DOI: 10.1093/jaoacint/qsad087 ISSN: 1060-3271

A Green-and-White Integrative Analytical Strategy Combining Univariate and Chemometric Techniques for Quantifying Recently Approved Multi-Drug Eye Solution and Potentially Cancer-Causing Impurities: Application to the Aqueous Humor

Khalid A M Attia, Ahmed El-Olemy, Sherif M Eid, Ahmed Emad F Abbas
  • Pharmacology
  • Agronomy and Crop Science
  • Environmental Chemistry
  • Food Science
  • Analytical Chemistry



Drug impurities are now seen as a major threat to the production of pharmaceuticals around the world and a major part of the global contamination problem, especially when it comes to carcinogenic impurities.


We present the first spectrophotometric strategy based on a combination of univariate and multivariate methods as impurity profiling methods for the estimation of lignocaine (LIG) and fluorescein (FLS) with their carcinogenic impurities: 2,6-xylidine (XYL) and benzene-1,3-diol (BZD).


The data processing strategy depends on overcoming unresolved bands by employing five affordable, accurate, selective, and sensitive methods. The methods applied were a direct UV univariate spectrophotometric analysis (D0) and four multivariate chemometric methods, including classical least squares (CLS), principal component regression (PCR), partial least squares (PLS), and genetic algorithm (GA-PLS). FLS analysis (1–16 μg/mL) was performed using the D0 method at 478 nm; then, the application of the ratio subtraction method (RSM) allowed the removal of interference caused by the FLS spectrum. From the resulting ratio spectra, LIG, XYL, and BZD can be efficiently determined by chemometrics. The calibration set was carefully selected at five concentration levels using a partial factorial training design, resulting in 25 mixtures with central levels of 160, 40, and 3 μg/mL for LIG, XYL, and BZD, respectively. Another 13 samples were applied to validate the predictive ability.


The statistical parameters demonstrated exceptional recoveries and smaller prediction errors, confirming the experimental model’s predictive power.


The proposed approach was effectively tested using newly FDA-approved LIG and FLS pharmaceutical preparation and aqueous humor. Additionally, it was effectively assessed for whiteness, greenness, and sustainability using five assessment tools.


With its remarkable analytical performance, sustainability, affordability, simplicity, and cost-efficiency, the proposed strategy is an indispensable tool for quality control and in situ analysis in little-equipped laboratories, increasing the proposed approach’s surveillance ability.

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