SIMPLE METHOD FOR OPTIMIZATION OF HEPARIN AND ENOXAPARIN DETECTION USING AGAROSE GEL ELECTROPHORESIS
Objective: A simple gel electrophoresis method for low-molecular-weight heparins (LMWH) is required for use in a variety of laboratories to allow further identification and purification. This study aimed to optimize the detection of heparin and enoxaparin (low-molecular-weight heparin by gel electrophoresis.
Methods: Several gel electrophoresis conditions were tested to optimize the detection of enoxaparin by using a simple method with a modified Volpi’s approach. Multiple gel thicknesses, voltage settings, and enoxaparin concentrations were tested in the optimization procedure. Enoxaparin was purchased from a local supplier as pre-filled pharmaceutical injections. Highly purified 0.5% and 1.0% agarose gels were prepared and a series of enoxaparin concentrations was added to both gels for comparison and optimization. The 0.2% toluidine blue stain was prepared by the addition of 1 ml in an ethanol-water-acetic acid mixture (50:49:1; v/v/w). The staining process comprised two steps: first, toluidine blue was added for 30 min and destained overnight in the solvent mixture. Subsequently, the following morning, the second step was conducted, in which the gel was restained for 30 min with the same concentration of toluidine blue. We continued to stain the gel until the bands were visible.
Results: The gel electrophoresis results showed that clearest and sharpest bands were obtained using 65–75 mAh and 85 V settings. At 95 mAh, the bands were slightly washed out.
Conclusion: This study successfully facilitated the detection of enoxaparin, a LMWH, and heparin in the laboratory by using simple tools and techniques available in most laboratories.
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