Calibration internal standards

An internal standard is a known amount of a nonanalyte element or compound that is added to all samples, blanks, and standard solutions. Calibration with internal standardization is a technique that uses the signal from the internal standard element or compound to correct for interferences in an analysis. Calibration with internal standardization improves the accuracy and precision of an analysis by compensating for several sources of error. 

Concentration ratio (A/S) in sample Concentration ratio (A/S) in standard 

The method of internal standardization is widely used in spectroscopy, chromatography, MS, and other instrumental methods. The use of internal standards can correct for losses of analyte during sample preparation, for mechanical or electrical drift in the instrument during analysis, for volume change due to evaporation and other types of interferences. The internal standard must be chosen carefully, usually so that the chemical and physical behavior of the internal standard is 

Time of Measurement Signal Counts 10 ppb Pb, m/e = 208 Signal Counts 20 ppb BI, m/e = 209 Ratio of Counts Pb/Bi 

---

A single-point internal standardization has the same limitations as a singlepoint normal calibration. To construct an internal standard calibration curve, it is necessary to prepare several standards containing different concentrations of analyte. These standards are usually prepared such that the internal standard s concentration is constant. Under these conditions a calibration curve of (SA/Sis)stand versus Ca is linear with a slope of K/Cis-... 

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

Using the Regression Equation Once the regression equation is known, we can use it to determine the concentration of analyte in a sample. When using a normal calibration curve with external standards or an internal standards calibration curve, we measure an average signal for our sample, Yx, and use it to calculate the value of X... 

Matrix effects were evaluated by Gago-Ferrero et al. [23]. Both signal suppression and signal enhancement were observed. The extent of these effects was found to be fairly dependent on the UV filter. Thus, quantification should be performed by standard addition or internal standard calibration. Since standard addition is a high time-consuming procedure, internal standard calibration with appropriate isotopi-cally labeled compounds is the best option. 

Dunnivant, F.M. and A.W. Elzerman. 1988. Determination of polychlorinated biphenyls in sediments, using sonication extraction and capillary column gas chromatography-electron capture detection with internal standard calibration. Jour. Assoc. Offic. Anal. Chem. 71 551-556. 

Even once a method is standardized, erroneous results can still be generated. As a result, it is critical to have robust quality control procedures in place. Here, careful attention should be paid to identify opportunity for in-process control measures such as internal standards, calibration, control plates, replicates and so on as opposed to post-processing data review steps. Inline QC approaches allow sources of error to be identified and remedied much more rapidly and help limit costly re-tests, or the possibility of erroneous data leaving the laboratory. 

Here is a student procedure to measure nicotine in urine. A 1.00-mL sample of biological fluid was placed in a 12-mL vial containing 0.7 g Na2CO , powder. After 5.00 pig of the internal standard 5-aminoquinoline were injected, the vial was capped with a Teflon-coated silicone rubber septum. The vial was heated to 80°C for 20 min and then a solid-phase microextraction needle was passed through the septum and left in the headspace for 5.00 min. The fiber was retracted and inserted into a gas chromatograph. Volatile substances were desorbed from the fiber at 250°C for 9.5 min in the injection port while the column was at 60°C. The column temperature was then raised to 260°C at 25°C/min and eluate was monitored by electron ionization mass spectrometry with selected ion monitoring at m/z 84 for nicotine and m/z 144 for internal standard. Calibration data from replicate... 

Instruments must be calibrated to the appropriate site instrument calibration procedure using calibration and test equipment traceable to accepted national or international standards. Calibration procedures should be produced for each unique type of instrument. An instrument calibration procedure should ... 

It may be noted that for calibration each analyte is needed to be spiked with its labeled analog. This enhances the cost of the analysis. High cost and often unavailability of labeled analogs are the major drawbacks of isotope dilution method as compared with external and internal standard calibration methods. The isotope dilution method should be, theoretically, more accurate than the internal standard method, as the chromatograpic response and the retention times of the labeled analogs are closest to the compounds. 

The RF should be determined using standards at various concentrations, and an average RF value should be used in the calculation. Both the external standard and internal standard calibration methods for GC analysis are fully discussed in Chapter 1.3. 

The extract should be then injected onto the GC column for GC or GC/MS analysis. If internal standard calibration is performed, spike three or more internal standards into the sample extract. Any organophosphorus pesticides whose analysis is not required or which is not found to be present in the screening test may be used as an internal standard. The chromatographic columns and conditions are presented below ... 

