Quantitation by Internal Standardization

Internal standardization circumvents the effects of time-variant instrument response, but does not compensate for different ionization efficiencies of analyte and standard. For internal standardization, a compound exhibiting close similarity in terms of ionization efficiency and retention time is added to the sample at a known level of concentration, e.g., an isomer or a homolog eluting closely to the analyte may serve that purpose. 

It is important to add the internal standard before any clean-up procedure in order not to alter the concentration of the analyte without affecting that of the standard in the very same way. For reliable results, the relative concentration of analyte and standard should not differ by more than a factor of about ten. 

Virtually identical ionization efficiency for a pair of compounds is only given for isotopologs. As these differ from the nonlabeled target compound in mass, they can be added to the mixture at known concentration to result in a special case of internal standardization, hence the term isotope dilution [55]. The ratio of intensities of the peaks corresponding to target compound and labeled standard as delivered by the RICs, SIM, or MRM traces is then taken as the relative concentration of labeled internal standard and target compound. As the absolute concentration of the standard added before the analysis is known, the concentration of the analyte can reliably be calculated. 

Example I A potential drug (M) and its metabolite (M ) in a liver sample are quantified by LC-ESI-MS with internal standardization using trideuterated standards for each analyte (Fig. 14.7) [27]. Under these conditions it neither presents a problem that both analytes and their isotopic standards are almost co-eluting from the LC column, nor does the completely unspecific TIC play a role. If required for sensitivity reasons, this analysis could also have been performed in the SIM mode using the m/z values of the RICs shown. 

Example II The number of m/z values to be monitored in SIM is limited. Such limitations are more severe when additional lock mass peaks have to be included in case of HR-SIM. Therefore, it is commonplace to monitor different sets of SIM traces during consecutive time windows leading to a sequence of different SIM setups during a single chromatographic separation. The quantitation of halo-genated dibenzo-p-dioxins in municipal waste incinerator fly ash at concentrations in the ppb to low-ppm range requires such a setup (Fig. 14.8) [47]. Here, a combined approach of external standardization for the BrCls-species and internal standardization for the CLj-species has been realized. 

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Quantitation by Internal Standard. Quantitation by internal standard provides the highest precision because uncertainties introduced by sample injection are avoided. In this quantitation technique, a known quantity of internal standard is introduced into each sample and standard solutions. As in the external standard quantitation, chromatograms of the standard and sample solutions are integrated to determine peak heights or peak areas. The ratio of the peak height or area of the analyte to an internal standard is determined. The ratios of the standards... 

Appropriate sampling procedures are to be followed. This test method is not suitable for liquified petroleum gases. Volatile liquids to be analyzed by this test method shall be sampled using the procedures outlined in Practice D 4057. A sufficient quantity of sample should be taken for multiple analyses to be performed (at least 10 to 20 g for quantitation by internal standardization). Store all samples and standard blends at a temperature of 7 to 15 C (45 to 60 F). Do not open the sample or standard container at temperatures above 15 C (60T). 

Pd removal was determined as follows. An aliquot of a representative liquid or solid sample was accurately weighed and subsequently digested by refluxing in nitric and/or hydrochloric acid using a closed vessel microwave procedure (CEM MARS5 Xpress or Milestone Ethos EZ). Cooled, digested samples were diluted, matrix matched to standards, and referenced to a linear calibration curve for quantitation an internal standard was employed to improve quantitation. All samples were analyzed by an Inductively Coupled Plasma Mass Spectrometer or ICP/MS (Perkin Elmer SCIEX Elan DRCII) operated in the standard mode. 

ABA is detected and quantified by enzyme immunoassay with a monoclonal antibody.578 The detection limit of immunoassay with a monoclonal antibody is about 0.02 pmol however, enzyme immunoassay cannot exclude cross-reactivity with unknown substances, so GC- or LC-SIM analysis using the stable isotope dilution method is recommended for correct quantitation. As internal standards labeled with the stable isotope, deuterated derivatives of ABA such as [3, 5, 5, 7, 7, 7 -2H6]ABA (ABA-//6)579 and [7, 7, 7 -2H3] derivatives of phaseic and dihydrophaseic acids580 are used. 

