Development and Validation of the Stability of p-SCN-Bn-Df via the Reversed-Phase Chromatography Method: Practical Experiences †
Department of Nuclear Medicine, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 206014, UP, India
*
Authors to whom correspondence should be addressed.
†
Presented at the 28th International Electronic Conference on Synthetic Organic Chemistry (ECSOC-28), 15–30 November 2024; Available online: https://sciforum.net/event/ecsoc-28.
Chem. Proc. 2024, 16(1), 39; https://doi.org/10.3390/ecsoc-28-20175
Published: 14 November 2024
(This article belongs to the Proceedings of The 28th International Electronic Conference on Synthetic Organic Chemistry)
Abstract
:The DFO, a special hexadentate chelator with three hydroxamate moieties, is a bifunctional 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)- 6,11,17, 22- tetraazaheptaeicosine] thiourea (p-SCN-Bn-Df), a significant next-generation ligand. The presence of the thiocyanate (-SCN) group makes it capable of hydrolysis and the protonation process. In this study aims to optimize the HPLC protocol for 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(n-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosine] thiourea (p-SCN-Bn-Df) via the Reversed-Phase Chromatography (RP-HPLC) method. A variety of mobile phases were tested in various ratios of solvent constituents such as methanol/water, acetonitrile/water, and phosphate buffer along with at variable pH concentrations. However, when employing a mobile phase consisting of water to acetonitrile containing 0.1% TFA (05:95, v/v) in an isocratic manner, satisfactory separation and symmetric peaks were observed. This method utilized an Eclipsed C-18 column (5 μm, 4.6 × 250 mm) column with a flow rate of 0.5 mL/min. The maximum absorption of p-SCN-Bn-Dfat 254 nm wavelength was selected as the detection wavelength. The Retention time (tR) of p-SCN-Bn-Df was found at 5.205 min. The ICH guideline was used to evaluate the linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), specificity, and system appropriateness criteria to validate the optimized chromatographic and spectrophotometric procedures. For accurate compound separation in pharmaceutical and environmental analyses, this phase is adaptable and often used. This study is useful for the evaluation of p-SCN-Bn-Df QC parameters and chelation rates with different radioisotopes e.g., Zirconuim-89 (Zr-89).
1. Introduction
p-SCN-Bn-Df, or p-Isothiocyanatobenzyl-desferrioxamine, is a bifunctional chelator widely used in the field of radiopharmaceuticals, particularly in the labeling of biomolecules with radiometals for diagnostic and therapeutic applications [1]. The molecule consists of a benzyl group functionalized with an isothiocyanate (-SCN) group and deferoxamine (Df), a high-affinity chelator for various radiometal ions, notably gallium-68 (Ga-68) and zirconium-89 (Zr-89) [2]. The isothiocyanate group (-SCN) allows for facile conjugation to primary amines on proteins, peptides, or other biomolecules, forming stable thiourea linkages [3]. This capability makes p-SCN-Bn-Df highly versatile for labeling a range of targeting vectors, such as monoclonal antibodies, peptides, and small molecules, enabling their use in positron emission tomography (PET) imaging and radio-immunotherapy.
Deferoxamine (Df) is a siderophore with a strong affinity for metal ions, forming stable and inert complexes [4,5,6]. The incorporation of Df into the p-SCN-Bn-Df structure allows it to sequester radiometals efficiently, ensuring high radiochemical purity and the stability of the radiolabeled conjugates in biological systems [7]. This property is crucial for minimizing non-specific binding and enhancing the targeting specificity and sensitivity of the radiopharmaceuticals. One of the prominent applications of p-SCN-Bn-Df is in the development of Ga-68- and Zr-89-labeled antibodies and peptides for PET imaging. Ga-68, with its favorable half-life and positron emission properties, is suitable for rapid imaging protocols, while Zr-89, with a longer half-life, is ideal for tracking the biodistribution of monoclonal antibodies over extended periods [7].
In summary, p-SCN-Bn-Df is a critical component in the toolkit of radiopharmaceutical chemistry, enabling the creation of highly specific and stable radiolabeled compounds for advanced diagnostic and therapeutic applications in nuclear medicine [8,9]. Its ability to conjugate to a wide range of biomolecules and form stable complexes with radiometals underpins its widespread use and importance in the field employed in pharmaceuticals [10,11,12,13,14,15]. It functions based on differential interactions between the components of the sample, a mobile phase, and a stationary phase. The separation of components in a sample occurs when the mobile phase flows through the stationary phase at various rates. In this research, the RP-HPLC method for p-SCN-Bn-Df was successfully developed under optimized chromatographic conditions. This method demonstrated excellent resolution, symmetric peak shapes, and linearity across a broad concentration range, confirming its suitability for quantitative analysis.
