Quantification of Delta Toxin by HPLC-MS

Delta toxin was quantified as described previously.^{20 (link)} Briefly, 5 μL of culture supernatant was infused into a C8 column (ZORBAX SB-C8, 2.1 × 30 mm, 3.5 μm, Agilent, Santa Clara, CA, USA) connected to Waters 2795 separation module (Waters, Milford, MA, USA). Delta toxin was eluted by 0.05% trifluoroacetic acid (TFA) in double distilled water (eluent A) and 0.05% TFA in acetonitrile (eluent B) at a flow rate of 0.3 mL min⁻¹ for 10 min. The gradient program for elution is as follows: 0% eluent B for 1.5 min; 0% to 50% of eluent B for 1 min; 50% to 100% of eluent B for 4 min; 100% eluent B for 2.5 min; 0% eluent B for 1 min. The separated delta toxin was ionized and analyzed by the conjunct Waters ZQ 2000 MS system (Waters, Milford, MA, USA) equipped with an electrospray ionization (ESI) source. Ion source parameters are as follows: capillary voltage, 3.3 kV; cone voltage, 50 V; source temperature, 120 °C; desolvation temperature, 350 °C; desolvation gas flow, 900 L h⁻¹; cone gas flow, 50 L h⁻¹. Ions were analyzed in the positive ion full scan mode for the range of m/z 600 to 1600. Scan time is 1 s with an inter-scan delay of 0.1 s following parameters. The production of delta toxin was measured by integrating the extracted ion chromatograms based on the m/z 1504 and 1003 of doubly- and triply-charged ions, respectively.