Hplc grade

Low molecular mass solutes were extracted from freeze-dried cell pellets with 80 % ethanol (HPLC grade, Roth, Germany) at 68 °C for 2 h. For GC analysis, a defined amount of sorbitol was added as an internal standard. The extracts were centrifuged (13,000g, 5 min, 20 °C) and the supernatant was lyophilized. The dry extract was resuspended in 500 μl ethanol (99 % HPLC grade, Roth, Germany) and centrifuged. The subsequent supernatant was again dried and then resuspended in 500 μl deionized water (HPLC grade, Carl Roth, Karlsruhe, Germany). After drying, the final extract was dissolved in pyridine, silylated, and analyzed by gas chromatography (GC) according to Hagemann et al. [68 (link)].

For the extraction of cereulide, bacteria grown in LB medium were pelletized by centrifugation (8.000 g, 23°C, 10 min) and 50 mg of bacterial biomass was resuspended in 1 ml acetonitrile (99%, HPLC grade, Carl Roth). After incubation for 16 h on a rocking table, the pellet was centrifuged and the supernatant was directly transferred to a HPLC vial and stored at room temperature. MS measurements were performed using a QTRAP^® 5500 MS/MS system (Applied Biosystems, Foster City, CA, USA) equipped with a TurboV electrospray ionization (ESI) source and a 1290 series UHPLC system (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was performed at 25°C on a Gemini^® C₁₈ – column, 150 × 4.6 mm i.d., 5 μm particle size, equipped with a C₁₈ security guard cartridge, 4× 3 mm i.d. (all from Phenomenex, Torrance, CA, USA). Stock solutions of 500 ppm cereulide (Chiralix, Nijmegen, The Netherlands) and 100 ppm ¹³C₆- cereulide (Chiralix) were prepared for the standard curve and internal standard, respectively. MS-analysis was performed as described by Bauer et al. (2010) (link) and quantitation of isocereulides was carried out as reported previously (Marxen et al., 2015c (link)). All samples were measured in two different dilutions as duplicates. Mean values and standard deviations were calculated from two independent experiments.

Acetic acid and methanol (Optima LC/MS Grade) as well as acetonitrile (HPLC-MS grade) were obtained from Fisher Scientific (Schwerte, Germany) and ammonium acetate (p.a.) was purchased from Merck (Darmstadt, Germany). Methyl tert-butyl ether and n-hexane (HPLC grade) were obtained from Carl Roth (Karlsruhe, Germany). Methyl tricosanoate (FAME C23:0) was obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Oxylipin and deuterated oxylipin standards were purchased from Cayman Chemicals (local distributor: Biomol, Hamburg, Germany). Further oxylipin standards (Epoxy octadecadienoic acids (EpODEs) and dihydroxy octadecadienoic acids (DiHODEs)) were a kind gift from the laboratory of Bruce Hammock (UC Davis, CA, USA). Ethyl acetate, methyl formate and all other chemicals were purchased from Sigma Aldrich (Taufkirchen, Germany).