Journal of Advanced Pharmaceutical Technology & Research

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 11  |  Issue : 1  |  Page : 6--12

The purity identification and radiolabeling of α-mangostin with technetium-99m


Muchtaridi Muchtaridi, Luthfi Utami Setyawati, Risda Rahmi Islamiaty, Kevin Reinard Lie, Wiwit Nurhidayah 
 Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Bandung, Indonesia

Correspondence Address:
Prof. Muchtaridi Muchtaridi
Jl. Bandung-Sumedang KM 21, Jatinangor, 45363
Indonesia

Abstract

Alpha-mangostin (AM) is a natural compound that has the greatest activity in breast cancer. Radiolabeling AM with technetium-99 m (Tc-99m) has a function as breast cancer radiotracer. This study is aimed to identify the purity of Tc-99m-labeled AM. The identification method was conducted by a validated radio-high-performance liquid chromatography (HPLC) to confirm the chemical purity of the compound when the thin layer of chromatography and paper chromatography were used to find out the radiochemical purity (RCP). The validated radio-HPLC method obtained was C18 column with methanol:water (90:10) as the mobile phase and ultraviolet (243 nm) tandem radioactive detector (Gabi Star). The result showed that the RCP was 70.6% ± 2.87%. The analytical method met the validation criteria according to ICH Q2 (R1); thus, it could be applied in the identification. Unfortunately, the99mTc-AM identification using radio-HPLC showed that the expected complex was not yet formed perfectly because of chemical impurities.



How to cite this article:
Muchtaridi M, Setyawati LU, Islamiaty RR, Lie KR, Nurhidayah W. The purity identification and radiolabeling of α-mangostin with technetium-99m.J Adv Pharm Technol Res 2020;11:6-12


How to cite this URL:
Muchtaridi M, Setyawati LU, Islamiaty RR, Lie KR, Nurhidayah W. The purity identification and radiolabeling of α-mangostin with technetium-99m. J Adv Pharm Technol Res [serial online] 2020 [cited 2022 Dec 7 ];11:6-12
Available from: https://www.japtr.org/text.asp?2020/11/1/6/277929


Full Text



 Introduction



Alpha-mangostin (AM) (1, 3, 6-trihydroxy-7-methoxy-2, 8-bis [3 methyl-2-butenyl]-9Hxanten-9-on) is one of the natural compounds containing xanthon derivatives which is isolated from mangosteen pericarp (Garcinia mangostana Linn.). AM is considerably appreciable to treat cancer. Muchtaridi and Wijaya reviewed the potentiality of AM as an anticancer.[1] The AM plays a role as anti-proliferative that suppresses tumor growth and metastasis in breast cancer of model rat. Breast cancer cells MCF-7 is inhibited by AM.[2] It induces apoptosis of cancer cells through mitochondrial pathways, cell cycle retention through induction of p21cip1, and Akt dephosphorylation on breast cancer cells. Moreover, it inhibits the invasion and migration of cancer cells in the breast gland. The anti-proliferative activity of this compound against MC-7 adenocarcinoma cell apoptosis is demonstrated with IC50 value of 20 μM.[3],[4] AM is a potentially anti-breast cancer agent with antagonistic activity to estrogen receptor α using in silico study.[5]

Based on the chemical structure, AM has an electron donor group in its molecule, thereby making it possible to be labeled with radioisotope technetium-99 m (Tc-99m).[6] The use of radioisotope Tc-99 m for radiotracer purposes has a significant advantage in comparison due to Tc-99m that has ideal properties with half-life of 6 h and low energy at 140 keV, easily obtainable, and has efficient price.[7]

In the development of radiopharmaceuticals, it is very important to have a high purity of labeled compound to get an effective product. A good radiopharmaceutical should have high radiochemical purity (RCP) (>90%).[8] On the other hand, the impurity might be occurred because of the presence of free radioisotopes, degradation products, or waste products during the reaction process. Therefore, besides the quality control of the products, the positive result of labeling process should be considered, for example, by using chromatography and electrophoresis. The chromatographies which are commonly used to separate radiolabeled compounds are thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC).[9],[10]

The objective of this study is to identify the purity of Tc-99m-labeled AM as a breast cancer radiotracer by using radio-HPLC. The radio-HPLC method should be validated initially; thus, the analytical method will always fulfill the expected results.[11]

 Materials and Methods



Materials

The materials used in this study were AM standard (isolated by the Faculty of Pharmacy Padjadjaran University with purity of 95%),99m TcO4− (from99m Tc/99 Mo generator was obtained from Hasan Sadikin Hospital), Na2 EDTA (Merck, Germany), SnCl2(Sigma-Aldrich, USA), sodium hydroxide (Merck, Germany), Whatman 31ET paper, and TLC-SG F254(Merck, Germany). All solvents were purchased from Merck (Germany), except sodium chloride 0.9% and sterile aqua bidest (IPHA). Meanwhile, phosphate buffer pH 7.4 was produced in house.

