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  • Stability of Clozapine Stored in Oral Suspension Vehicles at Room Temperature: METHODS

Chromatographic Analysis

The liquid chromatographic system consisted of a solvent delivery pump (model P4000, Thermo Separation Products, San Jose, California), which pumped a mixture of 31.5% acetonitrile (EM Science, distributed by BDH, Toronto, Ontario, catalogue no. AX0142-1) and 68.5% 0.05 mol/L potassium phosphate monobasic at pH 4.1 (Fisher Scientific, Toronto, Ontario, catalogue no. P286) through a 15 cm x 4.6 mm reverse-phase Q8, 3-pm column (Supelcosil ABZ plus, Supelco, Oakville, Ontario) at 1.0 mL/min. Samples were injected directly onto the liquid chromatography column, in duplicate, using an autoinjector (Ultra WISP 715, Waters Limited, Toronto, Ontario). The column effluent was monitored with a variable-wavelength ultraviolet detector (UV 6000, Thermo Separation Products) at 230 nm. Signals from the detector were integrated and recorded with a PC1000 chromatography data system (Thermo Separa­tion Products). The area under the clozapine peak at 230 nm was subjected to least-squares linear regres­sion, and the actual clozapine concentration in each sample was determined by interpolation from the stan­dard curve. These concentrations were then converted to a concentration in milligrams per millilitre by use of the dilution factor (100). The quantitative resolution (standard deviation of duplicates/slope) for standards observed during assay validation was never greater than 0.0035 mg/mL. On the basis of the quantitative resolution, clozapine concentrations were recorded to the nearest 0.001 mg/mL but were reported to the nearest 0.01 mg/mL.

Assay Validation

Following development of the chromatographic system for clozapine, the suitability of this method for use as a stability-indicating assay was tested by analyzing samples of clozapine that had been subjected to accelerated degradation with sodium hypochlorite. Clozapine 12.5 mg (Sigma-Aldrich Canada Limited, Oakville, Ontario, lot 10K1204) was dissolved in 25 mL of methanol to make a 0.5 mg/mL stock solution. One millilitre of this stock solution was placed in a 5-mL glass test tube. Various volumes (between 5 pL and 100 pL) of sodium hypochlorite solution (1% available chlorine; Hygeol, Wampole Canada Inc, Scarborough, Ontario, lot 8A037A) were added to this solution. The mixture was combined with a vortex mixer (Fisher Vortex Genie 2, Fisher Scientific) and chromatography was performed immediately. Chromatograms were inspected for the appearance of additional peaks, and the clozapine peak was compared between samples for changes in concentration, retention time, and shape.
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After this first phase of evaluation and validation, the accuracy (the percentage deviation from the known concentration) and reproducibility (the coefficient of variation, determined as standard deviation/mean, expressed as a percentage) of the standard curves and quality control samples were tested over 4 days, and system suitability criteria (theoretical plates, tailing, and retention time) were developed to ensure consistent chromatographic performance on each study day.

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