Abstract
Although the absolute retention time is a fundamental value in HPLC, there is usually some small variation in every chromatogram, even though the operating conditions are the same. For this reason, the most widespread method used today is the internal standard methd, in which the retention time of a solute is measured relative to that of a reference solute (internal standard). An internal standard added in the sample is also useful as a visible reference during the HPLC run. Such situat.ons often exist in clinical studies such as those of abnormal insulinemia or hyperproinsu linemia, in which the sample concentrations are usually too low to be detected by spectrophotometry.
We prepared three peptide fragments of cytochrome-C by trypsin or protease digestion for use as internal standards for HPLC of human insulin, proinsulin and C-peptide. Under our experimental conditions, coefficients of variability (C.V.) of the absolte retention time were 1.46% for insulin, 1.36% for proinsulin and 0.91% for C-peptide, while the C.V. values of the relaive retention time were much lower: 0.84%, 0.56% and 0.65%, respectively. These results suggest the usefulness of the internal stadard method for HPLC of these hormones.
