Carotenoid Quantification by HPLC-MS

Absorption UV–visible spectra were obtained at room temperature on a Cary 60 UV-Vis spectrophotometer (Agilent, Santa Clara, CA, USA) using the scan mode, with a 300–800 nm absorbance range. Acetone was used as blank and baseline correction.
The HPLC analysis of carotenoids in acetone was performed using a Zorbax extended -C18 column (Agilent, Santa Clara, CA, USA) (2.1–50 mm, 1.8 m) on an Agilent 1200 series system (Santa Clara, CA, USA). The optimization and validation of HPLC parameters were determined in previous experiments [16 (link)]. To determine the mass spectra of the different compounds, a 6490 Triple Quad LC/MS system (Agilent, Santa Clara, CA, USA) was used equipped with an electrospray ionization source (ESI) Jet stream operating in positive scan mode (m/z range of 300–900), with 0.1 a.m.u. (atomic mass unit). precision, and controlled by MassHunter Workstation Software (Agilent, B.05.00, Santa Clara, CA, USA). The following specific working conditions were used: capillary voltage 3000 V, gas flow rate 11 L min⁻¹, gas temperature 290 °C, sheath gas flow rate 12 L min⁻¹, sheath gas temperature 300 °C and nebulizer pressure 35 psi.