TRIAZINE HERBICIDES and THEIR METABOLITES: METHOD 8315, Issue 1, dated 15 March 2003 - page 3 of 6 SAMPLE PREPARATION: 3. W eigh 0.7 g sodium chloride into screw-top culture tubes. 4. W eigh 0.5 g sod ium bicarbon ate into eac h of these tubes. Cap these tubes an d then they can be sto red fo r at least tw o weeks and m ost like ly for as long as necessary. 5. Label two culture tubes (one empty and one containing the two salts) and one centrifuge tube with a unique n um ber for ea ch sam ple. NOTE: The labelling system is unimportant as long as there is no danger of the samples being m ixed-up and s o a record is ke pt of which sam ple correspond s to which tube num ber. 6. Thaw s am ples to roo m tem perature. U sing warm water is oka y. 7. Mix thoroughly as urine becomes inhomogeneous upon freezing. 8. Transfer 5 m L of sam ple to its culture tube conta ining the salts . Leave the caps off th e tubes until samples have been dispensed to every tube and any CO 2 evolved has dispersed. 9. Sp in on R oto -torque for about one m inute on settin g 4 Hig h to dissolve salt. So m e salt w ill rem ain in the tube. 10. Dispense 5 mL ethyl ether into each of the urine tubes. Again leave caps off or on loosely to allow for gas to disperse. 11. Roto-torque for fifteen minutes on setting 4 High. 12. Centrifuge samples for five minutes at 3000 rpm. 13. Rem ove ether layer (top) with short transfer pipet to the second labelled culture tube. Remove all the ether; taking some aqueous phase does not appear to be detrimental to the analyses. 14. Dispense 5 mL ethyl acetate into each sample tube containing the remaining aqueous phase. 15. Roto-torque for fifteen minutes on setting 4 High. 16. Centrifuge samples for five minutes at 3000 rpm. 17. Rem ove organic layer (top) with short transfer pipet and add to the ether layer. Again, remove all the organic layer. 18. Fill the filtration columns about 3/4 full of anhydrous sodium sulfate. 19. Balance the column on top of the labelled 15-mL centrifuge tubes. 20. Using a transfer pipet, transfer the combined extract to the appropriate filtration column. 21. W ash the tube which held the organic phases with one pipetful ethyl ether and add this to the filtration column. 22. W ash the filtration column with another 2 m L ethyl ether. 23. Allow th e filtration colum ns to drain com pletely. 24. Place the centrifuge tubes in the analytical evaporator and gently evaporate the solvent with a stream of nitrogen until the tube is dry. NOTE: A waterb ath helps dissipate the co ld, but it is im porta nt the bath be k ept at room tem pera ture. Raising the bath temperature to 30 oC m ay cause marked decreases in the recoveries of analyte s (5) and (6 ) an d m ay well affe ct th e othe r an alyte s as well. 25. Rinse the sides o f the tubes with about 0.5 m L ethyl ether. 26. Evaporate to dryness again. 27. Add 10 :L internal standard solution to each centrifuge tube. 28. Add 90 :L ethyl acetate to each tube and m ix well. 29. Let the extracts sit for at least thirty minutes. 30. Label autosampler vials and place an insert into each vial. Have the caps ready for the vials. 31. Trans fer the entire e xtrac t to the c orrect autosa m pler vial with a long tran sfer pipet. 32. Cap the vial and place in approp riate tray position in the GC autosam pler.
CALIBRATION AND QUALITY CONTRO L: 33. The stock solution of the six analytes is prepared from the neat solids to about 240 :mol/L by accurately weighing 1.25 mg of each analyte into a 25 mL volumetric flask (larger flasks could be used with a concom itan t increase in the am ount of solid weighed out.) Fill the flas k to the line with ethyl acetate. It is generally necessary to sonicate the flask for complete and quick dissolution, after which the analytes stay in solution. Aliquot into autosampler vials, cap, and store this solution in the refrige rator. Calculate th e ex act c onc entra tion of eac h an alyte from the am oun t of so lid weigh ed o ut. In ethyl acetate these analyte s are quite stab le fo r ex ten ded periods of tim e. A t no point did degradation of the standard solutions become a problem, so they are stable for at least six months under these conditions. NIOSH Manual of Analytical Methods (NMAM), Fourth Edition
