Nucleodur 100 3 c18 column

Steroids without intrinsic absorption were converted into their Δ₄-3-keto-derivatives prior to RP-HPLC analysis, to allow photometric detection at 240 nm. 1 mL culture samples were boiled in water for 1 min. 20 µL cholesterol oxidase solution (5 mg cholesterol oxidase (Calbiochem) and 5 mg Na-cholate dissolved in 5 mL 50 mM HEPES buffer pH 7, containing 0.05% Tween-20) were added and the sample was incubated at 37°C with 1,000 rpm shaking for 1 h. For RP-HPLC analysis, the sample was extracted twice with 1 mL ethylacetate and the extract was dissolved in acetonitrile. Samples were analyzed isocratically with pure acetonitrile using a NUCLEODUR 100-3 C₁₈ column (Macherey & Nagel). Samples containing cholestenone and β-sitostenone were detected at 240 nm wavelength, 7-DHC at 280 nm. A flow rate of 1 mL/min was applied. Progesterone yield was calculated by using a progesterone calibration curve. 7-Dehydropregnenolone yield was determined by dividing product peak area by total substrate/product peak area. To purify pregnenolone for NMR analysis after whole-cell conversion, 200 mL culture medium were extracted and separated using a 250/8 NUCLEODUR 100-3 C₁₈ column and the product collected.

In order to determine the effect of the glucosinolate content on the infestation of plant genotypes by C. napi five randomly selected non-infested main stems per genotype were collected per plot just before release of adult weevils on March 23^rd for glucosinolate analyses. The glucosinolate content was analysed on six of nine genotypes (Campala, Express617, Visby, L16, S3, S30) because plant material of three genotypes was not sufficient for the analyses. The stems were immediately frozen on dry ice and stored at -20°C. After freeze-drying for 96 hours, the stems were homogenised using a mill (Krups KM 75). Stem glucosinolates were separated and individual compounds were identified and quantified [48 ] using a Shimadzu Prominence LC20AT series HPLC (Shimadzu Deutschland GmbH) equipped with a Nucleodur 100–3 C18 column (Macherey Nagel‎). Desulfoglucosinolates were extracted as detailed in [13 ] and were separated using a water-acetonitrile gradient (solvent A water, solvent B acetonitrile; 0–20 min 1–20% B; 20–25 min 20% B; 25–27 min 20% B; 27–34 min 1% B) at a flow rate of 0.6 ml / min. Retention times of known standards were used to identify desulfoglucosinolates. The concentration of glucosinolates is expressed in μmol / g dry weight (DW).