Analyses Without Internal Standards

Lack of any internal standard leads to several sources of uncertainty and, with the exception of the Method of Standard Additions (Section 8.5.1b), it is emphasized that in general analyses without use of an appropriate internal standard are inherently less rehable and are fit for purpose only in less demanding apphcations. For convenience of discussion these approaches have been grouped into subcategories. 

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F. Qaisse, Accurate x-ray fluorescence analysis without internal standards, Norelco Reporter, 4(l) y-l (1957). 

Qualitative HPLC methods, using area percent, are used to monitor the disappearance of starting material and the formation of byproduct. Without the inclusion of an internal standard and the calculation of response factors, it is not possible to establish with certainty whether all of the starting material can be accounted for. An internal standard must be stable in the reaction mixture, must not co-elute with any of the components, and must be stable in the mobile phase. Ideally, the internal standard has a retention time about half that of the total analysis time. Internal standardization is extremely useful for kinetic studies. Added to the reaction vessel, samples that are withdrawn at various times will contain identical concentrations of internal standard, and chromatograms can be directly compared or adjusted to identical scales to correct for variation in injection volume. 

But for methods without internal standard, care must be taken to matrix-match the standards and the unknowns, and to work with constant weight in the sample cups. The alternative procedure is to account for the wedge effect and to use a full fundamental parameter approach to the analysis. 

In 2001 CEN TCI9 WG27 [I] performed a round robin test for the analysis of low sulfur levels in fuel, where 102 laboratories participated in the test Out of these, five laboratories have used EDXRF instruments (from one manufacturer) using polarized X-rays for excitation. Tables 3 and 4 show a comparison of the achieved analysis results for diesel and petrol samples in comparison to those results achieved with WDXRF instruments with and without internal standard. 

Stolker et al. " described an analytical method based on TFC-LC-MS/MS for the direct analysis of 11 veterinary drugs (belonging to seven different classes) in milk. The method was applied to a series of raw milk samples, and the analysis was carried out for albendazole, difloxacin, tetracycline, oxytetracycline, phenylbutazone, salinomycin-Na, spiramycin, and sulfamethazine in milk samples with various fat contents. Even without internal standards, results proved to be linear and quantitative in the concentration range of 50-500 (xg/1, as well as repeatable (RSD<14% sulfamethazine and difloxacin <20%). The limits of detection were between 0.1 and 5.2 xg/l, far below the maximum residue limits for milk set by the EU. While matrix effects, namely, ion suppression or enhancement, were observed for all the analytes, the method proved to be useful for screening purposes because of its detection limits, linearity, and repeatability. A set of blank and fortified raw milk samples was analyzed and no false-positive or falsenegative results were obtained. 

For the heavier petroleum fractions as well as crude oil, analysis problems are encountered as a result of the presence of solids and materials that will not elute from the column. To account for the total sample, an internal standard mixtme is used. For heavy refinery cuts, an internal standard mixture, such as C10-C12 n-paraffins, can be used such that they elute before the sample. This allows analysis of the sample in a single run. Because of the complexity of crude oils, there are no sections in the sample chromatogram that are void of sample components. Thus an analysis of the sample with and without internal standard (C14-C17 normal hydrocarbons) is required. Typically, results are reported for these samples up to 538°C (1(X)0°F). Use of a Dexsil 300 colunm allows for determination of final boiling points up to 600°C. This extends the limit of the analysis to C40-C60 hydrocarbons. 

In the next step in the preparation of a validation, three (or preferably more) samples with the envisaged lowest concentration after sample preparation considering the estimated recovery rate should be analyzed. If it is at all possible, an internal standard should be added to the samples. A final decision as to whether to use a final method with or without internal standardization can then be taken later. An internal standard is generally the best quality control possibility. Each analysis sample will then be individually controlled with virtually no additional effort. 

In a second vial, dissolve approximately the same amount of dried sample as 11.3.5 with an approximately equal volume of carbon disulfide. Use this solution for the separate crude oil without internal standard analysis (see 11.4.4). 

An internal standard is desirable in any quantitative trace environmental analysis. The ideal internal standard should behave in a manner identical to that of the analyte in all the procedures followed for isolation, purification, and determination without producing interference. This is a difficult requirement to meet for nitrosamines, especially for NDMA. 

The use of Equation (22) is very general, but it is also possible, with accurate measurements and data treatment, to perform the quantitative phase analysis in semi-crystalline materials without using any internal standard. This procedure is possible only if the chemical compositions of all the phases, including the amorphous one, are known. If the composition of the amorphous phase is unknown, the quantitative analysis without using any internal standard can still be used provided that the chemical composition of the whole sample is available [51]. This approach, until now, has been developed only for the XRD with Bragg-Brentano geometry that is one of the most diffused techniques in powder diffraction laboratories. 