The equipment to carry out this research consists of dose calibrator (Victoreen), vortex, single-channel analyzer (SCA) (Ortec), paper chromatography (PPC) apparatus, paper electrophoresis device, and radio-HPLC with ultraviolet (UV) detector tandem radioactive detector (Gabi Star).

Synthesis of99m technetium-alpha-mangostin

Labeling Tc-AM was based on indirect labeling using Na2 EDTA as co-ligand and SnCl2 as reducing agent. To obtain an optimum condition, the experiment was carried out by various parameters, including the pH level, the amount of reducing agent (SnCl2), the amount of ethylenediaminetetraacetic acid (EDTA), the amount of AM, and the incubation time. The optimum results for each parameter determined other parameters until the optimum formula obtained. The optimum formula was carried out by adding 300 μg AM, 90 μg SnCl2·2H2O and 150 μg Na2 EDTA, and 10 μL chloride acid 0.1 M (to set the optimum pH 9). After the addition of all reagents, 400 μL99m Tc-pertechnetate (99m TcO4−) with activity of ± 0.3 mCi was put into a vial. The final volume was carried out into 1 mL by adding NaCl 0.9% and the incubation lasted for 5 min in room temperature.

Radiochemical purity analysis of technetium-α-mangostin

RCP was determined using a TLC and PPC. To separate impurities of99m Tc-reduced (99m TcO2) at Rf = 0, TLC-SG F254 (10 cm × 1 cm) was used as stationary phase, whereas ethanol: water: ammonia (2:7:1) as mobile phase. With that regard, to separate the impurities of99m Tc-pertechnetate (99m TcO4−) at Rf = 1, Whatman 31ET paper was used as a stationary phase and acetonitrile: water (1:1) as mobile phase. Both TLC and PPC were marked every 1 cm, and 2 ml of the labeled compound was spotted on the strip and eluted. Every 1 cm segment of chromatogram strips was cut and measured by SCA with NaI (Tl) scintillation counter to determine the distribution of radioactivity.

[INLINE:1]

Paper electrophoresis

Paper electrophoresis was carried out using cellulose acetate paper (13 cm × 1 cm). Two microliters of the labeled compound was spotted in the middle of the paper. Electrophoresis was observed for 1 h at 200 V and 10 A per paper. Every 1 cm segment of cellulose acetate papers was cut and measured by SCA to determine the distribution of radioactivity.

Identifying chemical purity using radio-high-performance liquid chromatography

The radio-HPLC method was initially validated by measuring some parameters according to ICH (2005) criteria, such as system suitability, specificity, linearity, precision, accuracy, limit of detection (LoD), and limit of quantification (LoQ).

The validated HPLC system (Agilent 1200 Infinity Series) with UV and radioactive (Gabi Star Raytest, Germany) detectors was set at maximum wavelength 243 nm. The SGE Analytical Enduro® C18 column (4.6 mm × 250 mm, 5 μm) was used as the stationary phase and methanol: water (90:10 v/v) as the mobile phase with 1.0 mL/min flow rate and 20 μL for injection volume in 15 min. The Tc-AM was injected as well as the controls (AM, Tc-99m and99m Tc-EDTA) to the HPLC system.

 Results and Discussion



The radiochemical purity analysis of technetium-alpha-mangostin

The optimum conditions of Tc-AM radiolabeling must be done to obtain the high RCP of the labeling compounds. Some factors affect the purity of the labeled compounds, including the reaction pH, the amount of SnCl2-EDTA and AM, and the incubation time. The result of the optimum conditions of Tc-AM radiolabeling is shown in [Table 1].{Table 1}

The optimal pH must be within the acceptable pH range in the form of intravenous injection preparations between 3 and 10.5. The optimum pH used was set at 9 with a clear appearance.

