Effect of needle gauge on TLC performance
The amount of a sample deposited on the chromatographic support for thin-layer chromatography is essential for the efficient separation of species present in a radiopharmaceutical. Different SPCs and pharmacopoeial monographs recommend spotting between 2 and 5 µL, and up to 10 µL per sample. Small nuclear medicine centres which employ radiopharmaceutical kits often do not have a laboratory equipped with pipettes that are checked and calibrated on a regular basis. Thus, quality control of radiopharmaceuticals is performed by withdrawing a small volume of radiolabelled product using a sterile syringe and needle and spotting a drop on an iTLC strip. The drop size distribution as a function of needle gauge summarised in Table 1 showed that the smallest 25G needle produced a 10 µL drop, which is the largest amount of sample recommended for iTLC by QC guidelines. In clinical nuclear medicine, needle sizes ranging from gauge 20G to 26G are used, and are routinely available to professionals carrying out the QC of [99mTc]Tc-NanoHSA. Needles 25G or 27G are recommended for subcutaneous injections for lymphoscintigraphy with radiotracers which include nanocolloidal HAS (Bluemel et al. 2015). In this study, only a 25G needle was shown to provide 10 µL, equivalent to the volume deposited with a pipette. This means that caution should be taken when interpreting iTLC results obtained with larger needles.The determination of free pertechnetate in partially reduced eluate as a function of volume spotted for iTLC showed a trend towards an apparent decline of the free pertechnetate fraction with increased sample volume: from 1.57 ± 0.20% when spotted with 2 µL and up to 0.23 ± 0.03% when spotted with 20 µL. These small numbers obtained with iTLC do not provide the most robust conclusions regarding the effect of sample volume on chromatographic separation and should be considered indicative results only. However, it must be noted that chromatograms of larger sample volumes – with 15 and 20 µL spots – had to be analysed with the TLC scanner once they had been left to decay by one half-life. When using the same settings as for the 2–10 µL spots immediately after developing the strips, the 15 and 20 µL spots saturated the radioactivity detector, thus compromising the quantification.
Thin-layer chromatography
According to the SPC, the methods recommended for determining the RCP of [99mTc]Tc-NanoHSA employ thin-layer chromatography with SG-60 on aluminium stationary phase in acetone as mobile phase, or iTLC-SA stationary phase in methyl ethyl ketone. For both methods 99mTc-labelled human serum albumin nanocolloid remains at the origin, while free unbound pertechnetate [99mTc]TcO4− migrates with the solvent front. These methods are widely recommended in the literature. However, for some time now, acetone has not been recommended for this purpose because it tends to overestimate the free [99mTc]TcO4− fraction due to its higher water content (Decristoforo and Zolle 2007). Methyl ethyl ketone is the ultimate mobile phase to enable an accurate separation of free pertechnetate with TLC (World Health Organisation 2019).
Therefore, we selected three alternative methods and verified their suitability for the QC of [99mTc]Tc-NanoHSA in a coherent, reproducible fashion with a minimal number of replicates. We chose iTLC-SG chromatographic paper for MEK mobile phase because this stationary phase is more widely used in practise compared to iTLC-SA, and it is easier to handle because it does not require thermal activation prior to use, unlike iTLC-SA. Additionally, we chose methanol/water 85/15 mobile phase for iTLC-SG because it enables the migration of small hydrophilic species along with the free pertechnetate [99mTc]TcO4−. Methanol may contribute to the chemical degradation of delicate biological molecules such as human serum albumin. Therefore, iTLC was also carried out in a 0.9% NaCl solution as mobile phase with iTLC-SA stationary phase (World Health Organisation. The International Pharmacopoeia. Technetium (99mTc) pentetate complex injection (Technetii (99mTc) pentetatis multiplex injectio); 2019.
The QC of the 99mTc-labelled NanoHSA using MEK showed quantitative RCP in all radiolabellings, with the free [99mTc]TcO4− fraction below 1% shortly after reconstitution of the kit. To determine the presence of other potential soluble impurities, more polar, aqueous mobile phases are necessary. iTLC-SG in 85% methanol showed quantitative radiolabelling of NanoHSA, however a slightly lower RCP after 10 min incubation (96.2 ± 0.3%). After 30 min incubation the RCP increased to over 98%. Although the initial result fulfilled the QC requirements of the SPC, it suggested the presence of a labile hydrophilic species carrying 99mTc that did not migrate in MEK. iTLC-SA in 0.9% NaCl showed remarkably similar results with methanol/water (Table 3). The RCP after 10 min incubation was 96.5 ± 0.3%, comparable to methanol/water (96.2 ± 0.3%) but slightly lower than with MEK (99.7 ± 0.2%). After 30 min incubation, the RCP increased to 98%, same as methanol/water. A comparison of the results of three iTLC methods revealed that MEK overestimated the RCP of [99mTc]Tc-NanoHSA, identifying only free pertechnetate impurity. Aqueous mobile phases identified approximately 1–2% of additional hydrophilic impurities, which consistently tended to decline within the timeframe of experiment (10–60 min).
