External-Standard Method

The role of a standard is to determine the mathematical relationship, in the concentration range to be measured, between the selected signal intensities and the mixture composition. 

This external standard method consists of preparing a synthetic sample containing a known quantity of the molecule to be measured (Mste), then introducing a precise volume of this solution into the spectrometer and recording the intensity of the response signal (/ste). 

without any modification of the analytical conditions, an equal volume of the solution containing the molecule to be quantified (Mx) is introduced into the spectrometer and the intensity of its response signal (Ix) is measured. Since the volumes that are introduced are equal, there is a proportionality between the response intensities and the quantities as long as the response signal intensity remains linear with respect to the concentration and as long as the signal intensity is zero at zero concentration. That is, 

In electron ionization, the response is normally linear with respect to the concentration over a wide range, often six orders of magnitude. This is not true for the other ionization techniques because of the influence which the sample quantity can have on the number of ions that is produced and on the fragmentation, and thus on the production yield of the various ionic species. For instance, in the chemical ionization mode the formation of adducts appearing at higher sample pressures changes the relative intensities in the spectrum. 

verification by a calibration curve is necessary. This calibration curve allows one to calculate the quantities of a compound to be measured in the unknown samples, to confirm the method specificity and also to define its sensitivity. In order to do this, equal volumes of a series of synthetic samples containing an increasing quantity of the molecule to be measured are introduced into the mass spectrometer and the intensity of their response signal is recorded. 

In the external standard method the detector response for pure compound is foimd by determining the peak area per unit concentration of pure compoimd by obtaining chromatograms from injections of solutions of the reference standard at known concentrations and measuring peak areas. This detector response may then be used to calculate how much pure compound is in a solution of a sample by considering the peak 

The accuracy and precision of this method are limited because the standards and the samples are analyzed at two different times, and within that space of time, the mass spectrometry response might drift. Also, the matrix of the calibration standards is difficult to match with the sample matrix. 

The three main methods of analysis differ in what is used as a reference line (1) external standard method (a line from pure a), (2) direct comparison method (a line from another phase in the mixture), and (3) internal standard method (a line from a foreign material mixed with the specimen). 

In all methods, the absorption coefficient of the mixture is itself a function of Cj, and can have a large effect on the measured intensity Alexander and Klug [14.6] were the first to clearly recognize this effect and to work out the equations needed in analysis. 

To put Eq. (14-2) in a useful form, we must express p in terms of the concentration. From Eq. (1-12) we have 

This equation relates the intensity of a diffraction line from one phase to the volume fraction of that phase and the linear absorption coefficients of both phases. 

We can put Eq. (14-4) on a weight basis by considering unit mass of the mixture. The volume of the contained ot is wjp and the volume of is wpjpp. Therefore, 

The absolute response factor (not to be confused with the partition coefficient), is not an intrinsic parameter of the compound since it depends upon the tuning of the chromatograph. To calculate the response factor AT, according to expression 4.5, it is essential that both the area A, and the mass m, of compound i injected on the column, are known. However, this mass is difficult to determine with precision since it relies simultaneously upon the syringe, upon the injector type (in GC), or upon the injection loop (in HPLC). This is why most chromatographic methods utilized for quantitative analyses, whether pre-programmed into an integrated recorder or in the multiplicity of available software, do not make use of the absolute response factors, K.  

To determine the areas of the peaks appropriate chromatographic software is used which also ensures not only the control and working of the chromatograph but also the data treatment to furnish a report corresponding to one of the pre-programmed methods of quantitative analysis. 

This method allows the measurement of the concentration (or percentage in mass) of one or more components that appear as resolved peaks on the chromatogram, even in the presence of other compounds yielding unresolved peaks. Easy to use, this method corresponds to the application of a principle common to many quantitative analysis techniques. 

