Hip 1290 sampler

Cordyceps extracts were separated on a Poroshell 120 EC-C18 column (2.1 × 150 mm; 1.9 µm) (Agilent Technologies, Santa Clara, CA, USA) maintained at 35 °C connected to an Agilent HiP 1290 Sampler, Agilent 1290 Infinity pump, and Agilent 6545 Accurate Mass Q-TOF dual AJS-ESI mass spectrometer operated in negative ion mode. Source gas temperature was set to 250 °C, with a gas (N2) flow of 12 L/min and a nebulizer pressure of 35 psi. Sheath gas temperature was 325 °C, sheath gas (N2) flow of 11 L/min, capillary voltage is 3500 V, and nozzle voltage 0 V. Mobile phase A consisted of acetonitrile (ACN):water (5:95 v/v), mobile phase B consisted of ACN:water (95:5 v/v), and both contained 0.1% formic acid. The chromatography gradient started at 10% mobile phase B, holds for 1 min, increases to 100% B from 1 to 10 min, where it is held until 12 min and then returned to starting conditions. Post-time was 3 min and the flowrate was 0.4 mL/min throughout. Injection volume was 10 µL and the samples were analyzed in a randomized order. Myriocin was monitored using accurate mass m/z 400.2705 of [M − H]⁻ with the retention time established by the standard, and quantitated based on the ratio to the myriocin standard injected separately.