The optimization of reducing agent used is the important step because the more redactor is used, the more99m TcO2 is formed as a result of reduction process of99m TcO4− to the lower oxidation state. Nonetheless, if less redactor is used, the reduction process will not run perfectly; therefore, the99m TcO4− is still original.[12]

The optimum amount of SnCl2·2H2O was 90 μg and EDTA was 150 μg. If the amount of SnCl2 is more than 90 μg, it will produce more99m TcO2 impurities because more Tc (VII) will be reduced and form Tc (IV) and vice versa.[13] However, the strength of this reducing agent will generate impurities of radiocolloids during the labeling process by this method.[14],[15]

The optimum amount of AM was 300 μg. RCP will increase with the presence of ligand (AM) when compared to labeling process without AM (99m Tc-EDTA as control). It means that the absence of AM increases the amount of99m TcO2 impurities because Tc (VII) will be reduced and unbind to the ligand (AM). As a result synthesis of99m Tc-Ketoconazol, the amount of additional ketoconazole poor, thereby producing more impurities which make the efficiency of radiolabeling decreased.[16]

Finally, the best incubation time for this radiolabeling reaction was set in 5 min, with purity 70.60% ± 2.87%. The fast incubation time on strong reducing agent leads to the increase of99m TcO2 impurities.[12] On the other hand, pH 9 showed a clear solution, making it eligible for intravenous preparation.

The RCP analysis of the optimum conditions of Tc-AM was measured through the PPC and TLC. The RCP graphics for both are shown in [Figure 1] and [Figure 2]. As shown in both figures, TLC and PPC could not separate99m Tc-AM and99m Tc-EDTA. Furthermore, electrophoresis and HPLC were needed to ensure that99m Tc-AM and99m Tc-EDTA were separated.{Figure 1}{Figure 2}

The electrophoresis was conducted by analyzing Tc-AM and99m TcO4− and99m Tc-EDTA as controls. The result was a radioactive migration pattern of the sample which consequently compared to the controls. The results showed that the three samples had different migration pattern; hence, it can be concluded that the formation of compounds was different [Figure 3].{Figure 3}

The peak of control99m TcO4− at the range of migration 4 cm, the peak of Tc-AM at range of migration 0 cm, while Tc-EDTA control showed several peaks. To sum up, Tc-AM has been formed. The differences in migration patterns are based on electronegative sample. The negative charge compound will move toward the anode and vice versa.[17] However, electrophoresis still could not ensure this separation; thus, HPLC has played an important role to confirm that the separation has been succeed. Prior to use HPLC as a method, it has to be validated by measuring the validation parameters, such as system suitability, specificity, linearity, accuracy, precision, LoD, and LoQ.

Validation method and characterization of technetium-α-mangostin using radio-high-performance liquid chromatography

The results of system suitability and validation parameters are shown in [Table 2] and [Table 3]. The chromatogram of specificity test between AM and blank is shown in [Figure 4].{Table 2}{Table 3}{Figure 4}

Interestingly, the radio-HPLC can be used to identify the chemical purity of Tc-AM. The chemical impurities include nonradioactive materials, such as raw materials, solvents, and other compounds within the preparation process.[18] The HPLC analytical method should be validated first to attain the expected results. According to [Table 2], the measured system suitability with AM standard injection has consistent retention time with relative standard deviation 0.94% from six injections. While in [Table 3], another validation parameters seemingly meet the requirements criteria from the International Conference of Harmonization Q2 (R1);[19] thus, the HPLC method could be used to analyze the compounds.

The test results in [Table 4] show that the AM peak is still appeared in the Tc-AM chromatogram (in 6.68 min). It is apparent that Tc-AM has chemical impurity meaning that the reaction between ligands (AM) and radioisotope (Tc-99m) is not perfectly occurred. By comparing the migration pattern of each sample, it proves the application of the system to analyze the Tc-AM could not separate Tc-AM and99m Tc-EDTA control because both of them have the same chromatograms (at 1.60 and 1.67 min). At this point, the new HPLC system which could separate Tc-AM and99m Tc-EDTA should be found. The chromatograms of Tc-AM are shown in [Figure 5].{Table 4}{Figure 5}

 Conclusion



The analytical method meets the validation criteria according to ICH Q2 (R1); thus, it can be applied in the identification. However, the purity identification of Tc-AM using radio-HPLC shows that the expected complex is not yet formed perfectly because of chemicals impurities. This result is in line with the RCP analysis result that produces the purity of 70.6% ± 2.87%.

Acknowledgment

This study was financially supported by the Students Creativity Program for Research (PKM-PE) Grants and Academic Leadership Grants (ALG) no. 1373b/UN6.O/LT/2019. We would like to thank the Center for Applied Nuclear Science and Technology of National Nuclear Energy Agency (BATAN), Bandung, for supporting this study through the provision of radioactive compounds and research sites.

Financial support and sponsorship

The Directorate General of Higher Education of the Ministry of Research and Technology of Indonesia supported this study through Students Creativity Program for Research (PKM.PE) Grants and ALG 2019, Unpad that funding of this study.

