentages of the internal standard and of the sample in the mixture are calculated. The mixture is chromatographed, and the areas of the component peak and the internal standard peak are calculated by one of the methods described above. 

Unfortunately, the resistance of the internal standard method to the interference effect is, in principle, even less than for the set of standards method. The reason is that the sample components can change both the signal measured for the analyte and the signal measured for the internal standard. The chance that that both changes are the same (or at least similar) is very small, especially in trace analysis. Therefore, the method can be exploited only if the interferences (if present) are eliminated in one of the ways applied for calibration by the set of standards method. 

Chemical interference is practically non existent as a result of the high temperature of the plasma. On the other hand, physical interference may be observed. This stems from variations in the sample atomisation speed which is usually due to changes in nebulisation efficiency caused by differences in the physical properties of the solutions. Such effects may be caused by differences in viscosity or vapour tension between the sample solutions and the standards due, for example, to differences in acidity or total salt content. The technique most commonly used to correct this physical interference is the use of internal standards. In this technique a reference element is added at an identical concentration level to all the solutions under analysis, standards, blank and samples. For each element, the ratio of simultaneous measurements of the lines of the element and the internal standard is then determined in order to compensate for any deviation in the response of the plasma. If the internal standard behaves in the same way as the element to be determined, this method can be used to improve the reliability of the result by a factor of 2 to 5. It can also, however, introduce significant errors because not all elements behave in the same way. It is thus necessary to take care when using it. Alternatives to the internal standard method include incorporating the matrix into the standards and the blank, sample dilution, and the standard addition method. 

Analysis Using Method of Standard Addition. Sometimes it is not possible to overcome interference effects using standard calibration curves or internal standard methods of analysis. However, a third method involving the standard addition may be used to achieve reproducible results. Under these conditions all solutions will have the same matrix composition, so influence of the matrix will be the same. It must be emphasised that this method only corrects for the slope of the calibration curve i.e. element measurements and not for effects of sample plasma noise, shifts, etc. The oil... 

Many examples are known where the FIA technique is used for sample transport only and an example of this is where sample contains a concentration of interfering matrices. These samples can be injected in very small volumes (10 to 100 pi) into a carrier stream to minimise these interferences due to excessive dilution. Standard addition and internal standard methods can equally be applied to FIA techniques to reduce matrix, spectral and other potential interfering effects. Ion exchange columns connected in the sample feed... 

Standard addition is elegant if the sample amount is not limited. It allows calibration of the analysis under realistic conditions, i.e. not with a standard chromatogram which is free from interferences. The standard addition and internal standard methods can be combined. 

With attention to the pnrity of the standards and to the lack of interference of any solvent impnrities, the precision of the internal standard method is controlled by the ability to quantify peak size. That certainly qnalifies this technique as the most precise method of quantitative analysis by GC, and where... 

The method is based on the international standard ISO 4053/IV. A small amount of the radioactive tracer is injected instantaneously into the flare gas flow through e.g. a valve, representing the only physical interference with the process. Radiation detectors are mounted outside the pipe and the variation of tracer concentration with time is recorded as the tracer moves with the gas stream and passes by the detectors. A control, supply and data registration unit including PC is used for on site data treatment... 

When possible, quantitative analyses are best conducted using external standards. Emission intensity, however, is affected significantly by many parameters, including the temperature of the excitation source and the efficiency of atomization. An increase in temperature of 10 K, for example, results in a 4% change in the fraction of Na atoms present in the 3p excited state. The method of internal standards can be used when variations in source parameters are difficult to control. In this case an internal standard is selected that has an emission line close to that of the analyte to compensate for changes in the temperature of the excitation source. In addition, the internal standard should be subject to the same chemical interferences to compensate for changes in atomization efficiency. To accurately compensate for these errors, the analyte and internal standard emission lines must be monitored simultaneously. The method of standard additions also can be used. 

Various colorimetric methods have been employed for measuring ozone residuals, although most of these ate susceptible to significant interferences (142). The indigo trisulfonate method (143), however, has been approved by the Standard Methods Committee of the American Pubfle Health Association (141) and the International Ozone Association for ozone residual measurement. 

Errors due to nonspectral interferences can be reduced via matrix matching, the method of standard additions (and its multivariant extensions), and the use of internal standards. ... 

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. 

