Use of internal standards

If it is not clear whether the reaction pH is correctly adjusted to around 11.5 by addition of the borate buffer, the use of an internal standard is recommended. Tlris especially holds true if hydrolysates are to be analysed. Although small amounts may be present in particulate matter, non-protein amino acids such as a-amino butyric acid or norleudne should be employed, the former having the advantage of being well separated in the HPLC system under discussion (Fig. 26-4). The internal standard should be added before the reagent. The amount added depends upon the type of analysis to be carried out and may vary from 25 to 500 pmol per injection volume. Variations in the response of the internal standard compared with a calibration run allow correction for differences in reaction pH, time and temperature. However, if these parameters have been kept constant for both calibration and sample analyses, the response should be reproducible to within about 2 %. 

Commercially available calibration mixtures contain various combinations of individual amino acids, usually 2.5 /tmol/mL. For most DFAA determinations a standard corresponding to a protein hydrolysate is suffcient. Amino adds such as glutamine or asparagine may 

Peaks are identified by comparison of elution times in samples with those established for standards in some cases comparison of elution patterns will further confirm peak identity. 

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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. ... 

The most interesting and important use of internal standards is to compensate absorption and enhancement effects the Class I deviations of Section 7.8. A discussion of this function of an internal standard can be profitably begun by considering two statements (1) An internal standard is fully satisfactory when the intensity ratio obeys the relation... 

Recent results from the authors laboratory69 on the x-ray emission spectrography of tungsten or molybdenum in solution illustrate some of the points made in Section 7.13. The also show the usefulness of internal standards (7.12). Finally, the work on tungsten is closely related to the experiments on the absorption effect in sodium tungstate solutions, the results of which are summarized in Table 7-2. 

There are three commonly encountered methods of employing these standards, namely the use of external standards, the use of standard additions and the use of internal standards. 

It is clear that neither NMEA nor NDPA is appropriate for an internal standard in NDMA determination if criteria are interpreted strictly, but both compounds have been used for this purpose. Addition of a nitrosamine, not normally present in the sample, is helpful in detecting any gross errors in the procedure, but the addition should not be considered to be internal standardization. Utilization of NMEA or NDPA to indicate recovery of NDMA can lead to significant errors. In most reports of the application of these "internal standards", recovery of all nitrosamines was close to 100%. Under these conditions, any added compound would appear to be a good internal standard, but none is necessary. NDMA is a particularly difficult compound for use of internal standardization because of its anomalous distribution behavior. I mass j ectrometry is employed for quantitative determination, H- or N-labeled NDMA could be added as internal standard. Because the labeled material would coelute from GC columns with the unlabeled NDMA, this approach is unworkable when GC-TEA is employed or when high resolution MS selected ion monitoring is used with the equipment described above. 

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. 

Although cSFC shows relatively poor figures of merit (speed, sensitivity, detection dynamic range and sample capacity) as well as a limited application area, its applications tend to be unique. These include solutes that can be solvated with pure SCCO2 and quantified with FID. Linear density programs typical in cSFC are ideal for homologous series found in surfactants, many prepolymers, etc. Selectivity in cSFC, which can be achieved by mobile phase density and temperature programming, relies on selective interactions with the stationary phase. Quantitative analysis in cSFC may be rendered difficult by small injected volumes the use of internal standards is recommended. 

Recovery tests or use of internal standards for pre-treatment and use of reference materials for analytical calibration and traceability [5]. 

Matrix effects can influence significantly the extraction efficiency and signal intensity. For heavily contaminated samples such as sewage sludge, this problem is particularly relevant, and therefore the use of internal standards is essential in these applications. Internal standards applied in the extraction procedures for non-ionic surfactants include perinaphtenone [8], 4-fluoro-4,-hydroxyl-benzophenone [5] and 4-bro-mophenyl acetic acid [9]. More appropriate are internal standards from the same compound class butylphenol [10], heptylphenol [11],... 

Because pi, mobility, and viscosity are all affected by temperature, the use of internal standards is recommended. However, the use of synthetic pi standards to estimate pis should be approached with caution because the pis of the protein sample and standards may not be affected equally by temperature. Temperature has a direct effect on viscosity and therefore all effects of viscosity on the CIEF process apply as temperature changes. 

