Independent concentration extraction as a novel green approach resolving overlapped UV–Vis binary spectra and HPTLC-densitometric methods for concurrent determination of levocloperastine and chlorpheniramine
Authors: Mohamed Ekram H. , Batakoushy Hany A. , Ibrahim Adel Ehab , Nasr Zeinab Adel , Soliman Marwa M. , Barghash Sona S. , Mohamed Tahany F. , Fouad Fatma A. 
Published: August 2024

Abstract
Background
The proposed research study introduces independent concentration extraction (ICE) as a novel UV–Vis spectrophotometric approach. The approach can be used for extracting the concentration of two analytes with severely overlapped spectra from their binary mixtures. ICE is based on spectral extraction platform involving simple smart successive methods that can directly extract the original zero order spectra of the analytes at their characteristic (λmax). Chlorpheniramine maleate (CPM) and Levocloperastine fendizoate (LCF) are two commonly co-formulated drugs in cough preparations. The combined mixture was used to confirm the validity of the developed ICE tool. Another less green HPTLC was developed for the first time to separate both drugs and help also in confirming the proposed tool.

Methods
For the simultaneous determination of CPM and LCF, two ecologically friendly techniques were employed. The first approach encompasses the use of the ICE spectrophotometric method that could be successively applied for extracting the concentration of two analytes with severely overlapped unresolved spectra in their binary mixtures. Other complementary methods aiming at original spectral extraction; including spectrum subtraction (SS) and unity subtraction (US) were also successfully employed to resolve the zero order spectra of the combined drugs with all their characteristic features and peaks. The second technique used, a high-performance TLC-densitometric one, was performed on silica plates with silica plates F254 and a mobile phase with a ratio of 3:3:3:1 by volume of toluene, ethanol, acetone, and ammonia as a developing system at 230 nm.

Results
The presented extraction approach was executed without any optimization steps or sample pretreatment for the simultaneous determination of CPM and LCF. The method was found to be valid for their determination within concentration range of 3.0–30.0 μg mL−1 for both drugs. For HPTLC method, the resulting Rf values of CPM and LCF were 0.37 and 0.78, within concentration ranges of 0.3–4.0 μg/spot and 0.8–10.0 μg/spot, respectively. Greenness assessment of both developed methodologies showed that the HPTLC method is less green than the spectrophotometric method, yet with comparable sustainability when it comes to the used technique.

Conclusion
The procedures were found to be selective, accurate, and precise for analysis of the studied binary mixture. Furthermore, the environmental impact of the introduced methods was assessed using novel greenness metrics, namely AGREE and Green Analytical Procedure Index (GAPI) to prove their ecological safety. In addition, white analytical chemistry (WAC) evaluation metric was employed to ensure the synergy and coherence of analytical, practical, and ecological attributes.