Quantification of Hydroxyproline and Isorhamnetin via UHPLC-MS/MS

HYP content was quantified using UHPLC-MS/MS system, which includes Agilent 1290 Infinity UHPLC, and Agilent 6460 Triple Quadrupole, equipped with an electrospray ionization interface used to generate positive ions for the determination of HYP. The compounds were analyzed by using the Agilent Zorbax Eclipse Plus C-18 column with a particle size of 1.8 μM (flow rate: 0.35 mL/min). The mobile phase was set as follows: mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in ACN): 0–3 min, 10–30% B; 3–4 min, 70% B; 4–5 min, 30–70% B; 5–6 min, 70-10% B. The column and auto-sampler temperature were maintained at 30 °C and 4 °C, respectively. Data were analyzed by using Agilent MassHunter Workstation software B.01.03. The gas temperature was set at 300 °C with a flow rate of 11 L/min. Gases were set at 30 psi for the nebulizer, capillary, at 4000 V. For the HYP, the fragment was 170. The collision energy was set at 30; The mass transitions were as follows based on multiple reaction monitoring: m/z 464.38 → 299.9. For the Isorhamnetin (ISO), the fragment was 150. The collision energy was set at 30; The mass transitions were as follows based on multiple reaction monitoring: m/z 315.4 → 300.1, The measurements of HYP were done using the standard and linear least-squares regression curve.