Methylene blue-enhanced stability of (99mTc)HMPAO and simplified quality control — a comparative investigation

Applied Radiation and Isotopes 54 (2001) 633–636

Technical note

Methylene blue-enhanced stability of (99mTc)HMPAO and simplified quality control } a comparative investigation Grazyna Sobal*, Helmut Sinzinger Radiopharmacology Unit, Department of Nuclear Medicine, University of Vienna, Waehringer Gurtel 18–20, 1090, Vienna, Austria Received 20 March 2000; received in revised form 23 May 2000; accepted 25 May 2000

Abstract (99mTc)HMPAO is a radiopharmaceutical used for SPECT imaging of regional cerebral perfusion and for labeling cellular blood elements. The addition of methylene blue enhanced the stability of lipophilic (99mTc)HMPAO complex up to 3 h after reconstitution, 86.94.2% compared to 49.88.9% for the non-stabilised complex and persists over time (86.23.5% after 15 min compared to 78.24.0% after 3 h). The method widely used for estimation of radiochemical purity is a standard chromatographic procedure which is quite time-consuming taking about 30 min. Comparison of the more rapid and simple solvent extraction method with octanol, which needs only about 10 min to complete, showed a good correlation with the chromatographic method (84.44.4% compared to 89.14.3%). Using ethyl acetate as solvent instead of octanol gave a slightly higher extraction rate of the lipophilic complex (91.55.5% compared to 89.14.3%). Further, the ethyl acetate extraction method results in an overestimation, extracting partly secondary complex. It is confirmed, that the stability of the lipophilic (99mTc)HMPAO complex can be increased with methylene blue. The octanol extraction method, using higher extraction volume (up to 3 ml), is recommended as a fast and efficient approach for the quality control in daily clinical routine. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: (99mTc)HMPAO; Methylene blue-stabilisation; Quality control; Instant thin-layer chromatography (ITLC) method; Octanol extraction; Ethyl acetate extraction; Tracer stability

1. Introduction Technetium-99 m d,l-hexamethyl propyleneamine oxime (99mTc)HMPAO (99mTc-exametazime) is a widely used radiopharmaceutical for SPECT imaging of regional cerebral perfusion in patients with neurological diseases (Barthel et al., 1999; Sharp et al., 1986; Costa et al., 1986; Holmes et al., 1985) and for labeling cellular blood elements to image infections, inflammatory diseases and thrombosis (Kao et al., 1999; McAfee et al., 1987; Becker et al., 1987; Peters et al., 1986). The ability of (99mTc)HMPAO to pass the blood–brain barrier is believed to be due to its lipophilic property (Neirickx et al., 1987; Ceretec TM Package Insert, 1989). *Corresponding author. Tel.: +431-40400-5537, fax: +43140400-5552. E-mail address: [email protected] (G. Sobal).

The use of the radiopharmaceutical in brain scintigraphy is limited by its decomposition from the primary lipophilic (99mTc)HMPAO to a secondary, less lipophilic (hydrophilic) complex which is unable to cross the blood–brain barrier (Ceretec TM Package Insert, 1989; Reichman et al., 1986). Due to the formation of a hydrophilic secondary complex, the shelf life of the radiopharmaceutical is only about 30 min after reconstitution (Ceretec Product Monograph, 1990). As the 99mTc labeling of freeze-dried HMPAO kit produces a lot of impurities (pertechnetate, reducedhydrolysed technetium and secondary complex), the proportions of which change with time, the use of this radiopharmaceutical is often limited. Also the labeling procedure and subsequent quality control exceed by far this 30 min limit. In general, there are two methods for quality control of labelled HMPAO: the standard instant thin-layer chromatography (ITLC) (Neirickx

0969-8043/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 4 3 ( 0 0 ) 0 0 3 0 0 - 6

