Development, Analytical Validation and Stability Study of Low-Dose Atropine Eye Drops Prepared from Pharmaceutical-Grade Atropine Sulfate Powder

Objective
The shortage of commercially available single-dose 1% atropine eye drops (Faure^®) prompted the development of a compounding process for low-dose atropine ophthalmic solutions using pharmaceutical-grade atropine sulfate powder. The aim of this study was to develop a preparation process compatible with our aseptic compounding facilities and to evaluate the physicochemical and microbiological stability of the resulting formulations.

Materials and Methods
A 2 mg/mL atropine stock solution was prepared by dissolving 5 g of pharmaceutical-grade atropine sulfate powder (Inresa^®) in 0.9% sodium chloride under continuous stirring in a Grade B cleanroom. The stock solution was sterilized by membrane filtration during aseptic filling into infusion bags. Low-dose atropine eye drops were subsequently prepared by dilution of the stock solution to final concentrations of 0.1 mg/mL, 0.25 mg/mL, and 0.5 mg/mL.
Atropine quantification was performed by HPLC-UV using a Kinetex XB column and a mobile phase consisting of 25 mM phosphate buffer (pH 2.5) and acetonitrile (88:12, v/v), delivered at 1.0 mL/min. Detection was carried out at 210 nm with an injection volume of 20 µL. The retention time of atropine was 5.4 min. The analytical method was validated over a calibration range of 25–60 µg/mL using three quality control levels (37.5, 50.0, and 57.5 µg/mL) analyzed on three consecutive days. Method performance was assessed using the accuracy profile approach with a β-expectation level of 85%. Final formulations (0.1 mg/mL and 0.5 mg/mL; n = 6) were assayed. A 2-month stability study was conducted, including determination of pH, osmolality, atropine content (HPLC), subvisible particulate contamination, and sterility.

Results
The HPLC-UV method demonstrated excellent linearity over the validated concentration range (R² ≥ 0.99). Accuracy, repeatability, and intermediate precision met the predefined acceptance criteria (CV < 4%). The accuracy profile demonstrated that the β-expectation tolerance intervals (β = 85%) were entirely included within the predefined acceptance limits across the full calibration range. Assay results obtained for the compounded formulations confirmed the reliability of the validated analytical method.
During the 2-month stability study, osmolality remained stable at 289 mOsm/kg, while pH remained constant at 5.0. No significant changes in atropine concentration were observed throughout the study period. No increase in subvisible particulate contamination was detected, and all preparations remained sterile.

Discussion and Conclusion
The proposed compounding and quality control protocol enables the preparation of low-dose atropine eye drops from pharmaceutical-grade atropine sulfate powder, providing an effective alternative during supply shortages. The results obtained over 2 months demonstrate the physicochemical and microbiological stability of the formulations. However, the stability study is still ongoing, and additional data are required to establish the maximum shelf life of these compounded eye drops.

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