Dataset: Dissolved B-vitamins and vitamers
Deployment: Malaspina_2010_Hesperides

Sampling protocol:
Samples were collected using a Teflon tow-fish sampling system deployed at approximately 3-meters (m) depth utilizing established trace metal-clean techniques. After sample collection, seawater was filtered on board through acid-washed 0.2-micrometer (μm) filter cartridges and frozen until analysis.

Preconcentration protocol:
Samples for dissolved vitamin analysis were preconcentrated as previously described (Okbamichael and Sañudo-Wilhelmy, 2004; Okbamichael and Sañudo-Wilhelmy, 2005; Sañudo-Wilhelmy et al., 2012; Suffridge et al., 2017). Briefly, preconcentration columns were prepared by pouring 1:1 MeOH: C18 resin (HF Bondesil (Agilent Technologies)) slurry into Poly-Prep Columns (Biorad). The resin was allowed to settle, and the excess MeOH was drained, leaving 7 milliliters (ml) of resin. Thawed samples were adjusted to pH 6.5 using dilute HCl and passed over the preconcentration column at 1 ml per minute. The residual salt was rinsed off the resin using 30 ml of LC/MS grade water. The target analytes were then eluted from the resin using 12 ml of LC/MS grade methanol into methanol-rinsed 15 ml conical centrifuge tubes. Eluted samples were then further concentrated via evaporation in a nitrogen dryer using 5-20 PSI compressed N2 gas, in a dark, at room temperature. Samples were allowed to evaporate until 250 microliters (μl) remained, and then stored at -20 degrees Celsius (°C) until LC/MS analysis, which occurred within 24 hours. Prior to LC/MS analysis, samples were adjusted to pH 6.5 using 10 μl of dilute NaOH.

LC/MS Analysis:
Quantification of vitamin B7 (biotin); four different chemical forms of vitamin B12 ((adenosyl (AB12)-cyano (CB12)-hydroxy (HB12), and methyl (MB12) B12; vitamin B1 (thiamin); two of its precursors (HMP,( 4-Amino-5-hydroxymethyl-2-methylpyrimidine) and cHET (5-(2-Hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid) as well as HET (4-Methyl-5-thiazoleethanol), precursor of cHET and AmMP (4-amino-5-aminomethyl-2-methylpyrimidine), a salvage compound for the HMP synthesis, was carried out with a Thermo TSQ Altis Plus triple quadrupole mass spectrometer, coupled to a Vanquish Flex UHPLC system. The LC system used a stable-bond C18 reversed-phase column (Discovery HS C18 10cm x 2.1mm, 5μm column, Supelco Analytical) with a 50 μl sample loop. The computer software Trace Finder General 5.2 Quan and TSQ Altis Plus 3.4 Tune (Thermo Scientific) were used for data acquisition and analysis. A 12-minute gradient flow was used with mobile phases of methanol (MeOH) and LC/MS grade water, both buffered to pH 4 with 0.5% acetic acid. The flow rate was set at 230 microliters per minute (μl/min) throughout the run, with a gradient starting at 93% LC/MS water: 7% MeOH for two minutes, changing to 100% MeOH by seven minutes, and continuing at 100% MeOH until nine minutes and returning to initial conditions until the gradient completes at twelve minutes. All peaks were identified using standards dissolved in LC/MS grade water. The mass spectrometer was run in Selected Reaction Monitoring (SRM) mode with positive polarity with a well time of 100 milliseconds (ms) per transition. The resolution of the mass filters used for quadrupoles 1 and 2 were 0.7 and 0.1 m/z, respectively. The ESI spray voltage was 4000V, sheath gas (N2) pressure was 30 PSI, the auxiliary gas (Ar) pressure was 3 PSI, the capillary temperature was 269°C, and the collision pressure was 2.1 torr. B vitamin and vitamer values reported as 0.00 should be interpreted as "non-detectable".