Chapter 12
Sampling, sample preparation and dosing
To test SPE recovery and to assure concentration addition as mixture model (see Chapter 8) you prepare a stock of 100 chemicals each in a concentration of 1 mg/L in methanol.
To each of the water samples (from exercise 12B2) you spike 200 µL of this methanol stock (called “Mix”) before you do the extraction. You calculate the EFSPE with respect to the water sample but suddenly you realise that you might have a problem because each spike sample has the same amount of chemicals added but the volumes of water sample differ. Can you deal with this and still calculate the recovery or do you have to spike in a different way, e.g., with a fixed concentration of water volume to spiked volume?
| Water sampled | SPE extract | |
|---|---|---|
| WWTP influent | 0.5L+200 µL mix | 500 µL |
| WWTP effluent | 1L+200 µL mix | 500 µL |
| Surface water | 2L+200 µL mix | 500 µL |
| Drinking water | 4L+200 µL mix | 500 µL |
| Mix | Methanolic stock of 100 chemicals in 1 mg/L |
You also test your mix from 12E1 directly in the assay (in form of its methanolic stock). What is the virtual enrichment factor EF of this mix if it had been treated like a water sample (100% recovery assumed)? Is the virtual EF of the mix the same for all water samples? You have only one attempt to test the spike mix, can you still calculate the recovery in relation to all water samples?
You prepared three process blanks with 100 mL of LC water passed through your SPE cartridge. The samples are the same as in 12B2 (see table below) Normally you never see any effects of your blank but for once you are surprised. You dosed 10 µL of the SPE blank extract in methanol in 1 mL of medium (just like you did with all samples) and this highest concentration showed 30% of activation of the estrogen receptor.
| Water sampled | SPE extract (methanol) | |
|---|---|---|
| WWTP influent | 0.5L | 500 µL |
| WWTP effluent | 1L | 500 µL |
| Surface water | 2L | 500 µL |
| Drinking water | 4L | 500 µL |
| SPE Blank | 100 mL LC water | 500 µL |
Bummer! Your samples are very valuable, and you do not want to throw them away because of the poor blank. Can you save the samples? You remember that you have read in chapter 11.3.3.5 that a blank correction is possible on the toxic unit (TU) or effect unit (EU) level, provided the blank does not have more than 50% of the TU or EU of the sample. You reason that you might be able to do a blank correction also on the level of estradiol equivalent concentrations. But how do you calculate the EEQ of the blanks for blank subtraction?
Note that this is a mock example - you should never ever have a positive blank in an estrogenicity assay.