Internal standard for quantitative analysis

FIGURE 1.16 Configuration and operational details of online introduction of internal standard for quantitative analysis of drugs from biological matrices.118 (Reproduced with permission from the American Chemical Society and the authors.)... 

These are the retention times and/or mass spectra or other spectra for qualitative analysis and peak areas, including that of the internal standard, for quantitative analysis. For quantitative analysis, the peak area ratio is calculated as with the standards. 

Amounts of injected compounds are proportional to the peak area. BA concentrations are calculated by comparison to standard calibration curves. This method does not need an internal standard for quantitative analysis. 

Quantitative analysis by isotope dilution. In isotope dilution, a known amount of an unusual isotope (called the spike) is added to an unknown as an internal standard for quantitative analysis. After the mixture has been homogenized, some of the element of interest must be isolated. The ratio of the isotopes is then measured. From this ratio, the quantity of the element in the original unknown can be calculated. 

II. APPLICATIONS OF STABLE-LABELED COMPOUNDS A. Internal Standards for Quantitative Analysis... 

If we look back at Fig. 3 the elution of n-hexadecane was at 30.10 min which does not interfere with any component in dried flowers extraction (Fig. 2), it was later selected as the internal standard for quantitative analysis. The tenderly odour of the flowers still exists in most of the dried flowers purchased from traditional medicine stores in Thailand. Some active constituents of which the pure chemicals are commercial available were determined to represent existing components in the dried flowers, which will be described in section 5 "Quantitive Determination". There are more than 50 components identified in M. elengi Some components in the fresh flowers and 4-week dried flowers were compared as can be seen in Table 1. 

Figure 6.5 Representative two-dimensional mass spectrometric analyses of sphingomyelin molecular species in the alkaline-treated lipid extracts of mouse spinal cord in the positive-ion mode in the presence of LiOH. Lipid extracts from spinal cord of mice at 48 days of age were prepared by using a Bligh-Dyer extraction procedure as previously described [27]. A part of each lipid extract was treated with lithium methoxide as described previously [28] and the residual lipid extract was reconstituted with 50 pL of 1 1 CHClj/MeOH per milligram of original tissue protein. Each of the reconstituted lipid extracts was diluted 10 times prior to addition of a small amount of LiOH (10 pmol LiOH/pL). Positive-ion ESI mass spectrum in the full MS scan mode was acquired in the mass range from m/z 600 to 900 from the diluted lipid extract of mouse spinal cord, which displayed very low abundance ions corresponding to lithiated SM species. Neutral loss scans (NLS) as indicated were also acquired from the diluted lipid extract in the mass range. All scans were displayed after normalization to the base peak in individual scan. The ion at m/z 653.6 corresponds to the selected internal standard for quantitative analysis of SM species.

Radiochemical methods of analysis take advantage of the decay of radioactive isotopes. A direct measurement of the rate at which a radioactive isotope decays may be used to determine its concentration in a sample. For analytes that are not naturally radioactive, neutron activation often can be used to induce radioactivity. Isotope dilution, in which a radioactively labeled form of an analyte is spiked into the sample, can be used as an internal standard for quantitative work. 

This method requires about 40 g of tobacco which are extracted with ethyl acetate in the presence of ascorbic acid. A trace amount of C-NDELA is added as an internal standard for quantitative analytical work. The filtered extract is concentrated and NDELA is enriched by column chromatography of the concentrate on silica gel. The residues of fractions with p-activity are pooled and redissolved in acetonitrile. Initially, we attempted to separate NDELA on a 3% OV-225 Chromosorb W HP column at 210 C using a GC-TEA system with direct interface similar to the technique developed by Edwards a. for the analysis of NDELA in urine (18). We found this method satisfactory for reference compounds however, it was not useful for an optimal separation of NDELA from the crude concentrate of the tobacco extract (Figure 4). Therefore, we silylated the crude concentrate with BSTFA and an aliquot was analyzed by GC-TEA with direct interface. The chromatographic conditions were 6 ft glass column filled with 3% OV-... 

It appears that purification of commercially available solvents is sometimes required for the complete elimination of impurity resonances. Occasionally, these impurities may be turned into advantage, as in the case of C2D2CI4 where the (known) C2DHCI4 content may be used as an internal standard for quantitation. Thus, removal of every impurity peak is not always essential for identification and quantitative analysis of stabilisers in PE. Determination of the concentration of additives in a polymer sample can also be accomplished by incorporation of an internal NMR standard to the dissolution prepared for analysis. The internal standard (preferably aromatic) should be stable at the temperature of the NMR experiment, and could be any high-boiling compound which does not generate conflicting NMR resonances, and for which the proton spin-lattice relaxation times are known. 1,3,5-Trichlorobenzene meets the requirements for an internal NMR standard [48]. The concentration should be comparable to that of the analytes to be determined. 

