*Article* **Establishing a Metabolite Extraction Method to Study the Metabolome of** *Blastocystis* **Using NMR**

**Jamie M. Newton 1,2, Emma L. Betts 1, Lyto Yiangou 1, Jose Ortega Roldan 2, Anastasios D. Tsaousis 1,\* and Gary S. Thompson 2,\***


**Abstract:** *Blastocystis* is an opportunistic parasite commonly found in the intestines of humans and other animals. Despite its high prevalence, knowledge regarding *Blastocystis* biology within and outside the host is limited. Analysis of the metabolites produced by this anaerobe could provide insights that can help map its metabolism and determine its role in both health and disease. Due to its controversial pathogenicity, these metabolites could define its deterministic role in microbiome's "health" and/or subsequently resolve *Blastocystis*' potential impact in gastrointestinal health. A common method for elucidating the presence of these metabolites is through 1H nuclear magnetic resonance (NMR). However, there are currently no described benchmarked methods available to extract metabolites from *Blastocystis* for 1H NMR analysis. Herein, several extraction solvents, lysis methods and incubation temperatures were compared for their usefulness as an extraction protocol for this protozoan. Following extraction, the samples were freeze-dried, re-solubilized and analysed with 1H NMR. The results demonstrate that carrying out the procedure at room temperature using methanol as an extraction solvent and bead bashing as a lysis technique provides a consistent, reproducible and efficient method to extract metabolites from *Blastocystis* for NMR.

**Keywords:** *Blastocystis*; 1H NMR; metabolite extraction, metabolomics