Solid-phase extraction
Solid-phase extraction is used as an alternative to TLC for the quality control of some radiopharmaceuticals. Sep-Pak® cartridges with C18 hydrophobic reversed phase retain non-polar compounds and particles (Straub et al. 2018; Ramirez et al. 2000). Colloidal [99mTc]TcO2 is also retained on the Sep-Pak® and we hypothesised that larger colloidal particles (≤ 80 nm) of human serum albumin labelled with 99mTc could also be retained on the cartridge. The RCP of [99mTc]Tc-NanoHSA determined with SPE was similar to iTLC in methanol/water and in saline (Table 3), showing that Sep-Pak® makes it possible to separate the same hydrophilic impurities. Furthermore, this method may be used as a less expensive and rapid alternative for QC in the absence of a TLC scanner. However, the manual handling of SPE cartridges during this QC can result in a higher radiation dose to the operator and should be avoided. Furthermore, uncontrolled, manual elution may result in inaccurate and irreproducible results due to variations in the speed of elution.
Identification of a hydrophilic
99m
Tc impurity
Examining the results of the TLC and SPE of [99mTc]Tc-NanoHSA suggested the presence of a labile hydrophilic species carrying 99mTc. Although such species in no way impairs the final quality of the radiopharmaceutical, it is of interest to speculate on its nature. NanoHSA from ROTOP Pharmaka is a freeze-dried radiopharmaceutical kit containing 0.5 mg of colloidal particles of human serum albumin. According to the SPC, after reconstituting the kit, 95% of the particles sized ≤ 80 nm are radiolabelled with 99mTc. The excipients present in the kit include: tin chloride dihydrate as a reducing agent, glucose as a filling agent, an antifoaming agent called poloxamer 238, a sodium phosphate di-hydrate buffering agent, and a sodium phytate chelating agent for scavenging unbound 99mTc.Considering the chemical properties of each excipient, the hypothetical 99mTc-labelled species can be narrowed down to phytate or glucose. Phytic acid is a strong chelating molecule that forms stable complexes with multivalent transition metal ions (Vasca et al. 2002). Applications of phytate labelled with 99mTc have been reported in the literature, and a radiopharmaceutical kit (Phytacis®, IBA Molecular) is commercially available for diagnostic applications (Rao et al. 1990; Tavares et al. 2001; Takei et al. 2006). The chromatographic behaviour of [99mTc]Tc-phytate does not exhibit migration with solvent front in methanol/water mobile phase according to the QC method described in the SPC. Thus, [99mTc]Tc-phytate can be excluded as a hydrophilic impurity observed in this study.
Native non-functionalised glucose is only a very weak ligand for transition metals, and there are no stable complexes reported with 99mTc (Klufers and Kunte 2001; Bowen and Orvig 2008). Furthermore, 99mTc chemistry exhibits a strong preference towards N and S atoms when forming radiolabelled compounds (Alberto et al. 2020). Nevertheless, the imaging of glucose distribution on a TLC of the hydrophilic fraction A separated from [99mTc]Tc-NanoHSA using a Sep-Pak® cartridge showed that the glucose followed the distribution of 99mTc radioactivity in 0.9% NaCl and in 85% methanol mobile phases (Figs. 2 and 3). A large excess of glucose in the kit (15 mg) compared to nanocolloidal albumin (0.5 g) and 99mTc (1 GBq of 99mTc equivalent to 5 × 10–9 g) could enable the formation of a transient, elusive complex of [99mTc]Tc-glucose, sufficient to be picked up with TLC. This type of intermediate reaction supports our finding that the RCP of [99mTc]Tc-NanoHSA tended to increase between 10 and 60 min incubation time. In experiments with radiolabelling of glucose, the fraction of mobile 99mTc-carrying species decreased from 39 to 12% within 1 h incubation time (Table 5), which can be attributed to the formation of insoluble [99mTc]TcO2 colloid owing to dissociation of the labile [99mTc]Tc-glucose compound. Furthermore, varying distribution of radioactivity in the different fractions following the SPE of radiolabelled glucose (Table 6) showed an irreproducible behaviour, which was expected as a result of the weak, non-specific interaction of glucose with 99mTc. Another alternative explanation may be that a [99mTc]Tc-gluconate complex forms when a small fraction of the glucose in the kit becomes oxidised to gluconic acid.