Method optimization of the simultaneous detection of B₁₂ congeners leading to the detection of a novel isomer of hydroxycobalamin in seawater

Abstract

Rationale More than half of surveyed microalgae and over 90% of harmful algae have an obligate requirement for vitamin B12, but methods for directly measuring dissolved B12 in seawater are scarce due to low concentrations and rapid light‐induced hydrolysis. Methods We present a method to detect and measure the four main congeners of vitamin B12 dissolved in seawater. The method includes solid‐phase extraction, separation by ultrahigh‐performance liquid chromatography and detection by triple‐quadrupole tandem mass spectrometry utilizing an electrospray ion source. This method was applied to coastal field samples collected in the German Bay, Baltic Sea and the Danish Limfjord system. Results The total dissolved B12 pool ranged between 0.5 and 2.1 pM. Under ambient conditions methyl‐B12 and adenosyl‐B12 were nearly fully hydrolyzed to hydroxy‐B12 in less than 1 h. Hydroxy‐B12 and a novel, corresponding isomer were the main forms of B12 found at all field sites. This isomer eluted well after the OH‐B12 peak and was also detected in commercially available OH‐B12. Both compounds showed very high similarity in their collision‐induced dissociation spectra. Conclusions The high instability of the biologically active forms of Me‐B12 and Ado‐B12 towards hydrolysis was shown, highlighting the importance of reducing the duration of the extraction protocol. In addition, the vitamin B12 pool in the study area was mostly comprised of a previously undescribed isomer of OH‐B12. Further studies into the structure of this isomer and its bioavailability are needed.

Shedding light on cobalamin photodegradation in the ocean

“…Finally, a recent publication suggested that the dominant form of cobalamin in the North Sea was a putative OH-B 12 isoform that had similar fragmentation to OH-B 12 but eluted later (Möller et al 2022). We have not detected this putative isoform here or in any marine water column samples to date, despite our method's suitability for detecting this putative isoform's reported molecular ion in the proposed elution order (Supplemental Methods; Fig.…”

“…In addition, calculating concentrations from response factors are difficult to replicate without exceptionally detailed experimental procedure reporting and data transparency. Quantification based on calibration curves performed in water instead of representative, pooled samples are common (Suffridge et al 2017;Möller et al 2022) and could significantly alter measurements due to intense matrix effects (Heal et al 2014;Boysen et al 2018). Third, Me-and Ado-B 12 standards are also subject to this rapid photodegradation and if great care is not taken to protect them from light during calibration curve construction, this degradation could lead to an overestimate of in situ concentrations.…”

“…Cobalamin, or vitamin B 12 , is recognized as a critical currency in marine microbial communities (Fig. 1) (Lewin 1954;Cowey 1956;Daisley and Fisher 1958;Vishniac and Riley 1961;Menzel and Spaeth 1962;Carlucci and Silbernagel 1969;Fiala and Oriol 1984;Okbamichael and Sañudo-Wilhelmy 2004;Sañudo-Wilhelmy et al 2006, 2012Panzeca et al 2008Panzeca et al , 2009Koch et al 2011Koch et al , 2012Koch et al , 2013Su arez-Su arez et al 2011;Bonnet et al 2013;Heal et al 2014;Cohen et al 2017;Suffridge et al 2017Suffridge et al , 2018Joglar et al 2020Joglar et al , 2021Barber-Lluch et al 2021;Bruns et al 2022;Möller et al 2022), over half of surveyed eukaryotic phytoplankton require cobalamin but cannot synthesize it (Croft et al 2006). Cobalamin biosynthesis is restricted to an estimated 37% of bacterial species and select archaea and requires $ 30 enzymes (Rodionov et al 2003;Doxey et al 2015;Shelton et al 2019).…”

“…OH-B 12 and CN-B 12 are not biologically active and are converted to Me-or Ado-B 12 for use by cells. Ado-, Me-, and CN-B 12 convert to OH-B 12 in seconds to minutes of light exposure in blood, plasma, and slightly acidified water (Juzeniene and Nizauskaite 2013;Vaid et al 2018;Möller et al 2022). From this, it is expected that, in the presence of light, OH-B 12 would be the dominant form in aqueous solutions, including seawater.…”

Quantitative analysis and calibration of temperature-related deviations in the seawater total nitrogen analyzer

“…Figure 1 The flowchart of the measurement equipment According to the Beer-Lambert Law: A=εbc=log(I0/IT), when the path length is fixed, the quantity concentration of a substance is directly proportional to its absorbance. By measuring I0 and IT, and applying them to the standard curve, the quantity concentration of the substance can be calculated [4] . Based on the absorption curves of reaction products, suitable detection wavelengths are chosen, typically selecting wavelengths with strong absorption peaks.…”