Uvd 340 u

To determine precisely the different iturin A homologs produced by B. amyloliquefaciens, the extracted iturin A samples were analyzed by LC–MS using Shimadzu HPLC system LC-10AVP equipped with a photo diode array (PDA) detector and a reversed-phase C-18 column, Luna^® (250 mm × 4.6 mm internal diameter, 5 μ particle size, Phenomenex, USA). The samples (50 μl) were injected and two different solvent systems were used for effective separation and detection of iturin A homologs. The mobile phase consisting of water (eluent A) and acetonitrile (eluent B) supplemented with 0.035% (v/v) formic acid was used at a gradient flow rate of 10 ml min⁻¹. The gradient conditions were as follows: firstly, starting at 90% eluent A and 10% eluent B, eluent A was linearly decreased to 55% with the increase of eluent B to 45% within the first 2.5 min; then, eluent A was linearly decreased to 0% with the increase of eluent B to 100% in the next 2.5 min and then maintained for 3 min; finally, eluent A was linearly increased to 90% with the decrease of eluent B to 10% in the next 1 min and then maintained for 1 min. The eluants were read at 220 nm using PDA detector (UVD340U, Dionex, USA). The LC-MS analyses were performed using an LTQ-XL instrument (Thermo Scientific, Germany) equipped with Xcalibur software.

LC‐MS analyses were performed using a Dionex HPLC fitted with a UV detector (UVD340U with microflow cell) and connected to an MSQplus MS detector (Thermoscientific, Waltham, MA, USA). Chromatographic separation was obtained using a Zorbax SB C18 column (2.1 × 50 mm, Phenomex, Værlose, Denmark). The mobile phases were A: 50 μm NaCl, 0.1% HCOOH; B: 50 μm NaCl, 0.1% HCOOH, 80% MeCN. The gradient used was 0% B to 70% B (linear) in 30 min, 70% B to 100% B in 5 min (flow rate: 0.2 mL/min). The column was rinsed with 100% B and re‐equilibrated with A for 6 min (flow rate: 0.3 mL/min). Mass spectrometry was performed in the positive ionization mode ESI (cone voltage: 75 V, needle voltage: 3.5 kV). Samples were analysed in full‐scan mode (m/z 100–800).

For determination of the radiochemical purity of the [⁹⁰Y]Y-DTPA-Bn-CHX-A″-anti-CD66-mAb, a system comprising an HPLC-pump (UltiMate 3000, Thermo-Fisher, Waltham, MA, USA), a BioSep SEC-s3000 column, a UV-detector (UVD340U, Thermo-Fisher, Waltham, MA, USA), a Gabi gamma-HPLC-detector (Elysia-raytest, Straubenhardt, Germany) and a PC with Chromeleon (ver. 7.2.9) was used. A PBS solution with a pH between 6.6 and 6.8 (adjusted with hydrochloric acid (30%), Honeywell) was used as the mobile phase with a flow rate of 1 mL/min. Additionally, the radiochemical purity was monitored using thin-layer chromatography (TLC) using RP-18 aluminum plates (silica-gel RP-18 F254, Merck-Millipore, Darmstadt, Germany) as stationary phase and 0.1 M ammonium acetate (pH = 5.7) with EDTA (50 mM) as mobile phase. It is known that the mAb remains at the starting point (R_f = 0), whereas ⁹⁰Y moves with the mobile phase front (R_f = 1). For evaluation of the TLC plates, an FLA-3000 phosphorimager (Fuji Photo Film Co., Ltd., Tokyo, Japan) in combination with the control software BASReader (version 3.14, Elysia-raytest, Straubenhardt, Germany) and the image analyzer software AIDA (version 4.24.036, Elysia-raytest, Straubenhardt, Germany) was used.