The liquid chromatographic system consisted of an isocratic solvent delivery pump (model P4000, Thermo
Separation Products, Fremont, California), which pumped a mixture of methanol (OmniSolv, EMD Chemicals Inc, Gibbstown, New Jersey) and 0.05 mmol/L phosphoric acid (catalogue no. P286, Fisher Scientific, Toronto, Ontario) through a 15 cm x 4.6 mm reverse-phase C18, 3-pm column (Supelcosil, catalogue no. 58985, Supelco, Oakville, Ontario) at 0.5 mL/min. The ratio of methanol to phosphoric acid (67:33) was held constant during each chromatographic run. The samples were introduced into the liquid chromato- graphic system using an autoinjector (WISP 712, Waters Scientific, Toronto, Ontario).
The column effluent was monitored with a variable- wavelength ultraviolet detector (UV6000, Thermo Separation Products) at 232 nm. The signal from the detector was integrated and recorded with a chromatography data system (ChromQuest, Thermo Separation Products). The area under the docetaxel peak at 232 nm was subjected to least-squares linear regression and the actual docetaxel concentration in each sample determined by interpolation from the standard curve. Docetaxel concentrations were recorded to the nearest 0.01 mg/mL.
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The concentration of the degradation products of docetaxel (described below) could not be measured quantitatively because of a lack of standards for each of the degradation products. Instead, chromatograms were inspected on each study day for the appearance of degradation products, and the area of these peaks was monitored and compared between days for changes. Change in the amount of degradation product is considered a sensitive indicator of degradation.
After development of the chromatographic system for docetaxel (see above), the suitability of this method for use as a stability-indicating assay was tested by analyzing solutions obtained by accelerated degradation of docetaxel. A 0.5-mL sample of a 0.5 mg/mL solution of docetaxel (Taxotere, Aventis Pharma Inc, Laval, Quebec; lot 3P732, expiry October 2005; diluted in distilled water, pH 5.0) was placed in a glass vial, which was in turn placed in the autoinjector of the chromato- graphic system. Two-microlitre samples of this solution were injected and directly chromatographed just before addition of 10 pL of 1% sodium hypochlorite and at 8 other times over a 705-min period (10, 23, 48, 70, 92, 142, 210, and 705 min) after addition of the sodium hypochlorite. A second sample (25 mL) of the 0.5 mg/mL solution (diluted in distilled water, pH 5.0) was placed in a glass vial and incubated at 80°C in a water bath. Two-microlitre samples were drawn just before incubation began and at 9 other times over a 67-h period (0.5, 1, 2.25, 3, 4, 5, 20, 25, and 67 h) and were directly chromatographed. The chromatograms were inspected for the appearance of additional peaks, and the docetaxel peak was compared between samples for changes in concentration, retention time, peak shape, and ultraviolet spectral purity (200 nm to 320 nm) relative to a fresh undegraded sample.
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Following this first phase of evaluation and validation, the accuracy and reproducibility of standard curves were tested over 5 days, and system suitability criteria (theoretical plates, tailing, and retention time) were developed to ensure consistent chromatographic performance. A standard curve was prepared daily from a fresh vial of docetaxel (Taxotere, lot 3P732, expiry October 2005). Each vial supplied by the manufacturer (80 mg in 2 mL) actually contains 2.36 mL of a 40 mg/mL solution of docetaxel, which was diluted according to the manufacturer’s instructions with 7.33 mL of a 13% ethanol solution, also supplied by the manufacturer. This produced approximately 10 mL of a 10 mg/mL solution. Samples of this stock solution were further diluted in methanol and water (60:40) to obtain standards with final concentrations of 1.00, 0.75, 0.38, 0.25, 0.13, and 0.06 mg/mL. When combined with a blank, these standards served to construct a standard curve. A 2-pL sample of each standard was chromatographed in duplicate. Also, 2 quality control samples of docetaxel (concentrations of 0.50 and 0.19 mg/mL) were chromatographed in duplicate each day, with their concentrations determined and compared to the known concentrations. Intraday and interday errors were assessed by the coefficients of variation (CVs; standard deviation divided by the mean) of the peak areas of both quality control samples and standards.