Standards, quantitation Internal

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... 

Guo et al. [24, 25] analyzed these in positive-ion mode APPI with transitions m/z 271—>-213 for DHEA and 273—>213 for the dideutero internal standard. Quantitative data from these studies are reported by Holst et al. [30]. 

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. 

Acceptable laboratory method blanks must not contain any chemical interference or electronic noise at the m/z of the specified unlabeled PCDD/PCDF ions that is greater than 5 percent of the signal of the appropriate internal standard quantitation ion. 

All positive samples associated with a contaminated method blank and any samples which contain peaks that do not meet all of the qualitative identification criteria in Section 11 associated with a contaminated method blank must be reextracted and reanalyzed. Acceptable laboratory method blanks must not contain any chemical interference or electronic noise at the m/z of the specified unlabeled PCDD/PCDF ions that is greater than five percent of the signal of the appropriate internal standard quantitation ion. A peak that meets identification criteria in the method blank must not exceed two percent of the signal of the appropriate internal standard. 

Internal standardization Quantitative measurements may be made by an internal standard method using heptadecanoic acid which can be added to the samples before transesterification. The amount added is dependent on the concentration of free fatty acids in the sample. 

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]. 

A 2000-fold increase in concentration was achieved for traces of the mutagenic HAA PBTA-1 (32a) and PBTA-2 (32b) in 0.5 L samples of polluted river water by SPE on an Empore disk (C18) after drying the disk it was extracted with 5 mL of MeOH, the solution was evaporated to 0.25 mL and 2.5 ng of diazinon-d 0 (73) was added as internal standard. Quantitative recoveries were observed for both pollutants from spiked river water at 5 to 10 ngL 1 levels. Aliquots of 10 p,L were analyzed by HPLC-MS (Section IV.B.1)95. 

An example of internal standard quantitation is given in Table 10.1. 

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... 

LC-MS method to measure the concentration of vitamin B5 in human urine. Hopantenic acid (HOPA) is used as internal standard. Quantitative MS detection is performed in the single-ion monitoring mode. A linear calibration curve was obtained with F = 0.999 in the concentration range of 0.25-10 p-g/ml. The lower limit of detection is 0.1 pg/ml, with an intraassay coefficient of variation <5% and recoveries between 96 and 108%. 

A sixth spectrophotometric method for the quantitative determination of Pb + levels in blood uses CQ+ as an internal standard. A standard containing 1.75 ppb Pb + and 2.25 ppb CQ+ yields a ratio of Sa/Sis of 2.37. A sample of blood is spiked with the same concentration of Cu +, giving a signal ratio of 1.80. Determine the concentration of Pb + in the sample of blood. 

A seventh spectrophotometric method for the quantitative determination of Pb + levels in blood gives a linear internal standards calibration curve for which... 

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... 

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... 

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. 

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 ... 

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. 

To produce a quantitative result, chromatographic peak areas of identified target compounds are compared with peak areas of the internal standards, which are of known concentration. 

Quantitative mass spectrometry, also used for pharmaceutical appHcations, involves the use of isotopicaHy labeled internal standards for method calibration and the calculation of percent recoveries (9). Maximum sensitivity is obtained when the mass spectrometer is set to monitor only a few ions, which are characteristic of the target compounds to be quantified, a procedure known as the selected ion monitoring mode (sim). When chlorinated species are to be detected, then two ions from the isotopic envelope can be monitored, and confirmation of the target compound can be based not only on the gc retention time and the mass, but on the ratio of the two ion abundances being close to the theoretically expected value. The spectrometer cycles through the ions in the shortest possible time. This avoids compromising the chromatographic resolution of the gc, because even after extraction the sample contains many compounds in addition to the analyte. To increase sensitivity, some methods use sample concentration techniques. 

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. 

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