Agilent zorbax 300 sb c18

The TMJM peptide was synthesized by F-moc chemistry by Thermo Fisher (Waltham, MA), and purity (>95%) was assessed by MALDI-TOF and HPLC. The cysteine residue of TMJM was conjugated to Alexa Fluor 488 (Molecular Probes, Eugene OR) or Cyanine5 (Cy5) (Fluoroprobes, Scottsdale AZ), using a C₅ maleimide moiety. The reaction was carried out by adding a molar excess (peptide:dye of 1:1.1 mol) of dye dissolved in 100 mM sodium phosphate pH 7.6 to a peptide stock in 2,2,2-trifluoroethanol (TFE). Unreacted dye was removed by HPLC, by injecting the TFE mixture onto a semipreparative Agilent Zorbax 300 SB-C18 column on an Agilent 1200 HPLC system (Santa Clara, CA). The gradient of water +0.05% trifluoroacetic acid (TFA) to acetonitrile +0.05% TFA was 30 min from 0% to 100% acetonitrile. The conjugated peptide eluted around 95% acetonitrile. The collected fractions from HPLC were frozen and lyophilized. The dry conjugated peptide was resuspended in hexafluoroisopropanol (HFIP).

The drug solution of ¹⁷⁵Lu-DOTA-PSMA-GUL was prepared according to the method provided by Cellbion Co. (Seoul, Korea). Briefly, ¹⁷⁵Lu-DOTA-PSMA-GUL was synthesized by mixing DOTA-PSMA-GUL solution and ¹⁷⁵LuCl₃ solution. DOTA-PSMA-GUL solution was prepared by dissolving 50 mg of DOTA-PSMA-GUL powder in 20 mL of 0.5 M sodium acetate buffer (pH 4.5). ¹⁷⁵LuCl₃ solution was prepared by dissolving 19 mg of ¹⁷⁵LuCl₃ in 10 mL of 0.04 M HCl. Then, 20 mL of DOTA-PSMA-GUL and 8.5 mL of ¹⁷⁵LuCl₃ were mixed for 20 min at 40 °C in a shaking water bath. The mixture was finally tested for the purity of ¹⁷⁵Lu-DOTA-PSMA-GUL via HPLC–UV method using Waters 2695 separation module coupled with Waters 2487 dual wavelength absorbance detector (Waters, Milford, MA, USA). ¹⁷⁵Lu-DOTA-PSMA-GUL was separated on an Agilent Zorbax 300SB-C18 (4.6 × 250 mm i.d., 5 μm, Agilent, Santa Clara, CA, USA) and detected at 244 nm.