Breast milk samples were thawed to room temperature on a shaker and thoroughly homogenized by vigorous shaking. After thorough mixing, ~1 g of each sample were mixed with 2 mL nitric acid (65% suprapur, Merck, Darmstadt, Germany) and 3 mL deionized water, and thereafter heated at 250° C for 30 min in a Milestone ultraCLAVE II microwave digestion system (EMLS, Leutkirch, Germany). Digested samples (completely clear, colorless, and homogenous solutions) were transferred to acid-washed polyethylene tubes (SARSTEDT, Nümbrecht, Germany) and diluted with deionized water to a nitric acid concentration of 20%.
The obtained solutions were measured for 32 elements using inductively coupled plasma mass spectrometry (ICPMS; Agilent 7700x, Agilent Technologies,Tokyo, Japan) with a collision/reaction cell system, in order to minimize the influence of possible interferences, ICPMS autosampler (ASX-500 series; Agilent Technologies, USA), and a quartz MicroMist nebulizer. Standard solutions for the external calibrations (ICP Multi Element Standard Solution VI CertiPUR; Merck, Darmstadt, Germany) were prepared freshly before every run in 20% nitric acid (suprapur, Merck, Darmstadt, Germany).
Aluminum (27 Al), arsenic (75 As), cadmium (111 Cd), chromium (52 Cr), cobalt (59 Co), copper (65 Cu), iron (56 Fe), magnesium (24 Mg), manganese (55 Mn), molybdenum (95 Mo), nickel (60 Ni), phosphorous (31 P), potassium (39 K), rubidium (85 Rb), sodium (23 Na), strontium (88 Sr), sulphur (34 S), vanadium (51 V) and zinc (66 Zn) were measured in helium mode, calcium (40 Ca) and selenium (78 Se) in hydrogen mode, and antimony (121 Sb), barium (137 Ba), beryllium (9 Be), boron (11 B), cesium (133 Cs), lead (208 Pb), lithium (7 Li), silver (107 Ag), thorium (232 Th), tin (118 Sn) and uranium (238 U) in standard mode.
Internal standards, 72Ge (Li, Be, B, Na, Mg, Al, P, S, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se), 103Rh (Mo, Cd, Sb), and 193Ir (Rb, Sr, Ag, Sn, Cs, Ba, Pb, Th, U) were used.
Speciation of arsenic in breast milk samples with highest total arsenic concentrations (2-4 μg As/L; n = 3) was performed according to Fangstrom et al. . A density of 1.03 Kg/L has been reported for breast milk , which is in accordance with our measurements in the third week of lactation of 1.01 Kg/L, Thus, we approximate mass with volume when presenting our element concentrations and comparing with literature data.
Analytical quality control included sampling material chemical blanks and reference materials. In order to evaluate contamination from the bottles used for storing the milk (250 mL PYREX), three empty bottles were filled with 100 mL deionized water and placed in −20°C freezers for a week. The thawed water was then included in the analysis. The bottle blanks held <0.1% of the average concentrations of the analyzed samples for all elements except for Mo (0.5%) and U (0.2%).
No suitable reference material for human milk was available. Instead, certified reference materials included 1) Infant/Adult Nutritional Formula, NIST 1849 (reference values for Ca, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, P, Se, Zn), 2) Toxic metals in bovine blood, Level 1, NIST SRM 966 (reference values for Cd and Pb), 3) SeronormTM Trace Elements Whole Blood L-1, REF 201505, LOT MR4206 (reference values for all 32 elements analyzed) and 4) SeronormTM Trace Elements Whole Blood L-2, REF 201605, LOT 0503109 (reference values for all 32 elements analyzed). The NIST materials were purchased from the National Institute of Standards and Technology, Gaithersburg, MD and the SeronormTM materials from SERO AS, Billingstad, Norway. All sample preparation and analyses were carried out at the Unit of Metals and Health, Institute of Environmental Medicine, Karolinska Institutet, Sweden.
The limit of detection (LOD) was set at 3 times the standard deviation (SD) of the mean blank values for each element, based on 5 blank samples which were included in the analytical run.