Dodson MH (1963) A theoretical study of the use of internal standards for precise isotopic analysis by the surface ionization technique Part I General first-order algebraic solutions. J Sci Instrum 40 289-295 Douglas DJ (1989) Some current perspectives on ICP-MS. Canad J Spectrosc 34 38-49 Douglas DJ, French JB (1986) An improved interface for inductively coupled plasma-mass spectrometry (ICP-MS). Spectrochim Acta 41B 197-204... 

Repeatability is automatically tested during robustness testing. It should be better than 2% RSD for the area of a main peak without IS, less than 1 % using relative PAs or analyte/IS, and better than 10% RSD for a trace impurity. Repeatability in CE is generally lower than in HPLC because of the small injection volume (between 2 and 20 nl), but can be significantly improved by the use of internal standard. 

Sample extraction and hydrolysis details e.g., solvent extraction after freeze drying, with optimized acid or enzymatic hydrolysis Preparation of flavonoid standards and use of internal standards Chromatographic separation and detection method used, ideally RP-HPLC with UV or fluorescent detection Outline of quality assurance procedures employed... 

The main prerequisite for the quantification of low-molecular weight compounds by MALDl MS is the use of internal standards and optimized analysis protocols. For example, the quantification of alanine using 1- C-alanine as labeled internal standard in the system [C4CiIm][PFg]/CCA was possible with acceptable precision [14]. The method is therefore suited for screening processes, for example, for the comparison of enzyme activities in ILs. The detectability of peptides and proteins itself in this context also allows for an... 

Upon use of structurally modified variants as internal standards for the particular analytes, the relative quantificahon of oligonucleotides, peptides, and small proteins was demonstrated [44]. The potential of the ILM to allow quantitative analyses of peptides without the use of internal standards was presented recently [43]. Linear correlahons between peptide amount and signal intensities could be found upon applicahon of increased matrix-to-analyte ratios between 25,000 and 250,000 (mokmol). The dynamic range of linearity thus spanned one order of magnitude. Unfortunately, the importance of the M/A ratio prevents the use of this method in samples with unknown orders of concentration, for example, in a proteomics environment. On the other hand, the method is applicable for the screening of enzyme-catalyzed reactions because the starting concentrahons of the peptides are generally known in such assays. 

Internal Standard. The use of internal standard is critical in bioanalytical methods to improve precision and accuracy. The role of internal standard is to mimic the analyte of interest. It should be added before sample preparation/extraction to account for losses and errors introduced during the process. The more sample handling steps there are, the greater the error becomes, because errors are additive. In this case, the use of internal standard minimizes errors significantly. 

There are vast differences in the quoted relative fluorescences of the fluorophores obtained from adrenaline and noradrenaline (i.e. adrenolutin and noradrenolutin). With one exception (Anton and Sayre252), noradrenolutin is reported to be less fluorescent (on a w/w basis) then adrenolutin. This, however, is not true, since experiments with crystalline noradrenolutin70,71 have shown that (i) noradrenolutin is approximately twice as fluorescent as adrenolutin,255 and (ii) it is somewhat more stable than adrenolutin in aqueous solution 255 (cf. ref. 256). The use of internal standards has, however, allowed the method to function, more or less satisfactorily, in most cases. 

While the use of internal standards is generally recommended for gas chromatographic analysis, this technique is not as uniformly applied in trace analysis. In many cases, the chromatogram of a processed sample is too complex to provide for the additional peak from an internal standard. In cases where selective detectors are used, an internal standard which is comfort-... 

It should be strongly stressed here that the chemist obtain a set of "normal" values, for the so-called "normal" may vary somewhat in different regions due to ambient conditions as well as differences in instrumentation and technique. Proper technique, and use of internal standards, should result in agreement among laboratories. The methods in this chapter represent the current state of the art in clinical GC. Nevertheless, results are not guaranteed, and the validity of the results is the responsibility of the individual laboratory employing the procedure. Each chemist should determine the applicability of the methods and the verification of materials and standards used in his own laboratory. 

Concentration of odorants in sample being analyzed, determined by use of internal standards, GC-MS in selected ion monitoring (SIM) mode or a stable isotope dilution assay for trace quantities (see unitgu). 

