Appendix D: Analytical Methods 



2. To each tissue sample in the centrifuge tube add: (a) 35 ml of CH2CL2, (b) Standard 

 solution. Make certain that the solutions are placed into the CH2CL2. 



3. For each set of samples prepare a spiked blank ("reagent spike") by adding to a 

 centrifuge tube containing 35 ml of CH2CL2 and Standard solution. 



4. If the sample set requires a field blank ("tissue blank"), prepare this by washing down 

 the empty sample container 3 times with 10 ml of CH2CL2 each time and adding the 

 combined washings to an empty centrifuge tube. Add 5 ml more of CH2CL2 to the 

 tube, and proceed as in the next step starting at (b). 



5. For each set of samples prepare a blank ("reagent blank") by adding to an empty 

 centrifuge tube: (a) 35 ml of CH2CL2, (b) Standard solution. 



6. Prepare 2 AH/PES analyte-calibration solutions. 



7. Add 25 g Na2S04 to each tube from steps 2-5. Macerate/extract the sample in the tube 

 with the Tissumizer. Avoid spattering the tissue. 



8. Wash down the probe with CH2CL2, collecting the washings in the centrifuge tube. 

 Centrifuge the sample. 



9 . Decant the extract into a labeled flask. 



10. Add 35 ml of CH2CL2 to the tube. Repeat steps 7-9 once. 



1 1 . Wash the Na2S04/sample mass by adding 10 ml of CH2CL2 to the tube, and mixing on 

 the Vortex Genie for 5-10 seconds at setting 5-6. 



12. Repeat steps 8-9 once. 



4.3 Concentration of extract 



Initial concentration to small volume should be performed by rotary evaporation. For both 

 extraction procedures the final extracts are concentrated in a Kuderna-Danish concentrator. 

 Concentrate extract to near 1 ml with the concentrator and adjust extract volume to exactly 1 ml by 

 a gentle stream of clean dry nitrogen. Record the volume accurately. This can be done by weight. 



Caution: too strong a N2 stream may remove the compounds to be analyzed. 



4.4 Clean-up by Alumina and High Performance Liquid Chromatography 



Prior to GC-ECD, fats and other interfering substances are removed and the compounds of 

 interest are separated into different fractions. This is accomplished in a two step operation: one 

 involving Alumina solid-liquid adsorption chromatography which is followed by clean-up and 

 separation using normal phase HPLC. 



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