• 22
    Sep
  • Stability of Clozapine Stored in Oral Suspension Vehicles at Room Temperature: METHODS part 2

Physical Inspection

On study days 0, 3, 6, 14, 28, and 63, samples drawn from each of the 18 sample containers for the purpose of liquid chromatography were inspected visually for caking and consistency as well as for changes in colour.

Clozapine Analysis

On each study day (days 0, 3, 6, 14, 28, and 63), standard curves were prepared by dissolving 10.0 mg of clozapine (Sigma-Aldrich Co, lot 10K1204) in 25 mL of methanol to make a 0.4 mg/mL stock solution. Samples of this stock solution were further diluted with distilled water to obtain standards with final concentrations of 0.3, 0.2, 0.15, 0.10, 0.075, and 0.050 mg/mL. These standards, along with a blank, were used to construct a standard curve. One-microlitre samples of each standard or blank were chromatographed in duplicate. In addition, 2 quality control samples of clozapine (concentrations 0.075 and 0.30 mg/mL) were chromatographed in duplicate each day; the concentrations of the quality control samples were determined and compared with the known concentrations. Intra-day and inter-day errors were assessed by the coefficients of variation of the peak areas of both quality control samples and standards.

On each study day, suspension samples drawn from each container were assayed for clozapine content. All samples initially had a nominal clozapine concentration of 20 mg/mL. Samples were prepared for assay by dissolving 1.0 mL of each suspension in 100 mL of methanol to pre­pare a solution with nominal concentration of 0.20 mg/mL. This solution was mixed with a vortex mixer and sonicat­ed until no particulate matter was evident. One microlitre of each diluted sample solution was injected directly onto the liquid chromatography system without further preparation. All samples were analyzed in duplicate. The concentration of clozapine in each of the replicates was determined by interpolation from a standard curve of 6 standards plus a blank. These concentrations were then multiplied by the dilution factor (100) to determine the clozapine concentration in milligrams per millilitre, recorded to the nearest 0.001 mg/mL.
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Data Reduction and Statistical Analysis

After the coefficient of variation of the assay had been determined, a power calculation indicated that duplicate injection had the ability to distinguish between concentrations that differed by at least 10%. Means were calculated for replicated analyses and are reported in Table 1. Mean results from different days for each container were compared statistically (by linear regression) to determine if there was an association between the observed result and time. The lower limit of the regression line, based on a 95% confidence interval (CI), was also determined. Clozapine concentrations were considered acceptable or within acceptable limits if the concentration on any day of analysis was greater than 90% of the initial concentration (on day 0) and the lower limit of the 95% CI exceeded 90% remaining. Analysis of variance was used to test differences in degradation rate between different vehicles. Multiple linear regression was also used to detect any effect of time and suspension vehicle on concentration during the study period (by means of SPSS for Windows, release 10.0.5, 1999). The 5% level was used as the a priori cut-off for significance.

Table 1. Observed Concentration (as Mean Percent of Initial Concentration) of Clozapine after Storage in Various Suspending Vehicles


Study Day


Ora-Sweet


Ora-Plus


1:1
Mixture


HSC


Simple


Guy’s


Ora-Sweet and


Suspending


Syrup


Pediatric


Ora-Plus


Vehicle


Mixture


Initial concentration


(mg/mL)*


19.98 ±0.17


19.86 ±0.18


19.84 ±0.30


18.43 ±0.06t


19.37 ±0.19


20.14 ±0.06


Day
3


100.21 ±1.52


100.09 ± 2.95


102.38 ±2.56


101.96 ±1.38


103.95 ± 0.67


99.94 ±
0.67


Day
6


99.09 ±1.54


97.44 ±6.19


99.98 ±1.72


98.40 ± 4.49


99.80 ±1.31


102.68 ±2.03


Day 14


96.16 ±1.72


99.22 ±
2.39


100.65 ±1.90


95.86 ±1.57


95.80 ±1.24


103.68 ±1.20


Day 28


97.89 ±2.12


100.65 ± 0.79


101.30 ±1.82


101.68 ±5.16


101.91 ±1.08


96.71 ± 0.82


Day
63


96.97 ±1.85


96.01 ± 0.70


100.78 ±0.63


103.91 ± 0.64


103.92 ± 0.35


100.08 ± 2.03


Coefficient of


variation (%)*


1.68


1.82


0.89


2.86


3.05


2.42


% remaining on day 63


(estimated by regression)*


97.22


97.01


100.11


104.40


103.34


98.26


Lower limit of
95%
CI


for
% remaining on day
63§


93.98


93.97


99.13


98.71


97.65


94.64

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