Uncertainty for the Analysis of 2,4-D Herbicide in Urine by gc/ECD
Principle Samples of urine are acidified and extracted using toluene which contains a surrogate standard. The mixture is derivatised using diazomethane to form the methyl ester Analysis Procedure 1. Take 1 mL of each urine to be analysed 2. To each tube add 1 mL of HCl/NaCl mixture. 3. Add 2 mL of nanograde toluene containing the surrogate. 4. Stopper the tube and shake for 5 sec, then mix for 1 hour on rotating mixer. 5. Centrifuge the samples for 15 minutes at 3500 rpm. 6. Transfer 1 mL of the toluene extract to a clean quick-fit tube, add sodium sulfate (using a spatula) and shake to make sure that the Na2SO4 removes any water. 7. Add 1 mL of diazomethane reagent (see appendix for procedure on preparing diazomethane reagent). Mix the solution and let stand in a fume cupboard. 8. Leave the tubes in the fume cupboard overnight to allow for the complete decomposition of any unreacted diazomethane. 9. Transfer the toluene extract (~1 mL) to a gc vial and analyse using an EC detector. Standards Prepare separate stock standard solutions (0.1mg/ml) as follows: 1. Weigh accurately about 1.000-2.000mg of each of the standard 2,4-D, into a 10ml volumetric flask and make up to volume with methanol. 2. Prepare composite spiked urine standards as follows:
3. Using calibrated syringes (250, 100, 500 μL) dilute the amount given in Table 1 to 200 mls with blank urine in a volumetric flask.
Table 1 SPIKED URINE STANDARDS Herbicide Standard 1 Standard 2 2,4-D 500μl [250ppb] 250μl [125ppb]
Assumptions • The QC data was calculated using a real urine sample which had shown to be positive for 2,4-D 358.07μg/L. It had been analysed from 18/10/1999 to 25/10/2000 by different analysts 42 times • The uncertainty of making up the internal standard solution is not necessary as it is added to the standards and samples in the extracting solvent in the same way. The pipetting error is counted in the precision study of the repeat analysis of a positive sample • The uncertainty of the method from changes in urine matrixes is somewhat included in the standard curve uncertainty as different urines are used to make the standards over time • The uncertainty of the analyte, 2,4-D, being present in different chemical forms (eg. conjugated with a sugar) in a real sample compared to a spiked urine sample is not counted. It is considered from the literature and experience that after acid hydrolysis 2,4-D exists in the free acid form.
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