A simple, specific, precise and accurate reverse phase HPLC method has been developed for the simultaneous determination of preservatives (Methyl Para hydroxybenzoate, Ethyl para hydroxybenzoate, Propyl para hydroxybenzoate and Chlorocresol) in ointment. The chromatographic separation was achieved on Zorbax column, 150 mm × 4.6 mm, 5 µm, using PDA detector. The mobile phase was 50% Methanol (v:v), at a flow rate of 1.0 mL/min. All preservatives were detected at 230 nm, at 30oC temperature for column. The retention times were 2.15 min for Methyl para hydroxybenzoate, 2.70 min for Ethyl para hydroxybenzoate, 3.69 min for Propyl para hydroxybenzoate and 4.01 min for Chlorocresol, with incredibly good resolution between peaks. The method was validated according to the ICH guidelines with respect to specificity, linearity (r2 =0.999; 0.998; 0.999 and 0.998), accuracy (99 to 100.1%), precision (RSD<2%).
The biosensors are based on the electrons movement, i.e. electronic current determination as a reaction of enzyme-catalyzed redox reaction. Generally a normal contact voltage passes through the electrodes to analyze. In the enzymatic reaction which produces the substrate or product can transfer the electrons with the surface of electrodes to be reduced. As a result an alternate current flow can be measured. The substrate concentration is directly proportional to the magnitude of the current. The reduction of oxygen is acquired through the oxygen electrodes and it is a simple way to from an amperometric biosensor (Figure 2). The example is the determination of glucose by glucose. The above description is about the first generation of amperometric biosensor and it has a direct transfer of electrons which are released from the electrodes are having some difficulties. The second generation amperometric biosensors are developed in a mediator takes the electrons and transfer to the electrodes.
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