**5. Conclusions**

The clean-up of extracts from powdered nutraceutical samples is a challenge. Extracts of matcha infused in water have inheritably better relative standard deviation of recoveries of selected conazoles as only the water soluble components are extracted, however other conazoles still show poor recoveries [14]. Evaluation of a wider range of conazole fungicides and other structurally similar pesticides showed that there are similar issues when using PSE with poor extraction recoveries for the less commonly analyzed conazole fungicides. In general matrix suppression of the MS signal was moderate indicating a need for matrix matched or standard addition calibration. PSE with in-cell clean-up can be used to remove pigments from powdered nutraceutical products such as matcha. There are more commercially available products with high pigment levels such that new extraction and clean-up methods need to be developed. This analysis shows that conazole fungicides strongly adsorb to many of the sorbent materials used for pigment removal such that alternate sorbents from carbon based materials need to be evaluated in the future. Matcha is a very difficult sample matrix and selected conazole fungicides can be extracted directly into ethyl acetate with subsequent in-cell clean-up to remove chlorophyll. The first detection of flusilazole, prothioconazole-desthio and trace levels of tebuthiuron are presented. The MS signal suppression that is still present is attributed to the presence of polyphenols including catechins that future extraction and clean-up methods need to address. This work shows that issues with MS signal suppression are also caused by other components of matcha than pigment. Further reducing the polarity of the extraction solvent while maintaining good recoveries for conazole fungicides, and clean-up with Florisil®, polymeric or alternative green sorbent materials should be evaluated. Of the carbon based sorbents and other sorbents evaluated Anasorb 747 was best able to remove high amounts of the green pigment from ethyl acetate extracts such that filtration was not required after the pre-concentration of extracts. Anasorb 747 also has potential for re-use after clean-up to reduce sample analysis costs. To improve the method development strategy utilizing green analytical approaches, improvements in PSE with in-cell clean-up are needed to deal with difficult sample matrices without the need for subsequent off-line clean-up steps [22,23]. To further reduce matrix effects caused by more polar matrix components, a membrane filter could be tested for use with PSE in-cell clean-up. Some membrane filters strongly absorb polyphenols, and this

may aid in reducing matrix issues. Pressurized solvent extraction is well suited to address filtration in the extraction cell as extracts are filtered as the solvent is pushed out of the extraction cell with nitrogen gas.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2305-6304/6/4/64/s1, Figure S1: Selected Reaction Monitoring Chromatograms of Diniconazole With and Without Matrix Added, Table S1: Method Detection Limit, Regression Coefficient of Matrix Matched Calibration Curve, and Percentage Matrix Effects for Target Analytes Analyzed by LC-ESI+-MS/MS.

**Author Contributions:** Conceptualization, methodology, data curation, writing-review, R.R.-F.; methodology and data curation, A.A.M.

**Funding:** This research was funded by Natural Sciences and Engineering Council of Canada (NSERC) Discovery Development Grant.

**Conflicts of Interest:** The authors declare no conflict of interest.
