The International Mussel Watch 



5.6 Quantification 



The principle methods for quantification of compounds use both internal and external 

 standards. In the external standard method, absolute response factors are determined by 

 independent chromatographic runs of standards in known concentrations. These response factors 

 are used to calculate the concentrations of compounds in the sample extract. The external standard 

 method can be used when peak areas or heights are highly reproducible. Internal standards are 

 added to the samples, preferably in a concentration range to produce a similar response on the ECD 

 as the contaminants present in the samples. Preliminary "range finding" analyses are necessary to 

 determine the amount of internal standard to be added to the samples. The addition of standard has 

 to take place in a highly reproducible way. The internal standard method is calibrated in terms of 

 response ratios, involving standards that do not occur in the environmental samples, do not 

 interfere with the compounds of interest, are stable to all procedures applied to the samples and 

 behave similarly to the compounds of interest. The recovery of the internal standard is used to 

 more accurately assess the fraction of total sample injected 



The reproducibility of area counts from the integrator using the splitless injection technique 

 «an be as good as a few percent relative standard deviation (RSD). Under such conditions, the 

 external standard method is reliable. Several injections should be made of standard solutions 

 containing a range of concentrations, allowing the determination of the linear range of detector 

 response and the response factor of each compound. The calibration mixture must be analyzed 

 under the same instrumental conditions used for the samples. Differences in sensitivity of the 

 detector for different compounds are accounted for by using the individual response factors. The 

 amount injected, therefore, has to be highly reproducible. This system has to be calibrated 

 frequently in order to minimize instrumental (column) drift. 



After comparison of the retention times of peaks in the sample chromatogram to those of a 

 corresponding standard, peak heights (or areas) are measured. The concentration of a particular 

 compound is then calculated using the following formulas. As an example, the concentration of a 

 compound in biological tissue, calculated as ng g~l dry weight or lipid weight as follows: 



First, the response of the ECD to each compound to be quantified is calculated from 

 injections of standards. The response factors (RF's) are calculated as: 



RF=C st xV st xl/h st 



where h st = peak height of the compound in the standard (mm) 

 V st = volume of the standard injected (|il) 



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