Suprapur nitric acid

To check the presence of zinc in our protein preparation, we used ICP-OES. 100 μM each of labeled and unlabeled His₆-MBP-2C was mixed with 10 ml of 1% Suprapur nitric acid (Merck Millipore). The mixture was filtered with a 0.2 μm filter. Then, the sample was loaded on the Agilent 5800 VDV ICP-OES (Agilent technologies, Santa Clara, California, US) machine and the emission corresponding to zinc was recorded (202 nm). Zinc acetate was used as a control.

Measurements of total arsenic in maternal breast milk, saliva, and erythrocytes were performed by inductively coupled plasma mass spectrometry (ICPMS; Agilent 7500ce series) with an integrated sample introduction system (Agilent Technologies, Waldbronn, Germany). Before the ICPMS analysis, breast milk and erythrocytes were acid digested with 65% concentrated suprapur nitric acid (Merck, Darmstadt, Germany) in microwave-assisted autoclave (UltraClave; EMLS, Leutkirch, Germany). For quality control, reference material (Seronom Trace Elements Whole Blood L-1, Lot MR4206; L-2, Lot 0503109; SERO AS, Billingstad, Norway) with a reference value of 1.8 ± 0.40 μg/L and 13.2 ± 1.3 μg/L, respectively. We obtained an average at 2.2 μg/L and 14 μg/L, respectively (n = 17).
We measured the sum of iAs and the methylated metabolites, here referred to as urinary arsenic (U-As), in maternal urine by hydride generation–atomic absorption spectroscopy (HG-AAS) (Vahter et al. 2006 (link)). Separation of the different As metabolites [As(III), As(V), MA, and DMA] in urine was performed by a high performance liquid chromatography (HPLC) system (Agilent 1100 series system; Agilent Technologies), equipped with a Hamilton PRP-X100 anion-exchange column 4.1 × 250 mm (Reno, NV, USA) and coupled to hydride generation (HG) and ICPMS. HG is commonly coupled to the detector to discriminate for organic arsenic species like arsenobetaine in the urine, because these species do not form volatile arsines as iAs and its metabolites do. The method and the equipment have been described in detail elsewhere (Lindberg et al. 2007 (link)). For quality control, a reference urine (NIES CRM no. 18; National Institute for Environmental Studies, Ibaraki, Japan) with a certified DMA concentration of 36 ± 9 μg/L was analyzed together with the collected urine samples. The obtained average (± SD) DMA concentration was 37 ± 1.7 μg/L (n = 5). The correlation between measured U-As by HG-AAS and calculated U-As by adding the different metabolites measured by HPLC–HG–ICPMS was 0.98 (n = 87), demonstrating good column recovery.
To determine the arsenic compounds in breast milk, we added 10 μL of concentrated formic acid (Fluka, Buchs, Switzerland) to an aliquot of 500 μL breast milk to precipitate the proteins. Thereafter, the samples were centrifuged for 15 min at 15,000 rpm (Microliter centrifuge; Hettich, Tuttlingen, Germany) to separate fat, proteins, and whey. For the arsenic measurements, the fat layer was removed and the whey was carefully transferred into the polypropylene vials sealed with rubber caps (both from Agilent, Waldbronn, Germany). The arsenic compounds were determined with an HPLC system (Agilent 1100 series system; Agilent Technologies) coupled to an ICPMS (7500c; Agilent) equipped with a Babington type nebulizer. The metabolites were separated on a Hamilton PRP-X100 anion-exchange column, 4.1 × 250 mm with 20 mM aqueous ammonium phosphate (NH₄H₂PO₄) solution at pH 6 [adjusted with ammonium hydroxide (NH₄OH)] at a flow rate of 1.5 mL/min and a column temperature of 40°C. The injection volume was set to 20 μL. For signal enhancement, methanol (MeOH) was pumped to the ICPMS spray chamber as described elsewhere (Kovaãeviã and Goessler 2005 ). The signal was recorded at m/z 75 (⁷⁵As) and m/z 77 (⁴⁰Ar³⁷Cl, ⁷⁷Se). To evaluate the data, we used the ICPMS chromatographic software version C.01.00 (Agilent). The limit of detection was < 0.01 μg/L for both blood and saliva. For breast milk and urine it was 0.2 μg/L for As(V) and 0.1 μg/L for As(III), MA, and DMA.
To compensate for variations in urine dilution, we adjusted the arsenic concentrations to the average specific gravity (SG), measured by a digital refractometer (EUROMEX RD 712 clinical refractometer; EUROMEX, Arnhem, Holland). U-As was adjusted to the overall mean SG value of 1.003 g/mL in the infant urine and 1.012 g/mL in maternal urine, according to U-As × [(1.003 – 1) or (1.012 –1)]/(measured SG – 1). SG adjustment is shown to be less influenced by body size, age, and arsenic exposure than is creatinine adjustment (Nermell et al. 2008 (link)). The SG of saliva was about the same (1.003–1.004 g/mL) in all samples, so we did not adjust arsenic concentrations in saliva.

The determinations were performed using flame (FAAS) and electrothermal (ETAAS) atomic absorption spectrometry. The content of Ca, Cu, Fe, K, Mg, Mn, Na, and Zn was determined directly from the digests with F-AAS SOLAAR M5 apparatus (Thermo Scientific, Waltham, MA, USA) using, if needed, 10-, 500-, and 5000-fold dilutions. Lanthanum trichloride at a final concentration of 10% in the analyzed sample was used as a spectral buffer during calcium determinations.
The levels of Cd, Cr, Ni, and Pb were determined by electrothermal technique of atomic absorption spectrometry with atomization in a L’vov platform graphite cuvette using a High-Resolution Continuum Source Atomic Absorption Spectrometer ContrAA 700 (Analytik, Jena, Germany). Stock solutions at the concentration of 60 ppb (µg/L) for lead, 5 ppb for cadmium, 60 ppb for nickel, and 50 ppb for chromium were prepared by the dilution of standard solutions at a concentration of 1000 ppm (Merck) in 0.5% nitric acid, previously prepared by diluting the 65% Suprapur nitric acid (Merck) in deionized water. The calibration curves for the elements were selected by the decomposition of residues method. The dilution factor was chosen automatically by the autosampler. Twenty-five microliters of each sample solution with the addition of 5 µL of Pd(NO₃)₂/Mg(NO₃)₂ matrix modifier (when necessary) was injected into the furnace.
As a reference material used to validate the spectroscopic determination (AAS), a mixture of flour and milk powder (in proportions 70:30), fortified with known concentrations of investigated elements, was used. The content of investigated elements in the flour and milk mixture resulting from the fortification is presented in Table 4 and Table 5 as a theoretical value, whereas the quantities of the respective elements determined in the measurement are shown in the “found value” line. The analysis of the investigated and reference material were performed simultaneously under the same conditions.
Based on calibration curves, the obtained values were calculated to provide the content of elements in fresh ginger rhizome and in dry mass. Operating parameters for FAAS and ETAAS analysis are presented in the Tables S2 and S3. The limits of detection (LOD), as well as the recovery values, are presented in Table 4 and Table 5.