Separation of oxidized human growth hormone variants by reversed-phase high-performance liquid chromatography. Effect of mobile phase pH and organic modifier.

Human growth hormone (hGH), a polypeptide of 191 amino acids, contains three methionine residues, two of which are susceptible to oxidation by both chemical and photochemical means (Met14 and Met125). To date, no method has existed for resolving the various mono- and di-oxidation products. We report on the resolution of these oxidized variants and native hGH at weakly (pH 3.5) acidic pH. Although all of the oxidized species can be resolved at pH 3.5, use of low pH and neutral pH mobile phases confer some advantages. For example, the Met14 oxidized variant (MetSO-14) and native hGH are best resolved at neutral pH and the mono-oxidized variants (MetSO-125 and MetSO-14) are best resolved at low pH. The effect of organic modifier on the separation of the oxidized variants was also evaluated. 1-Propanol was more effective than acetonitrile in the separation of MetSO-14 and native hGH while acetonitrile was more effective than 1-propanol in the separation of MetSO-125 and MetSO-14. In summary, mobile phase pH and organic modifier were shown to be important parameters in the separation of oxidized hGH variants.