Development of a highly sensitive mixed-polymeric EKC system for analysis of heparin and related substances

FLOR SABRINA; SCIOSCIA SILVIA; CONTIN MARIO; ESTEVEZ PABLO; SCASELLETTA FLORENCIA; TRIPODI VALERIA; LUCANGIOLI SILVIA

Simposio; 16th Latin American Symposium on Biotechnology, Biomedical, Biopharmaceutical and Industrial Applications of Capillary Electrophoresis and Microchip Technology. LACE 2010; 2010

Heparin is a natural linear complex polydisperse sulfated polysaccharide consisting of 1→4 linked pyranosyluronic acid and 2-amino–2-deoxycopyranose (with either N-sulfo or N-acetyl substitution) repeating units and it has been used clinically as an anticoagulant and antithrombotic agent for many years. Recently, oversulfated condoitin sulfate (OSCS) which is an unusual form of condroitin sulfate, was found in Heparin samples. Patients treated with OSCS contaminated Hep experienced angioedema, hypertension, swelling of the larynx, and in some cases, death. A mixed-polymeric electrokinetic chromatography system has been developed for the simultaneous determination of a contaminant like oversulfated condroitin sulfate (OSCS) and impurities expressed as dermatan (Der) in heparin (Hep) samples. The EKC system consisted of 0.5 % w/v polymeric β-cyclodextrin, 0.4 % w/v tetronic® 1107 and 400 mM tris-phosphate buffer at pH 3.5. The optimized electrophoretic conditions included the use of an uncoated-silica capillary of 50 cm of total length and 75 µm i.d., an applied voltage of -7 kV, a temperature of 30 ºC and 200 nm UV-detection. The highly sensitive method developed showed low values of LOD, 0.07 % w/w (0.07 µg/ml) (OSCS) and 0.1 % w/w (0.1 µg/ml) (Der), and values of LOQ 0.2 % w/w (0.2 µg/ml) (OSCS) and 0.3 % w/w (0.3 µg/ml) (Der) with a concentration level of heparin sample as low as 0.1 mg/mL. Additional parameters of validation such as specificity, linearity, accuracy, and robustness were evaluated according to international guidelines. Due to its simplicity, high sensitivity and reliability, the proposed method can be an advantageous alternative to the traditional methodologies for the analysis of heparin in raw material and in finished products because of the low amounts of heparin sample required.