Internal standard choice

Figure 7.6 Internal standard choice in reversed-phased LC. (a) Analyte, (b) Suitable internal standard if readily available similar physicochemical properties to analyte but probably enough difference in hydrophobicity to allow separation, (c) Unsuitable, different pAg from analyte, therefore large difference in k at some pH values, (d) Unsuitable, not the best match for Lmaj and likely significantly larger k values, (e) Highly unsuitable, very different molar absorbance, (f) Highly unsuitable, different behaviour relative to analyte with changes in pH (i.e. neutral of basic analyte).

The method of choice for determining carboxyl groups in lignin is based on potentiometric titration in the presence of an internal standard, /)-hydroxybenzoic acid, using tetra- -butylammonium hydroxide as a titrant (42). The carboxyl contents of different lignins are shown in Table 6. In general, the carboxyl content of lignin increases upon oxidation. 

Often, the choice of an appropriate internal standard is very difficult but the following criteria should be considered ... 

Adequate precision and accuracy are only likely to be achieved if some standardization procedure is employed and the nature of this, internal or external standards or the method of standard additions, needs to be chosen carefully. If internal standardization procedures are adopted then appropriate compound(s) must be chosen and their effect on the chromatographic and mass spectrometry methods assessed. The ideal internal standard is an isotopically labelled analogue of the analyte but, although there are a number of commercial companies who produce a range of such molecules, these are not always readily available. An analytical laboratory is then faced with the choice of carrying out the synthesis of the internal standard themselves or choosing a less appropriate alternative with implications on the accuracy and precision of the method to be developed. 

Due to the lower injection precision of CE, it is not the method of first choice for the assay determination because the methods with higher precision are competing (HPLC, titration). However, a few examples in literature can be found and as a rule of thumb a well-developed CE method including internal standards should be able to obtain a repeatability of injections around 1% while values below 0.5% are generally expected for HPLC. 

After two decades of quantitative l.c. analyses, it has been established that, when proper precautions are taken, these methods can provide accurate and reproducible results.Quantitative l.c. measurements are usually as accurate as, and often more precise than, those obtained by spectrophotometric, " paper-chromatographic, and gas-liquid-chro-matographic methods. Both external and internal standardization have been used to translate peak height or areas into quantitative, solute-concentration values. Because peak heights are easy to measure, many methods use this parameter, and, when slightly overlapping peaks or unsteady baselines are encountered, it is the method of choice. With introduction of... 

The choice of reference compound(s) which can serve as internal standards is also crucial. It is important to choose a reference compound, which has the right physicochemical properties and its readout or affinity is in the mid-range of the... 

Quantification of the separated amino acids is usually performed by using external calibration or the internal standard method. Due to the large differences in chemical structure exhibited by the various amino acids, there is not a single ideal standard for the overall amino acid profile. Nevertheless, a suitable internal standard must be stable to hydrolysis and offer chromatographic resolution. The most popular choices comprise norleucine, norvaline, and a-amino-n-butanoic acid (AABA) [196]. 

Chemical shifts of a probe nucleus involved in the titration process are the weighted averages of its chemical shifts in the two species (e.g., B and BH ). It follows that reliable pKs will be obtained, provided that medium effects on both chemical shifts be small or properly corrected. These effects may be appreciably reduced by a careful choice of the internal standards. 

The precision of MS assays is in the range typical of most clinical assays (i.e., under 5-15%). The best choice of internal standard is the stable-isotope-labeled form (preferably 13C) of the compound of interest (e.g., P-hydroxy myristic acid or muramic acid). Specific trace detection of chemical markers in complex matrices requires appropriate negative controls. Procedures are often described that do not employ the mass spectrometer and false positives are often reported. The mere analysis of blank filters or water blanks is not satisfactory since chemical noise contributed by the sample is much greater and is not accounted for with this form of control. 

Accreditation is the procedure by which the competence of a laboratory to perform a specified range of tests or measurements is assessed against a national or international standard. The accreditation covers the kinds of materials tested or measured, the procedures or methods used, the equipment and personnel used in those procedures, and all relevant systems that the laboratory has in place. Once accredited, the laboratory is entitled to endorse test results with their accreditation status which, if it has any validity, is an imprimatur of some degree of quality and gives the client added confidence in the results. Accreditation therefore benefits the laboratory, by allowing the laboratory to demonstrate competence in particular tests, and the client, by providing a choice of accredited laboratories that are deemed competent. 

The right internal standard should have similar chemical and physical properties to match those of the analyte of interest. In LC-MS and LC-MS/MS assays, the coelution of the internal standard represent the ideal situation. Therefore, both structural analogs and stable labeled isotope forms of the analyte can be used. Stable labeled isotope internal standards are an ideal choice of every mass spectrometric-based assay because they offer the highest precision. However, not all the stable labeled isotope forms of the analyte behave in the same way when used as an internal standard. Actually, the analog compounds may behave better than the wrongly labeled isotope internal standards. 

The choice of an internal standard is fundamental to accuracy in mass spectrometric procedures. Ideally, the internal standard should not differ in basic structure from the analyte so stable labeled analogs are preferred, with mass differences preferably of more than 4 Da. However, full availability of labeled internal standards for steroid quantification has still not been achieved and non-biological steroids of similar structures may have to be used. This must change soon as too many published methods are compromised by the use of nonidentical internal standards. 

This method is more precise if several injections of the standard and the samples are carried out. The internal standard method is of general use and is reproducible but relies on the proper choice of compound for the internal standard, which should have the following characteristics ... 

Choice of an Internal Standard. One of the difficulties in the spec-trometric trace analysis of coal ash samples, in addition to choosing a suitable comparison standard matrix, is choosing an internal standard. The first choice in both analytical methods was indium, which was used as a constant internal standard added to the graphite powder diluent-buffer. The results obtained had poor reproducibility, as previously... 

Quantitation using unlabeled compounds as internal standards and GC-FID detection lacks the high sensitivity and the high selectivity required for aroma compounds present in the ppb level. For chemically stable compounds and those in higher concentration (i.e., >1000 ppb), however, this method gives reliable data. In all other cases, using isotope labeled compounds as internal standards is the method of choice if the they are available. 

The opposite scheme is to employ a C]8 resin (e.g., C 8 Sep-Pak from Waters Associates) in which the eluent will contain the amino acids while lipids and large proteins will be retained on the column. This procedure has been called into question, however (22). It has been suggested that partial retention of the hydrophobic amino acids can occur. This is especially a problem if norleucine (or any other hydrophobic amino acid, e.g., norvaline) is employed as an internal standard. Its partial retention on the C18 column will skew the apparent recoveries for all the other amino acids. This points to a particular problem for amino acid analysis There is no ideal choice for an internal standard. A subsequent section of this chapter will address the issue of internal standards in more detail. 

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. 

Since no single internal standard can possibly mimic the chemistry of all the amino acids (for overall profile), the choice of internal standard has been based primarily on two criteria. The first is chemical stability. The internal standard must not be labile under the conditions employed. The second is that the internal standard must offer chromatographic resolution. This is not easy, since the overall profile produces a chromatogram that is already very cluttered. A review of the literature reveals that three internal standards are the overwhelming popular choices norleucine, norvaline, and a-amino-n-butanoic acid (AABA). It should be noted that norleucine and norva-line are very hydrophobic amino acids, whereas AABA is relatively hydrophilic. How these standards might behave during sample preparation steps (e.g., filtration) as a function of their hy-drophobicity should be taken into consideration. 

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