Peaks are identified from absolute or relative retention times by comparison with data from previously run standards stored in RAM or in libraries on disk. To take account of the variability of retention times from successive runs, retention time windows are used. These are defined as being /R x% for a standard, the unknown being positively identified if its retention time falls within the specified range. The size of the window can be varied by the user to conform with the degree of certainty required. Reference peaks can be selected for the calculation of relative retention times or as internal standards in quantitative analysis (pp. 9, 114). 

Choi BK, Gusev AI, Hercules DM (1999) Postcolumn introduction of an internal standard for quantitative LC-MS analysis. Anal Chem 71 4107-4110... 

Targeted analysis refers to metabolome analysis that targets one, or a few metabolites, and typically uses an internal standard for quantitation. The most common method is isotope dilution mass spectrometry (IDMS) [34], which relies on the use of stable isotope internal standards to enable the absolute quantitation of metabolites. This method has proven highly effective and has been successfully used in numerous studies. 

However, chemical ionization is sensitive to source parameters and matrix effects, and these problems are exacerbated by the direct introduction of a complex mixture into the source. The effects can be ccnpensated to seme degree by the use of an isotopically labelled internal standard for quantitative work. In the analysis of unknowns in canplex mixtures, the nature of the source chemistry should be a constant concern. 

Using internal standards in quantitative analysis is advantageous, for instance, in cases where the sample thickness cannot be determined exactly, or in gaseous samples whose total pressure is unknown. The compound which serves as an internal standard should have a simple spectrum which does not interfere with the bands of the analyte. It should be a stable substance, readily available, and non-toxic. The following substances have been recommended as internal standards by Colthup et al. (1975)... 

Although quantitative analysis of endogenous plant hormones by traditional GC has serious limitations, isotopic dilution analysis by GC-SIM using a single internal standard labelled with a stable isotope, such as H, C or N, is a completely different proposition [3-6]. Because the cost of a simple, computer-controlled, quadrupole-mass spectrometer has fallen substantially, and many highly enriched, isotopically-labelled compounds suitable for use as internal standards in quantitative analysis, can be either synthesized (1) or purchased from commercial sources (see Table 1), capillary GC-SIM is now the quantitative assay of choice in the vast majority of laboratories in which endogenous plant hormones are analysed on a routine basis. 

Figure 5 Mass spectra and chemical structures of secobarbital (A), Hs-secobarbital (B), and C4-secobarbital (C) (All as methyl-derivatives). (Reproduced with permission from Chang W-T, Smith J, and Liu RH (2002) Isotopic analogues as internal standards for quantitative GC/MS analysis - molecular abundance and retention time difference as interference factors. Journal of Forensic Sciences 47 873-881.)...

Chang WT, Smith J, and Liu RH (2002) Isotopic analogs as internal standards for quantitative GC/MS analysis -molecular abundance and retention time difference as interference factors. Journal of Forensic Science 47 873-881. 

Retinyl acetate is sometimes used as internal standard for retinol analysis it is not ideal because it is an ester, not a free alcohol (and is hydrolyzed by saponification processes), but superior in principle to the use of tocol or other nonretinoid forms for retinoid quantitation. Substituted retinal oximes have been used as internal standards (93). 15-Methylretinol (94) is conceptually an excellent internal standard for retinol analysis, because of its very similar chemical structure and properties. 

Standards in use for quantitation are essentially employed in three ways. With the internal standard technique, known quantities of a carefully selected (usually high purity) substance, the internal standard, are added to both samples and standards. The internal standard (preferably a non-commercial product) should have similar chemical and physical properties to the analyte, in particular, volatility and functional groups, in order to react in the same way to changes in the chemical environment (e.g. dinonyl adipate may serve as an internal standard for the determination of di(2-ethylhexyl) adipate). Solutions of pure additives used as standards may be unsatisfactory due to the difference in the evaporation profile between pure additives and those blended in the polymer samples. For example, a pure Permanax WSP sample evaporates in the ion source from about 30 to 150°C, whereas Permanax WSP blended in PE evaporates from about 120 C (m.p. of PE) to 350°C [17]. If one opts for polymer-based calibration standards the homogeneity of the samples is of crucial importance. Using internal standards in quantitative analysis is advantageous, for instance, in cases where the sample thickness cannot be determined exactly, or in gaseous samples with unknown total pressure. 

R. T. M. Fraser, Sodium Azide as an Internal Standard for Quantitative Infrared Analysis, Anal, Chem. 31, 1602, 1959. 

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