Analysis of size characterized manganese species from liver extracts using capillary zone electrophoresis coupled to inductively coupled plasma mass spectrometry (CZE-ICP-MS)

This paper focuses on experiments for getting more information on Mn species in liver extracts. The investigations are performed with respect to (1) a size characterization and (2) a subsequent identification of the Mn species in liver extracts using preparative size exclusion chromatography (SEC) followed by capillary zone electrophoresis coupled to inductively coupled plasma mass spectrometry (CZE-ICP-MS). First, extracts were analyzed using a mass calibrated SEC column coupled to ICP-MS detection. The chromatogram showed the 55Mn-trace and proved main Mn elution between ca. 60-150 kDa. Second, liver extracts were fractionated on the same SEC column, however, now the effluent was directed to a fraction collector. This resulted in fractions containing pre-purified, size characterized Mn species per fraction. It turned out that the Mn concentrations per fraction reflected roughly the previous on-line Mn trace. Third, the fractions were subject to CZE-ICP-MS, where the MS was operated additionally with dynamic reaction cell (DRC) technique. From size characterization (with SEC coupled on-line to ICP-MS or connected to a fraction collector and subsequent Mn determination in fractions) it was shown that most Mn species from liver extract were of high molecular mass (HMM) nature as they eluted mostly between 50 and 80 min, corresponding to ca. 60-150 kDa. With the two-dimensional speciation approach employing first SEC and then CZE-ICP-DRC-MS together with standard addition method, a series of Mn species was identified. Mn species predominantly were Mn-enzymes e.g. arginase, isocitric dehydrogenase, galactosyltransferase, prolidase, pyruvate carboxylase and oxalate oxidase. A typical Mn-transporter - Mn-albumin - was also seen, whilst Mn-transferrin obviously was degraded during SEC separation. This Mn-compound (independent whether as a standard or from liver extract) was not stable during SEC even at the finally chosen physiological conditions.