Under such conditions transfer of the analyte from the bulk solution to the electrode surface is principally by diffusion. A typical DC polarogram is presented in Figure 1. The half wave potential (Eyx) is characteristic of the analyte and hence yields qualitative analytical data. The analyte reduction current, produced at the electrode surface, is the limiting diffusion current (U) which is proportional to the concentration of the analyte in the solution, hence providing the quantitative information. Calibration is performed by internal standard additions or the construction of a calibration curve. 

Using isotope dilution MS method, folates were detected in positive ESI SRM mode and accnrately quantified by internal standards [102-104]. formic acid and 5-CH3-H4folate were added into various food matrices as internal standards for the quantification of folic acid and 5-CH3-H4folate, respectively [102-107]. For quantitative purposes, the CID transitions monitored were m/z 442 295, 447 295, 460 313, and 465 313 for folic acid, 5 folic acid, 5-CH3-H4folate, and 5 5-CH3-H4folate, respectively. 

FAB or LSIMS using a probe inlet does not readily lend itself to quantitative work. Firstly, it is not possible to know how much of the sample has been consumed in the analysis. Secondly, discrimination effects (see section 12.3.3) prevent the comparison of intensities between species of differing surface activity. Semiquantitative results may be readily obtained if discrimination effects are assumed to be constant for the species of interest, for example the determination of homologue distributions in a mixture. For accurate quantitation an internal standard of an isotopically enriched analogue of the analyte should be used. For example, in the determination of cationic surfactants in environmental samples [10], quantitation was achieved by using an internal standard of a trideuterated form of the analyte. In this way the standard will be subject to the same level of discrimination as the analyte. Discrimination effects between different cationic species may also be reduced by adding to the sample an excess of a highly surface-active anionic surfactant. The anionic species will dominate the matrix surface and attract cations into the surface monolayer [10]. 

For a given spectrometer, a set of relative values of S can be developed for the elements of interest. Note that the ratio fIS is directly proportional to the concentration n on the surface. The quantity / is usually taken as the peak area, although peak heights are also used. Often, for quantitative work, internal standards are used. Relative precisions of about 5% are typical. For the analysis of solids and liquids, it is necessary to assume that the surface composition of the sample is the same as its bulk composition. For many applications this assumption can lead to significant errors. Detection of an clement by XPS requires that it be present at a level of at least 0.1 %. Quantitative analysis can usually be performed if 5% of the element is present. 

Fixed volume pipettes of 100 nL and 200 nL are available in the form of platinum-iridium capillaries melted in a glass holder. Repeatability of the volume dispensed by such a nanopipette is very good (< 1 %), but the absolute volume accuracy is only about 5%. This means that for quantitative analysis, all samples and calibration standards on one plate must be applied with the same nanopipette unless calibration is by internal standard. 

Caffeine is extracted from beverages by a solid-phase microextraction using an uncoated fused silica fiber. The fiber is suspended in the sample for 5 min and the sample stirred to assist the mass transfer of analyte to the fiber. Immediately after removing the fiber from the sample it is transferred to the gas chromatograph s injection port where the analyte is thermally desorbed. Quantitation is accomplished by using a C3 caffeine solution as an internal standard. 

When possible, quantitative analyses are best conducted using external standards. Emission intensity, however, is affected significantly by many parameters, including the temperature of the excitation source and the efficiency of atomization. An increase in temperature of 10 K, for example, results in a 4% change in the fraction of Na atoms present in the 3p excited state. The method of internal standards can be used when variations in source parameters are difficult to control. In this case an internal standard is selected that has an emission line close to that of the analyte to compensate for changes in the temperature of the excitation source. In addition, the internal standard should be subject to the same chemical interferences to compensate for changes in atomization efficiency. To accurately compensate for these errors, the analyte and internal standard emission lines must be monitored simultaneously. The method of standard additions also can be used. 

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... 

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. 

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. 

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