Sample collection and solid phase extraction:
Surface water samples (500 milliliters (mL)) were collected at all river stations via a submersible pump lowered ~1.0 meter (m) below the water's surface. For the collection of submarine groundwater samples, U.S. Geological Survey small boat operations along the West Florida Shelf (WFS) were conducted. Surface water, bottom water, and submarine groundwater samples (2 liters (L)) were collected via peristaltic pump along three coastal transects.
Samples were filtered through 0.2 micrometer (um) filters (polyethersulfone) into acid-cleaned polypropylene bottles to 2L. The sample was then acidified to a pH of 2-3 with hydrochloric acid (trace metal grade) and pumped at 25 milliliters per minute (mL/min) through Bond-Elut PPL solid phase extraction (SPE) columns (1 gram (g), 6 mL, Agilent Technologies) that had been previously activated by passing 6 mL each of methanol (MeOH, Optima LCMS grade, Fisher Scientific) and ultrapure water (qH₂O, 18.2 MΩ) through the column. SPE columns were rinsed with 10 mL acidified qH₂O (pH = 2) to remove salts and frozen at -20 degrees Celsius (°C) immediately after sample collection and returned to the laboratory for further processing. Sample columns were gravity eluted with 10 mL LCMS Optima grade methanol. The eluent was collected in 15 mL polypropylene centrifuge tube and evaporated in a SpeedVac to concentrate to a volume of ~ 0.2 mL. The eluent was transferred to a 2 mL centrifuge tube and brought up to a final volume of 2 mL with qH₂O. Each sample was spiked with 20 microliters (µL) of 100 micromolar (µM) B12 stock for a concentration of 1 µM cyanocobalamin. Solid phase extraction process blank samples were prepared with the same procedure without sample loading.
Analysis by LCMS:
Tubes were centrifuged at max speed for 5 minutes to sediment any particles, and then the spiked samples were transferred to plastic microcentrifuge tubes for storage in freezer, 1 mL was used for analysis on Orbitrap in 2 mL plastic autosampler vials. A pooled sample from each batch of samples for each Orbitrap run was prepared by combining 15 µL from each sample. Chromatographic separation was performed using a Phenyl-Hexyl column (Waters ACQUITY Premier CSH) with dimensions of 2.1 x 100 millimeters (mm) and a 1.7 µm particle size. The column temperature was maintained at 30.0 °C throughout the analytical run. Mobile phase A was composed of water supplemented with 0.1% formic acid, while mobile phase B consisted of methanol with 0.1% formic acid. The system operated at a constant flow rate of 200 µL/min with a 30-minute gradient starting from 95% solvent A and 5% solvent B to 5% solvent A and 95% solvent B and a 5-minute hold at 95% solvent B. Solvents were then switched back to the initial condition of 95% solvent A and held for 6 minutes to re-equilibrate the column. The total injection volume for each sample was 20 µL. Mass spectral data were acquired using an Orbitrap IQ-X Tribrid Mass Spectrometer (Thermo Fisher Scientific) operating in positive electrospray ionization (ESI) mode. The ESI source parameters were optimized with a spray voltage of 3500 volts (V), a sheath gas flow of 50 (Arb), an auxiliary gas flow of 10 (Arb), and a sweep gas flow of 1 (Arb). The ion transfer tube and vaporizer temperatures were set to 325 °C and 350 °C, respectively. Full MS scans were recorded in the Orbitrap mass analyzer across a mass range of 100 to 1000 m/z. The resolution was set to 500,000 at m/z 200 to ensure high mass accuracy for the analytes. Data acquisition settings included the collection of 1 microscan per data point with a maximum injection time of 1014 milliseconds (ms).