The modus operandi is based upon the comparison of two chromatograms obtained successively without changing the control settings of the chromatograph 

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Ethanol concentration in the fermentation broth is determined by using gas chromatography (HP 5890 series II with HP Chemstation data processing software, Hewlett-Packard, Avondale, PA) with a Poropak Q Column, and a Hewlett-Packard model 3380A integrator. A flame ionisation detector (FID) is used to determine ethanol. The oven temperature is maintained at 180 °C, and the injector and detector temperature are maintained at 240 °C. The sample taken from the fermentation media has to be filtered and any internal standard must be added for analysis based on internal standard methods otherwise, the area under the peak must be compared with known standard samples for calculation based on external standard methods. 

External standard method (linear calibration curve of peak areas)... 

The external standard method requires that the standard is chromatographed separately from the sample and thus, the chromatographic conditions must be maintained extremely constant. The great advantage of the external standard method is that the reference standard (or standards) can be identical to the solute (or solutes) of interest in the sample. Thus, a synthetic mixture can be made up in which the concentration of the components is closely similar to those of the sample. 

The determination of the relationship between detector response and the sample concentration is termed the calibration of the method. There are two types of methods in use for the quantitative analysis of a sample, i.e., the external standard and the internal standard method. An external standard method is a direct comparison of the detector response of a pure compound (standard) to a sample.2 The calibration of the method is performed by preparing standards of varying concentration and analyzing them by a developed method. Method 1 (below) was developed for toluene, and standards of varying concentration were prepared and analyzed. The results obtained are summarized in Table 2 see Figure 3. 

Table 2 Calibration Data for External Standard Method...

Figure 4 Calibration of external and internal standard method. Chromatographic conditions — column 30 cm x 3.9 mm p-Bondapak C18 (10-pm particle size) mobile phase water acetonitrile (50 50) flow rate 1.5 ml/min column temperature ambient detector wavelength 254 nm. (A) External standard method, (B) internal standard method.

An internal standard method gives more reliable results when elaborate sample preparation is required, as in extraction of a drug substance from biological fluids, or extraction of pesticides and herbicides from soil and plant matter. The addition of internal standard (IS) to the sample and standard acts as a marker to give accurate values of the recovery of the desired compound(s). Since the determination of wt% involves the ratio of the detector responses in the two chromatograms, the injection volume is not critical as in an external standard method. 

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. 

Each reaction step was monitored qualitatively by TLC using hex-ane ethyl acetate as the developing solvent and quantitatively by GC. Impurity peaks were identified by GC/MS. An HPLC external standard method (Method 2) was developed and used to determine the purity of the final isolated product (RWJ-26240). The following rugged HPLC method was developed to optimize scheme 1, step 6 ... 

To determine the weight percent of each compound in the reaction sample, a standard and sample of known weight concentration were prepared and analyzed. The weight percent of each component purity was determined as described in the External Standard Method. 

The preferred method of analysis for imparity qnantification is through the comparison to an external standard. External standard methods rely on a stable chromatographic system and the ability to obtain a reference standard for the imparity nnder test. 

The dissolution method for the immediate-release/sustained-release tablet requires the following parameters USP paddle method, 900 ml of water, SO rpm paddle speed, 37°C, and samphng points at 20 minutes, 40 minutes, 1, 2, 4, 6, 8 and 10 hours. Robotically, ahquots (8 ml) were removed, filtered through a 10 jm polyethylene filter and transferred to the storage rack. The volume (8 ml) was replaced with heated media. The samples were assayed hy HPLC using the external standard method. 

Reduced Linear Range. Unlike the area percent and high-low methods, which use the response of the drug substance in sample injections for calculation, an external standard method uses a standard curve. Typically, the concentration range of the calibration curve is similar to that of related substances in the sample (e.g., 1 to 5% of the nominal sample concentration). Therefore, this method requires a small linear range. 

Reference Standard. One of the limitations of the external standard method is that a well-characterized reference standard is essential. In addition, each analysis requires accurate weighings of small quantities (e.g., 10 mg) of reference standard. Therefore, weighing error can affect method precision and accuracy. 

