Dataset for "Targeted and Untargeted Mass Spectrometry Do Not Detect Grayanane-type Toxins in Monotropa uniflora L.": UPLC-MS Analysis of Grayanotoxin I and II standards in Negative Mode

Grayanotoxin I and II standards were serially diluted in methanol for analysis with Ultra-high Pressure (UP) LC-MS on both an orbitrap and triple-quadrupole mass spectrometer. For the orbitrap, Grayanotoxin I and II standards were serially diluted in methanol from 1.0 ug/mL to 0.03 ug/mL. Data were acquired using an Orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoScientific, Waltham, MA, U.S.A.) with an electrospray ionization source coupled to a Vanquish UHPLC system (ThermoScientific). 5 μL injections were separated by reverse-phase UPLC using an Acquity BEH C18 column (150 × 1 mm, 1.7 μm particle size (Waters Corp., Milford, MA, U.S.A.)) held at 55 °C with a flow rate of 100 μL/min. The following binary solvent gradient was employed with solvent A (LC-MS grade water with 0.1% formic acid), and solvent B (LC-MS grade acetonitrile with 0.1% formic acid): initial isocratic composition of 97:3 (A:B), increasing linearly to 55:45 over 10 min, increasing linearly to 25:75 over 2 min, increasing linearly to 0:100 over 0.5 min followed by an isocratic hold at 0:100 for 4 min, gradient returned to 97:3 over 0.2 min and held for 3.3 min. The negative ionization mode was utilized over a full scan of m/z 100–1000 with the following settings: spray voltage, 2.5 kV; IT tube temperature, 275 °C; vaporizer temperature, 75 °C; sheath gas and auxiliary gas flow, 25 and 5 units, respectively. Tandem MS acquisition was performed using a data-dependent acquisition method with the following settings: intensity threshold, 2.5e4; collision energies, 15,30,45 eV; cycle time, 0.6sec; mass tolerance, 10ppm. Each concentration of standard was run in triplicate. Mass spectral data were converted to mzML format using MSConvert.

For the triple-quadrupole mass spectrometer, grayanotoxin I and II standards were serially diluted in methanol from 1 ug/mL to 0.003 ug/mL for analysis. Ultra-high Pressure (UP) LC-MS data were acquired using a Thermo TSQ Quantis Plus mass spectrometer (ThermoScientific, Waltham, MA, U.S.A.) with an electrospray ionization source coupled to a Vanquish UHPLC system (ThermoScientific). 5 μL injections were separated on a reverse-phase Acquity BEH C18 column (50 × 2.1 mm, 1.7 μm (Waters Corp., Milford, MA, U.S.A.)) held at 50 °C with a flow rate of 300 μL/min. The following binary solvent gradient was employed with solvent A (LC-MS grade water with 0.1% formic acid), and solvent B (LC-MS grade acetonitrile with 0.1% formic acid): initial isocratic composition of 97:3 (A:B) held for 0.5 min, increasing linearly to 25:75 over 2.5 min, followed by an isocratic hold at 25:75 for 0.75 min, gradient returned to 97:3 over 0.25 min and held for 1 min. The negative ionization mode was utilized over a scan range of m/z 300–500 with the following settings: scan rate, 1000 Da/sec; spray voltage, 2.5 kV; IT tube temperature, 375 °C; vaporizer temperature, 350 °C; sheath gas and auxiliary gas flow, 50 and 10 units, respectively. Selective ion monitoring (SIM) was performed with a retention time window of 1 minute during the method for the following ions: m/z 411.2 at a retention time of 2.2 minutes (Grayanotoxin I [M-H]-), m/z 457.2 at a retention time of 2.2 minutes (Grayanotoxin I [M+FA]-), m/z 351.2 at a retention time of 2.5 minutes (Grayanotoxin II [M-H]-), and m/z 397.2 at a retention time of 2.5 minutes (Grayanotoxin I [M+FA]-). Each concentration of standard was run in triplicate. Mass spectral data were converted to mzML format using MSConvert.

To control for possible degradation of grayanotoxins during the sample extraction process, a simulation of the extraction process was conducted with aliquots of grayanotoxin I and II standards. These standards were then analyzed on both orbitrap and triple quadrupole mass spectrometers.