A word of caution concerning the use of internal standards is also warranted. Detailed guidelines for the selection of internal standards have been published (13,14). The most crucial and most often overlooked of these criteria is the necessity for the internal standard and the analyte to possess similar physicochemical properties, including similar responses to the extraction and chromatographic conditions. Like recoveries from spiked samples, the internal standard should be added to the samples at the beginning of the extraction in order that it be subjected to the same extraction, separation, and quantitation conditions as the analyte of interest. 

Although laser-ablation sample preparation and analysis are conducted with relative ease, quantification of data can prove challenging. With liquid samples, the amount of material introduced into the ICP-MS remains relatively constant, and instrument drift is usually corrected through the use of internal standards. However, in LA-ICP-MS, conditions such as the texture of the sample, ablation time, the location of the sample within the laser cell, surface topography, laser... 

Gas chromatography was used to determine n-paraffin distribution in the oil and wax samples. An F and M Instrument Company Model 500 chromatograph was used with an uncompensated single column, a helium carrier gas flow rate of 25 ml/min., and a thermal conductivity detector. The column was 4.8 mm in diameter and 3.3 m in length, and was packed with 3% Dexil 300 on Chromo-sorb P. The block and injection port temperatures were maintained at 673 K. The column was temperature-programmed from 348 K to 673 K at a rate of 5.7 K per minute. Peak identification was aided by the use of internal standards of decane, dodecane, and hexadecane. The baseline was determined by heating without sample injection. Response values were not available for the various areas on the traces, so the analyses were reported as % by area. 

Normally, odd-numbered fatty acids are used as internal standards. While the use of internal standards ensures the correctness of the extraction procedure, it does not guarantee the completeness of extraction for different fatty acids. Due to this reason, a comparison between the methods is essential to truly determine the efficacy of extraction. Chavarri et al. (1997) compared two sample preparation procedures. The first method was the direct method developed by de Jong and Badings (1990), described above. The second method involved saponification with TMAH as described by Martin-Hemandez et al. (1988) and the formation of methyl esters in the injector prior to analysis. The authors found that separation of the FFAs from the triglycerides prior to derivatization improved the analysis. Another comparative study by Ardo and Polychroniadou (1999) reported that the saponification method described above (Martin-Hernandez et al., 1988) was found suitable for both low and high FFA levels in cheese. 

Use of internal standards. Mann and Jaworski (31) reported that when the recovery of 1-TI C IAA is monitored during a sample purification procedure, considerable loss of IAA can be detected. Bandurski and Schulze (32) suggested the use of reverse isotope dilution to help quantify the actual loss of IAA during sample analysis. In this procedure, one adds a trace amount of radio-labeled compound which ideally is identical to the compound being monitored. High specific activity is required so that statistically significant amounts of isotope can be detected without having to add an excessive quantity (mass) of internal standard. The amount of internal standard must be less than the amount of PGS. One may then accurately determine the recovery efficiency of the internal standard and thus of the PGS (32). 

The recovery of PGS at the nanogram level is sensitive to losses by adsorption, while at the microgram level adsorption is insignificant. This also indicates that estimation of recovery by use of internal standards requires that the standard should be added at levels approximating those of the sample. 

In order to harmonize SPS measures on as wide a basis as possible, the SPS agreement encourages members to base their SPS measures on international standards, guidelines, or recommendations. Thus, the SPS agreement, like the new TBT agreement, encourages use of international standards. The SPS agreement refers specifically to standards established by the Codex Alimen-tarius Commission, as discussed below. 

Internal standards could be used in external calibration, matrix-matched external calibration, and standard addition calibration [2], However, the use of internal standards in LC-MS quantitative methods should not be confused with internal calibration in which an internal standard is employed as a calibrant and the concentration of a unknown sample is calculated from the concentration of this internal standard and its analyte/IS signal ratio, i.e., the concentration of the unknown sample is calculated without the need for a calibration curve [3], The use of internal standards in most LC-MS quantitative methods belongs to signal-ratio calibration or internal standardization [2,4], In fact, the majority of bioanalytical LC-MS methods use matrix-matched signal-ratio external calibration. 

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