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

Monotropa uniflora samples were collected from 12 sites from public and private land across Pennsylvania, as well as from four states throughout its U.S. range: Oregon, Vermont, North Carolina, and Michigan. The aerial portions (stem and flower) of three biological individuals were collected at each site. Samples were placed approximately 40 mL aqueous ethanol (80% w/v ethanol + 1% w/v formic acid) per 10 g fresh weight immediately after collection and allowed to steep for eight weeks in the dark at room temperature (20 ℃). The resulting maceration was filtered using Whatman #1 paper, and residual solvent was removed by rotary evaporation (Büchi Corporation, New Castle, DE). Herbarium voucher specimens were prepared to confirm taxonomic identity and deposited at the Penn State Herbarium (PAC). Monotropa uniflora extracts were dissolved in MeOH at 1mg/mL.

Ultra-high Pressure (UP) LC-MS data were first 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. To rule out the possibility of a failure to detect a real presence of grayanotoxins in Monotropa uniflora at biologically relevant levels, the pooled individual Monotropa uniflora sample extracts from each of the 16 sample sites ("QC") were spiked with both grayanotoxin I and II to a concentration of 230 mg/kg dry weight each. These spiked samples were also analyzed with LC-MS using the above method. Mass spectral data were converted to mzML format using MSConvert

Ultra-high Pressure (UP) LC-MS data were also 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 (Chambers, et al. 2012).