Zorbax eclipse c18 column

LC-MS analysis was done by using HPLC-ESI-MS-NEG-PHENOMENEX in negative ionization mode. System was equipped with binary pump, auto sampler, thermostated column compartment and iFunnel quadrapole time-of-flight spectrometer (Q-TOF). Zorbax eclipse C18 column (4.6 × 250 mm, 5 μm particle size) was used for compounds separation at 25°C temperature. In the present study, 0.1% (v/v) formic acid (A) and acetonitrile (B) was used in gradient elution. Gradient was initiated at 80% A and 20% B to 30% B (after 10 min), followed by 40% B (40 min), 60% B (60 min) and 90% B (80 min) and finally returned to the initial conditions. Solvent system B was injected with flow rate of 0.8 ml/min. Mass spectrometer was operated in range of 100–1,000 m/z. N₂ gas was used as a nebulizer. Drying gas flow rate was 8 L/min at 325°C and nebulizer gas at 25 psi with fragmentor voltage 150 V (Patel and Ghane, 2021 (link)). For data analysis, mass hunter qualitative analysis software package (Agilent Technologies) was used. Detected compounds were validated on the basis of molecular formula, molecular mass, retention time and m/z ratio. For the authentication of compounds, details were compared with available literature and Metline personal metabolites database.

Sugars and glycerol were analyzed by using a Shimadzu HPLC (Kyoto, Japan) and a Zorbax carbohydrate column (150 × 4.6 mm, Agilent, Santa Clara, CA, USA) connected to an evaporative light scattering detector (ELSD) with 80% acetonitrile as a mobile phase [4 (link)]. Ethanol, organic acids, phenolic acids, and alkaloids were quantified by a Shimadzu HPLC (Kyoto, Japan), detailed in our previous study [4 (link)]. Phenolic acids and alkaloids were measured by HPLC connected with a Zorbax Eclipse C18 column under gradient elution with mobile phase A (0.1% v/v acetic acid in H₂O) and mobile phase B (100% methanol) and connected with a PDA detector at 320 nm. Free amino compounds were analyzed by ARACUS Amio Acid Analyzer (MembraPure, Berlin, Germany) under a hydrolysate separation program [4 (link)].

Monitoring PFOA concentrations in the water samples was carried out by direct injection of 5 µL of water samples into a Zorbax Eclipse C18 column (2.1 × 100 mm, 3.5 μm), maintained at a temperature of 30 °C. PFOA was eluted from the chromatographic column, using Aq 5 mM ammonium acetate/MeOH (20/80 v/v) as a mobile phase. The mobile phase flow rate was 0.2 mL/min and was eluted in an isocratic manner. Multiple reaction monitoring was used as the MS/MS detection mode.
The determination of PFOA concentrations accumulated in fish organs and tissues, as well as the formed metabolites, was carried out using the same LC-MS/MS system. The separation of the compounds was achieved using the same Zorbax Eclipse C18 column (2.1 × 100 mm, 3.5 μm), thermostated at 30 °C. The mobile phase used consisted of Aq 5 mM ammonium acetate (A) and MeOH, in the gradient mode (Table S2), at a 0.2 mL/min flow rate, using an injection volume of 20 µL aqueous extract. The EIS parameters were the following: drying gas temperature: 300 °C; drying gas flow rate: 8 L/min; nebulizer pressure: 50 psi; and voltage on the capillary: 2500 V. The elution of the two analytes was achieved in less than 3.5 min. The operational parameters of the mass spectrometer and the MRM chromatogram obtained for PFOA, and its transformation products are shown in Supplementary Information (Table S3 and Figure S1).