Appendix D: Analytical Methods 



C st = concentration of the standard (ng |il"l) 



The RF's are, thus, tabulated as ng mm"!; or if an area is measured h st is replaced by a st 

 and the RF's are tabulated as ng per unit area. 



Second, from a known aliquot of the sample injected the response of the ECD to each peak 

 to be quantified is measured (peak height in mm, h sam ). Note that integrator areas are usually 



reported independently of the chart attenuation. But if peak heights or areas are measured by hand, 



then the chart attenuation must be the same between standard and sample injections or an 



appropriate correction in the calculations. 



Third, perform the following calculation for each peak: 



Csam = n sam x Rr x _o_ x _L 

 VsamM 



where h sarn = peak height of the compound in the sample (mm) 

 V sam = volume of the sample extract injected (|il) 

 M = dry wt. or lipid wt. of the sample (g) 

 A = total volume of the sample extract (|il) 

 C sam = concentration of the compound in the sample (ng g"l dry wt., or ng g _1 lipid) 



Again, h sam can be replaced with a sam and the RF expressed in area units applied. 



The quantity (A/Vsam) is tne inverse fraction of the total sample analyzed and is termed the dilution 

 factor. 



An internal standard can be used to correct for losses of analyte throughout the analytical 

 procedure and for errors in volume measurement (which increase as sample volume is decreased) 

 and for slight variations in detector responses. The response factor for the internal standard 

 (RFjs) is determined as in equation 1 from several injections of the IS under identical 



chromatographic conditions as for samples. Sufficient amount of the internal standard is added to 

 the sample before extraction to result in a measurable peak in the final chromatogram of the extract. 

 The peak height of the internal standard measured in the sample chromatogram (h j s ) is then used to 



compute a corrected dilution factor as follows: 



IS x l/RFi s x l/hj s = XFj s 



where IS is the total amount of internal standard that was added to the sample (in ng). This 

 corrected dilution factor (dimensionless, ng ng"*) replaces XF (dimensionless, vol vol'l) in the 

 calculations applied to sample peaks: 



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