Rapid Determination of the Radiochemical Purity of ^99mTc-Antimony Trisulfide Colloid Prepared by Standard and Alternative Heating Methods

General

^99mTc colloidal antimony sulfide injection was prepared with a commercial kit (Lymph-Flo; RAH Radiopharmacy), the contents of which were described previously (12). Sodium ^99mTc-pertechnetate was obtained from the daily elution of a ⁹⁹Mo-^99mTc generator (Gentech; Australian Radioisotopes). All preparations were made in triplicate unless stated otherwise. Results are presented as mean ± SD. Linear regression analysis was performed to compare the results determined with the cartridge method and the ITLC method.

Preparation of ^99mTc-ATC with Boiling Water Bath

^99mTc-ATC was prepared according to the manufacturer's instructions. In brief, after the addition of ^99mTc-pertechnetate (900–1,000 MBq) in saline (1.0 mL) and hydrochloric acid (0.1 mL, 1.0 mol/L) to the antimony trisulfide vial (1.0 mL), the breather needle was removed and the vial was submerged in boiling water. After 30 min, the vial was removed from the bath and allowed to cool for 5 min in an aluminum block; this step was followed by the addition of phosphate buffer (0.5 mL) and subsequent filtration (0.2 μm) into an empty vial.

In a second group of preparations, the procedure was repeated as described previously, except that the duration of heating in the boiling water bath was increased to 35 min.

Alternative Preparation of ^99mTc-ATC with Dry Block Heater

^99mTc-ATC was prepared as described previously, except that a temperature-controlled dry block heater (DBH20D; Ratek Instruments) was used to heat the reaction vial at different temperatures and for variable durations. The digital temperature readout was validated with a thermometer. Two different custom aluminum heating blocks (Ratek Instruments) were inserted into the heating apparatus. The 2 blocks differed in the diameter and depth of the 6 holes that could accommodate the 20-mm-diameter reaction vials. One block featured 21-mm-diameter holes to a depth of 40 mm, and the other block had 26-mm-diameter holes to a depth of 43 mm.

For a direct comparison with the manufacturer's recommended method, ATC was mixed with 900–1,000 MBq of ^99mTc-pertechnetate and acid and then heated in the 21-mm dry block at 100°C or 115°C for 30 min.

For all further heating block experiments, 200–400 MBq of ^99mTc-pertechnetate in saline (1.0 mL) were used. The 26-mm heating block was trialled by heating at 115°C for 30 or 35 min. In a second group of experiments, the radiolabeling conditions were kept constant by heating in the dry block (21-mm) at 115°C for 35 min (n = 25). In a third set of experiments, the heating temperature was kept constant at 115°C while the heating duration was varied from 15 to 35 min. In a fourth group of experiments, the heating duration was kept constant at 35 min while the heating temperature was varied from 70°C to 100°C.

Quality Control Analysis

The quality control analysis of the various preparations took place after filtration of the colloid. Each preparation was analyzed for the RCP of ^99mTc-ATC by use of ascending ITLC with a silica gel–impregnated glass fiber strip (ITLC-SG; 1 × 16 cm; Gelman Sciences) and 0.9% saline as the solvent or by use of a strong anion-exchange minicolumn (Amprep SAX; 100 mg, 1 mL; Amersham Biosciences) with saline as the eluent. The 2 quality control tests were performed concurrently.

Radiochemical Purity of ^99mTc-ATC Determined by ITLC

Each ITLC strip was marked at 2 cm from one end of the strip for the origin and every 1 cm until the solvent front was marked at 10 cm from the origin. A small sample from the kit was diluted with saline (to 0.1 mL), and a single drop was spotted onto the ITLC strip at the origin. The strip was developed in saline and then cut into 1-cm segments, which were placed in a γ-counter (Packard Auto-γ-5650; Hewlett-Packard) for counting over a ^99mTc window (70−210 keV). The percentage of ^99mTc-pertechnetate was calculated to be the combined activity of sections with R_f ≥ 0.7 divided by the total activity, and the percentage of ^99mTc-ATC was calculated to be the percentage of sections with R_f < 0.7.

Radiochemical Purity of ^99mTc-ATC Determined by Anion-Exchange Minicolumn Chromatography

The SAX minicolumn was rinsed with saline (2 mL) before use. A sample (<0.05 mL, 5–15 MBq) of the filtered solution was added dropwise onto the sorbent bed. The cartridge was eluted with saline (4 mL) at a flow rate of 2 or 3 drops per second, followed by an air space (1–2 mL), into a collection vial. The contents of the cartridge and the vial were counted separately in a validated counting unit (Atomlab 100⁺ dose calibrator; Biodex Medical Systems). All sample counts were background corrected.

Elution Profile of ^99mTc-Pertechnetate

^99mTc-pertechnetate (∼10 MBq) in saline (0.05 mL) was loaded onto the sorbent bed of 2 different anion-exchange cartridges, the Amprep SAX cartridge described earlier and the Oasis MAX cartridge (30 mg, 1 mL; Waters Corp.). The cartridges were eluted with saline (4 mL) at a flow rate of 3 drops per second into empty glass vials. The contents of the cartridges and collection vials were counted in the dose calibrator as described previously.

