Summary
Reversed-phase high-performance liquid chromatography (HPLC) is examined as a method
for separating pancreatic peptides. The method was based on gradient elution with
acetonitrile in an acid phosphate buffer (pH 3.10). Apart from human and porcine insulin
all the other peptide standards tested (thyrotropin-releasing factor, vasoactive intestinal
polypeptide, human C-peptide, porcine C-peptide, somatostatin, porcine glucagon, porcine
proinsulin and porcine pancreatic polypeptide) could be separated simultaneously in
40 minutes with a binary gradient composed of five linear segments and increasing
from 0 to 60% acetonitrile. Human and porcine insulin could be almost completely resolved
by a minimal reduction in the steepness of the acetonitrile gradient. Repeated injections
of human C-peptide and porcine insulin resulted in a coefficient of variation of less
than 1.5% in the retention times. The use of 125I-labelled peptides gave recoveries exceeding 90%. HPLC of acid ethanol extracts of
autopsy pancreases from three infants showed that the immunoreactivity of the peptides
measured remained unaffected by the chromatography. Both immunoreactive C-peptide
and immunoreactive insulin (IRI) were recovered in two peaks, the second common peak
representing proinsulin and amounting to 6.5 to 8.4% of total IRI. Immunoreactive
glucagon waseluted in a single peak. Chromatography of plasma extracts from two infants
of diabetic mothers demonstrated that proinsulin accounted for 59-63% of total IRI,
while insulin was separated into two peaks corresponding to the standards of human
insulin and porcine insulin. These results indicate that reversed-phase HPLC is a
method with a good reproducibility and a high recovery applicable to the rapid and
effective separation of pancreatic peptides from biological extracts.
Key-Words:
Pancreatic Peptides
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High-Performance Liquid Chromatography
