Calibration of Internal Standard

Stock solutions of 1-acetylpyrrolidine, and an internal standard (1-propionylpyrrolidine or 1-methylnaphthalene) in dioxane at a concentration of 10 mg ml-1 are prepared. A mixture of 1ml each of the 1-acetylpyrrolidine and internal standard solutions is used to establish the GC response factor. The calibration is made by injecting a 2-/A aliquot of the mixture into the gas chromatograph and measuring the peak areas. 

Wood meals or ground pulp samples (Note 2) equivalent to about 25 mg of lignin are weighed accurately into a 20-ml glass centrifuge tube (Note 3). After 

The washed, reduced sample is dried by solvent exchange (Note 5) with acetone (7ml) followed by pyridine (7 ml). Each solvent wash is repeated three times. 

Lignin. A 20-mg sample in a 20-ml pear-shaped flask is acetylated with 1 ml of a pyridine-acetic anhydride (1 1, v/v) solution at ambient temperature for 15 h. The mixture, after the addition of 8 ml of 50% aqueous methanol, is evaporated to dryness under vacuum. The solvent treatment is repeated three times with toluene (3 x 8 ml), and once with methanol (8 ml). The acetylated sample is dried in vacuo at 50 °C overnight. 

The acetylated lignin, dissolved in 1.0 ml of dioxane containing 5 mg of an internal standard, is treated with 1.0ml of a dioxane-pyrrolidine (1 1, v/v) solution which initiates the aminolysis reaction. The rate of the 1-acetylpyrrolidine formation is followed by periodically injecting 1-2/d of the reaction mixture into the gas chromatograph. The initial rapid phase is generally complete within 20 min, but a total of 60min is required to complete the deacetylation process. 

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The procedure comprises the addition of a constant amount of internal standard to a fixed volume of several synthetic mixtures which contain varying known amounts of the component to be determined. The resulting mixtures are chromatographed and a calibration curve is constructed of the percentage of component in the mixtures against the ratio of component peak area/standard peak area. The analysis of the unknown mixture is carried out by addition of the same amount of internal standard to the specified volume of the mixture from the observed ratio of peak areas the solute concentration is read off using the calibration curve. 

ISO (1990) ISO/IEC 25 General requirements for the competence of calibration and testing laboratories and ISO/IEC DIS 17025 General requirements for the competence of testing and calibration laboratories. International Standards Organization, Geneva. 

For the example of toluene given above, the external standard method can be converted into an internal standard method by adding anisole (an appropriate internal standard) to both standard and sample. The retention time of anisole is 4.5 minutes if analyzed by the method above. To calibrate the internal standard method for toluene, toluene standards of concentration 0.3 to 1.5 mg/ml containing 0.5 mg/ml anisole were prepared. The detector response as a function of the amount of sample injected is shown in Figure 4B. 

As mentioned in the last section, when a related substance is added to both the chemical standards and to the samples problems with variations in injection volumes are removed. There is another use for internal standards of this kind, i.e. where the standard acts as an internal calibrant. The internal standard has to behave in the same way as the sample in relation to the measurement process, except that the signals can be distinguished from each other. When the related substance is added early on in the measurement process, any losses of analyte as a result of the measurement process are equally likely to affect the chemical standard and the analyte. Thus, no adjustment to the result, to compensate, e.g. for poor recovery, is necessary. The concentration of the sample is obtained from the ratio of the two signals (one from the standard and one from the sample). 

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

Quantitative PCR has been widely used to determine the amount (number of molecules) of DNA molecules in a test sample. The best quantitative PCR method involves the addition of known amounts of a similar DNA or RNA fragment, such as one containing a short deletion or specific mutation, to the test sample before amplification. Such internal standards must be precisely calibrated to ensure that they are amplified and detected in a form and manner that are similar to the test sample. The ratio of the internal standard and the targeted template will depend on the amount of internal standard added and allows for the determination of the amount of the targeted molecule in the test sample. Therefore, the ideal standard for quantitative amplification based assays should have a structure that is comparable to the template of interest and which allows for the simultaneous amplification of both template and standard using a single primer pair. 

Assays based on the use of an internal standard use response factors to compare the sample solution with the calibration solution. In this case a simple one-point calibration is used. The concentration of betamethasone can be ignored since it is the same in Solutions 1 and 3 it should usually be the case that the same concentration of internal standard is present in the calibration and sample solutions. If this is the case then for the assay described above ... 

The amount of internal standard added should be similar to the amount of analyte contained in the sample. In fact, errors are minimized when the relative responses for analyte and internal standard are comparable. If a wide concentration range is to be measured, the amount of internal standard should be selected to maximize precision where it is critically important. As a general rule, with a six-level calibration curve, the internal standard concentration, should be between the second and third calibration standard concentration depending on the relative instrument response for analyte and internal standard and precision at the LLOQ level (i.e., internal standard should be closer to LLOQ if the precision of the LLOQ is low). Many bioanalytical laboratories use a written policy for rejecting sample with low internal standard even though it is not suggested by the 2000 Washington conference final report. 

