Abstract
Recombinant (r-) hirudins and PEG-hirudin are currently tested for anticoagulant therapy.
For their concentration measurement, radioimmunoassay and HPLC methods are available.
The separation of r- and PEG-hirudin is currently performed by HPLC. However, the
sensitivity of the method is low. Capillary electrophoresis is a rapid, selective
technique that requires low sample amounts. Our aim was the development of a capillary
electrophoresis method to measure r- and PEG-hirudin. The results are as follows:
In a borate solution (0.3% boric acid and 0.4% sodium tetraborate, pH 9.5) r-hirudin
was separated from PEG-hirudin in a purified system using a fused silica capillary
(50 cm long and 75-μm i.d. and reversed polarity). A neutral capillary with a 20 mM
tricine buffer (pH 8.0, field strength 500 V/cm) was also effective in resolving r-
from PEG-hirudin. A linear correlation was found between the peak area and the concentration
between 20 μg/mL and 10 mg/mL for hirudin (r2 = 0.99) and between 1.25 and 10 mg/mL for PEG-hirudin (r2 = 0.99). In human plasma mixtures, r- and PEG-hirudin were completely separated.
The linear correlation between the peak area and the concentration was r2 = 0.99.
Conclusion: In a fused silica capillary, r- and PEG-hirudin are separated in a purified
system. Capillary electrophoresis which is performed in a neutral capillary, resolves
r- from PEG-hirudin in a purified system, in plasma and in urine. The sensitivities
of the methods are comparable. Capillary electrophoresis separates r- from PEG-hirudin
and may be applied to biologic systems to measure the concentration and purity of
r- and PEG-hirudin.
Keywords:
Thrombin inhibitor - capillary electrophoresis - r-hirudin - PEG-hirudin
