Internal standardization, quantitation analysis

A quantitative analysis for vitamin Bi was carried out using this procedure. When a solution of 100.0 ppm Bi and 100.0 ppm o-ethoxybenzamide was analyzed, the peak area for vitamin Bi was 71 % of that for the internal standard. The analysis of a 0.125-g vitamin B complex tablet gave a peak area for vitamin Bi that was 1.82 times as great as that for the internal standard. How many milligrams of vitamin Bi are in the tablet ... 

Too often, the analyst omits a very important step in method development. A standard should be selected which will assure that the desired separation is reproducible on a day-to-day, month-to-month basis. The internal standard (quantitative) discussed above is not satisfactory for this purpose because it is not one of the compounds that is a "potential" compound to be resolved. Consider, if you will, a situation wherein a method has been developed which will resolve a drug from a given metabolite. This metabolite is a product indicative of some toxic reaction within the body. Let us assume that the drug produces this reaction and, hence, the metabolite occurs only rarely in some people. Obviously, the internal standard (quantitative) must be resolved from the drug and the metabolite. However, because the toxic reaction occurs rarely, in most analyses, the metabolite will not be present. Could the analyst assume that because the retention volumes of the internal standard (quantitative), and the drug Standard, are about the same as they were the last time the analysis was done, that the metabolite would still be resolved, if present The answer is apparent The analyst must include a metabolite "standard" to reaffirm selectivity and sensitivity. 

Several recommendations arose from the interlaboratory smdy to minimize analytical challenges and to ensure data quality. As discussed above, it is recommended that mass labelled PFCs be employed as internal standards [93, 97]. It should be noted, however, that some electrospray ionization suppression may still occur if these internal standards are used at high concentrations [97]. Matrix effects can also be minimized by employing matrix-matched calibration standards in lieu of solvent-based calibration standards [97]. Unfortunately, matrix-matched standards can be impractical when an appropriate clean matrix cannot be found [94]. Other quality assurance and quality control measures, such as spike and recovery analyses of an analyte added to the sample matrix, repetitive analysis of samples to determine precision and comparison of internal standard quantitation to quantitation via standard additions, are also useful in determining data quality [94]. 

The first and second reactions proceed quantitatively under mild conditions whereas the third reaction is so slow that it can be neglected. To determine quantitatively the degree of branching of polyethyleneimine, the cyanethylation reaction was performed in sealed ampoules at 38°C for 3h. Umeacted acrylonitrile was analysed by GC with pyridine as an internal standard. The analysis was accomplished in a column (150 m x 0.4 cm I.D.) containing 10% polyethylene glycol 3000 on Chromosorb W. The results obtained agreed well with spectroscopic results [59]. 

The VOCs in the inside air are enriched by means of active sampling on different sorption tubes, which are thermodesorbed or desorbed with a solvent. After adding internal standards, the analysis is done by capillary GCYMS. The hundred compounds of the chromatogram with the most intensive signals are identified by mass spectroscopy, and are then semi-quantitatively evaluated with toluene as the substance of reference. The toluene equivalents are summed (Wensing, 1996a), and this result serves as a semi-quantitative estimation of the total VOC concentration (TVOC). 

A more recent example of this GC-CI-MS approach (Longo 1995) described a quantitative method for NDMA in beer samples. The analyte was isolated by distillation and subsequent extraction from the distillate using dichloromethane, and quantitation was achieved using [dg]-NDMA as internal standard. The analysis monitored the (M- -H)+ ions of analyte m/z 75) and internal standard (ni/z 81) at unit mass resolution using a single quadrupole analyzer this low resolution detection had low selectivity, as indicated by the SIM chromatogram for m/z... 

Three internal standards are utilized in the analysis. Table 3 lists each analyte and the recommended internal standard. Quantitation should be performed using analyte to internal standard ratios against the known concentrations of the calibration curve. 

Relatively few studies have been made on the feasibility of quantitative FAB analysis. Riley et al. [217] have described a quantification procedure to monitor the paint additive Tinuvin 770 in two coating systems (acrylic melamine and a hydroxy ester melamine). Tinuvin 770 proved to be well suited for FAB analysis in coating extracts on glycerol basis using an internal standardisation procedure. Lay et al. [218] have developed a FAB-MS method for the quantitative analysis of plasticisers (DEHP, including any isomeric dioctyl phthalates) in baby PVC pacifiers that does not require sample extraction, clean-up, or chromatographic separation. A reference material, didecylphthalate (DDP), was added to a solution of the PVC sample in THF as an internal standard. Quantitation was based on the relative... 

Standardization—External standards, standard additions, and internal standards are a common feature of many quantitative analyses. Suggested experiments using these standardization methods are found in later chapters. A good project experiment for introducing external standardization, standard additions, and the importance of the sample s matrix is to explore the effect of pH on the quantitative analysis of an acid-base indicator. Using bromothymol blue as an example, external standards can be prepared in a pH 9 buffer and used to analyze samples buffered to different pHs in the range of 6-10. Results can be compared with those obtained using a standard addition. 

Troost and Olavesen investigated the application of an internal standardization to the quantitative analysis of polynuclear aromatic hydrocarbons. The following results were obtained for the analysis of the analyte phenanthrene using isotopically labeled phenanthrene as an internal standard... 

