B03

An integrated method combining solid-phase extraction (SPE) with ultra-performance liquid tandem mass spectrometry (UPLC-MS/MS) has been established for quantifying bacitracin (BTC), bacitracin zinc (BZ), and bacitracin methylene disalicylate (BMD) in animal feed. A pretreatment procedure that can effectively, quickly, and simultaneously extract and purify BTC, BZ, or BMD in feed was developed for the first time through the optimization of extraction and SPE conditions. After extraction with acetonitrile + methanol + 15 % ammonia solution (1:1:1, v:v:v) and dilution with EDTA solution (1.5 mmol/L, pH 7.0), a SPE procedure was carried out with C₁₈ cartridge. Following LC-MS/MS analysis utilized a Waters Peptide BEH C₁₈ column with a gradient elution of 0.1 % formic acid in water/acetonitrile with. This method demonstrated a strong linear correlation (R² > 0.9980) across a 0.01-1.0 mg/L concentration span, based on a matrix-matched standard curve. Satisfactory recoveries of BTC (bacitracin A, B₁, B₂, and B₃), BZ, and BMD in different feeds were obtained from 80.7 % to 108.4 %, with relative standard deviations below 15.7 %. Low limits of quantification ranging within 7.2-20 μg/kg were achieved for bacitracin A, B₁, B₂, and B₃. This method provided an effective and reliable detection method to prevent the addition of BTC and different BTC formulations in feeds.

Optimal maternal vitamin status during pregnancy and lactation is essential to support maternal and infant health. For instance, vitamin D₃ is involved in infant bone development, and B-vitamins are involved in various metabolic processes, including energy production. Through a double-blind randomised controlled trial, we investigated the effects of maternal supplementation from preconception throughout pregnancy until birth on human milk (HM) concentrations of vitamin D₃ and B-vitamins. In addition, we aimed to characterise longitudinal changes in milk concentrations of these vitamins.

Both control and intervention supplements contained calcium, iodine, iron, β-carotene, and folic acid, while the intervention also contained zinc, vitamins B₂, B₆, B₁₂, and D₃, probiotics, and myo-inositol. HM samples were collected across 4 time points from 1 week to 3 months post-delivery from 158 mothers in Singapore, and 7 time points from 1 week to 12 months from 180 mothers in New Zealand. HM vitamin D was quantified using supercritical fluid chromatography and B-vitamins with mass spectrometry. Potential intervention effects on HM vitamins D₃, B₂, B₆, and B₉, as well as other B-vitamin (B₁ and B₃) concentrations were assessed using linear mixed models with a repeated measures design.

Over the first 3 months of lactation, HM 25-hydroxyvitamin D₃ concentrations were 20% (95% CI 8%, 33%, P = 0.001) higher in the intervention group, with more marked effects in New Zealand. There were no observed intervention effects on HM concentrations of vitamins B₁, B₂, B₃, B₆, and B₉. In New Zealand mothers, longitudinally, vitamin D₃ concentrations gradually increased from early lactation up to 12 months, while vitamins B₁ and B₂ peaked at 6 weeks, B₃ at 3 weeks, and B₆ and B₉ at 3 months.

Maternal supplementation during preconception and pregnancy increased HM vitamin D, but not B-vitamin concentrations in lactation. Further studies are required to examine the discrete benefits of vitamin D supplementation starting preconception vs during pregnancy, and to further characterise the effects of supplementation on later offspring health outcomes.

Registered at ClinicalTrials.gov on the 16 July 2015 (identifier NCT02509988); Universal Trial Number U1111-1171-8056. This study was academic-led by the EpiGen Global Research Consortium.