A reverse-phase HPLC method for the analysis of tryptic digests of recombinant porcine growth hormone (pGH) has been developed and validated. Digestion was performed at 4 degrees C for a 20-hr period with TPCK-treated trypsin at a 1:20 (w/w) trypsin:pGH ratio. Gradient elution HPLC, using an Aquapore RP300 C8 column, was incorporated for separation of the digestion products and peak identification was carried out by mass spectrometry (MS). The digestion procedure and subsequent chromatography were linear in the initial concentration range of 4.55-45.46 microM (100 to 1000 micrograms/mL) pGH. The variability in the fragment retention times was low and the normalized peak area variability was less than 5% for all but three of the fragments. The utility of the trypsin digestion and chromatography procedures has been demonstrated by assessing chemical changes in pGH induced by incubation at elevated pH. Upon incubation of pGH in 0.2 M Tris buffer at pH 9 (ionic strength adjusted to 0.5 with NaCl) and 37 degrees C over a period of 400 hr, significant degradation in the regions corresponding to the digestion fragments T23-T25 (residues 181-182 linked by a disulfide bond to residues 184-191), T9 (residues 96-108), and T5-T18 (residues 43-64 linked by a disulfide bond to residues 158-166) was observed. The disappearance of the peaks corresponding to fragments T23-T25 and T9 both displayed apparent first-order degradation kinetics over the time period investigated with half-lives of 131 and 154 hr, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)