Green Chromatographic Method for Determination of Active Pharmaceutical Ingredient, Preservative, and Antioxidant in an Injectable Formulation: Robustness by Design Expert

Abstract

We report an efficient HPLC method for simultaneous qualitative and quantitative analysis of lincosamide antibiotic injectable formulations containing Clindamycin phosphate (CMN), benzyl alcohol (BA), and ethylenediaminetetraacetic acid (EDTA) as major ingredients. The three components were separated by Phenomenex prodigy C8 (250 mm × 4.6 mm, 5 μm) HPLC column, flow rate 1.1 mL/min, injection volume 30 μL, and column temperature 35 °C, using 0.05 M sodium acetate buffer (pH 4.5) with acetonitrile (ACN) in the ratio of 80:20 (v/v). The detection wavelength was set as 240 nm. The method was validated as per International Conference on Harmonization (ICH) guidelines and was confirmed to be specific, precise, accurate, and linear. Method robustness was executed by utilizing quality in the design of the experiment. Accuracy results were found to be 99.3-100.5% for CMN, 99.3-100.8% for BA, and 99.1-100.3% for EDTA. Precision results were obtained as % relative standard deviation (RSD): 0.6% for CMN, 0.4% for BA, and 0.4% for EDTA. Correlation coefficient (r ²) values were obtained as >0.999 for the three components. Analytical solutions are stable for 48 h at benchtop and refrigerator conditions. The greenness of the analytical method was evaluated by the Green Analytical Procedure Index (GAPI), National Environmental Method Index (NEMI), analytical eco-scale, and Analytical Greenness (AGREE) tools to confirm that the method is eco-friendly.

Figure 1

Structures of clindamycin phosphate (A), EDTA (B), and benzyl alcohol (C).

Scheme 1. EDTA UV-Active Complex Formation by Reaction with Copper Acetate

Figure 2

Final chromatogram with optimized conditions, flow rate 1.1 mL/min, column temperature 35 °C, injection volume 30 μL, and wavelength 240 nm with Phenomenex prodigy C8 (250 mm × 4.6 mm, 5 μm) column.

Figure 3

Precision chromatogram: 6 individual sample preparations from a homogeneous sample expressing the closeness of agreement between series of measurements.

Figure 4

Linearity Chromatogram: EDTA linearity from 0.1 μg/mL to 0.8 μg/mL, BA linearity from 2.4 μg/mL to 14.2 μg/mL, and CMN linearity from 37.5 μg/mL to 225.0 μg/mL.

Figure 5

(A) Half normal plot, Contour plot, and 3D plot of CMN Retention time. (B) Half normal plot, Contour plot, and 3D plot of BA Retention time. (C) Half normal plot, Contour plot, and 3D plot of EDTA Retention time.

Figure 6

NEMI, GAPI, and AGREE pictograms of the current method. In NMEI, GAPI and AGREE are marked green, yellow, or red depending on the impact on the environment. Waste disposal was not discussed; hence waste was not addressed in NEMI.