Manganese speciation in human milk using size exclusion chromatography combined with strong anion exchange chromatography and inductively coupled plasma mass spectrometry detection

Abstract

Human milk was investigated concerning Mn speciation. In a first step subfractions were produced by centrifugation and analysed for manganese by inductively coupled plasma atomic emission spectrometry (ICP-AES). It turned out that Mn concentrations were approximately 3 µg L⁻¹ (human milk), 2.85 µg L⁻¹ in the defatted fraction, 0.25 µg L⁻¹ in the pellet fraction and 2.6 µg L⁻¹ in the low molecular weight (LMW) -supernatant fraction. The defatted fraction was investigated further by on-line coupling of size exclusion chromatography (SEC) to inductively coupled plasma mass spectrometry (ICP-MS). The columns had either a separation range between 10 and 150 kDa or 100–2000 Da. It is shown that manganese was found predominantly in the LMW fraction. The mainpeak in LMW-SEC eluted at 95 min, which was assigned to masses around 300 Da due to the mass calibration of the TSK column. Subsequent investigations analysed respective SEC-fractions by strong anion exchange (SAX)-ICP-MS and compared chromatograms with those of Mn standard compounds. Inorganic manganese species and Mn–citrate complex were identified. Some further Mn compounds of low concentration were seen, but could not be assigned to specific standard compounds.