Cobalamin Concentrations in Fetal Liver Show Gender Differences: A Result from Using a High-Pressure Liquid Chromatography−Inductively Coupled Plasma Mass Spectrometry as an Ultratrace Cobalt Speciation Method

Maternal diet and lifestyle choices may affect placental transfer of cobalamin (Cbl) to the fetus. Fetal liver concentration of Cbl reflects nutritional status with regards to vitamin B12, but at these low concentration current Cbl measurement methods lack robustness. An analytical method based on enzymatic extraction with subsequent reversed-phasehigh-pressure liquid chromatography (RP-HPLC) separation and parallel ICPMS and electrospray ionization (ESI)-Orbitrap-MS to determine specifically Cbl species in liver samples of only 10−50 mg was developed using 14 pig livers. Subsequently 55 human fetal livers were analyzed. HPLC−ICPMS analysis for cobalt (Co) and Cbl gave detection limits of 0.18 ng/g and 0.88 ng/g d.m. in liver samples, respectively, with a recovery of >95%. Total Co (Cot) concentration did not reflect the amount of Cbl or vitamin B12 in the liver. Cbl bound Co contributes only 45 ± 15% to Cot. XRF mapping and μXANES analysis confirmed the occurrence of non-Cbl cobalt in pig liver hot spots indicating particular Co. No correlations of total cobalt nor Cbl with fetal weight or weeks of gestation were found for the human fetal livers. Although no gender difference could be identified for total Co concentration, female livers were significantly higher in Cbl concentration (24.1 ± 7.8 ng/g) than those from male fetuses (19.8 ± 7.1 ng/g) (p = 0.04). This HPLC−ICPMS method was able to quantify total Cot and Cbl in fetus liver, and it was sensitive and precise enough to identify this gender difference.