Range. Ideally, linearity should be established from 50% of the ICH reporting limit to the nominal concentration of drug substance in the sample solution (for area percent method). If the linearity does not support such a wide range of concentration, determine the linearity from 50% of the ICH reporting level to 150% of the proposed shelf life specifications of the related substance (for the high-low and external standard methods) as a minimum. This will ensure a linear response for related substances at all concentration levels to be detected during stability. 

Figure 3.12. Overall accuracy (external standard method).

For the external standard method, measure creatinine in the urine samples and calculate the results per mol of creatinine (oxalate, citrate, glycolate, and glycerate = mmol/mol creatinine sulfate = mol/mol creatinine). 

Calibration is carried out using the external standard method. The calibration curve was linear for each of nine BAs in the range of 5-100 nmol/ml. The resulting concentrations were extrapolated by the plotted curve. 

The internal standard ratio method for quench correction is tedious and time-consuming and it destroys the sample, so it is not an ideal method. Scintillation counters are equipped with a standard radiation source inside the instrument but outside the scintillation solution. The radiation source, usually a gamma emitter, is mechanically moved into a position next to the vial containing the sample, and the combined system of standard and sample is counted. Gamma rays from the standard excite solvent molecules in the sample, and the scintillation process occurs as previously described. However, the instrument is adjusted to register only scintillations due to y particle collisions with solvent molecules. This method for quench correction, called the external standard method, is fast and precise. 

Analytes are identified on the basis of retention time compared to standards and with the addition of the suspected compound to the sample (55). The diode array detector has been used recently as an additional aid in the identification of sweeteners and the determination of peak purity (56). Quantification is performed by the internal or external standard method on the basis of peak height or area. 

The internal standard method is more reliable than the external standard method. Equal amounts of one or more internal standards are added onto equal volumes of sample extracts and the calibration standards. The response factor (RF) is then calculated as follows ... 

A 25-g soil sample was extracted with methylene chloride and the extract was concentrated to a final volume of 2 mL. A 10-component alkane mixture at a concentration of each component as 50 pg/mL was used in quantitation performed by external standard method. 1 pL of the extract and the standard were injected onto the column for analysis. Determine the concentration of diesel range organics in the sample from the following data ... 

Quantitation. The organic species identified by the GC-MS analyses were quantitated by GC analysis using internal and external standardization methods. Pure compounds representative of the various compound classes identified by GC-MS were selected as standards and methylated. A specific amount of each standard was co-injected with each sample to confirm the GC-MS identifications. For quantitation purposes, each standard was injected onto the gas chromatograph prior to and following sample analyses. The response factor of each standard was calculated under analytical conditions identical to those of the sample analyses. 

Quantitation. Combined GC-SICM has been used mainly for quantitation. For a particular GA the absolute intensity of a characteristic ion in its mass spectrum is related to the amount of GA present, using standards to calibrate the instrument. Frydman et al. (35) used this "external standard method" to measure the levels of a number of GAs throughout the development of pea seeds. An alternative and preferable approach employs... 

Integration of a peak is simply the first step in data manipulation for the determination of component concentrations in a sample. Peak integration is performed in order to convert the detector signal into numerical data. There are four principal techniques for determining relative composition information about the sample, all of which rely on the construction of calibration curves. These methods are normalization, the internal standard method, the external standard method, and the method of standard additions. 

The external standard method may be applied in a number of ways. The methods operate on the same principle but vary in the amount of work required and therefore in the accuracy of the results. In all cases, standard solutions containing the solutes of interest are prepared, preferably at three different, known concentrations. The standard solutions are then analyzed, and calibration plots of peak area (height) versus concentration are constructed for each solute, as shown in Figure 7.19. From the calibration curves, the unknown concentrations of solutes in the sample can be determined. 

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