Xbridge c18 5 μm

[³H]A-740003 was synthesized in a similar manner as previously described for [¹¹C]A-740003 [23 (link)]. Briefly, desmethyl precursor was reacted with [³H]methyl nosylate in the presence of tetrabutylammonium hydroxide for 16 h at room temperature. HPLC purification was performed on a Reprospher 100 C18-DE (50 × 8 mm, 5 μm) column (Dr. Maisch GmbH, Ammerbuch, Germany) using MeCN/H₂O/diisopropylamine (22:78:0.1, v/v/v) as eluent at a flow rate of 3 mL.min⁻¹. Fractions containing product (t_R = 16 min) were collected and formulated in 1.5 mL 96% EtOH. Radioactivity concentration (10.9 kBq.μL⁻¹) was determined by liquid scintillation counting (LSC) (LKB/Wallac 1219 Rackbeta, Mount Waverley, Australia) using 5 mL of Optiphase “Highsafe 3” scintillation liquid (PerkinElmer, Waltham, MA, USA). The identity of the product was confirmed with analytical HPLC by coinjection of the product and non-labeled A-740003 (t_R = 10 min), and the (radiochemical) purity was > 99%. Analytical isocratic HPLC was performed on an Xbridge C18 5 μm (100 × 4.6 mm) column (Waters, Milford, MA, USA) using MeCN/H₂O (30:70, v/v) as eluent at a flow rate of 1 mL.min⁻¹.

HPLC
was primarily performed using a Varian Pro Star HPLC system, with
a 230 pump and a 310 UV detector. The autosampler used was a 410 model,
with a 100 μL injection loop. The column (4.6 × 250 mm²) was a Waters XBridge C18 5 μm. The detector was operated
at 280 nm, and the system was run at a flow rate of 1.0 mL min^–1. Samples of ∼1 mg were dissolved in ∼1
cm³ of acetonitrile, and injected into the column. A 50:50
acetonitrile:water mixture was used for 19 min, whereupon the system
was moved to 100% acetonitrile for a further 10 min.
Due to
maintenance, the HPLC analysis for PY306 was performed on a Waters
2695 separation module, with a 2487 dual lambda absorbance detector.
The column and method were the same as those used with the Varian
system.