Hlb cartridge

HKE2 (1 μM) was dissolved in 2.7 ml of RPMI and placed in a cell culture incubator at 37°C. At 0, 1, 2, 4, and 8 an aliquot of 250 μl was removed and extracted using a preconditioned 30 mg Waters HLB cartridge. The cartridge was eluted with methanol and evaporated. The sample was dissolved in 40 μl of acetonitrile/water (1:1, by vol.) and analyzed by LC-ESI-MS in the negative ion mode.

Recombinant human COX-2 (0.2 μM) was added to 2 ml of 100 mM Tris-HCl pH 8 containing 1 μM hematin and 0.5 mM phenol. The solution was warmed to 37°C, and 30 μg 5S-HETE were added. The reaction was kept at 37°C for 5 min followed by incubation at room temperature for 45 min. To the reaction were added 16 μl glacial acetic acid and 50 μl methanol before extraction using a preconditioned 30 mg Waters HLB cartridge. The cartridge was preconditioned by washing with 2 ml of methanol followed by 2 ml of water. Samples were loaded, washed with water, and eluted with 0.5 ml of ethyl acetate followed by 0.5 ml of methanol into a conical vial. The water bubble in the bottom of the vial after ethyl acetate elution was removed with a pointy glass syringe prior to elution of the cartridge with methanol. The solvents were evaporated under a stream of N₂, and the sample was reconstituted in 100 μl of methanol for HPLC analysis. For preparative isolation of HKs 10 or 20 2 ml-reactions were conducted in parallel, and two reactions each were extracted using one HLB cartridge.

As described elsewhere (37 (link)), NM600 was radiolabeled with ⁸⁶YCl₃ as no-carrier-added formulation to obtain [⁸⁶Y]Y-DOTA-NM600 (hereafter simplified to ⁸⁶Y-NM600) for positron emission tomography (PET) imaging and dosimetry; for therapeutic treatments, NM600 was radiolabeled with 90YCl₃ to obtain ⁹⁰Y-DOTA-NM600 (hereafter simplified to ⁹⁰Y-NM600). Radiolabeling of NM600 with ^86/90Y was performed by mixing 185–370 MBq (5 −10 mCi) of ^86/90Y and 54–81nmol/GBq (10–15 nmol/mCi) of NM600 in 0.1 M NaOAc (pH = 5.5) buffer. After incubation at 90°C for 30 min under constant shaking (500 rpm), ^86/90Y-NM600 was purified by solid phase extraction using an HLB cartridge (Waters^™ Corp., Milford, MA). For in vivo use, ^86/90Y-NM600 was formulated in vehicle consisting of normal saline containing 0.4% v/v Tween^™ 20 and sodium ascorbate (0.5% w/v). Yields and radiochemical purity were consistently >95%, and a similar apparent molar activity of 18 GBq/μmol was obtained for both ⁸⁶Y-NM600 and ⁹⁰Y-NM600. Additionally, radiotracer stability in vivo has been demonstrated from mouse serum samples up to 48 h, with no significant radio peaks corresponding to metabolites observed (37 (link)).