High SA of 111In-labelled peptides and antibodies is required to administer a tracer dose of peptide or protein, preventing target saturation and/or side effects, while administering high activity doses required for imaging. Acetate buffers are routinely used for the labelling of DTPA- and DOTA-conjugated compounds with 111In. Here, we examined the effect of the buffer used during the radiolabelling: HEPES and MES, and compared this with the most commonly used acetate buffers: sodium acetate and ammonium acetate, and showed that an increased SA could be obtained when DTPA- and DOTA-conjugated compounds were labelled in MES or HEPES buffer. Moreover, the labelling efficiency was not affected by Cd2+ concentrations up to 0.1 nM when the labelling was performed in MES and HEPES, whereas a drastic effect was observed when the labelling was performed in acetate buffers. In line with these results, the ageing of the 111InCl3 stock had only a minor effect on the labelling efficiency 14 days after the production of 111InCl3 when compounds were labelled in MES and HEPES.
The use of MES as a buffer for radiolabelling resulted in a SA of all DTPA-conjugated compounds that was approximately two to three times higher when compared to radiolabelling in ammonium acetate and sodium acetate, respectively. When HEPES was used, an even higher SA of the DTPA-conjugated compounds was observed: four times higher than the labelling performed in ammonium acetate. The effect was less pronounced when the DOTA-conjugated compounds were labelled with 111In. Overall, radiolabelling in HEPES and MES was more efficient than that in acetate buffers in most cases and at least as efficient as in the case of DOTA-octreotide. Labelling of DOTA-conjugated compounds resulted in 5 to 20 times lower SA than that of DTPA-conjugated compounds. Most likely, this is due to the interference of contaminating metals with DOTA chelation, which might play a role to a lesser extent when labelling DTPA-conjugated compounds.
The decay product of 111In, 111Cd, can also be chelated by DTPA or DOTA, and it is therefore expected that the complexation of 111In is less efficient over time due to increasing amounts of Cd2+. Indeed, this phenomenon was observed when sodium acetate and ammonium acetate were used for the 111In-labelling of DTPA-exendin. Lower labelling efficiencies were observed as early as 7 days after the production of 111InCl3, and threefold lower SA were obtained when the labelling was performed with 111InCl3 11 days after the production date. This effect was not observed for the labelling of DTPA-exendin-3 in MES and HEPES with a maximal decrease in SA of 10% and 4% at day 11, respectively. Even the decrease in SA 14 days after the calibration date of 111InCl3 was not more than 18% for 111In-labelling in MES and 5% for HEPES. These latter results could explain the differences in SA of the six compounds used in this study since the labelling of these compounds was performed with 111In 9 days after production. Generally, 111InCl3 is used from 7 to 11 days after the production day, which could lead to reduced specific activities at later time points when acetate buffers are used. To overcome this problem, HEPES or MES buffer could be used for radiolabelling, with high specific activities at time points up to 14 days after 111InCl3 production. This could have an impact on experiment planning since experiments which require high-SA-labelled compounds are only available early after 111In production when acetate buffers are used, whereas the time point is not relevant when MES or HEPES is used. These results suggest that increasing amounts of Cd2+ contamination, due to the ageing of the 111InCl3 stock, do not influence the labelling of DTPA-conjugated compounds when MES and HEPES are used as a buffer for radiolabelling.
The suggested effect of cadmium on the 111In-labelling of DTPA-conjugated compounds was confirmed when increasing amounts of Cd2+ were added to the 111In-labelling mixture of DTPA-exendin. In HEPES and MES buffer, a 100-fold higher amount of cadmium could be added to the labelling mixture without reducing the labelling efficiency than in acetate buffer. The decreased labelling efficiency at low concentrations of cadmium might be due to the efficient formation of coordination complexes of Cd2+ with acetate, allowing efficient 'transchelation' of Cd2+, whereas no coordination complex with HEPES or MES is formed , and transchelation of Cd2+ to DTPA or DOTA is less efficient.
It has been postulated that coordination complex formation of 111In with acetate buffers is necessary for efficient labelling of DTPA- and DOTA-conjugated compounds  since it is assumed that the coordination complex formation prevents the formation of insoluble 111In-hydroxide. This study suggests that coordination complex formation of the buffer with 111In is less important for efficient labelling of DTPA- and DOTA-conjugated compounds since the labelling in the weakly coordinating buffers MES and HEPES was more efficient than that in acetate buffers in most cases or at least equivocal in the case of DOTA-octreotide.
Breeman et al. described the effect of contaminants on the labelling of DOTA-octreotide with 111In, 177Lu and 90Y, and found a similar result of the effect of cadmium contamination on the radiolabelling . The labelling procedures described in the latter study were performed in sodium acetate, and these findings are in line with the findings in our study, where a pronounced effect of Cd2+ on the labelling of DTPA-exendin-3 is observed when sodium acetate is used as buffer for radiolabelling.
The purification of 111InCl3 by an anion exchange method was described to improve the labelling of DTPA- and DOTA-conjugated compounds caused by the removal of contaminants, mainly Cd2+, present in the 111InCl3 solution . By using HEPES or MES buffer for the labelling of the compounds, this could omit a time-consuming purification method.