1260 infinity hplc instrument

HPLC–DAD-Q-TOF–MS/MS analyses were carried out using 1260 Infinity HPLC instrument (Agilent Technologies, Santa Clara, CA, USA) coupled with a 6520 Q-TOF mass spectrometer (Agilent Technologies) equipped with a dual electrospray ionization (ESI) source. The mobile phase consisted of water-0.01% formic acid (A) and acetonitrile-0.01% formic acid (B). Gradient elution conditions were: 0–30 min, 8–25% B; 30–80 min, 25–45% B; 80–100 min, 45–76% B and then returned to the initial condition. The flow rate was 1 ml/min. Chromatographic separation was carried out at 30 °C on a Venusil C₁₈ column (250 mm × 4.6 mm, 5 μm). Samples were detected at 203 nm. Parameters of MS detection were optimized as follows: drying gas temperature, 325 °C; flow rate of drying gas (N₂), 8.0 l/min; fragmentor voltage, 120 V; nebulizer, 40 psi; capillary, 3500 V; skimmer, 65 V; Oct RFV, 750 V. The sample collision energy was set at 35 V. All acquisition and analyses of data were controlled by Mass Hunter software (Agilent Technologies). Mass spectra were recorded in the range m/z 100–1700 with accurate mass measurement of all peaks. Each sample was analyzed in negative mode.

Organic acids as well as fructose and maltotriose were detected and quantified by HPLC, according to the method described by Marsili et al. (1981) (link) with the following modifications. Samples (1.00 g) were mixed with water (MilliQ), 0.5 M H₂SO₄ and acetonitrile in a MultiRS-60 BIOSAN turner (Montebello Diagnostics A/S, Oslo, Norway) operated at 30 rpm for 30 min. Samples were centrifuged for 15 min at 1470 × g using a Kubota 2010 centrifuge (Kubota Corporation, Tokyo, Japan) prior to filtration through 0.2 μm PTFE membrane (Acrodisc CR 13 mm Syringe Filter, PALL, United Kingdom). Organic acids were separated on an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA, United States) with 0.05 M H₂SO₄ as mobile phase and a flow rate of 0.4 mL/min. The column, operated at 30°C, was connected to a 1260 Infinity HPLC instrument (Agilent Technologies, Singapore) with pump, autosampler, column oven, RI-detector (refractive index, used for acetic acid, fructose and maltotriose) and diode array detector-ultra violet (DAD-UV) detector, used for the other organic acids. Openlab CDS software (Agilent Technologies) was used to operate the system and detection and quantification were done according to calibration with external standards. Maltose, sucrose and glucose were quantified by the K-MASUG enzymatic kit (Megazyme, Wicklow, Ireland), used according to the instructions.

To all tissue samples weighing 50 mg, 200 μL of methanol and the same amount of water (MiliQ) were added in a 1:1 ratio. Then, the tissues were homogenised and proline concentration was determined using the combination of the aTRAQ™ Kit for amino acid analysis (SCIEX, Framingham, MA, USA) and LC-MS/MS analysis. Samples for the aTRAQ™ Kit were prepared in accordance with the manufacturer’s protocol. LC-MS/MS analysis was performed using a 1260 Infinity HPLC instrument (Agilent Technologies, Santa Clara, CA, USA) and a 4000 QTRAP mass spectrometer (SCIEX, Framingham, MA, USA) equipped with an electrospray ionisation source. Sample analyses were performed in a random order. Detailed specifications of sample preparation and LC-MS/MS parameters have been described in our previous publications [48 (link),49 (link)]. Data acquisition and management were conducted using Analyst 1.5.2 software (Sciex, Framingham, MA, USA).