1260 infinity hplc

As previously described, total sphingolipids were analyzed using their released LCB (Markham et al., 2006 (link)). The samples (fruit mesocarp and epicarp) were standardized by adding C20-4-SPH (d20:1) as the internal standard. The hydrolysis of the sphingolipids followed Morrison and Hay (1970) (link), modified after Cahoon and Lynch (1991) (link), and o-phthaldialdehyde was used for derivatization. Agilent Technologies HPLC Infinity 1260 were used for the HPLC analyses using reverse-phase HPLC on a 2.1 mm × 150 mm Eclipse XBD-C18 Narrow-bore column (Agilent Technologies, Inc., Palo Alto, CA, United States). The samples were eluted (at 0.4 ml/min) with 20% RA solvent (5 mM potassium phosphate, pH 7.0), 80% solvent RB (100% methanol) for 7 min, rising to 90% solvent RB at 15 min, after which isocratic flow was applied for 10 min prior to increasing to 100% solvent RB at 30 min and a 3-min 100% solvent RB wash preceding a return to 80% solvent RB and 2 min of re-equilibration. At 340 nm, fluorescence was excited and then detected at 455 nm. The results were both analyzed as well as integrated by Openlab.

The samples (2.0 mL) collected during fermentation were placed in Eppendorf tubes (duplicate) previously dried at incubator (Labor) at 105°C for 4 hours and weighed to determine cellular concentration by gravimetry. Subsequently the samples were centrifuged at Centrifuge 5810 R (Eppendorf) with 3,000 rpm at 25°C for 15 minutes. Afterward they were filtered with Millipore membrane 0.45 μm pores and diluted with deionized water.
HPLC Infinity 1260 (Agilent Technologies) and the column Aminex HPX-87 H (BIO RAD) were used. The operating conditions were temperature of 25°C, 5.0 mM sulfuric acid as mobile phase, and flow rate of 0.6 mL/min. The mobile phase was vacuum-filtered with Millipore membrane 0.45 μm pores and degassed in an ultrasound bath for 30 minutes to prevent bubble formation, which may damage the fuel injection pump or even produce false peaks while passing through the detector. The detector was a refractive index with temperature of 25°C. The method was calibrated with standard solutions of sucrose, glucose, fructose, and lactic acid.
To determine cellular concentration, the precipitates obtained after samples centrifugation for analytical determination were washed with distilled water and centrifuged twice with the same conditions. Following that, the tubes were dried once again at 105°C for 24 hours and weighted to obtain the dry cellular weight.

Radiochemical analyses were performed using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). TLC analyses were performed using Agilent Technologies instant TLC-SA (iTLC) chromatography paper (Santa Clara, CA, USA) developed using a 1:1 mixture of 1.0 M ammonium acetate and methanol. TLC plates were read using an Eckert & Ziegler AR-2000 radioTLC system (Hopkington, MA, USA) and Eckert & Ziegler Winscan 3.0 software (Hopkington, MA, USA) was used to define regions of interest (ROI) and integrate peaks. HPLC analyses were performed on an Agilent Technologies HPLC (Infinity 1260, Santa Clara, CA, USA) equipped with a multi-wavelength diode-array detector followed by an in-line LabLogic detector (Flow-RAM Galbi NaI, Brandon, FL, USA) using a Thermo Scientific C18 analytical column (BetaBasic, 150 mm × 4.6 mm × 5 μm, 150 A, Waltham, MA, USA). LabLogic Laura™ radiochromatography software was used for HPLC data collection and analysis. Before any measurement by HPLC, solvents (HPLC grade) were degassed and water used for HPLC mobile phase was passed through a 0.22 μm filter. The HPLC method involved use of two solvents (A & B). Solvent A was acetonitrile (ACN) with 0.1% trifluoroacetic acid (TFA) and Solvent B was water with 0.1% TFA. The gradient elution began at 5%(A):95%(B) and changed linearly to 80%(A):20%(B) over 20 min at 1.0 mL/min.