*2.3. ATR–FTIR Analysis (Preformulation Study)*

ATR–FTIR analysis was carried out on the pure drug (methotrexate) and various physical mixtures of patch formulations (F1 to F9) to investigate possible interactions. A total of 32 scans were observed for each spectrum at a resolution of 4 cm−<sup>1</sup> from 4000 to 600 cm<sup>−</sup>1.

#### *2.4. Physicochemical Evaluation of Patches*

The physicochemical properties of the formulated patches were evaluated using the following parameters.

#### 2.4.1. Surface pH

The surface pH of the formulated patches was evaluated by placing a 1 cm<sup>2</sup> portion of a patch in 1 mL of distilled water for 2 h at room temperature (25 ± 2 ◦C) in a test tube. Excess water from the test tube was removed by the filtration process. A pH meter (InoLab®, Xylem Analytics, Dr. Karl Slevogt Street 1. Weilheim 82362, Germany) was used for the identification of the surface pH of the formulated patches. The pH meter was placed at the swollen part of the patch, and three readings were recorded for the average (mean ± SD) result [22].

#### 2.4.2. Physical Appearance

All formulated patches were physically inspected for smoothness, color, clarity, transparency, and homogeneity.

### 2.4.3. Thickness

The uniformity of thickness was evaluated for all formulated patches. A vernier caliper (Germany) was used for the evaluation of the thickness of the formulated patches. The thickness of patches was evaluated at 6 different places, and then the average was calculated [23].

#### 2.4.4. Weight Uniformity

All formulated patches were weighed individually for weight uniformity. An analytical weighing balance (Shimadzu AX 200, Kyoto, Japan) was used for the determination of weight. Individual weight was compared with average weight [24].

#### 2.4.5. Folding Endurance

The efficacy of the plasticizer was investigated with the folding endurance test. The folding of a patch at the same point until a break or crack appears shows the folding endurance capacity of a patch. At the same point, a patch was folded several times without cracking or breaking defines the value of folding endurance. The folding endurance test was conducted for all formulated patches [24].

#### 2.4.6. Moisture Uptake

The formulated patches were weighed accurately for the determination of percent moisture uptake. Aluminum chloride and the patches were placed in a desiccator to maintain humid conditions. After 3 days, the patches were taken out of the desiccator. The patches were weighed again. The difference between the initial and final weights of the patches gave the value of percent moisture uptake. Finally, average percent moisture uptake was calculated [25].

$$\text{\textbullet Moisture Uptake} = \text{(wf} - \text{wi)} / \text{wi} \times 100 \tag{1}$$

where wf is the final patch weight, and wi is the initial patch weight.

#### 2.4.7. Moisture Loss

All formulated patches were weighed individually for the determination of moisture loss. The patches, along with anhydrous calcium chloride, were placed in the desiccator at 37 ◦C in order to maintain dry conditions. After 3 days, the patches were taken out of the desiccator. The patches were weighed again. The difference between the initial and final weights of the patches gave the value of percent moisture loss. Finally, average percent moisture loss was calculated [14].

$$\% \text{Moisture Loss} = (\text{wi} - \text{wf})/\text{wi} \times 100 \tag{2}$$

where wi is the initial weight, and wf is the final weight.

#### 2.4.8. Moisture Content

The formulated patches were weighed accurately for the determination of moisture content. The patches, along with silica, were placed in the desiccator at room temperature for 24 h. The patches were taken out of the desiccator and weighed again until a constant weight was calculated. Percent moisture content was calculated using the following equation [26].

$$\% \text{Moisture Loss} = (\text{wi} - \text{wf}) / \text{wi} \times 100 \tag{3}$$

whereas wi is the initial patch weight, and wf is the final patch weight

#### 2.4.9. Tensile Strength and Percent Elongation at Break

The mechanical properties of the formulated patches were determined using a pulley system. A scale was used for the identification of the initial patch length. One end of the patch was tied with a thread, while the second end was tied with a rope crossing over the pulley. A weighing pan was attached to the hanging side of the thread. Gradually, weight was added until a crack or break appeared in the patch. The total weight in the pan was calculated for the tensile strength. The thread pointer indicated percent elongation of work. From the following equation, the total amount of force (tensile strength, kg/cm2) required to break a patch was calculated.

$$\text{Tensile Strength} = \text{F} / (\text{a.b} (1 + \text{L} / \text{I})) \tag{4}$$

Where,

F is the force needed to break a patch, a is the patch width (cm) and b is patch thickness (cm).

L is patch length (cm), and I is patch elongation before patch breakage (cm). The percent elongation of the patches was determined from the following equation [27].

$$\% \text{ Elongation} = (\text{Lf} - \text{Li}) / \text{Li} \times 100 \tag{5}$$

where Lf is the final patch length before breaking, and Li is the initial patch length.

#### 2.4.10. Drug Content Uniformity

Drug content uniformity was evaluated for the formulated patches. A patch was placed in a volumetric flask filled with phosphate buffer (pH = 7.4) and then placed in a sonicator for 8 h. After sonication, the solutions were filtered. A double-beam UV–visible spectrophotometer (Shimadzu 1601, Kyoto, Japan) was used for the identification of drug content using a 303 nm wavelength [11].

#### 2.4.11. Water Vapor Transmission Rate

Oven-dried and properly washed equal-diameter glass vials were used as transmission cells. In the transmission cells, 1 g of anhydrous calcium chloride was kept. At brim, the formulated patches were fixed. Transmission cells were weighed and then placed in the desiccator. Potassium chloride solution was kept closed in the desiccator to maintain 84% humidity. After predetermined time intervals, i.e., 6, 12, 24, 36, 48 and 72 h, cells were removed from the desiccator. Then, the cells were weighed again for the identification of the water vapor transmission rate [28].