2. Materials and Methods
Chemicals and Instruments
The compound 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosine]thiourea (p-SCN-Bn-Deferoxamine, B-705, ≥94%) Figure 1, was obtained from Macrocyclics, Inc. (Plano, TX, USA). The DMSO as a solvent and trifluoroacetic acid (TFA) were sourced from Sigma Aldrich India Pvt. Ltd. (Bangalore, India). The mobile phase was prepared using HPLC-grade acetonitrile and water, both procured from Merck India Pvt. Ltd. (Bangalore, India). All the chemicals were of analytical grade and used without further modification.
The RP-HPLC was performed on Agilent Technologies, Santa Clara, CA, USA 1260 Infinity equipped with a UV detector and quaternary pump, and VWD detector. It also included an automated sample injector with a 100 μL injector loop and an Eclipse Plus C18 (4.6 × 250 mm, 5 μm) column. The pH measurements were carried out using a pH meter (EUTECH INSTUMENTS 2700 pH/mV/°C/°F meter). Nylon membrane filters of pore size 0.45 µm diameter 25 mm) were used for the filtration of the mobile phase. The analytes were fully separated in under 15 min. Several solvents were tested to achieve good symmetric UV peaks and the optimal solvent system was determined to be water/acetonitrile (5:95, v/v) containing 0.1% TFA, with a flow rate of 0.5 mL/min. Using this solvent system, the resolution and peak symmetry were satisfactory, with well-resolved peaks displaying good symmetry and sharpness. The mobile phases were degassed using an ultrasonic power 120W bench top ultrasonic bath (Model: HBS-30A, Helix Biosciences, New Delhi, India).
3. Results and Discussion
A compound p-SCN-Bn-Df of 2mg/mL in DMSO was taken in an Eppendorf tube and the mixture was sonicated for 10 min for complete dissolution. After that, the compound was filtered through a 0.45 μm membrane filter and made a solution of 2 mg/mL. This solution (corresponding to 100%) was used for linearity, precision, and sensitivity tests and thesequence was run starting from 100 μL from each stock sample (0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, 1 mg/mL, 1.2 mg/mL, 1.4 mg/mL, and 1.6 mg/mL) injected into the HPLC system. The resulting solutions were analyzed in triplicate and the peak areas were normalized and treated to a 6-point linearity regression curve.The slope, y-intercept, and coefficient of determination (r2) were used as the measures of linearity. The optimized method for the HPLC is as follows (Table 1):
The prepared homogeneous samples were injected in triplicate into the HPLC using the previously described technique to assess system suitability. The retention time for p-SCN-Bn-Df was approximately 5.1 min. The mean peak area was 18,848.5, with a standard deviation of 877.6, resulting in a relative standard deviation (RSD) of 4.656%. The peak areas of the triplicate injections were recorded. A chromatogram demonstrating the system suitability is shown in Figure 2.
Precision was evaluated by assessing the method’s intra day variations, and HPLC repeatability was determined by analyzing the standard solution on the same day. The precision study involved injecting the standard solution twice at four different concentrations: 10, 20, 30, and 40 µg/µL (Table 2).
The standard calibration curve was generated using eight standard solutions with concentrations ranging from 0.2 to 1.6 mg/mL. Under optimized chromatographic conditions, each standard solution was injected three times with a run time of 15 min per injection. The method’s linearity was assessed by performing a least squares linear regression analysis on the average peak area plotted against concentration (Figure 3).
The representative chromatogram obtained for p-SCN-Bn-Df is shown in Figure 2 with those of the marketed formulations. The calibration curve was linear over the concentration range of 0.2–1.6 mg/mL (Table 3) and the regression equation was found to be y = 25,417x − 50 with a correlation coefficient of 0.9995 (See Table 3). The RSD in precision studies was 0.44–1.10% (Intra-day) (Table 3). The LOQ was found to be 36.87 mcg/mL and the LOD was found to be 12.16 mcg/mL.
