Hplc ms ms

Fungal growth: at the end of incubation, two perpendicular diameters of the fungal colony (mm) were measured. Then, the colonies were removed, by gently scraping the mycelium from the cheese surface, and put in a Falcon^® plastic vial with a screw cap. Both the Falcon vials and the cheese blocks were stored at −20 °C until use.
Mycotoxin Analysis: the chemicals and solvents used for the extraction and clean-up solutions were ACS grade or equivalent (Carlo Erba, Milan, Italy), mycotoxin standards were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mycotoxin analyses were performed separately both on the mycelium colonies and the cheese blocks. Rind cheese and fungal mycelium were ground and homogenised before analysis. All samples were subdivided for consistency in three aliquots for mycotoxin extraction using CH₃CN:H₂O (80:20 v/v) for MPA, PR-toxin, ROQ-C, STC and PA, CH₃CN:10 mM H₃PO₄ (70:30 v/v) for CIT and CH₃OH:3% NaHCO₃ (50:50 v/v) for OTA. Toxin analyses were performed by HPLC-MS/MS (Thermo-Fisher Scientific, San Jose, CA, USA) and standards for STC, CIT, ROQ, MPA, OTA, PR toxin, and PA were prepared, as described by Camardo Leggieri et al. [1 (link)]. Data were reported as total ng of mycotoxin on cheese block or mycelium. Limits of detection (LODs) were 1 ng for ROQ-C, 5 ng for MPA, PA, STC, OTA, and CIT, 10 ng for PR-toxin.

Forty milligrams of plant tissue were ground into powder in liquid nitrogen, and a mixture of ethyl acetate and BHT-butylhydroxytoluene was added. The suspension was vortexed for 15 min and sonicated in an ice bath for 15 min. After centrifuging at 13,000× g for 10 min at 4 °C, the supernatant was gently blow-dried with nitrogen and then redissolved in 200 μL of 70% methanol. After being swirled for 5 min, sonicated at 0 °C for 5 min, and centrifuged at 12,000 rpm at 4 °C, 100 μL of supernatant was obtained to determine the contents of IAA and ABA by HPLC-MS/MS (Thermo Scientific, Germering, Germany).

HPLC-MS/MS (Thermo Fisher) was used to analyze urolithin A at 305 nm by using a ZORBAX SB-C18 column (250 × 4.6 mm, 5.0 μm) (Agilent Technologies, Santa Clara, CA, USA) and HR-ESI-MS (high-resolution electrospray ionization mass spectroscopy) can range from 150.0 to 2000.0 Da. Sheath gas-flow rate, 45 arb; aux gas-flow rate, 15 arb; capillary temp: 320 °C; and S-Lens RF Level 50 T. The column temperature was 30 °C. The mobile phase consisted of solvent A (0.1% formic acid water) and solvent B (acetonitrile). The gradient elution was as follows: 0~10.5 min, 10~100% solvent B; 10.5~12.5 min, 100% solvent B; 12.5~12.6 min, 100%~10% solvent B; 12.6~16.5 min, 10% solvent B for re-equilibration. Urolithin A in the fermentation broth was first identified through HPLC and further identified by comparing the molecular mass of the compound obtained with that of a pure standard by using HPLC-MS/MS.

Aliquot amount of each standard was weighed and solubilized in 100% chloroform. Stock solution was consequently diluted by n-hexane/propan-2-ol (1 : 1, v/v) for calibration by HPLC-MS/MS (ThermoFisher Scientific, Franklin, MA).

Bacillus amyloliquefaciens LZ-5 was activated using nutritious broth and then was added to landy medium with 5% inoculation, and then cultured with shaking of 180 rpm for 72 h at 33 °C in order to obtain the fermentation broth. The fermentation broth was centrifuged at 10,000 rpm/min for 20 min and the supernatant was collected. The supernatant was adjusted to pH 2 and stored at 4 °C overnight. Then, the solution was centrifuged at 10,000 rpm/min for 10 min and the supernatant was removed. The supernatant was discarded and the precipitated lipopeptides were extracted for three times by anhydrous ethanol. Iturin A was identified and measured by reversed-phase high-performance liquid chromatography (HPLC; C18 column, ODS 4.6 mm × 250 mm, AGILENT 1100 series) with UV detectors and HPLC-MS/MS (Thermo Electron Corporation, San Jose, CA, USA). The eluent was methyl cyanides at a flow rate of 0.6 mL/min. The injection volume of the sample was 20 μL.

The bioactive substance was isolated by acid precipitation. Activated culture of B. amyloliquefaciens LZ-5 was incubated (5%, v/v) in modified landy medium, and shaken at 180 rpm at 30°C for 72 h. And then the fermentation broth was centrifuged at 10,000 g, 4°C for 20 min. The supernatant collected was adjusted to pH 2 using 6 N HCl, followed by centrifugation at 8000 g for 10 min. The supernatant was discarded and the precipitated lipopeptides were extracted for three times by anhydrous ethanol. To study the bioactive substance purified from fermentation broth, Iturin A was identified and measured by reversed-phase high-performance liquid chromatography (HPLC; C18 column, ODS 4.6 mm × 250 mm, AGILENT 1100 series) with UV detectors and HPLC-MS/MS (Thermo Electron Corporation, San Jose, CA, United States). The eluent was methyl cyanides at a flow rate of 0.6 ml/min. The injection volume of the sample was 20 μL. For its application in the orange juice, the bioactive substance was decolorized using 0.5% (w/v) activated carbon and gently shaken for 4 h, and then it was concentrated and freeze-dried. Then anti-yeast test of Iturin A in Orange Juice was carried out as following.