An integrated analytical method for the separation, identification and quantification of carotenoids in Rhodobacter capsulatus.

The analysis and quantification of microbial carotenoids remain challenging due to the complexity of biological matrices and the high cost or limited availability of commercial standards. Herein, we present a cost-effective and reproducible workflow for the extraction, isolation, identification, and quantification of carotenoids in Rhodobacter capsulatus cultures. The method integrates light-protected pigment extraction, screening by thin-layer chromatography, and column chromatography for the isolation of individual carotenoids. Structural assignment is supported by UV-Vis spectroscopy, nuclear magnetic resonance (NMR), and LC-APCI-QTOF-HRMS exact-mass/isotopic-cluster evidence, while quantification is performed without commercial standards using the Lambert-Beer law with compound-specific molar extinction coefficients and HPLC-based calibration curves from internally isolated carotenoid-enriched fractions. The workflow showed strong calibration linearity (R² > 0.99), satisfactory reproducibility, with RSD values below 7 % for individual carotenoids, and high extraction efficiency recovering approximately 99 % of the total quantified carotenoids. The workflow facilitates comprehensive characterization of microbial carotenoid profiles, supporting both fundamental research and biotechnological applications. Future applications should evaluate its adaptation to other pigment-producing microorganisms and complex biological matrices.