4. Conclusions
In conclusion, the development and validation of a stability-indicating RP-HPLC method for p-SCN-Bn-Df was successfully achieved using optimized chromatographic conditions. The method exhibited satisfactory resolution, symmetric peaks, and linearity over a wide concentration range, demonstrating its effectiveness for quantitative analysis. Additionally, the method was validated by the ICH guidelines, ensuring its precision and accuracy. This method proves to be highly suitable for evaluating the stability of p-SCN-Bn-Df in pharmaceutical formulations, as well as for its quality control and chelation efficiency with radiometals like Zr-89, which are crucial for radiopharmaceutical applications. The RP-HPLC method described here provides a reliable analytical tool for both routine quality control and research purposes in the field of radiopharmaceutical chemistry.
Author Contributions
A.S.: investigation, methodology, original draft, and data correction. M.F.: original draft, editing, and communication. V.P.: data correction and editing. M.D.: supervision, review, and editing. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data are contained within the article.
Conflicts of Interest
The authors declare no conflicts of interest.
References
- Yang, D.; Severin, G.W.; Dougherty, C.A.; Lombardi, R.; Chen, D.; Van Dort, M.E.; Barnhart, T.E.; Ross, B.D.; Mazar, A.P.; Hong, H. Antibody-based PET of uPA/uPAR signaling with broad applicability for cancer imaging. Oncotarget 2016, 7, 73912. [Google Scholar] [CrossRef] [PubMed]
- Nagy, P.; Wang, X.; Lemma, K.; Ashby, M.T. Reactive sulfur species: Hydrolysis of hypothiocyanite to give thiocarbamate-S-oxide. J. Am. Chem. Soc. 2007, 129, 15756–15757. [Google Scholar] [CrossRef]
- Davey, P.R.; Paterson, B.M. Modern developments in bifunctional chelator design for gallium radiopharmaceuticals. Molecules 2022, 28, 203. [Google Scholar] [CrossRef]
- Christenson Emily, A. Effects of Complexation with the Siderophore Desferrioxamine b on Transition Metal Removal from Seawater. Master’s Thesis, University of Maryland, College Park, MD, USA, 2013. [Google Scholar]
- Jones, K.E.; Batchler, K.L.; Zalouk, C.; Valentine, A.M. Ti (IV) and the siderophore desferrioxamine B: A tight complex has biological and environmental implications. Inorg. Chem. 2017, 56, 1264–1272. [Google Scholar] [CrossRef] [PubMed]
- Mular, A.; Shanzer, A.; Kozłowski, H.; Hubmann, I.; Misslinger, M.; Krzywik, J.; Decristoforo, C.; Gumienna-Kontecka, E. Cyclic analogs of desferrioxamine e siderophore for 68Ga nuclear imaging: Coordination chemistry and biological activity in staphylococcus aureus. Inorg. Chem. 2021, 60, 17846–17857. [Google Scholar] [CrossRef] [PubMed]
- Vosjan, M.J.; Perk, L.R.; Visser, G.W.; Budde, M.; Jurek, P.; Kiefer, G.E.; Van Dongen, G.A. Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine. Nat. Protoc. 2010, 5, 739–743. [Google Scholar] [CrossRef] [PubMed]
- Deri, M.A.; Ponnala, S.; Kozlowski, P.; Burton-Pye, B.P.; Cicek, H.T.; Hu, C.; Lewis, J.S.; Francesconi, L.C. p-SCN-Bn-HOPO: A superior bifunctional chelator for 89Zr immunoPET. Bioconjugate Chem. 2015, 26, 2579–2591. [Google Scholar] [CrossRef] [PubMed]
- Gohar, M.S.; Rahman, T.U.; Bahadur, A.; Ali, A.; Alharthi, S.; Al-Shaalan, N.H. Development and Validation of Novel HPLC Methods for Quantitative Determination of Vitamin D3 in Tablet Dosage Form. Pharmaceuticals 2024, 17, 505. [Google Scholar] [CrossRef] [PubMed]
- Bhupathiraju, N.V.S.D.K.; Younes, A.; Cao, M.; Ali, J.; Cicek, H.T.; Tully, K.M.; Ponnala, S.; Babich, J.W.; Deri, M.A.; Lewis, J.S.; et al. Improved synthesis of the bifunctional chelator p-SCN-Bn-HOPO. Org. Biomol. Chem. 2019, 17, 6866–6871. [Google Scholar] [CrossRef] [PubMed]
- Nikolin, B.; Imamović, B.; Medanhodžić-Vuk, S.; Sober, M. High perfomance liquid chromatography in pharmaceutical analyses. Bosn. J. Basic Med. Sci. 2004, 4, 5. [Google Scholar] [CrossRef]
- Ali, A.H. High-performance liquid chromatography (HPLC): A review. Ann. Adv. Chem. 2022, 6, 010–020. [Google Scholar]
- Liu, L.X.; Zhang, Y.; Zhou, Y.; Li, G.H.; Yang, G.J.; Feng, X.S. The application of supercritical fluid chromatography in food quality and food safety: An overview. Crit. Rev. Anal. Chem. 2020, 50, 136–160. [Google Scholar] [CrossRef] [PubMed]
- Di Stefano, V.; Avellone, G.; Bongiorno, D.; Cunsolo, V.; Muccilli, V.; Sforza, S.; Dossena, A.; Drahos, L.; Vékey, K. Applications of liquid chromatography–mass spectrometry for food analysis. J. Chromatogr. A 2012, 1259, 74–85. [Google Scholar] [CrossRef] [PubMed]
- Li, S.; Tian, Y.; Jiang, P.; Lin, Y.; Liu, X.; Yang, H. Recent advances in the application of metabolomics for food safety control and food quality analyses. Crit. Rev. Food Sci. Nutr. 2021, 61, 1448–1469. [Google Scholar] [CrossRef] [PubMed]
Figure 1.
