Fruit samples were collected for metabolic profiling when the fruits were ripe in 2020. Referring to Shen et al. (2018) (link), all samples were ground into fine powders in liquid nitrogen. After mixing, 5g of samples were weighed into a test tube, 10 mL of extraction solution (aqueous hydrochloric acid solution with pH 1 was added, and ultrasonic extraction was performed for 30 min. The supernatant was collected after centrifugation, the residue was added to 10 mL of extraction solution again, and ultrasonic extraction was performed for 30 min. The supernatant was collected after centrifugation and combined with the supernatant obtained previously, and 25 mL of the supernatant was taken and filtered with a syringe filter. The sample is ready for HPLC. Agilent 1100 high-performance liquid chromatograph equipped with Xtimate XB-C18 column (250mm*4.6mm, 5μm) was used for HPLC in this study (Agilent, USA). The condition of HPLC was set up as follows: the mobile phase: 0.01 moI/L of potassium dihydrogen phosphate solvent; G4212-60008 diode Array detector, detection wavelength: 215nm, the control flow rate: 0.8ml/min, column temperature: 40°C.
Agilent 1100 high performance liquid chromatograph
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1100 high-performance liquid chromatograph is a laboratory instrument designed for the separation, identification, and quantification of chemical compounds in complex mixtures. It utilizes high-pressure liquid chromatography (HPLC) technology to achieve efficient separation of the sample components.
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4 protocols using agilent 1100 high performance liquid chromatograph
1
Metabolic Profiling of Ripe Fruit Samples
2
Quantification of DNA Methylation
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Extraction of DNA was conducted using the conventional phenol/chloroform/isoamyl alcohol method. Hydrolysis of DNA was performed as previously described.19 (link) In brief DNA was hydrolyzed to nucleosides using nuclease P1, phosphodiesterase I and alkaline phosphatase (all from Sigma, St. Louis, MO, USA). Thereafter, internal standards were added to samples. The isotope-labeled internal standard for deoxycytidine was [15N3]-2′-deoxycytidine, while that of 5-methyl-deoxycytidine and 5-hydroxymethyl-deoxycytidine were (methyl-d3, ring-6-d1)-5′-methyl-2′-deoxycytidine (both from Cambridge Isotopes Laboratories, Inc., Andover, MA, USA). The DNA hydrolysates were separated by an Agilent 1100 high-performance liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA) and masses were detected through a 3200 Q Trap MS-MS system (Applied Biosystem, Concord, ON, Canada). Using the known masses of isotope-labeled internal standards added to each sample and the area of each peak the absolute amount of unmodified cytosine, 5′-methylcytosine and 5′-hydroxymethylcytosine per 1 μg of DNA can be calculated. Thereafter each amount was expressed as a percentage of total cytosine as we previously described.20 (link)
3
HPLC-FLD for ZON Quantification
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High performance liquid chromatography equipment combined with multiwave detector and fluorescent detector (HPLC-FLD) was used to perform both qualitative and quantitative measurements. All chromatographic measurements were performed, using a quaternary pump Agilent 1100 high performance liquid chromatograph (Santa Clara, CA, USA) using a C18 ZORBAX column (150 mm × 4.6 mm, 3–5 µm) by Agilent as stationary phase. The mobile phase used for chromatographic analysis consisted of acetonitrile containing 0.1% (v/v) TFA, as solvent A, and Milli-Q water containing 0.1% (v/v) TFA as solvent B. The analysis was performed in 50:50 isocratic mode (A:B) at a flow rate of 1 mL min−1. All compounds were detected at λexc = 270 nm, λem = 452 nm and the column temperature was set at 20 °C. The injection volume was 10 µL. The ZON eluted at around 7 min.
4
Aflatoxin B1 Quantification by HPLC-FLD
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The analytical detection was carried out by an accredited laboratory (Wessling Hungary Ltd., Hungary) and the remained AFB1 concentration was detected by HPLC-FLD. After ultrasonic homogenization, the pre-column derivatization was accomplished at 55 °C for 15 min with the mixture of trifluoroacetic acid:acetic acid:distilled water (1:1:8). For the measurement, Agilent 1100 High-Performance-Liquid-Chromatograph (Agilent Technologies, California, USA) was used and equipped with Restek C18 (150 mm × 4.6 mm × 5 μm) column (Restek Corporation, Pennsylvania, USA). The detection was completed by fluorescent detector at the wavelengths of 365 (extinction) and 440 nm (emission). Depending on the AFB1 concentration, 200 or 10 μL sample were mixed with 800 or 990 μL water:acetonitrile:methanol (68:16:16) eluent agent. An amount of 100 μL of the mixture was injected into the equipment under isocratic conditions. The retention time was 17 min and the applied column temperature was 30 °C. As a standard, Supelco Aflatoxin Mix Kit (Supelco Inc., Pennsylvania, USA) was used.
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