Development and validation of the enzymatic kinetic photometric procedure for determination of residual quantities of dequalinium chloride on the surface of the pharmaceutical equipment

Abstract

Aim. To develop and validate the new enzymatic kinetic photometric procedure for analysis of dequalinium chloride based on application of enzymatic acetylcholine hydrolysis to determine the residual quantities of dequalinium chloride while monitoring the completeness of cleaning of the pharmaceutical equipment. Results and discussion. The optimal conditions for the enzymatic reaction course were determined – the order of mixing and the concentration of acetylcholine (0.05 mg/mL), cholinesterase (0.4 mg/mL), hydrogen peroxide (10 %) and p-phenetedine (1 %), the time of the reaction mixture maintaining (20 min), pH (8.35), the effect of the nature of the buffer solution. Validation of the procedure developed was carried out – the application range of the procedure was proposed (40–160 % in the normalized coordinates, the maximum acceptable concentration of dequalinium chloride in washes from the pharmaceutical equipment хcrit = 0.5 μg/mL), the number of concentration levels (n = 7); the order of their location within the application range, taking these data into account the maximum acceptable uncertainty of the procedure (maxΔx = 3.05 %) and the acceptability criteria for linear dependence parameters, systematic and random errors were calculated. The validation characteristics of the procedure developed were shown to correspond to the acceptability criteria calculated. The degree of extraction of dequalinium chloride from swabs with flushing from the pharmaceutical equipment was determined. Experimental part. The residual quantities of dequalinium chloride on the surface of the pharmaceutical equipment were determined by the degree of inhibition of the enzymatic reaction assessed by the unreacted residue of the substrate of the biochemical reaction, such as acetylcholine. The residual quantities of acetylcholine in the reaction mixture was determined by the kinetic photometric method by the indicator reaction of p-phenetidine oxidation with peracetic acid (formed during the auxiliary perhydrolysis reaction when adding an excess of hydrogen peroxide to the reaction mixture) in the way of registering the light absorbance of the resulting reaction product – azoxifenetole (λmax = 358 nm) for a definite period of time. Conclusions. The enzymatic kinetic photometric procedure has been developed to determine the residual quantities of dequalinium chloride on the surface of the pharmaceutical equipment after its cleaning. The procedure developed has been validated by such parameters as linearity, accuracy, precision and the limit of quantification.

Keywords

• dequalinium chloride
• cholinesterase
• acetylcholine
• validation