Simple and reliable chromatographic technique for the determination of HEPES in Ga-68-labeled pharmaceuticals

Objectives Ga-68 radiolabeled somatostatin analogues like DOTAtoc have found their place in nuclear medicine. Various buffers like HEPES, acetate, succinate, have been tested whereas HEPES provides a better results. Since HEPES is not registered as a pharmaceutical, it will be not listed in the pharmacopoeia and thus prior to clinical use the radiopharmaceutical quality controls must be conducted ensuring that HEPES does not exceed the maximum dose of 200 µg mL^-1. The purpose of this study was the development of a chromatographic technique to determine HEPES in Ga-68-DOTAtoc after labeling.

Methods We tested 10 Ga-68 DOTAtoc products. The HPLC separation was achieved on a ProntoSil C18 column (250 mm×4.0 mm, 5µm). The detector has a discrete channel set at 220 nm for quantification. The mobile phase consisted of water (pH 7.0) at a flow rate of 0.7 mL^-1. 33.3 µg DOTAtoc was labeled using 2,2 ml generator eluate (0.6 M HCl) containing 1000 MBq. The reaction mixture was adjusted to pH 3.5 using 500 mg HEPES and allowed to react for 7 min at 95°C under gentle stirring

Results The purification step with the SepPak C18 showed a reduction of the HEPES content of > 99.8 % ((0.58 ± 0.14) mg mL^-1). An additionally inserted rinsing step with 2 mL water leads to an reduction of > 99.9 % ((0.04 ± 0.02) mg mL^-1) which is far below the maximum value 200 µg mL^-1 . In addition, the lower level of detection is 0.015 mg per mL.

Conclusions We present a HPLC method that determines HEPES contents after standard SepPak purification to be less than 99.9 %. This corresponds to a concentration below the recommended limit of the maximum dose of 200 µg^-1 . Beside the purpose to keep the pH at 3.5 previous studies showed benefits like radical scavengers abilities and degradation protection. Therefore, we suggest that HEPES in combination with a routine SepPak purification has to be included into the European Pharmacopei for the routine QC.