C18 solid phase extraction cartridge

Melatonin was quantified following a previously reported protocol [30 (link)]. Tomato leaves were ground into a fine powder and melatonin was extracted with chloroform at 4 °C in the dark for 12 h. Then the samples were centrifuged at 4000× g for 5 min and the chloroform fraction was purified using a C18 solid-phase extraction (SPE) cartridge (Waters, Milford, MA, USA). The extract was evaporated to dryness and dissolved in methanol for analysis using HPLC.

To measure the endogenous plant melatonin concentration, a direct sampling extraction procedure was used as described (Arnao and Hernandez-Ruiz, 2009a (link)) with modifications. Fresh leaf or root samples (0.3 g) were cut into small sections and placed into a 15 mL tube containing 6 mL of chloroform, which was shaken overnight at 4°C in the dark. The sections were extracted again using 2 mL of chloroform. The supernatant was transferred to a C¹⁸ solid-phase extraction (SPE) cartridge (Waters, Milford, MA, USA) for the purification of melatonin. The extract was then concentrated to dryness under nitrogen. The residue was dissolved in 2 mL of the mobile phase for HPLC analysis as described (Korkmaz et al., 2014 (link)).

For analysis, three hops and three capsules of each food supplement were randomly selected and homogenized. Then, 100 mg of sample was weighed and extracted with 10 mL of methanol by sonication for 30 min at 25 °C using an ultrasonic bath (Branson 5510, Branson Ultrasonic Corp., Danbury, CT, USA). The extracts yielded were diluted (when required) and filtered through a 0.22 µm PTFE membrane filters before HPLC analysis. For beer analyses, samples were first degassed by sonication for 1 h. Then, 10 mL of each sample was loaded on a C18 solid-phase extraction (SPE) cartridge (Waters Corporation, Dublin, Ireland) after conditioning with 5 mL methanol and 5 mL water. Next, the SPE cartridge was washed with 5 mL of water and the fraction of interest was eluted with 5 mL of methanol. A portion of the extract was filtered with a 0.22 µm PTFE membrane filter before HPLC injection. Fractions yielded from samples G and MS were evaporated to dryness under a stream of nitrogen at 40 °C, using an evaporator system Labconco Rapid Vertex-Evaporator (Labconco Corporation, Kansas City, MO, USA) to concentrate ten times, and fractions yielded from samples M and B were concentrated 2 times under the same procedure. The residues were finally redissolved in methanol and filtered through 0.22 µm PTFE membrane filters for further HPLC analysis. All determinations were performed in triplicate.

The total biodegradation reaction volume was 5 mL containing 14.8 mg/L MC-YR and 103.6 mg/L protein in PBS in a 10 mL centrifuge tube. The reaction was at 30°C with the shake rate of 200 r/min. The samples of 0.5 mL for each were taken at 0, 1.5, 3, 5, 10 and 24 hr, respectively, and then 0.05 mL of concentrated hydrochloric acid (36%, v/v) was added to each sample to stop the reaction. All samples were centrifuged at 12,000 r/min for 10 min, and then the supernatant was used to measure MC-YR and its products with HPLC.
In order to identify the products of MC-YR, the samples of 0.5 mL were passed through a C₁₈ solid-phase extraction cartridge (Waters, OASISTMHLB, USA, 30 mg/mL). Methanol of 0.5 mL was used to elute the products of MC-YR with the rate of 1 mL/min. The elution was used to measure the mass/charge (m/z) of products with LC-MS/MS (API-3000, Applied Biosystems, USA).

Melatonin was extracted using the method described by Pothinuch and Tongchitpakdee [69 (link)]. Embryo samples (0.2 g) were pulverized with liquid nitrogen and homogenized in 5 mL of methanol. The homogenates were centrifuged at 10,000× g for 15 min at 4 °C, after ultrasonication (80 Hz) at 45 °C for 40 min. The extracts were dissolved in 1 mL of 5% methanol and purified using a C18 solid phase extraction cartridge (Waters). The sample solution was eluted through a 0.1 μm syringe filter by 1 mL of 80% methanol, and then assayed by UHPLC-ESI-MS/MS (UHPLC-1290 Series and a 6460 QqQ-MS/MS; Agilent Technologies). The excitation and emission wavelengths were at 285 and 345 nm, respectively. Melatonin content was calculated by comparing the peak area (% fluorescence) of the sample with that of its standard curve.