Samples of analyte are dissolved in a suitable solvent and placed on the IR card. After the solvent evaporates, the sample s spectrum is obtained. Because the thickness of the PE or PTEE film is not uniform, the primary use for IR cards has been for qualitative analysis. Zhao and Malinowski showed how a quantitative analysis for polystyrene could be performed by adding an internal standard of KSCN to the sample. Polystyrene was monitored at 1494 cm- and KSCN at 2064 cm-. Standard solutions were prepared by placing weighed portions of polystyrene in a 10-mL volumetric flask and diluting to volume with a solution of 10 g/L KSCN in... 

Precision The precision of a gas chromatographic analysis includes contributions from sampling, sample preparation, and the instrument. The relative standard deviation due to the gas chromatographic portion of the analysis is typically 1-5%, although it can be significantly higher. The principal limitations to precision are detector noise and the reproducibility of injection volumes. In quantitative work, the use of an internal standard compensates for any variability in injection volumes. 

This experiment describes a quantitative analysis for caffeine, theobromine, and theophylline in tea, pain killers, and cocoa. Separations are accomplished by MEKC using a pH 8.25 borate-phosphate buffer with added SDS. A UV detector set to 214 nm is used to record the electropherogram. An internal standard of phenobarbital is included for quantitative work. 

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. 

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory dmg (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by Hquid-Hquid extraction at an acidic pH. After evaporation of the organic layer, the dmg and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after adrninistration of therapeutic doses. 

Liquid chromatography was performed on symmetry 5 p.m (100 X 4.6 mm i.d) column at 40°C. The mobile phase consisted of acetronitrile 0.043 M H PO (36 63, v/v) adjusted to pH 6.7 with 5 M NaOH and pumped at a flow rate of 1.2 ml/min. Detection of clarithromycin and azithromycin as an internal standard (I.S) was monitored on an electrochemical detector operated at a potential of 0.85 Volt. Each analysis required no longer than 14 min. Quantitation over the range of 0.05 - 5.0 p.g/ml was made by correlating peak area ratio of the dmg to that of the I.S versus concentration. A linear relationship was verified as indicated by a correlation coefficient, r, better than 0.999. 

Because of the complex nature of the discharge conditions, GD-OES is a comparative analytical method and standard reference materials must be used to establish a unique relationship between the measured line intensities and the elemental concentration. In quantitative bulk analysis, which has been developed to very high standards, calibration is performed with a set of calibration samples of composition similar to the unknown samples. Normally, a major element is used as reference and the internal standard method is applied. This approach is not generally applicable in depth-profile analysis, because the different layers encountered in a depth profile of ten comprise widely different types of material which means that a common reference element is not available. 

High performance liquid chromatography is used for the separation and quantitative analysis of a wide variety of mixtures, especially those in which the components are insufficiently volatile and/or thermally stable to be separated by gas chromatography. This is illustrated by the following method which may be used for the quantitative determination of aspirin and caffeine in the common analgesic tablets, using phenacetin as internal standard where APC tablets are available the phenacetin can also be determined by this procedure. 

Quantitative analysis using the internal standard method. The height and area of chromatographic peaks are affected not only by the amount of sample but also by fluctuations of the carrier gas flow rate, the column and detector temperatures, etc., i.e. by variations of those factors which influence the sensitivity and response of the detector. The effect of such variations can be eliminated by use of the internal standard method in which a known amount of a reference substance is added to the sample to be analysed before injection into the column. The requirements for an effective internal standard (Section 4.5) may be summarised as follows ... 

It is possible to carry out a chromatographic separation, collect all, or selected, fractions and then, after removal of the majority of the volatile solvent, transfer the analyte to the mass spectrometer by using the conventional inlet (probe) for solid analytes. The direct coupling of the two techniques is advantageous in many respects, including the speed of analysis, the convenience, particularly for the analysis of multi-component mixtures, the reduced possibility of sample loss, the ability to carry out accurate quantitation using isotopically labelled internal standards, and the ability to carry out certain tasks, such as the evaluation of peak purity, which would not otherwise be possible. 

HPA catalyzed liquid phase nitration was eairied out in a Teflon-lined stainless autoclave of 200 mL equipped with a magnetic stirrer. Reactants and HPA were quantitatively added to the autoclave, which was sealed and heated in an oil-bath. Products were analyzed by GC with OV-101 30 m capillary column and FID detector by using calibrated area normalization and internal standard method. All products were confirmed by GC-MASS analysis. 

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-... 

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. 

The method using GC/MS with selected ion monitoring (SIM) in the electron ionization (El) mode can determine concentrations of alachlor, acetochlor, and metolachlor and other major corn herbicides in raw and finished surface water and groundwater samples. This GC/MS method eliminates interferences and provides similar sensitivity and superior specificity compared with conventional methods such as GC/ECD or GC/NPD, eliminating the need for a confirmatory method by collection of data on numerous ions simultaneously. If there are interferences with the quantitation ion, a confirmation ion is substituted for quantitation purposes. Deuterated analogs of each analyte may be used as internal standards, which compensate for matrix effects and allow for the correction of losses that occur during the analytical procedure. A known amount of the deuterium-labeled compound, which is an ideal internal standard because its chemical and physical properties are essentially identical with those of the unlabeled compound, is carried through the analytical procedure. SPE is required to concentrate the water samples before analysis to determine concentrations reliably at or below 0.05 qg (ppb) and to recover/extract the various analytes from the water samples into a suitable solvent for GC analysis. 

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