Cobalamin, vitamin B₁₂, is an important micronutrient that has been investigated for decades in the marine context because it is required for phytoplankton growth. The biologically active forms (Me-B₁₂, Ado-B₁₂) and the synthetic form (CN-B₁₂) quickly convert to OH-B₁₂ after light exposure in various aqueous solutions, but puzzlingly have been frequently reported to dominate dissolved cobalamin pools in the sunlit ocean. Here we document photodegradation timescales for these cobalamin forms in natural seawater using targeted mass spectrometry, providing quantitative evidence that OH-B₁₂ is expected to be the dominant dissolved form in irradiated seawater. Then, through high resolution mass spectrometry, we identify four photodegradation products of OH-B₁₂ which represent potential building blocks microbes could salvage and remodel to satisfy cellular cobalamin requirements. Taken together, these results clarify the impact of light on marine cobalamin dynamics, laying a foundation for a more quantitative understanding of the role of cobalamin in microbial communities and biogeochemical cycles.

Analytical cobalamin standards

Cobalamin standards CN-B₁₂ (≥ 98%, Fisher BioReagents), Me-B₁₂ (≥ 97%, Sigma-Aldrich), Ado-B₁₂ (≥ 97%, Sigma-Aldrich) and OH-B₁₂ (≥ 95%, Supelco) were obtained and primary stock solutions were prepared by dissolving 1 mg of each compound in 1 mL of Optima LC-MS grade water and stored at –80°C in the dark until use.

Seawater collection

We collected seawater from CTD rosette bottles into amber HDPE bottles while protecting from light, then filtered (0.2 µm pore-size nylon filters) while protected from light and stored at -20°C in acid-washed, milli-Q water rinsed, and sample-rinsed amber HDPE bottles until processing. Surface seawater (< 5 m) samples were collected from Lat: 43.1822; Lon: -62.0983 (Station HL05) on May 26^th, 2022, and Lat: 41.4100; Lon: -60.6772 (Station HL12) on May 23^rd, 2022, in the Northwest Atlantic.

Photodegradation experiments  

Thirty-minute experiment: we added 8 pM of Me- and Ado-, and 5 pM of CN-B₁₂ and OH-B₁₂ into 15 mL of seawater, collected at HL05, individually in UV-penetrable quartz vials (QP059, Cuvet.Co). Duplicate samples (15 mL) were taken directly after the addition of cobalamins at time 0. Then, duplicate quartz vials were exposed to direct, natural sunlight then 15 mL sample was transferred into clean amber vials in the dark at -20 °C after 5, 10, 15, and 30 minutes on June 7^th, 2022 (Ado-, Me- and CN-B₁₂) (35.1°C; 1450 µmol/s/m²) and July 21^st, 2022 (OH-B₁₂) (26.6 °C; 1400 µmol/s/m²). Four-day experiment: we added 4 pM OH-B₁₂ in 20 mL seawater, collected at HL12, in quartz vials, took duplicate T₀ samples then exposed to direct sunlight in May 2022. We transferred duplicate vials daily at noon into clean amber vials then stored in the dark at -20 °C until processing. Temperature (Logger, RC-5, Elitech) and light (Digital Light Meter, LUX29TK, TekcoPlus) were monitored (Table S2). For the 4-day experiment, mean day length was 11 hours, mean air temperature was 23.1 °C and average daytime light ~950 µmol/s/m² with maximum 1400 µmol/s/m². We collected dark controls that were supplemented with the same concentrations of cobalamin forms, wrapped in opaque black bags, exposed to similar temperatures, and sampled identically. We also processed control seawater samples in an identical manner to determine endogenous concentrations of cobalamins (Table S2).