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 standard calibration is used when the changes in the analytical system are known to be frequent and substantial. To compensate for these changes, internal standards at known concentrations are added to all calibration standards, field samples, and laboratory QC samples prior to analysis. Internal standards are synthetic analogs of specific target compounds or compounds that are similar in nature to the target analytes and that are not found in environmental samples. Internal standard calibration is a requirement of GC/MS methods. Laboratories sometimes use it for GC methods as it significantly improves the accuracy of compound quantitation. 

During the preparation of standards for internal standard calibration, the same volume and concentration of the internal standard is added to every calibration... 

To perform an external or internal standard calibration procedure and to calculate sample results, the laboratory conducts a series of calculations. Although these calculations are computerized at a modern laboratory, professional judgment and experience of the analyst are critical in the selection of the appropriate calibration model and for correct programming of the data acquisition system that collects the data and performs the calculations. 

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. 

Internal standard calibration can be used to compensate for variation in analyte recovery and absolute peak areas due to matrix effects and GC injection variability. Prior to the extraction, a known quantity of a known additional analyte is added to each sample and standard. This compound is called an internal standard. To prepare a calibration curve, shown in Figure 4.6b, the standards containing the internal standard are chromatographed. The peak areas of the analyte and internal standard are recorded. The ratio of areas of analyte to internal standard is plotted versus the concentrations of the known standards. For the analytes, this ratio is calculated and the actual analyte concentration is determined from the calibration graph. 

Although internal standard calibration compensates for some errors in external standard quantitation, there are several difficulties in method development. First, choosing an appropriate internal standard can often be difficult, as this compound must be available in extremely pure form and it must never appear in the samples of interest. Second, it cannot interfere in either the extraction or the chromatography of the analytes. Finally, it must be structurally similar to the analytes, so that it undergoes similar extraction and chromatography, otherwise, the compensation will be lost. 

International Standard, Calibration and testing laboratory accreditation systems D General requirements for operation and recognition, ISO/IEC Guide 58 1993. 

Compound analysis by GC. Individual compounds are determined by gas chromatography (GC), GC in combination with a Mass-Spectrometer (MS) is preferred, GC in combination with a Flame Ionisation Detector (FID) with internal standard calibration is an alternative. 

Similar methods were reported by others [35-37]. Frerichs et al. [35] performed a LLE and a linear gradient between 25 and 80% acetonitrile in a 5 mmol/1 acetate buffer. A RIT-analogue was applied as internal standard. Calibrated concentration ranges are summarized in Table 12.2. LC-MS analysis time is 3.5 min. The chromatogram obtained from a patient sample, after administration of AMP, RIT, and LOP is shown in Figure 12.2. 

Data processing and calibration. Use internal standard. Calibrate frequently. 

In the case of internal standard calibration this is not necessary and small variations in injection volume cease to be important. If sample preparation is complicated or demanding, the internal standard approach is strongly recommended. In this case the standard is added before the first preparation step has begun. The choice of a suitable internal standard may not be easy. It must be a pure, clearly defined compound with similar properties with respect to sample preparation, chromatographic separation and detection to the compound(s) of interest. If possible it should be eluted in a chromatogram gap and not at the very beginning or end. Examples can be found in Figures 6.11, 11.2, 11.6, 13.1, 13.2 and 22.4. 

Three primary methods are used for calibration external standard calibration, internal standard calibration, and standard addition. Figs. 1-3 show the typical response curves for each method. 

Internal standard calibration similarly requires the preparation of external standard solutions, but in addition a constant concentration of a second compound is added to each sample. The sample concentration is directly proportional to the ratio of the analyte to internal standard. This method is used for the analysis of biological samples and other more difficult analyses. 

Internal Standard Calibration, Plus Standard Deviation, 589 Chapter 20, Problem 18... 

FIGURE 10-16 Internal standard calibration curves with an tCP source. Here, an yttrium line at 242.2 nm served as an internal standard. NoUce the lack of interelement interference. (From V. A. Fassel. Science. 1978. 202,187. With permission Copyright 1978 by the American Association for the Advancement of Science.)... 

Quantitative analysis using external calibration, internal standard calibration, and the method of standard additions are all possible with SPME. Calibration is discussed in Section 1.5.2 and at greater length in Chapter 2. 

If the Bi internal standard signal is used and the ratio of the Pb signal to the Bi signal is calculated, the internal standard calibration factor is obtained ... 

---