# Development of a Mouthwash Using Freeze-Drying Technique: An Optimization Study
## 2 Materials and Methods
### Methods
#### 2.2.4 Characterization of Mouthwash Samples  
All samples were analyzed at an ambient temperature of 22 \(\pm\) 5 \({}^{\circ}\)C.  
The texture analysis was performed with the Brookfield CT3 Texture Analyzer (Brookfield Engineering, Middleboro, MA, USA) through compression tests on one freeze-dried tablet at a time. The sample was placed horizontally on the analysis surface and was subjected to a constant force applied by a TA10 probe with a speed of 0.1 mm/s and a load of 10 g until a target distance of 5 mm. For each test, three measurements were performed and the rigidity at two different distances, fracturability, load at target, mechanical work at the first fracture, deformation at the first fracture, mean load were determined using the TexturePro CT Software V 1.9 (Brookfield Engineering, Middleboro, MA, USA). For each parameter, the mean and standard deviation were calculated.  
Reconstitution times for each sample were measured by placing a lyophilize in 10 mL of distilled water followed by manual stirring. The time required for the complete dissolution of the lyophilized tablet was measured with a digital stopwatch. For each test, three experiments were performed, and the mean and standard deviation were determined.  
Weight Loss Percentage  
To quantify the integrity of the lyophilizes, weight loss for each sample was measured after removing it from the blister pocket. Firstly, the lyophilize was weighed into the alveolae, then it was removed and weighed separately, and finally after weighing the empty alveolae, we determined the weight difference. For each test, three experiments were performed, and the mean and standard deviation were determined.  
Viscosity Measurement of the Reconstituted Mouthwashes  
For the reconstituted mouthwashes, the rheological measurements were made using the Brookfield DV-III Ultra (Brookfield Engineering, Middleboro, MA, USA) equipped with LV-1 spindle, at a rotational speed of 200 rpm. For each test, three experiments were performed, and the mean and standard deviation were determined.  
Experimental Design  
To investigate the effects of the formulation variables on the lyophilized mouthwashes, a DoE was developed, with the possibility to highlight the interactions between factors, using the Mode 12 software (Umetrics, Umea, Sweden). A full factorial design with three factors that varies on two levels, thus a total of eight runs and three center points, was generated. As factors, a qualitative one was chosen, the filler type (X1) that varied between mannitol or sorbitol, and two quantitative ones: the filler ratio (X2) set from 3 to 5% and methylcellulose (MC) ratio (X3) from 0 to 0.5%. The composition of the formulations is detailed in Table 1. The studied responses derived from the previously described characterization methods: rigidity at 2 (Y1) and 4 mm (Y2), load at target (Y3), fracturability (Y4), first fracture work (Y5), first fracture deformation (Y6), mean load (Y7), the weight loss percent (Y8), reconstitution time (Y9), and viscosity (Y10). For optimizing the final formula, some constraints were applied: weight loss, reconstitution time wereminimized and fracturability (measured as hardness values of initial fractures) and viscosity were maximized.