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.