Chemical structure of p-SCN-Bn-Df.
Figure 2.
Chromatogram ofp-SCN-Bn-Df along with retention time at 254 nm.
Figure 3.
Linearity curve for p-SCN-Bn-Df at 254 nm HPLC.
Table 1.
Methods and parameter of HPLC for p-SCN-Bn-Df.
| Equipment | Agilent, California, USAhigh-performance liquid chromatography equipped with Auto Sampler and VWD detector. |
| Column | Eclipse Plus C18 (4.6 × 250 mm, 5 μm) |
| Flow rate | 0.5 mL/min |
| Wavelength | 254 nm |
| Injection volume | 100 μL |
| Column oven | Ambient |
| Run time | 15.0 min |
Table 2.
Determination of precision forp-SCN-Bn-Df at 254 nm.
| Sample No. | Conc. (μg/µL) | Mean Peak Area ± SD | RSD % |
|---|---|---|---|
| 1 | 10 | 5276.3 ± 58.12 | 1.102 |
| 2 | 20 | 11,998.3 ± 108.82 | 0.907 |
| 3 | 30 | 18,070.45 ± 91.56 | 0.507 |
| 4 | 40 | 25,065.3 ± 110.24 | 0.440 |
Table 3.
Optimum conditions, optical characteristics, and statistical data of the regression equation of p-SCN-Bn-Df.
Table 3.
Optimum conditions, optical characteristics, and statistical data of the regression equation of p-SCN-Bn-Df.
| Parameter | UV Method |
|---|---|
| λmax (nm) | 254 |
| Beer’s law limits (mcg/mL) | 20–40 |
| Molar extinction coefficient (L mol−1 cm−1) | 25 |
| Sandell’s sensitivity (mcg/cm2−0.001 absorbance units) | 0.0004 |
| Regression equation (Y) | y = 25,417x − 50 |
| Slope (b) | 25 |
| Correlation coefficient (r2) | 0.9995 |
| Precision (% RSD) | 0.739 |
| Limit of detection (mcg/mL) | 12.16 |
| Limit of quantitation (mcg/mL) | 36.87 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
MDPI and ACS Style
Shrivastav, A.; Faheem, M.; Pandey, V.; Dixit, M. Development and Validation of the Stability of p-SCN-Bn-Df via the Reversed-Phase Chromatography Method: Practical Experiences. Chem. Proc. 2024, 16, 39. https://doi.org/10.3390/ecsoc-28-20175
AMA Style
Shrivastav A, Faheem M, Pandey V, Dixit M. Development and Validation of the Stability of p-SCN-Bn-Df via the Reversed-Phase Chromatography Method: Practical Experiences. Chemistry Proceedings. 2024; 16(1):39. https://doi.org/10.3390/ecsoc-28-20175
Chicago/Turabian StyleShrivastav, Anjli, Mohd. Faheem, Vaibhav Pandey, and Manish Dixit. 2024. "Development and Validation of the Stability of p-SCN-Bn-Df via the Reversed-Phase Chromatography Method: Practical Experiences" Chemistry Proceedings 16, no. 1: 39. https://doi.org/10.3390/ecsoc-28-20175
APA StyleShrivastav, A., Faheem, M., Pandey, V., & Dixit, M. (2024). Development and Validation of the Stability of p-SCN-Bn-Df via the Reversed-Phase Chromatography Method: Practical Experiences. Chemistry Proceedings, 16(1), 39. https://doi.org/10.3390/ecsoc-28-20175
