*2.3. <sup>165</sup>Er Quality Control*

Holmium mass across the separation procedure was quantified using microwave plasma atomic emission spectrometry (MP-AES, MP4200, Agilent Technologies, Santa Clara, CA, USA). Holmium standard solutions of 0.1–50 ppm were made by dissolving holmium chloride hydrate (REaction® 99.99% (REO), Alfa Aesar, Haverhill, MA, USA) in water or holmium metal (US DOE Ames Laboratory MPC, Ames, IA, USA) in 11 M HCl, followed by dilution in 0.1 M HCl. The limit of detection for holmium was determined to be ~0.1 ppm in an undiluted sample solution. The MP-AES-analyzed samples were typically diluted by a factor of 2–10 in 0.1 M HCl. The Ho/Er SFs of the various separation steps and the overall separation procedure were calculated according to Equation (1) with *mHo,before/after* being the MP-AES-quantified holmium mass before/after the separation

step and *<sup>A</sup><sup>165</sup>Er, before/after* being the dose-calibrator-quantified <sup>165</sup>Er activity before/after the separation step.

$$SF\_{Ho/Er} = \frac{\frac{m\_{Ho, fefor}}{m\_{Ho,after}}}{\frac{A\_{16\%Er, hefor}}{A\_{16\%Er,after}}}\tag{1}$$

The apparent molar activity (AMA) of the final <sup>165</sup>Er solution was determined by titration using tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA, Macrocyclics Inc, Plano, TX, USA) and diethylenetriaminepentaacetic acid (DTPA, Acros Organics, Geel, Belgium) as previously described [46]. To polypropylene tubes, 0.5–3 MBq (20 µL) of <sup>165</sup>Er in 0.01 M HCl, 1 M NaOAc (100 µL, pH 4.7, 99.995% trace metals basis, Aldrich, St. Louis, MO), and DOTA or DTPA (100 µL, 0.03–3 µg/mL) in 18 MΩ·cm water were added. Following incubation at 85 ◦C for DOTA and 21 ◦C for DTPA for 1 h, each tube was assayed by thin layer chromatography (TLC) using silica-based stationary phase (J.T. Baker, Phillipsburg, NJ, USA) and 0.05 M disodium ethylenediaminetetraacetic acid (EDTA, Fisher Scientific Co., Pittsburgh, PA, USA) as mobile phase. Radioactivity distribution on the TLC plates was visualized using a Cyclone Plus phosphor plate reader (Perkin Elmer, Waltham, MA, USA). Free <sup>165</sup>Er had a retention factor (R<sup>f</sup> ) of ~1, chelated <sup>165</sup>Er-DOTA had an R<sup>f</sup> of ~0.2, and <sup>165</sup>Er-DTPA had an R<sup>f</sup> of ~0.7. To compute the AMA, <sup>165</sup>Er activity in MBq was divided by twice the number of moles of DOTA/DTPA required to complex 50% of the radioactivity, and the value was reported as MBq of <sup>165</sup>Er per nmol of ligand, or MBq/nmol (mean ± standard deviation, SD).