A new calibration curve must be implemented every time a new stock of internal standard solution is prepared, and at least twice per year. New calibration curves are validated by the following criteria for acceptability point-to-point comparison (<10% difference from the previous calibration curve), coefficient of linear regression (>0.99), intercept and slope (<10% difference from previous calibration curve). Normal and abnormal control samples are calculated against the new and the old curve and compared to the current quality control (QC) mean as the final step in the validation of the new curve. The new calibration curve is then used with subsequent runs if the curve validation is acceptable. Curves are unique to each instrument and therefore must be established for each instrument prior to clinical use. 

A quality assurance sample is run with every batch of samples and results should not differ daily by more than 15%. Profile analysis licensed for commercial clinical diagnostic use may have to meet special requirements. For example, quantification against a five-point standard curve was required for each compound. We prepare a series of calibrants with identical amounts of internal standard, but increasing amounts of reference analyte, typically covering 100-fold dynamic range. 

Concentration internal standard element determined by solution based calibration True concentration of internal standard element in the sample... 

Quantitative results were produced for each compound on the basis of internal standard method calculations. A three-point calibration curve was generated for each compound by using peak areas of a quantitation ion extracted from the mass spectrum of the compound. The ion was selected on the basis of it being a uniquely characteristic mass of the compound. The use of extracted ion quantitation produces more accurate results than total ion-current quantitation in cases in which two or more components are not completely resolved chromatographically. This situation is generally the case in complex mixture analysis. The quantitation ions selected for each of the compounds in the mix are listed in the box. 

An internal standard is a compound that is not present in the sample, but is chemically and physically similar to the analytes of interest. A fixed quantity is incorporated into the calibration solutions. The same concentration of internal standard is added to the samples during extraction to compensate for analyte recovery and injection variability. As seen in Figure F2.3.I, Echinenone, which is not typically found in foods, is used as the internal standard. Unfortunately, compounds which may be used as internal standards for carotenoid analysis are not readily available commercially. 

Calibration standards can be of two types external standards and internal standards. With external standards, multiple concentrations of the standards are injected, areas are measured, and a calibration curve is platted. Unknown samples are then injected, chromatograms run, and areas are calculated and compared with the calibration curves to determine amounts of each compound present. With internal standards, known amounts of an internal standard are added to each known concentration of standard compound and areas or peak height response factors relative to those of the internal standard are calculated. When unknowns are run, a known amount of internal standard is added to the unknown sample, response factors are calculated relative to the internal standards, and amounts of each unknown present are calculated from the standards calibration factors. Internal standards are usually used to correct for variations in injection size due to different operators and injection techniques. Internal standards can also be used to correct for extraction variation in GC/MS target compound quantitation, this standard is referred to as a surrogate standard. Generally, an internal standard is used for one purpose or the other, not both at the same time. 

Add Internal Standards—As part of internal standard calibration procedure, the analyst adds internal standards to the calibration standards and sample extracts prior to analysis. 

Internal standards are brominated, fluorinated or stable isotopically labelled analogs of specific target compounds or other closely related compounds not found in environmental samples that are added to all standards, field, and laboratory QC samples as part of internal standard calibration procedure. The addition of internal standards takes place after the samples have been prepared and before they are analyzed. 

As individual sample QC checks, internal standards are important in compound quantitation. They should be monitored with the same care as other QC checks. The deterioration of internal standard area counts (the area under a chromatographic peak) reflects the changes in the analytical system that may eventually degrade the quality of analysis to an unacceptable level. EPA Methods 8260 and 8270 require that the area for each internal standard be within the range of —50 to +100 percent of the area of the internal standards in the most recent CCV (EPA, 1996a). This requirement may be used as a general rule for all other methods that use internal standard calibration. 

The mutual recognition of international standards can be achieved by agreements between National Metrology Institutes (NMI). Calibration as an important activity in establishing traceability should be done by competent personnel. Therefore the competence should be assessed by a third party. Consequently agreements are also needed for accreditation bodies. 

GC-MS can be used to analyze organochlorine pesticides, for example, a-, [3-, y-HCH, HCB, and polychlorinated biphenyls (PCB). The components are quantified by using an internal standard. Furthermore, a calibration is performed with a standard mixture containing known concentrations of the components to be measured and one or more components not contained in the sample (internal standards). The calibration is followed by injection of the sample containing known amounts of internal standards. Quantification is relative to the internal standard. In this way, the sample extract volume will not be included in the calculations, and it is not necessary to accurately determine the final sample volume after evaporation of the injection volume. The GC-MS instrument should be calibrated every day. The sensitivity of the mass spectrometer can, for instance, be controlled daily by determining the signal-to-noise ratio for a given amount of a chosen component (PCB-101 could be one such component). For further details of the method, the reader is referred to different manuals and papers on the subject.417... 

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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