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et al., 1987; Ceretec TM Package Insert, 1989; Ceretec Product Monograph, 1990) and a solvent extraction method (Volkert et al., 1983; Piera et al., 1990; Hung et al., 1985). The ITLC method is based on the separation of impurities using three chromatography paper strips developed in three different solvents: methyl-ethyl ketone (MEK), 0.9% sodium chloride, and acetonitrile (ACN) and water 1 : 1. The solvent extraction method allows the separation of the primary lipophilic complex and impurites between an organic and aqueous phase. The most common organic solvents used are ethyl acetate or octanol (Ballinger et al., 1988; Andersen, 1989; Hung et al., 1987). The most commonly used aqueous solvent is 0.9% sodium chloride (saline). The ITLC method is more time-consuming and complex than the more simple and fast solvent extraction method. To improve the stability of the radiopharmaceutical the use of methylene blue is strongly recommended (Barthel et al., 1999). We investigated, whether this treatment indeed improves the stability of (99mTc)HMPAO. Further, different methods of quality control were compared. 2. Materials and methods 2.1. (99mTc) HMPAO preparation (99mTc)HMPAO was prepared by reconstitution of Ceretec kit containing 0.5 mg of HMPAO, 7.6 mg of stannous chloride dihydrate and 4.5 mg of sodium chloride with 99mTc (2000–4000 MBq) according to the manufacturer’s preparation procedure (Nycomed– Amersham, Buckinghamshire, UK). For stabilized (99mTc)HMPAO we used the same procedure including the addition of methylene blue stabilizing solution within 2 min of reconstitution. The injection solution was prepared by adding 0.5 ml of methylene blue injection USP (United States Pharmacopeia) (10 mg/ml) into a sterile syringe and injection into 4.5 ml vial of 0.003 M monobasic sodium phosphate USP and dibasic sodium phosphate USP in 0.9% sodium chloride injection USP. After gently vortexing 2 ml of methylene blue/phosphate buffer, the solution was added into 7 ml of reconstituted (99mTc)HMPAO. The final concentration of methylene blue in the solution was 0.222 mg/ml. Methylene blue/phosphate buffer solution was used within 30 min of preparation. 2.2. Quality control For quality control we used the following procedures: 1. Standard chromatographic method using paper (Whatman 1Chr, Maidstone, UK) and thin-layer strips

(ITLC-SG Gelman, Ann Arbor, MI, USA) in three solvent systems. (99mTc)HMPAO was spotted approximately 2 cm from the bottom of the strips and one of the ITLC-SG strips was developed in A } methyl-ethyl ketone (Merck Darmstadt, Germany) to a distance of 8– 10 cm. In this system primary (99mTc)HMPAO-complex and free pertechnetate migrated with the solvent front (Rf ¼ 0:8ÿ1:0), while the secondary (99mTc)HMPAO complex and reduced hydrolysed 99mTc remained at the origin. B } the second ITLC-SG strip was treated in the same way and developed in 0.9% sodium chloride. Free pertechnetate migrated with the solvent front (Rf ¼ 0:8ÿ1:0), while the primary and secondary (99mTc)HMPAO complex and reduced hydrolysed 99m Tc remained at the origin. C } the paper chromatography strip was developed in freshly prepared acetonitrile and water 1 : 1. In this system the primary and secondary (99mTc)HMPAO complex and free pertechnetate migrated with the solvent front, while the reduced hydrolysed 99mTc remained at the origin. The results obtained with chromatographic standard method were calculated according to the equation %D=100-(A+B+C)%, where A is the hydrophilic secondary complex; B the free pertechnetate; C the reduced-hydrolysed technetium; D the lipophilic complex. 2. Solvent extraction method using A } octanol: a few drops of (99mTc)HMPAO were added to a glass test tube containing 2 ml of octanol (Merck, Darmstadt, Germany) and 2 ml of 0.9% sodium chloride. The tube was closed with a cap, vortexed for 1 min and left for a few minutes to allow separation of the two phases. The top layer containing the lipophilic (99mTc)HMPAO was transferred by pipette to another tube. The radioactivity in both layers was measured. The percentage of lipophilic (99mTc)HMPAO was calculated as the activity in the top layer divided by the total activity in both layers. B } ethyl acetate (Merck, Darmstadt, Germany) performed in the same way as with octanol. To compare the results of both extraction methods (octanol and ethylacetate ), we examined the extracts by the ITLC method in the same way as (99mTc)HMPAO itself. Each fraction of the extract (top and bottom layers) was analyzed by the standard chromatographic method using three strips (ITLC-SG ) in three solvent systems. We assessed exactly the content of the extracts in both layers to evaluate the possible reason for lipophilic complex differences obtained by these two methods. 2.3. Statistical analysis The results are expressed as mean values S.D. Statistical analysis was performed using using one-way ANOVA; p50:05 was considered as statistically significant. Additionally, Student’s paired t-test was used.