Conflicts of interest

There are no conflicts of interest.

References

1Muchtaridi M, Wijaya CA. Anticancer potential of α-mangostinc. Asian J Pharm Clin Res2017;10:440-5.
2Won YS, Lee JH, Kwon SJ, Kim JY, Park KH, Lee MK. A-mangostin-induced apoptosis is mediated by estrogen receptor α in human breast cancer cells. Food Chem Toxicol 2014;66:158-65.
3Kurose H, Shibata MA, Iinuma M, Otsuki Y. Alterations in cell cycle and induction of apoptotic cell death in breast cancer cells treated with α-mangostin extracted from mangosteen pericarp. J Biomed Biotechnol 2012;2012:1-9.
4Setiawati A, Octa F, Riswanto D, Yuliani S. Anticancer activity of mangosteen pericarp dry extract against mcf-7 breast cancer cell line through estrogen receptor – α. Indones J Pharm 2014;25:119-24.
5Muchtaridi M, Dermawan D, Yusuf M. Molecular docking, 3d structure-based pharmacophore modeling, and adme prediction of α-mangostin and its derivatives against estrogen receptor alpha. J Young Pharm 2018;10:252.
6Shan T, Ma Q, Guo K, Liu J, Li W, Wang F, et al. Xanthones from mangosteen extracts as natural chemopreventive agents: Potential anticancer drugs. Curr Mol Med 2011;11:666-77.
7Komárek P, Kleisner I, Komárková I, Konopková M. The use of redox polymers in labelling procedures of proteins and peptides with 99mTc. II. Technique of preparation of kits for protein labelling by 99mTc and its effect on the stability and radiochemical purity. Nucl Med Rev Cent East Eur 2000;3:69-72.
8Satpati D, Korde A, Venkatesh M, Banerjee S. Preparation and bioevaluation of a 99mTc-labeled chlorambucil analog as a tumor targeting agent. Appl Radiat Isot 2009;67:1644-9.
9Dixit M, Shi J, Wei L, Afari G, Bhattacharyya S. Synthesis of clinical-grade [(18)F]-fluoroestradiol as a surrogate PET biomarker for the evaluation of estrogen receptor-targeting therapeutic drug. Int J Mol Imaging 2013;2013:1-10.
10Parjapath R, Sali V, Kannan S, Kurapati A, Vasanthi H. Hydroxycitric acid-induced activation of peroxisome proliferator-activated receptors in 3t3-l1 adipocyte cells. Pharmacognosy Res2018;10:275-81.
11Chauhan A, Mittu AB, Chauhan P. Analytical method development and validation: A concise review. J Anal Bioanal 2015;6:1-5.
12Zolle I. Technetium-99m Pharmaceuticals. Berlin-Heidelberg: Springer; 2006.
13Khan NU, Naqvi SA, Roohi S, Sherazi TA, Khan ZA, Zahoor AF. Technetium-99m radiolabeling and biological study of epirubicin for in vivo imaging of multi-drug-resistant staphylococcus aureus infections via single photon emission computed tomography. Chem Biol Drug Des 2019;93:154-62.
14De K, Bhowmik A, Behera A, Banerjee I, Ghosh MK, Misra M. Synthesis, radiolabeling, and preclinical evaluation of a new octreotide analog for somatostatin receptor-positive tumor scintigraphy. J Pept Sci 2012;18:720-30.
15Motaleb MA, Adli AS, El-Tawoosy M, Sanad MH, AbdAllah M. An easy and effective method for synthesis and radiolabelling of risedronate as a model for bone imaging. J Labelled Comp Radiopharm 2016;59:157-63.
16Sriyani ME, Slamet IB, Ws H. Radiolabelling Optimization of the 99mTc-DTPA ketoconazole as a radiopharmaceutical for the detection of fungal infections. J Sains Teknol Nuklir Indones2013;14:11-22.
17Motaleb MA, Ibrahim IT, Rizq R, Elzanfaly ES. Preparation, chromatographic evaluation and biodistribution of 99m tc-procainamide as a radiopharmaceutical for heart imaging. Radiochimica Acta 2016;105:215-23. [doi: 10.1515/ract-2015-2558].
18Mihon M, Tuta C, Leonte R, Ion AC, Lavrie VD. An improved methodology for determination of radiochemical and chemical impurities in the synthesis process of 18f-fdg (2-[18f] fluoro-2-deoxy-d-glucose). Environ Eng Manage J 2015;14:289-96.
19International Council for Harmonisation. Validation of analytical procedures. In: Text and Methodology. London: International Council for Harmonisation; 2005.