ISSN print edition: 0366-6352 ISSN electronic edition: 1336-9075 Registr. No.: MK SR 9/7 Published monthly

Quantification of potential genotoxic impurity in caffeine: (Q)SAR assessment, metal chelation-assisted UPLC-ESI-MS/MS method, and its sustainability evaluation

Sriram Pepakayala, Venkata Nadh Ratnakaram, Yuvaraj Zunjarrao, Krishna Kumar A. Yadav, and Yogesh Kumar Lohia

Analytical Research and Development, IOL Chemicals and Pharmaceuticals Ltd, Barnala, India

E-mail: doctornadh@yahoo.co.in

Received: 2 December 2025  Accepted: 14 May 2026

Abstract:

Genotoxic impurities lead to mutations and can cause cancer. (Q)-SAR methodologies were used for the determination of the potential genotoxicity of 6-Amino-1,3-dimethyl-5-nitrosouracil (CAFE-III), an impurity in caffeine, to achieve SDG3 (Good Health and Well-Being). A straightforward UPLC-ESI-MS/MS method was developed to determine CAFE-III to realise SDG9 (Industry, Innovation, and Infrastructure). The problem of non-specific adsorption is minimized by adding citric acid to the mobile phase, which acts as a chelating agent to block metal sites and hence reduces the loss of analyte. The method used a Waters Acquity BEH C18 column (100 × 2.1 mm, 1.7 μm) at 45 °C, employing a gradient mobile phase at a flow rate of 0.3 mL/min. The mobile phases were Phase A (0.1% formic acid and 2 ppm citric acid in water) and Phase B (2 ppm citric acid in 95:5 v/v methanol:water), with a total run time of 12 min. The method was validated according to ICH (Q2 R2) guidelines. The current method enables the quantification of CAFE-III up to 0.34 ppm. Calibration curves displayed good linearity with correlation coefficients exceeding 0.99 across concentrations from the QL to 150% of the specification limit. Satisfactory recoveries for all selected impurities ranged between 87.97 and 112.12%. Greenness tools and CACI were used to assess the eco-friendliness and practicality of the method.

Graphical abstract

Keywords: Genotoxic impurity; UPLC-ESI-MS/MS; Caffeine; (Q)-SAR; Method development and validation; Analytical Greenness evaluation; Non-specific adsorption

Full paper is available at www.springerlink.com.

DOI: 10.1007/s11696-026-05067-y

Chemical Papers 80 (9) 11163–11178 (2026)