Nucleosil 100 5 c18 column

Quantitative analysis of samples was performed by HPLC on a C18 Nucleosil 100–5 column (250 mm × 4.6 mm) (Macherey Nagel, Germany). Elution was conducted with a linear gradient method using water (solvent A) and acetonitrile (solvent B) at 0.6 mL/min and ambient temperature. Total running time was 20 min during which the following proportions of solvent B were used: 0–15 min 28–72% and 28% 15–20 min. Detection was achieved by a PerkinElmer Flexar UV/VIS detector at 300 nm based on calibration curves prepared using standard solutions of the detected compounds in 1:1 water:acetonitrile.

Striata were homogenized with Branson Digital Sonifier (G. Heinemann Ultraschall- und Labortechnik, Schwäbisch-Gmünd, Germany) in ice-cold aqueous solution of H3PO4 (150 mM) and DTPA (500 μM). The homogenate was then centrifuged at 40700 g for 20 min at 4 °C. Aliquots (50 μl) of the obtained supernatant were chromatographed on a Nucleosil 100-5 C18 column (250 mm x 4.6 mm; 5 μm) (Macherey-Nagel, Düren, Germany). The separation was done in isocratic elution mode at room temperature using mobile phase containing 0.02 M sodium citrate, 0.1 mM EDTA, 0.01 M sodium phosphate, 0.003 M octanesulphonic acid, 0.003 M heptanesulphonic acid, 7 % acetonitrile, and 3 % methanol at a pH adjusted to 3.1 with diethylamine. For external standard, a stock solution containing 500 μg/ml dopamine, homovanillic acid, and 3,4-dihydrophenylacetic acid (Sigma-Aldrich, Steinheim, Germany) was prepared. The chromatography system consisted of an Agilent 1100 Series isocratic pump, a thermostatted autosampler, a thermostatted column compartment and a Bio-Rad 1640 electrochemical detector with glassy carbon electrode. The measurements were done at an electrode potential of +0.72 V versus the Ag/AgCl reference electrode. Results were normalized to reference naïve wt neurotransmitter level.

Isolation of the peptaibol from the extract was achieved by HPLC. Chromatographic (HPLC) analysis was performed using a Milichrom A-02 (ZAO Econova, Novosibirsk) chromatograph, Nucleosil-100-5-C18 column (Macherey-Nagel, Düren, Germany; L = 75.0 mm; D = 2.0 mm; d = 5 μm). Wavelength of detection used was 214 nm. Mobile phase included: A—H₂O (MQ) + 0.02% TFA (HPLC, Sigma-Aldrich, Darmstadt, Germany); B—MeCN (HPLC, Fisher Chemicals) +0.02% TFA (HPLC, Sigma-Aldrich, Germany). Injection volume was 15 μL; flow rate—100 μL/min; T = 35 °C. Gradient program X was used in workflow: X—from 0 to 100% B for 46 min.

Reversed phase high-performance liquid chromatography (RP-HPLC) for analytical purposes was conducted using a Waters HPLC setup consisting of a Waters Alliance e2695 equipped with a Waters 2998 PDA detector. The detection wavelength was chosen depending on the analyte between 214, 254, 280 and 301 nm. The eluent system for the HPLC system comprised eluent A (0.1% aq. TFA) and eluent B (99.9% acetonitrile containing 0.1% TFA). Unless otherwise specified analytical HPLC runs were conducted at a flow rate of 2 ml/min with an eluent gradient from 20 to 80% of eluent B in eluent A over 20 min. For analysis a Nucleosil 100–5 C18 column from Macherey–Nagel (5 µm, 100 Å) was used. Preparative purification of the peptide was performed on a Knauer Multokrom RP18 column 20 × 250 mm (5 μm, 100 Å) employing a flow rate of 9 mL/min and an acetonitril gradient from 0 to 50% without TFA in water over the course of 60 min.

The quantification of N-acetyltryptophanate was performed with a high performance liquid chromatography method. Samples were precipitated by adding 50 μl HSA sample to 250 μl methanol, vortexed, and cooled at -80°C for 20 minutes. After centrifugation (14000 g, 5 min) 20 μl of the supernatant was injected into the HPLC column (150 x 4.6 mm Nucleosil 100–5 C18 column combined with a 4 x 3 mm Nucleosil 100–5 C18 guard column; Macherey-Nagel, Düren, Germany). The temperature of the column oven was set to 35°C. The elution consisted of a linear gradient program from 30 to 50% methanol in water over 6.5 minutes, maintained at 50% methanol for 1 minute and returned to 30% methanol for 1 minute. The flow rate was 0.5 ml/min and absorbance detection at 280 nm was performed for NAT quantification. The amount of N-acetyltryptophanate was calculated in percent of the untreated 20% HSA-solution (Kedrion Biopharmaceuticals, Italy), and as negative control the stabilizer-free HSA (Sigma Aldrich, USA) was analyzed. The recovery of NTA using this HPLC procedure is 72.3 ± 0.5% (S1 Fig). It was determined by comparing the peak areas between NTA diluted in methanol and NTA spiked in stabilizer-free albumin solution.