Internal standards for corrections

An alternative to MSA in ICP-MS analysis is the internal standard technique. One or more elements not present in the samples and verified not to cause an interelement spectral interference are added to the digested samples, standards, and blanks. Yttrium, scandium, and other rarely occurring elements or isotopes are used for this purpose. Their response serves as an internal standard for correcting the target analyte response in the calibration standards and for target analyte quantitation in the samples. This technique is very useful in overcoming matrix interferences, especially in high solids matrices. 

Physical interferences include differences in viscosity, surface tension and dissolved solids between sample and calibration standards. To minimise these effects, solid levels in analytical samples should not exceed 0.2% w/v, which requires the dilution of the digest before analysis. Use of several internal standards for correction of physical interferences is suitable when carrying out analysis of soil digest. 

These markers are not of use with low pH separations where most analytes migrate faster than the EOF, which is extremely low. If a peak is still required as an internal standard for correction of migration time, any compound with a fast cathodic electrophoretic mobility will suffice as a frontal marker. We have found that a synthetic peptide containing seven lysine residues and a single tryptophan (K3WK4) functions adequately for this purpose at pH 2.5. A frontal marker may also be useful at higher pH (e.g., when the neutral marker comigrates with a species of interest), where cationic species such as normetanephrine can suffice. 

The functional correlation of the analytical response showed determination coefficients (r ) for harmane of 0.9915 within a plate, and of 0.9488 spread over several plates and a measuring period of several days with the MS [83]. For isopropylthioxanthone, the correlation coefficient (r) of the calibration curve was 0.9983 [73]. Using an internal standard for correction of the manual plate positioning, the determination coefficient for caffeine was 0.9998 [87]. 

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. 

The use of internal standards is somewhat controversial.115 There is agreement that an internal standard may be used as a correction for injection volume or to correct for pipetting errors. If an internal standard is included before sample hydrolysis or derivatization, it must be verified that the recovery of the internal standard peak is highly predictable. Ideally, the internal standard is unaffected by sample handling. Using an internal standard to correct for adsorptive or chemical losses is not generally approved, since the concentration of the standard may be altered by the conditions of sample preparation. An example of internal vs. external standards is given in Chapter 4. 

The key step in the internal standard method is to choose an appropriate internal standard, which has polarity similar to the analyte, is inert to the conditions of extraction and processing, and elutes before or well after the peak of interest. An internal standard method is useful only for correcting for losses due to transfer or variability in dilution or injection, and it is inappropriate to use an internal standard to correct for losses due to degradation.57 This technique gives reliable, accurate, and precise results. If the internal standard is truly inert, the method is useful for determining the rate of analyte conversion in a chemical reaction. 

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. 

Vandecasteele et al. [745] studied signal suppression in ICP-MS of beryllium, aluminium, zinc, rubidium, indium, and lead in multielement solutions, and in the presence of increasing amounts of sodium chloride (up to 9 g/1). The suppression effects were the same for all of the analyte elements under consideration, and it was therefore possible to use one particular element, 115indium, as an internal standard to correct for the suppressive matrix effect, which significantly improved experimental precision. To study the causes of matrix effect, 0.154 M solutions of ammonium chloride, sodium chloride, and caesium chloride were compared. Ammonium chloride exhibited the least suppressive effect, and caesium chloride the most. The results had implications for trace element determinations in seawater (35 g sodium chloride per litre). 

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. 

Drifts of migration times can partly be compensated by calculating the mobility for analyte identification and using corrected PAs or internal standards for quantitation. 

In recognition of the work of Strehlow and coworkers, Gagne, Koval, and Lisen-sky [7] recommended that ferrocene be used as an internal standard for all potential measurements in nonaqueous solvents and that its aqueous potential of +0.400 V relative to the standard hydrogen electrode (SFIE) could be considered to represent a consistent correction factor in referencing all nonaqueous potential measurements to aqueous SHE. This recommendation is somewhat in disagreement with the conclusion of Strehlow and coworkers who, on the basis of theoretical considerations, suggested that the potential of the ferrocene couple in acetonitrile should be... 

It is also possible to use an internal standard to correct for sample transport effects, instrumental drift and short-term noise, if a simultaneous multi-element detector is used. Simultaneous detection is necessary because the analyte and internal standard signals must be in-phase for effective correction. If a sequential instrument is used there will be a time lag between acquisition of the analyte signal and the internal standard signal, during which time short-term fluctuations in the signals will render the correction inaccurate, and could even lead to a degradation in precision. The element used as the internal standard should have similar chemical behaviour as the analyte of interest and the emission line should have similar excitation energy and should be the same species, i.e. ion or atom line, as the analyte emission line. 

