N-Acetyl-L-cysteine (NAC, Spectrum Chemical) was dissolved in milli-Q water, and synthetic honaucin A was solubilized in DMSO. Honaucin A was incubated with NAC in either 2-or 50-fold excess with stirring under argon for 2 h at room temperature (after Wang et al., 2013).22 (link) At the conclusion of the reaction, vial contents were passed over a Bond Elut-C18 SPE column (Agilent) that had been washed with 3 column volumes of methanol and then equilibrated with 3 column volumes of water. The reaction products were subsequently eluted with increasing percentages of methanol. Elutions were dried using rotary evaporation and then reconstituted at a concentration of 1 mg/mL in 50:50 methanol/water. This preparation was analyzed for the presence of the hypothesized addition products via high-resolution MS at the Molecular Mass Spectrometry Facility at the University of California, San Diego. Briefly, reaction products were separated via liquid chromatography using a Phenomenex Kinetex 5 μm EVO C-18 column prior to being introduced to an Agilent 6230 ESI-TOF-MS running in positive mode for high-resolution mass measurement.
Bond elut c18 spe column
Manufactured by Agilent Technologies
The Bond Elut-C18 SPE column is a solid-phase extraction (SPE) column designed for sample preparation and analyte purification. The column features a C18 stationary phase, which is commonly used for the extraction and purification of a wide range of organic compounds from various sample matrices.
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3 protocols using bond elut c18 spe column
1
Reaction of Honaucin A with N-Acetyl-L-cysteine
2
Solid-Phase Extraction of Polyamines
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Polyamines dissolved in the culture media were extracted using a solid-phase extraction (SPE) as described previously (73 (link)). A total of 10 ml of the supernatant from the initial centrifugation of cultures (described above) was used for each sample. Polyamines were extracted via gravity alone (nominal flow rate of 0.07 ml/min) onto a 1,000-mg Bond Elut-C18 SPE column (3 ml; Agilent) preconditioned with methanol and bicarbonate buffer at pH 12. Salts were removed from the column by washing three times with 1 ml of 0.1 M borate buffer, pH 12. Polyamines were eluted into cryovials with three washes of equal volumes of 1 M acetic acid and acetonitrile, final volume 5 ml. Greater than 85% recovery for CAD, PUT, NSD, and SPD was observed, while AGM had 48% recovery (Table S1 ), perhaps because of the high pKa of AGM (∼12) compared to that of the other four compounds (∼10). Artificial seawater (blanks) extracted using this method had only minimal concentrations (less than 4 nM) of NSD and SPD and none of the other three compounds (data not shown). Measured concentrations were not corrected for extraction recovery, so the values reported in this paper are conservative.
3
Polyamine Extraction from Cultures
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Polyamines dissolved in the culture media were extracted using a solid phase extraction (SPE) as described previously (64) . 10 mL of the supernatant from the initial centrifugation of cultures (described above) was used for each sample. Polyamines were extracted via gravity alone (nominal flow rate of 0.07 mL/min) onto a 1000 mg Bond Elut-C 18 SPE column (3 mL, Agilent) pre-conditioned with methanol and bicarbonate buffer at pH 12. Salts were removed from the column by washing three times with 1 mL of 0.1 M borate buffer, pH 12. Polyamines were eluted into cryovials with three washes of equal volumes of 1 M acetic acid and acetonitrile, final volume 5 mL. Greater than 85% recovery for CAD, PUT, NSD, and SPD was observed, while AGM had 48% recovery (Table S1), perhaps because of the high pKa of AGM (~12) compared to the other four compounds (~10). Artificial seawater (blanks) extracted using this method only had minimal concentrations (less than 4 nM) of NSD and SPD and none of the other three compounds (data not shown). Measured concentrations were not corrected for extraction recovery and so values reported in this paper are conservative.
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