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3. Results Using the standard chromatographic method, methylene blue was found to increase the stability of the lipophilic complex significantly over a period at least 3 h. Investigations on 11 preparations showed that the proportions of the methylene blue stabilized lipophilic complex remaining at 15 min and 3 h were 86.23.5 and 78.24.0%, respectively ( p50:0005, n ¼ 11). In contrast, the proportions of non-stabilized complex had fallen from 86.94.2% at 15 min to 49.88.9% at 3 h ( p50:0001; n ¼ 11). According to the European Pharmacopeia a purity below 80% is unacceptable for clinical use, time intervals longer than 3 h were not studied. Since the standard chromatographic method of analysis takes about 30 min to complete, a comparison was made with the simpler and more rapid solvent extraction procedure which can be completed in about 10 min. The results from studies on 26 methylene bluestabilized peparations at 15 min after reconstitution were standard chromatographic method: 84.44.4%; solvent extraction method (octanol): 89.14.3%; solvent extraction method (ethyl acetate): 91.55.5%. The differences between the standard chromatographic method and the extraction methods were statistically significant, p50:01 (n ¼ 26) for both solvents. The Students’ paired t-test gave value of a p ¼ 0:003 for octanol and p50:0005 for ethyl acetate. Using ethyl acetate as a solvent instead of octanol showed a slightly higher extraction of the lipophilic complex; however, the difference was not statistically significant.

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solvents. After extraction it is sufficient to separate 1 ml of the organic phase, measure the radioactivity and then calculate for the whole volume of 3 ml. Analyzing the aqueous and organic fractions obtained by the extraction procedure by the standard chromatographic method, we found some contamination of the secondary (hydrophilic) complex in the organic layer. Organic solvents should extract the primary lipophilic complex only. We found, however, that this is not absolutely true. Although we could observe this phenomenon using both extraction methods (octanol and ethyl acetate), the aliquot of the secondary (hydrophilic) complex appearing in the organic phase is higher in the ethyl acetate extract (4.3–12.5%) as compared to the octanol extract (3.5–10%). This could be the reason for slight overestimation of the lipophilic complex by the ethyl acetate extraction method. The stabilisation of the tracer with methylene blue is a great advantage. Our results confirm that in the presence of methylene blue the stability of the lipophilic (99mTc)HMPAO complex remains high (86.23.5% after 15 min compared to 78.24.0%, for up to 3 h). We thus conclude that the stability of the lipophilic (99mTc)HMPAO complex can be enhanced with methylene blue. The ethyl acetate extraction method is the most rapid method to perform the quality control for (99mTc)HMPAO, but may slightly overestimate. We recommend the octanol extraction method using a higher extraction volume of up to 3 ml for daily clinical routine.

References 4. Discussion The aim of the study was to compare different methods for quality control of (99mTc)HMPAO and to find the best, fastest and cheapest. The standard chromatographic method using instant thin-layer strips in three different solvents is reliable, but time consuming, taking about 30 min. This method gives comparable results with extraction methods, requiring only about 10 min and being much easier and faster. The extraction methods, however, may be subject to errors due to incomplete separation of the two layers (organic and aqueous). This problem is particulary relevant in the case of octanol extraction. The clean separation of the two layers is more difficult and needs time. This was the reason for changing octanol to ethyl acetate allowing better separation of the layers as already reported by many investigators (Volkert et al., 1983; Ballinger et al., 1988). According to our experience quite good results can be achieved by working with at least 3 ml volume of both (organic and inorganic)

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