Analysis of streptokinase by validated liquid chromatography methods and correlation with an in vitro bioassay.
Reversed-phase and size-exclusion liquid chromatography methods were validated for the assessment of streptokinase. The reversed-phase method was carried out on a Jupiter C4column (250 mm × 4.6 mm id) maintained at 25°C. The mobile phase consisted of 50 mM sodium sulfate solution pH 7.0 and methanol (90:10, v/v), run isocratically at a flow rate of 0.8 mL/min. The size-exclusion method was carried out on a Protein KW 802.5 column (300 mm × 8.0 mm id), at 25°C. The mobile phase consisted of 40 mM sodium acetate solution pH 7.0, run isocratically at a flow rate of 1.0 mL/min. Retention times were 19.3 min, and 14.1 min, and calibration curves were linear over the concentration range of 0.25-250 μg/mL (25.75-25 750 IU/mL) (r2= 0.9997) and 5-80 μg/mL (515-8240 IU/mL) (r2= 0.9996), respectively, for reversed-phase and size exclusion, with detection at 220 and 204 nm. Chromatographic methods were employed in conjunction with the in vitro bioassay for the content/potency assessment of Streptokinase, contributing to improve the quality control and ensure the efficacy of the biotherapeutic.
Postgraduate Program in Pharmaceutical Sciences, Federal University of Santa Maria, Santa Maria, RS, Brazil.
Clóvis Dervil Appratto
CD
Cardoso
Postgraduate Program in Pharmaceutical Sciences, Federal University of Santa Maria, Santa Maria, RS, Brazil.
Rafaela Ferreira
RF
Perobelli
Postgraduate Program in Pharmaceutical Sciences, Federal University of Santa Maria, Santa Maria, RS, Brazil.
Bruna
B
Xavier
Postgraduate Program in Pharmaceutical Sciences, Federal University of Santa Maria, Santa Maria, RS, Brazil.
Fernanda Pavani Stamm
FP
Maldaner
Postgraduate Program in Pharmaceutical Sciences, Federal University of Santa Maria, Santa Maria, RS, Brazil.
Francielle Santos
FS
da Silva
Department of Industrial Pharmacy.
Sérgio Luiz
SL
Dalmora
D001681:Biological Assay; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D057075:Enzyme Assays / Q000379:methods*; D011786:Quality Control; D015203:Reproducibility of Results; D013300:Streptokinase / Q000032:analysis* / Q000378:metabolism
D013300:Streptokinase
biotechnology; chromogenic bioassay; reversed-phase liquid chromatography; size-exclusion chromatography; streptokinase