Preliminary Evaluation of Anion-Exchange Cartridge Methods

^99mTc-ATC bound to the Amprep SAX sorbent, as observed visually by an orange band in the sorbent and a clear eluate in the collection vial. The time taken to perform the Amprep SAX cartridge method was 2–3 min. When ^99mTc-pertechnetate was added to the Amprep SAX cartridge, it eluted off the cartridge with 4 mL of saline such that 99.8% ± 0.2% of the activity was found in the eluate. Conversely, ^99mTc-pertechnetate was strongly retained by the Oasis MAX cartridge when it was eluted with 4 mL of saline such that 100% of the activity was found on the cartridge.

Accuracy of Amprep SAX Cartridge Method

The RCPs of ^99mTc-ATC samples heated for 30 or 35 min in a boiling water bath or dry block heater are shown in Table 1. When the samples were heated in a boiling water bath for 30 min, the RCP determined by the SAX cartridge method was 98.0% ± 0.8%, and that determined by the ITLC method was 97.6% ± 0.9%, with a mean absolute difference (|Δ|) of 0.4% ± 0.1%. Results comparable to those obtained with the boiling water bath preparation were obtained for the 21- and 26-mm heating block experiments after 30 min. When the samples were heated in the 21-mm dry block at 100°C, the RCPs determined by the SAX method and the ITLC method were 97.3% and 96.9%, respectively; these values increased approximately 2% to 99.3% for the SAX method and 99.1% for the ITLC method when the samples were heated at 115°C. The effect of using the slightly larger 26-mm dry block heating cavity was minimal when the samples were heated at 115°C for 30 min, with the SAX and ITLC methods recording RCPs of 98.3% and 98.0%, respectively. The |Δ| between the SAX and ITLC results was very low for triplicate experiments, ranging from 0.1% to 0.5%.

For the large series of ^99mTc-ATC samples prepared with a dry block heater (21-mm) set at 115°C for 35 min (n = 25), the mean quality control results (RCPs) obtained with the SAX and ITLC methods were 99.4% ± 0.3% and 99.2% ± 0.3%, respectively. The results obtained with the 2 methods for the individual samples differed by 0.0%−1.0%, with a |Δ| of 0.3% ± 0.2%.

The quality control results obtained with the SAX and ITLC methods for the samples heated in the dry block heater at 115°C for 15–35 min are shown in Figure 1. After 15 min, the RCP of ^99mTc-ATC determined by the cartridge method was 91.9% ± 0.4%; that determined by the ITLC method was 91.5% ± 0.5%. The RCPs at the 20-, 25-, 30-, and 35-min time points were 96.0% ± 0.5%, 97.9% ± 0.2%, 99.3% ± 0.1%, and 99.3% ± 0.3%, respectively, as determined by the cartridge method and 96.0% ± 0.5%, 97.7% ± 0.5%, 99.2% ± 0.5%, and 99.3% ± 0.4%, respectively, as determined by the ITLC method. The |Δ| between the 2 methods for the various time points ranged from 0.1% to 0.4%.

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FIGURE 1. 

RCPs, determined by SAX (○) and ITLC (♦) quality control methods, of ^99mTc-ATC samples (n = 3) prepared with dry block heater set at 115°C for various durations.

The quality control results obtained with the SAX and ITLC methods for the samples heated in the dry block heater for 35 min at 70°C–115°C are shown in Figure 2. When determined by the SAX cartridge method, the RCP rose from 85.0% ± 1.5% when the samples were prepared at 70°C to 90.1% ± 0.4%, 95.7% ± 0.3%, 97.4% ± 1.1%, 97.9% ± 0.3%, and 99.3% ± 0.3% when the samples were heated at 80°C, 90°C, 95°C, 100°C, and 115°C, respectively. Equivalent results were obtained with the ITLC method, which yielded RCPs of 83.7% ± 1.3%, 90.2% ± 0.3%, 95.1% ± 0.2%, 97.5% ± 1.0%, 97.6% ± 0.3%, and 99.3% ± 0.4% for the 70°C–115°C reactions, respectively. The |Δ| between the 2 methods for the various temperatures ranged from 0.2% to 1.3%.

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FIGURE 2. 

RCPs, determined by SAX (○) and ITLC (♦) quality control methods, of ^99mTc-ATC samples (n = 3) prepared with dry block heater set at various temperatures for set time of 35 min.

A plot of the RCPs obtained by the SAX method versus the RCPs obtained by the ITLC method for all samples (n = 55) is shown in Figure 3. The linear regression equation was as follows: % SAX = (0.95 × % ITLC) + 5.3%; the correlation coefficient was 0.99.

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FIGURE 3. 

Plot of RCPs determined by SAX method vs. RCPs determined by ITLC method for each ^99mTc-ATC sample tested in this study.

Precision of SAX Method

The precision of the SAX method was determined by testing a single vial of ^99mTc-ATC prepared by heating in a dry block heater for 35 min at 115°C. Five samples were assayed and yielded an RCP of 99.1% ± 0.1%.