Waters 2475

HPLC conditions were adopted from the instructions in the Aflaochraprep immunoaffinity columns manual supplied by R-Biopharm Rhone Ltd. (32 ). Reverse phase HPLC (Waters 2690^®, Waters Corp., MA, USA) coupled with a fluorescence detector (Waters 2475^®) and a HS C18 column (250 mm × 4.6 mm I.D., 5 μm particle diameter, Supelco Discovery^®) fitted with a C18 guard column (Supelco Supelguard^®, Sigma-Aldrich Co., MO, USA) at 25°C was used for analysis. The method for HPLC analysis was performed as described by the Aflaochra Prep IAC supplier R-Biopharm Rhone Ltd. Two mobile phases were used for analysis: solution A was composed of water: methanol (55:45 v:v), while solution B was composed of water: methanol (20:80 v:v). For both solutions, 119 mg potassium bromide and 350 μl of 4 M nitric acid were added for each 1 L. The mobile phases were prepared and filtrated on the same day of HPLC analysis, and they were passed at a flow rate of 0.8 ml/min. The sample injection volume was 100 μl and the wavelengths for excitation and emission at the beginning of the analysis were 365 and 442 nm, respectively, but after 17 min the excitation and emission were changed to 333 and 463 nm, respectively.

Melatonin and N-acetylserotonin were identified and quantified using high performance liquid chromatography (HPLC) (Waters 2695; Waters, USA) and a fluorescence detector (Waters 2475; Waters, USA). The solid phase comprised a reverse C18 column (Kromasil 100-5C18, 250 mm × 4.6 mm, 5 μm; AkzoNobel, Sweden). Separation was performed with a linear gradient using eluent A (water with 2% formic acid) and eluent B (acetonitrile with 2% formic acid). Arabidopsis leaves (200 mg) were ground to a fine powder and extracted with 6 ml of methanol. Cell pellets were collected by centrifugation, and extracted with 300 μl of ethyl acetate. Samples were centrifuged at 13,500×g for 10 min. Supernatants were fractionated according to the Kang method [9 (link)]. The fraction eluted in chloroform: methanol (30:1) solution was evaporated, and the residue was dissolved in 500 μl of methanol. The solution was directly subjected to HPLC for quantification.

To measure sulfide levels, we performed a fluorescent-based HPLC as reported previously^{62 (link)}. Briefly, brain tissues were homogenized in 100 mM Tris-HCl buffer (pH 9.5, 0.1 mM diethylenetriaminepentaacetic acid) and centrifuged at 15,000 × g × 10 min × 4 °C. Supernatant (30 µL) was added to the mixture of Tris-HCl buffer (70 µL) and monobromobimane (50 µL, 10 mM in acetonitrile), incubated at RT for 30 min in the dark, then 5-sulfosalicylic acid (50 µL, 200 mM) was added to stop the reaction and stabilize monobromobimane-labeled molecules. The solution was placed on ice for 10 min and centrifuged. The recovered supernatant was then analyzed using HPLC equipped with a multi λ fluorescence detector (Waters 2475, Waters, Inc.).

HPLC conditions were adopted from the instructions in the Aflaprep immunoaffinity columns manual supplied by R-Biopharm Rhone Ltd. (33 ). Reverse phase HPLC (Waters 2690^®, Waters Corp., MA, USA) coupled with a fluorescence detector (Waters 2475^®) and a HS C18 column (250 mm × 4.6 mm I.D., 5 μm particle diameter, Supelco Discovery^®) fitted with a C18 guard column (Supelco Supelguard^®, Sigma-Aldrich Co., MO, USA) at 40°C temperature was used for analysis. The method for HPLC analysis was performed as described by the Aflaprep IAC supplier R-Biopharm Rhone Ltd. A mobile phase made of water: methanol (60:40 v:v) with 119 mg potassium bromide and 350 μl of 4 M nitric acid per liter was used for analysis. The mobile phase was prepared and filtrated on the same day of HPLC analysis, and it was passed at a flow rate of 1.0 ml/min. The sample injection volume was 100 μl and the wavelengths for excitation and emission were 362 and 425 nm, respectively.

HPLC (Waters 2695, Waters Corp., Milford, MA, USA) equipped with a Waters 2475 fluorescence detector was used. The mobile phase was operated in isocratic mode (n-hexane: isopropanol: acetic acid (98.9:0.6:0.5, v:v:v)) with a flow rate of 1 mL/min. The normal-phase chromatographic column (LiChrosorb Si60, 250 × 4.6 mm, 5 µm; Suzhou, China) was held at 40 °C and the injection volume was 10 µL. The emission and excitation wavelengths were set at 330 nm and 290 nm, respectively. The system was controlled by a computer running the Waters-Empower 3 software. The chromatograms of the standards (60 µg/mL), refined sumac fruit oil sample, and sacha inchi oil sample were collected and are presented in Figure S3.

The amino acid compositions of the isolated PSC were identified using HPLC, following the method described by Jamilah et al. [20 ]. The isolated PSCs were first hydrolyzed with 5 mL of HCl (6 N) at 110 °C for 24 h. Then, 4 mL of internal standard α-aminobutyric acid (AABA) was added to the residue, and volume of the mixture was made up to 100 mL with deionized water. Derivatization was carried out using an AccQ-Fluor Reagent kit (Waters Co., Milford, MA, USA). Then, 10 µL of standard solution or the hydrolyzed samples was mixed with 70 µL of borate buffer and 20 µL of AccQ reagent and transferred to autosampler vials. The mixture was then incubated for 10 min at 55 °C. Peak separation was carried out by injecting 5 µL of the mixture into an AccQ Tag RP-column (3.9 × 150 mm, Waters Co., Milford, USA) using a gradient run. The eluent system consisted of two components: 40% AccQ Tag concentrate as Eluent A and 60% acetonitrile as Eluent B. The gradient condition was programmed as follows: 100% A in 0.5 min, 98% A in 14.5 min, 90% A in 4 min, 87% A in 13 min, 65% A in 2 min, and 0% A in 3 min, followed by 100% A for 13 min at a flow rate of 1 mL/min. Detection was achieved using a fluorescence detector (FD) (Waters 2475, Waters Co., Milford, USA). The amino acids’ determination was carried out using a Waters Auto analyzer (Waters 2690/5, Waters Co., Milford, USA).