This procedure allows quantitative phase analysis without using any internal standard, but it requires the knowledge of the composition of the sample and a careful treatment of the experimental data, which have to be corrected for the air scattering. 

Ehret-Henry et al. [220] have shown that H NMR spectra can be used without chromatographic analysis, to shorten the total identification time necessary, and as a fingerprint of all the extractable nonvolatile compounds present in food packaging material (safety control). Figure 5.10 shows a H NMR spectrum (in CDCI3 with TMS as internal standard) of a Soxhlet extract of a 35 pirn PP film (after solvent evaporation). The assignments of the resonances of Irgafos 168 and its decomposition products were confirmed by a 31P- H 2D correlation NMR experiment [220],... 

Lopez-Avila et al. [59] used microwave assisted extraction to assist the extraction of polyaromatic hydrocarbons from soils. Another extraction method was described by Hartmann [60] for the recovery of polyaromatic hydrocarbons in forest soils. The method included saponification of samples in an ultrasonic bath, partitioning of polyaromatic hydrocarbons into hexane, extract cleanup by using solid-phase extraction, and gas chromatography-mass spectrometric analysis using deuterated internal standards. Polyaromatic hydrocarbons were thermally desorbed from soils and sediments without pretreatment in another investigation [61]. 

In the absence of any added salts, the APCI-MS spectra were dominated by the Na+ adducts, as shown in Fig. 2.8.5. The NH4 and K+ adducts were present at lower intensities, the latter especially for the higher molecular weight analogues. Addition of CH3CO2NH4 did not simplify the adduct formation to [M + NH4]+ species as observed in ESI-MS and the best results for APCI-MS analysis were obtained without addition of any salt solutions. Application of this method to determinations of M2D-C3-0-(E0)n-Me recovery from solid substrates was achieved, using triethylene glycol monohexyl ether [C6(EO)3] as the internal standard (Fig. 2.8.5) [29],... 

Quantitative analysis is performed making use of the linearity of the detector response. In fact, peak areas of standards and samples are well correlated with the injected amount, and it is possible to build a calibration curve without the use of an internal standard. 

Despite these caveats, many analyses are done on land either because the sample can be stored without changing the concentrations of the analytes it contains or because the apparatus required for the analysis cannot be operated on board a ship. For instance, some radionuclides are measured on land for both reasons. Samples analyzed on land can be spiked immediately with another (artificial) isotope of the same element to fill the adsorption sites on container walls and to serve as an internal standard. The mass spectrometers required for isotope ratio analyses are often too sensitive to vibration and motion for shipboard use. Analytes present at greater than trace levels, or which can be stabilized with some pretreatment, may also be analyzed successfully on shore. 

The reduction of citral is performed in situ, in the same autoclave, without any exposure of the catalyst to air. After cooling down the reactor to room temperature and reducing the hydrogen pressure, a solution of 0.9 ml of citral and 0.4 ml of tetradecane (internal standard) in 10 ml of n-heptane is introduced under hydrogen in the autoclave. The temperature and the hydrogen pressure are then raised to respectively 340K and 7.6 MPa. The kinetic of the reaction is followed with time by analysis of samples of the liquide phase. The selectivity for a product X at 100% conversion (Sx) is defined by Sx = [X]10o/[Citral]0. (Citral]0 represents the initial concentration of Citral (2 and E) and and [X]iqo represents the concentration of X at 100% conversion. 

The methyl esters can be also determined by GC-FID. Using a 30 m x 0.32 mm ID x 0.25 pm (film thickness) capillary column, such as DB-1701 or equivalent, the compounds can be adequately separated and detected by FID. The recommended carrier gas (helium) flow rate is 35 cm/s, while that of the makeup gas (nitrogen) is 30 cm/min. All of the listed herbicides may be analyzed within 25 min. The oven temperature is programmed between 50 and 260°C, while the detector and injector temperatures should be 300 and 250°C, respectively. The herbicides may alternatively converted into their trimethylsilyl esters and analyzed by GC-FID under the same conditions. FID, however, gives a lower response as compared with ECD. The detection level ranges from 50 to 100 ng. For quantitation, either the external standard or the internal standard method may be applied. Any chlorinated compound stable under the above analytical conditions, which produces a sharp peak in the same RT range without coeluting with any analyte, may be used as an internal standard for GC-ECD analysis. U.S. EPA Method 8151 refers the use of 4,4,-dibromooctafluorobiphenyl and 1,4-dichlorobenzene as internal standards. The quantitation results are expressed as acid equivalent of esters. If pure chlorophenoxy acid neat compounds are esterified and used for calibration, the results would determine the actual concentrations of herbicides in the sample. Alternatively, if required, the herbicide acids can be stoichiometrically calculated as follows from the concentration of their methyl esters determined in the analysis ... 

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). 

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