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Boscolo et al
Thermo Scientific, Waltham, MA, USA) with a 50 mm × 2.1
mm reverse phase microcolumn C18 particle diameter 3.5
μm (XTerra C18 Waters Corp., Milford, MA, USA).
Chromatograms were processed using Breeze Software
(Waters, MI, USA). The mobile phase consisted of a mixture
of methanol and 0.030M disodium phosphate pH = 7.6
(40:60) with a flow rate of 0.3 mL/min. The column temperature was set at 25°C. Ten microliters of each sample were
introduced into the column using an automatic injector
(Thermo Scientific Spectra System AS3000). The column
effluent was monitored with a wavelength ultraviolet detector
set at 280 nm (Thermo Scientific Spectra System UV2000).
According to the stability study design, 2 aliquots were collected from each of the 3 containers for each temperature on
0, 7, 14, 30, 60, 90, 120, and 150 days.

of the method was evaluated by establishing a relationship
between the concentrations of OMZ and areas on the standard
chromatogram. This is shown by linear regression models
obtained for each of the 2 standard preparations. Linearity was
verified at different concentration levels (5.0, 10.0, 25.0, 50.0,
75.0, and 100.0 µg/mL) of OMZ, prepared in blank of excipients for each formulation and these were analyzed in duplicate
in 3 separate runs. LOD and LOQ were determined. Precision
was evaluated for intra-day (n = 6) and inter-day assays (n =
18), and it was expressed as relative standard deviation (RSD)
for retention times and areas. Accuracy was evaluated from
recovery studies. Placebo samples containing all excipients
present in the suspension were supplemented with OMZ at
80%, 100%, and 120% concentration levels of the nominal
values, and 3 replicates of each level were assayed.

Preparation of Standard Solutions

Microbiological Studies

Standard solution of OMZ. A stock standard solution of OMZ
containing 6 mg/mL was prepared in methanol and appropriately diluted in mobile phase to render a 8 µg/mL standard
solution.

Microbiological tests of formulations were performed at 0
and 150 days according to the USP monograph of non-sterile products.20 The microbial count was considered to be the
average number of colony forming units (cfu) found in agar.
Liquid oral formulations would meet microbial requirements. If the total aerobic microbial count was less than 102
cfu/mL, the total combined yeast/mold count less than 101
cfu/mL and confirmed the absence of Escherichia coli.

Sample preparation. The content of OMZ in the suspensions
was assayed. An OMZ suspension (2 mg/mL) was shaken vigorously by hand immediately before use. One gram was accurately weighed in a 10-mL volumetric flask and dissolved in
methanol, sonicated for 5 minutes, and centrifuged to 14 000
rpm for 5 minutes. A final dilution was performed from the
supernatant obtained in the previous centrifugation step, with
mobile phase to make a concentration of 7 µg/mL of OMZ.

Stress Conditions
Oxidation: 25 mL of a 3% v/v hydrogen peroxide solution
was added to 25 mg of OMZ accurately weighed. Acidic: 20
mg of OMZ and 100 mL of 0.5 M hydrochloric acid solution
were refluxed during 1 hour. Alkaline: similar condition to
the method described under "acid," using 0.5 M sodium
hydroxide solution. Light: 1 mg/mL of OMZ solution was
exposed to white light during 1 week. All samples were
diluted with diluent to obtain a final concentration of 8 µg/
mL of OMZ.

Validation of the Analytical Method
Validation of the method was performed by studying specificity, linearity, limits of detection (LOD) and limits of quantification (LOQ), precision, and accuracy. First, specificity was
mainly evaluated by subjecting OMZ standard solution to different possible degradation routes with acid, alkaline, oxidation, and light, which were then analyzed by micro-HPLC. In
addition, blank samples with all the excipients involved were
prepared and analyzed to check for interferences. The linearity

Results
Two different suspensions as oral liquid formulation have been
developed. One prepared from OMZ pellets (formulation A)
and the other from OMZ active (formulation B), both of them
at the same concentration 2 mg/mL. Results from different
physical, chemical, and microbiological studies are presented.
In the appearance test, after preparation (t = 0), both formulations resulted to be white suspensions, with characteristic odor of mint. For formulation A, there were no detectable
changes in color or odor in any sample during 150 days at
4°C and for 2 weeks at 25°C; formulation B remained stable
for 90 days at 4°C. In this sense, at 25°C, the color of the
suspension B after 1 day and suspension A after 14 days
changed from yellow to brown and finally violet.
In the taste test, the flavor of formulation A was similar to
point 3 of the scoring scale, corresponding to strong bitterness, for 60% of the volunteers and 80% for the formulation
B. Therefore, 80% of the volunteers preferred the formulation A prepared from the pellets.
Resuspendibility parameter can be observed for all formulations, because the sediments were easily redispersed
after 10 seconds of vigorous and manual agitation, resulting
in a homogeneous system for all temperatures and times.
The results of pH monitoring for both suspensions were
formulation A, 9.4 to 10.1 and formulation B, 9.5 to 10.1
during the study.

Hospital Pharmacy - October 2020

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