Internal standard (IS) calibration requires ratioing of an analytical signal to an IS which has very similar characteristics to that of the analyte of interest (an element which is similar to the analyte either in mass, ionisation potential or chemical behaviour). Quantitative analysis applying internal standardisation is the most popular calibration strategy in ICP-MS, as improvements in precision are obtained when the technique is appropriately used. Of course, the validity of this calibration method requires that one ensures a good selection of the correct internal standard. For this purpose it is possible to resort to chemometric methods [16]. 

In the photographic procedure, the lack of a suitable internal standard for exposure correction, the attempt to record and determine all elements on one generalized exposure, and the very high concentration of the trace elements in the ash (for some samples as much as 33 times the amount reported in the coal) caused a poor relative standard deviation. However, of the 13 elements determined, only Co, Ni, Cr, and V were less precise than 20%, a level which we feel is suitable for a photographic method. 

Brief mention needs to be made regarding the employment of internal standards. While it is desirable to employ an internal standard for procedures that involve significant sample preparation procedures, there is no ideal choice for an internal standard for amino acid analysis. This fact is due to the wide spectrum of chemistries exhibited by the various amino acids. If one is analyzing for a single amino acid (or class, e.g., the hydrophobic amino acids), it is possible to choose an internal standard that mimics the chemistry of that particular amino acid very well. However, for the overall amino acid profile, an internal standard will do nothing more than allow the analyst to make nonvolumetric solution transfers and correct for variability of the injection volume by the HPLC injector. Unfortunately, the employment of an internal standard can actually skew the apparent recoveries for the overall amino acid profile. 

New equipment should be carefully inspected to check that the plug has been correctly fitted, otherwise a live chassis will result. International standards for Great Britain and Europe stipulate the following colours for electric cables ... 

Wang, S., Cyronak, M., and Yang, E. (2007). Does a stable isotopically labeled internal standard always correct analyte response A matrix effect study on a LC/MS/MS method for the determination of carvedilol enantiomers in human plasma. J. Pharm. Biomed. Anal. 43 701-707. 

Use of Internal Standards The use of internal standards envisages different possibilities. The procedure described here is based on two internal standards. Once thawed, fish sample were dissolved in TMAH, ethylated with NaBEt4, extracted into iso-octane and subjected to GC-ICP-MS for the identification and quantification of Me-Hg and inorganic Hg2+. For the correction of procedural errors two internal standards were used. The sample pretreatment was corrected by the recovery factor of the spiked dibutyl-dipentyl-Sn (DBT-pe), while the GC-ICP-MS measurements were controlled by the signal stability of Xe added to the GC carrier gas [47], In another application propyl-Hg was used as an internal standard to correct for matrix-induced ion signal variation and instrumental drift [65]. 

The first coupling of a capillary gas chromatography to a sector field ICP-MS for the speciation of organometallic compounds present in a synthetic sample was described by de Smaele et cd Transient ion signals of °Sn, ° Hg+ and ° Pb+ were measured using Xe+ as an internal standard (to correct possible drifts of the magnetic field and plasma instabilities). 

Use of internal standards. With complex extraction procedures, it is preferable to use an internal standard to correct for both recovery and analytical variability. The internal standard should be chemically similar to the compounds of interest and be chemically stable. It should have similar detection characteristics as the unknown and should elute in a blank portion of the chromatogram preferably near the middle (or near specific peaks of interest), and be well resolved from adjacent peaks. To accurately reflect the recovery of the unknowns it must be added as near as possible to the start of the extraction procedure. The internal standard corrects mainly for dilution and sampling errors. The amount of internal standard added should be such that allowing for dilution it will give a prominent well resolved peak with a height of about 80% full scale under normal analytical conditions. 

We have developed a very sensitive assay which can quantify both 13-cis and all trans retinoic acid in the same plasma sample. Only 1 ml of plasma is necessary for analysis, with a limit of quantification of 0.5 ng/ml. The assay is linear for both cis and trans retinoic acid, and there is virtually no interconversion of the two isomers by assay manipulations. However, the assay does slightly underestimate the amount of all trans retinoic acid present due to the differential recovery of this isomer from plasma as opposed to recovery from PBS. This will be corrected in future work by the addition of a stable isotope labelled all trans retinoic acid internal standard for quantification. 

The method is based on the addition of a standard reference (internal standard) that is detected at a different wavelength from the analyte. The reference standard is added at the same concentration to samples and standards and diluted to mark in a volumetric flask. This technique uses the signal from the internal standard to correct for matrix interferences and is used with respect to precision and accuracy as well as eliminating the viscosity and matrix effects of the sample. 

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