The method using GC/MS with selected ion monitoring (SIM) in the electron ionization (El) mode can determine concentrations of alachlor, acetochlor, and metolachlor and other major corn herbicides in raw and finished surface water and groundwater samples. This GC/MS method eliminates interferences and provides similar sensitivity and superior specificity compared with conventional methods such as GC/ECD or GC/NPD, eliminating the need for a confirmatory method by collection of data on numerous ions simultaneously. If there are interferences with the quantitation ion, a confirmation ion is substituted for quantitation purposes. Deuterated analogs of each analyte may be used as internal standards, which compensate for matrix effects and allow for the correction of losses that occur during the analytical procedure. A known amount of the deuterium-labeled compound, which is an ideal internal standard because its chemical and physical properties are essentially identical with those of the unlabeled compound, is carried through the analytical procedure. SPE is required to concentrate the water samples before analysis to determine concentrations reliably at or below 0.05 qg (ppb) and to recover/extract the various analytes from the water samples into a suitable solvent for GC analysis. 

Unlike GC/MS methods, internai standards are not appropriate for immunoassays. Internal standards that would react with the antibody but would not interfere with the assay are nonexistent. In the place of internal standards, external QC must be maintained. 

Under some conditions, it is difficult to incorporate an internal standard into a method. If the chromatogram is very complex, an internal standard may interfere with quantitation of a peak of interest. The development of highly precise sample transfer techniques, including modem autoinjectors, reduces the dependence of the experimentalist on the use of an internal standard to correct for effects of dilution and transfer losses. In many cases, external standardization can be used effectively. The weight percent purity is determined by comparing the area of each peak in a chromatogram with those generated by separately injected pure standards of known concentration. 

In a bioanalytical method, analyses of blank samples (plasma, urine, or other matrix) should be obtained from at least six sources. Each blank sample should be tested for the possible interference of endogenous substances, metabolites, or degradation products. The response of the peaks interfering at the retention time of the analyte should be less than 20% of the response of a lower quantitation limit standard, and should be less than 5% of the response of the internal standard that was used [18, 19]. For dissolution studies, the dissolution media or excipients should not give a peak or spot that has an identical Rt or Rf value with the analyte [20]. 

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]. 

The first two points are best dealt with as part of the process for developing vahdated analytical methods. Vafidation should include testing the robustness of a method in repeated use over a period of time determining the precision and accuracy and study of potential interferences. As an example, it would be expected that in the capillary GC—TEA method for organic explosives, a peak should be at least three times the basefine noise to be counted as a real signal, and that the relative retention time should be within 1.0% of the standard for volatile compounds and within 0.5% for the rest. The relative retention time is simply the ratio of the analyte s retention time compared with that of an internal standard. Use of relative retention times significantly improves the repeatabdity of GC analysis... 

ICP-AES was validated for the simultaneous determination of Al, B, Ba, Be, Cd, Co, Cr, Cu, Fe, Li, Mn, Ni, Pb, Se, Sr and Zn in human serum in a clinical laboratory. The samples underwent digestion and yttrium was used as an internal standard. The LOD were as follows 0.002-0.003 (xM for Ba, Cd, Mn and Sr 0.014-0.07 (xM for Be, Co, Cr, Cu, Fe, Li, Ni, Pb and Zn and 0.2-0.9 (xM for Al, B and Se. The concentrations of Al, Be and Co in human serum were found to be above the LOD, while those of Cd, Cr, Ni and Pb were below the LOQ however, in case of acute intoxication with the latter elements the method is valid . Matrix effects were evaluated for ICP-AES analysis using solution nebulization and laser ablation (LA) techniques. The main matrix-related interferences stem from elements with a low second ionization potential however, these are drastically reduced when pure He is used as carrier gas. This points to Ar (the usual carrier) participation in the interference mechanism, probably by interacting with doubly charged species. ... 

Reference values of this approach are not different from those for other amino acid analyses. An example of a mass chromatogram, representing the plasma of a PKU patient, is shown in Fig. 2.1.1. When evaluating the results of MS/MS amino acid analyses, one has to reahze that the hquid chromatographic separation is by far less efficient that the AAA separation. For this reason, any amino acid may (partly) coelute with other amino acid(s), which potentially interferes with its mass spectromet-ric behavior. This effect is known as quenching. In order to overcome this as much as possible, stable-isotope-labeled internal standards (as many as possible) should be used. However, this matrix effect of ion suppression is the major pitfall in the MS/MS analysis of amino acids. Consequently, the MS/MS analysis of amino acids cannot be regarded as a reference method, similar to all other amino acid analytical methods. 

Figure 10.11—Optical arrangement of a Fourier transform IR spectrometer, a) A 90c Michelson interferometer including the details of the beam splitter (expanded view) b) optical diagram of a single beam spectrometer (based on a Nicolet model). A weak intensity HeNe laser (632.8 nm) is used as an internal standard to measure precisely the position of the moving mirror using an interference method (a simple sinusoidal interferogram caused by the laser is produced within the device). According to the Nyquist theorem, at least two points per period are needed to calculate the wavelength within the given spectrum.

---