Agilent 1100 capillary hplc system

Microbore HILIC separations were performed using an Agilent 1100 capillary HPLC system (Agilent, Tokyo, Japan) coupled either to ICP-MS detection (7500 cs instrument, Agilent) or to an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The column used for HILIC separation was a TSK gel amide 80 (250 mm × 1 mm i.d., 5 μm) from Tosoh Biosciences (Stuttgart, Germany). Gradient elution, at a flow rate of 50 µl min⁻¹, was carried out using eluent A, acetonitrile, and eluent B, 5 mM ammonium formate (pH 5.5). Samples were diluted with acetonitrile and water to obtain a 1:2, sample to acetonitrile ratio, and centrifuged. A 7 µl aliquot of the supernatant was injected into the HILIC column each time. To get accurate masses during HILIC/ESI MS analysis, MS and MS/MS spectra were recalibrated offline using precursor/fragment ions with known formula. Putative metal species were fragmented during a subsequent chromatographic run with collision induced dissociation (CID) mode at 35% energy. Signals were recorded at m/z corresponding to pseudopaline complexes with Ni and Zn (C₁₅H₂₁N₄O₈Ni⁺ and C₁₅H₂₁N₄O₈Zn⁺ respectively). Detailed procedures for chromatographic and MS analyses were described elsewhere^{17 (link)}.