Trace element analyses of diaspirin cross-linked hemoglobin solutions

A sensitive assay using inductively coupled plasma atomic emission spectroscopy (ICP-AES) has been applied to the measurement of trace elements in diaspirin cross-linked hemoglobin (DCLHb) solutions. Calcium, magnesium, zinc and iron were the only elements detected at greater than background levels. Ag, Al, As, B, Ba, Bi, Cd, Co, Cr, Cu, Mn, Mo, Pb, Sb, Se, Si, Sr, Ti, and V were present at concentrations either equal to the acid blanks or were not detected. Detection of non-heme iron (not incorporated into the hemoglobin porphyrin ring) in a 10 g/dL hemoglobin solution required the development of a special protocol. In this protocol a chelator, DTPA, was added to hemoglobin solutions to complex with both free and non-specifically bound non-heme iron. The resulting iron:DTPA complexes were separated from the hemoglobin molecules by ultrafiltration and the ultra-filtrate analyzed by ICP-AES. A modification of this assay in which the DTPA was omitted was used to measure the free non-heme iron in solution. Typical concentrations of chelatable (free and non-specifically bound) and solution (free) non-heme iron in DCLHb production lots at the completion of manufacture were 0.5-1.0 ppm and 0.1-0.3 ppm, respectively.