## 2 Materials and Methods
### Theoretical Modeling
#### 2.2.12 Drug Stability and Loss Analysis  
* Gentamicin (40 mg/mL)
* Plastic Syringe: After 12 h, an average drug loss of 9.7% was observed, with the formation of a light brown precipitate at 25 \({}^{\circ}\)C.
* Glass Syringe: After 12 h, an average drug loss of 7% was noted, with precipitate formation occurring at 12 h (at 25 \({}^{\circ}\)C). Method used to determine insulin concentration: ELISA (Enzyme-Linked Immunosorbent Assay). Principle:  
Gentamicin does not contain strong chromophores, but can be chemically derivatized (with FMOC-CI (Fluoreylmethyloxycarbonyl chloride is a chemical reagent derived from fluorene, mainly used for: derivatization of compounds containing amino groups (-NH\({}_{2}\)) labeling of compounds for fluorescent or UV detection in chromatography (e.g., HPLC)) to form fluorescent or UV (ultraviolet) detectable compounds, which can be analyzed by high-performance liquid chromatography (HPLC). HPLC-Fluorescence = HPLC with fluorescence detection. A fluorescence detector is used, which measures the light emitted by a fluorescent compound after it has been excited with a certain wavelength. It is much more sensitive than UV (can detect lower concentrations). It is used when the compound is fluorescent or can be derivatized to become fluorescent. Insulin (Density of Insulin 1.09 g/cm\({}^{3}\)). * Plastic Syringe: After 12 h, an average drug loss of 27.2% was observed, with the formation of a light brown precipitate at 25 \({}^{\circ}\)C [1]. * Glass Syringe: After 12 h, an average drug loss of 11% was noted, with precipitate formation occurring at 12 h (at 25 \({}^{\circ}\)C) [1]. Figure 8: Sampling methodology. Method used to determine insulin concentration:  
ELISA (Enzyme-Linked Immunosorbent Assay). It is the most used method for determining insulin in biological or pharmaceutical solutions, due to its excellent sensitivity and specificity. ELISA uses specific anti-insulin antibodies fixed in a microtiter plate (96 wells). Insulin in the sample binds to these antibodies. Then, an enzyme-labeled secondary antibody is added, which generates a signal (colorimetric) proportional to the insulin concentration. Experimental steps (Sandwich ELISA):  
Plate coated with anti-insulin antibody: binds insulin in the sample. Adding a sample or standard (known concentrations of insulin). Washing: removes unbound material. Adding a secondary antibody linked to an enzyme (e.g., peroxidase). Washing again. Adding enzyme substrate (e.g., TMB--tetramethylbenzidine), develops a color. Reading absorbance at 450 nm with a microplate reader. Comparing absorbance with a standard curve: determining the unknown concentration. Furosemide (Density of furosemide 1.606 g/cm\({}^{3}\))  
* Plastic Syringe: After 12 h, an average drug loss of 26.1% was observed, with the formation of a light brown precipitate at 25 \({}^{\circ}\)C.
* Glass Syringe: After 12 h, an average drug loss of 5% was noted, with precipitate formation occurring at 12 h (at 25 \({}^{\circ}\)C). The observed loss correlates with prolonged injection times, particularly when the solution was prepared 6 h before administration. Drug Concentration Variability in Syringe
* Increased attention was paid to insulin, where a (+16.3%) increase in drug concentration was detected in the lower section of the syringe when mounted in the syringe pump after 12 h.
* Within the proposed pump, a mechatronic homogenization system before sample collection significantly reduced concentration discrepancies across the syringe. The measured concentration differences between the upper, middle, and lower sections were reduced to 0.7-1.1%. Potential measurement errors were estimated at 0.3-0.4%, which is within an acceptable accuracy range. Furosemide concentration detection method: Furosemide absorbs UV light, especially around 230-270 nm, due to the aromatic and sulfonamide groups in the structure.