Measurement of vitamin D sulfates in breastmilk by liquid chromatography tandem mass spectrometry
Authors: Reynolds Carmen , Dyer Roy , Oberhelman-Eaton Sara , Konwinski Brianna , Singh Ravinder , Thacher Thomas 
Published: May 2023

Introduction: Vitamin D is an essential micronutrient for infant calcium metabolism and bone development. Concentrations of vitamin D in breastmilk are low, despite higher anti-rachitic activity and correlations between infant and maternal serum 25-hydroxyvitamin D (25D) (Taylor, et al., 2008; Oberhelman, et al., 2013). The sulfate-conjugated form of 25D is present in blood at concentrations approximately 40% of unmodified 25D. We hypothesized that sulfate conjugates, vitamin D3-sulfate (VitD-S) and 25-hydroxyvitamin D3-sulfate (25D-S), are present in milk and increase during supplementation. The objectives are (1) developing an LC-MS/MS assay to quantify VitD-S and 25D-S in milk, and (2) determine if these vitamin D sulfates increase with daily vitamin D supplementation. Methods: An LC-MS/MS method was developed to measure VitD-S and 25D-S in milk and in serum. Standards (Toronto Chemicals, Toronto, ON, CA) and internal standard (Glycomyr, Ames, IA) were used to optimize methods. Briefly, Milk (500 uL) and serum (250 uL) were extracted with methanol (MeOH) and resuspended in 150 uL 70% MeOH. Analytes were separated by C18 column with 70% to 100% MeOH with ammonium acetate (NH4OAc) organic phase gradient followed by MS/MS with optimized mass transitions and ESI in negative mode on SCIEX API 5000. An ongoing study is under way for breastmilk and serum collections from healthy lactating women consuming <600 IU vitamin D3/day at enrollment (day 1) and after 28 days of oral 5000 IU vitamin D3/day (day 28). This study was approved by the Mayo Clinic Institutional Review Board. Results: Preliminary analyses have been completed for 3 study participants. Serum 25D status had a trending increase from 30±2 ng/mL 25D on day 1 to 40±2 ng/mL 25D on day 28 ( P=0.1, paired t-test). Limit of detection [signal to noise (s/n) >3] for sulfates were 0.5 ng/mL 25D-S (s/n=7.3) and 0.25 ng/mL VitD-S (s/n=6.2). Upon enrollment, breastmilk sulfates were 3.25±0.12 ng/mL VitD-S (s/n=83.7±20.6) and 0.18±0.02 ng/mL 25D-S (s/n=13.6±3.3), and serum sulfates were 7.5±0.8 ng/mL VitD-S (s/n=39.4±22.5) and 53.2±10.4 ng/mL 25D-S (s/n=22.5±7.2). No statistical differences were observed between day 1 and day 28 in milk or serum sulfates for the 3 participants. However, VitD-S concentrations demonstrated a significant negative correlation between serum and milk ( r=0.87, P=0.03), and serum 25D-S was 21.3 ng/mL greater than unmodified serum 25D ( P=0.009, n=6). This study is ongoing, and data presented will include a greater number of study participants. Conclusions: We have developed an LC-MS/MS assay to measure VitD-S and 25D-S and found that VitD-S and 25D-S are present in freshly expressed breastmilk and in serum from women consuming <600 IU vitamin D3/day. F32 DK128987 & The Mayo Clinic Parker D. Sanders & Isabella G. Sanders Endowed Professorship. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.