We performed all extractions in a dark, windowless room with only a red headlamp as a light source. Samples were extracted on 100 mg HyperSep C18 solid phase extraction (SPE) cartridges (ThermoScientific, 60108-302) using a vacuum manifold. SPE cartridges were preconditioned with 2 × 0.5 mL MeOH then 2 × 0.5 mL deionized water and kept wet through the entire protocol. Samples were extracted at ~1 mL min^-1, washed with 2 × 0.5 mL milli-Q water then eluted with 2 × 0.85 mL MeOH into clean microtubes. Eluent was dried for ~2 hours under vacuum (Vacufuge, Eppendorf, Ontario), in the dark, then stored at –80 °C prior to analysis when it was resuspended with 100 µL of H₂O containing 0.1% formic acid.

Degradation product identification experiment

To investigate OH-B₁₂ degradation products, we prepared 1 µM of OH-B₁₂ in HPLC grade water and exposed it to ambient sunlight and temperature for 8 (EXP 1) and 4 (EXP 2) days. One sample from EXP 2 on day 4 was exposed to two 5-minute intervals of 254 nm UVc light from a 30-Watt lamp (EXP 2, Day 4+UV). We diluted samples 200-fold before LC-MS analysis as outlined below and in supplemental methods.

Mass spectrometry

Cobalamin photodegradation in seawater was quantified using a Dionex Ultimate-3000 LC system coupled to an electrospray ionization source of a TSQ Quantiva triple-stage quadrupole mass spectrometer (ThermoFisher) with transition list reported in Table S4. Photodegradation products were investigated on an Agilent 1290 Infinity II LC coupled to a Q Exactive HF Orbitrap mass spectrometer (ThermoFisher) with a heated electrospray ionization probe using both data dependent acquisition and full scan modes. Complete details of LC-MS analyses are provided in supplemental methods.

Cobalamin quantification

We combined equal portions of each sample to obtain quality control (QC) pools for the 30-min and 4-day experiments. Cobalamins were quantified using standard addition with calibration curves prepared in the QC pools using the LC-MS approach described in the supplemental methods. Duplicate injections were performed with 0, 2.5, 5, and 25 fmol on C18 column for all cobalamin forms (Fig S7) and R² for slopes were all >97%. SPE percent recovery for 100 mg HyperSep C18 cartridge (n = 2) was determined by spiking 15 mL seawater samples with each cobalamin form equivalent to 80 fmol on C18 column (106 pM) before or after preconcentration on SPE. All reported concentrations and limits of detection are corrected for percent recoveries. Raw files generated with Xcalibur software (ThermoFisher) were uploaded into Skyline Daily (University of Washington) and the transitions with the best signal to noise and lowest interference were selected for quantification (Table S4). Limits of blanks and detection were determined according to Armbruster and Pry, 2008 (Table S3)⁶².

Photodegradation product identification

We identified potential photodegradation products of OH-B₁₂ in pure water using three approaches. First, we selected peaks of interest based on their increasing peak area in samples during sunlight exposure by performing a ‘background subtract’ of the first samples (T₀) from T₄ and T₈ samples. Second, we assembled reports of cobalamin degradation products from the literature and searched for their accurate masses (±5 ppm) (Table S5). Third, we searched product ion spectra for masses that are known cobalamin fragments (m/z 147.0922 and m/z 359.1005 product ions, DMB and DMB sugar-phosphate respectively). All candidate products were absent in blank samples.  

We used exact mass to determine chemical formula of candidate degradation products by constraining upper limits with ring-double bond equivalent (<26), elemental composition (C₆₂H₉₀CoN₁₃O₁₅P), and MS run-specific mass error (~4.0 ppm) of the OH-B₁₂ standard. We present level 2 (diagnostic) probable chemical structures (Fig 3) if they were either previously reported in literature or we were able to obtain a conclusive product ion spectrum ⁴⁵.

Statistical Analysis

Two outliers (Ado-B₁₂: 15 min, CN-B₁₂: 30 min) were removed from the photodegradation experiment analyses during data analysis due to abnormally high B₁₂ concentrations (Grubbs test, p-value <0.05), likely due to contamination during extraction (Fig S1,). Differences between T₀ and dark controls at 30 min and 4 days (Table S4) as well as OH-B₁₂ at T₀ and T₃₀ (Fig 2D) were normally distributed and determined insignificant using a student t-test (Table S2).