Extraction with and without dSPE and Determination of Compound

Concentration within Sweet Potato Leaves

Section 3.2.2.4 demonstrated that the recovery of analyte spikes onto sweet potato leaf homogenate were not significantly lower for the extracts cleaned via dSPE than those that were not (i.e. compounds are not lost during dSPE). Since the target compounds are not lost during dSPE, the extracts of identically spiked homogenates should give the same ELISA response with or without dSPE; if they do not (as was observed), then the matrix components within the uncleaned extracts must be assumed to be confounding.

no-dSPE Sample dSPE Sample Caffeine concentration (ng/g) spiked onto homogenate Response Difference (ng/g) (no-dSPE less dSPE)

Sample 1 Sample 2 0 4.3E+01 8.8E+00 3.5E+01

Sample 3 Sample 4 5.01E+01 6.2E+01 3.0E+01 3.2E+01

Sample 5 Sample 6 2.51E+01 5.3E+01 2.1E+01 3.2E+01

Sample 7 Sample 8 5.01E+01 5.4E+01 2.9E+01 2.5E+01

Sample 9Sample 10 2.51E+01 5.6E+01 2.0E+01 3.6E+01

3.1E+01 3.5E+01 Caffeine "Recovered" from homogenate (ng/g) no-dSPE Caffeine Recovered from homogenate (ng/g) with dSPE

Average Response Difference Difference Unspiked Samples

Table 15: Determining the difference in caffeine ELISA response for extracts without dSPE cleanup vs. those with dSPE

Table 15 shows the caffeine ELISA responses of identically spiked homogenates with and without dSPE. The response (fit) of the spiked extracts that did not undergo dSPE are significantly greater (p=0.0007 when performing a paired t-test) than those that did with a mean difference 31.3 +/- 7.0 (Appendix C.5). Since the recovery rate (response above the

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appropriate unspiked sample) with and without dSPE are not significantly different (section 3.2.2.4) this observed difference is expected to arise from confounding matrix components in the uncleaned extract (i.e. caffeine ELISA responses for the extracts without dSPE are

giving, on average, a false response indicative of 32ng/g). The difference between the

unspiked samples (35ng/g) confirms these findings and the concentration of 8.8ng/g observed in the unspiked sample with dSPE is expected to be the actual concentration within the homogenate. Given the 1:1 ratio of leaves to LGW in the homogenate, this represents 17.6ng/g within the leaves. As mentioned in section 3.2.1.1, while few studies have reported observing caffeine in crops within the food distribution system, Calderón-Preciado et al. (2011) observed caffeine in alfalfa and apple tree leaves, (irrigated under field conditions with a variable river-water and reclaimed waste water mixture) at concentrations between 21- 55ng/g and 0.1-110ng/g respectively.

The same observation (similar recovery rates, with significantly elevated responses for the samples without dSPE) can be made for extracts analyzed for triclosan (Appendix C.5). Indeed, the response (fit) of the spiked extracts that did not undergo dSPE are

significantly greater (p=0.006 for unpaired t-test) than those that did with a mean difference of 4.2ng/g +/- 2.2 (Appendix C.5). Given that the response (fit) of the unspiked sample for the extract that did not undergo dSPE was 5.5ng/g (Appendix C.4) it was determined that the response was likely an artifact of confounding matrix components and that the below

measureable detection response observed for the sample cleaned with dSPE is more likely. For estradiol, the response (fit) of the spiked extracts that did not undergo dSPE are significantly greater (p= 0.005 for paired t-test) than those that did with mean difference and 95% confidence 85pg/g +/- 38 (Appendix C.5). The response of the unspiked sample that

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did undergo dSPE was below the LOD for the estradiol ELISA and expected to be a more accurate reflection of the estradiol concentration within the leaves.

In summary, the recovery rate observed (dSPE vs. no-dSPE) was not significantly different (p>0.05) for any of compounds (section 3.2.2.4), indicating that the analytes are not significantly lost during dSPE. At the same time, the response of the homogenate-spiked samples for the extracts that did not undergo dSPE were all significantly higher than the response than those that did. This indicates the presence of confounding matrix components within the uncleaned samples, and that samples without dSPE cleanup are likely to give an inaccurately large estimation of the concentration of compounds within any sample. Thus it was determined that dSPE should be utilized in the preparation of extracts for ELISA analysis.

3.2.3 Phase 3: Analysis of All Greenhouse Experimental Matrices Near Environmental Concentrations

In the months following the Phase 1 and Phase 2 initial investigations, numerous ELISAs were run on a variety of samples and many lessons learned. These lessons influenced the design of the final method compatibility and subsequent matrix extraction efficiency experiments. The following summarize some of the lessons learned and their design implications for the final compatibility and efficiency experiments:

• It was determined that serial dilutions of environmental samples should be at tight intervals (10 fold 20 fold 40 fold) rather than wide (10 fold 100 fold 1000 fold) since the apparent concentration of the unspiked homogenate extracts, once dilute 10 fold, do not appear to be an order of magnitude above the lower LOD for the ELISA kits. (i.e. 100 fold dilutions almost always drop the concentration out of range and confirmatory analysis is not accomplished).

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• It was determined that the spikes onto the homogenates and into the extracts should be designed at a level such that 10 fold and 20 fold serial dilutions would likewise be in range of the ELISA quantitation range (Table 5). Spikes requiring greater sample dilution eliminate the ability to determine the analyte concentration in the original sample behind the spikes delivered, and make it impossible to compare spiked samples to unspiked samples. Additionally large dilutions greatly magnify the effect of the 99% confidence intervals (CI) when using the requisite dilution correction factors to determine the concentration range of the undilute samples (i.e. if the 99% CI range for the dilute sample analyzed is 0.5 to 0.6 µg/L, this translates to a still relatively narrow 5-6 µg/L in the undilute extract for a designed 10 fold dilution, but a wide 500-600µg/L in the undilute extract for a designed 1000 fold dilution).

• It was determined that the working solutions used to spike onto the homogenates and into the final extracts should be diluted in LGW and analyzed with the ELISAs as a measure of quality control.

• It was observed that the calibration curves not only shift up and down but also change shape from day to day, kit to kit. Hence the greatest amount of data is achieved when running full duplicate standard curves on the day of analysis. While trends can be investigated between samples run on the same day using partial standard curves, inter-day comparisons of sample responses can only be satisfactorily complete for samples analyzed in tandem with full calibration curves.