The antiviral drug ganciclovir, also referred to as 1,3-dihydroxy-2-propoxymethylguanine (DHPG), is a cyclovir homologue that has demonstrated efficacy against various viruses including Epstein-Barr virus, varicella-zoster virus, herpes viruses, and cytomegalovirus [1].

Gancyclovir (GCV) [2] is a highly efficacious antiviral medication that is extensively employed for the management of viral infections, specifically cytomegalovirus (CMV) [3] retinitis in individuals with compromised immune systems, as well as for the prevention of CMV in recipients of organ transplants [4]. The administration of Ganciclovir occurs through intravenous means [5], while the prodrug Valganciclovir is administered orally and subsequently undergoes hydrolysis to convert into Ganciclovir after absorption [6]. It is worth noting that the bioavailability of a single dose of Valganciclovir is estimated to be around 60% [7].

GCV measurements hold significant importance in biological samples due to the drug’s substantial resemblance to endogenous substances [8]. Furthermore, GCV has been employed for the management of ocular infections [9], gastrointestinal infections, hepatitis B, compromised immune function, and the treatment of acquired immune deficiency syndrome (AIDS) [10]. The precise and dependable quantification of Gancyclovir holds significant significance in the fields of pharmaceutical analysis [11], clinical pharmacokinetics, and therapeutic drug monitoring, owing to its crucial therapeutic applications [12].

Several analytical techniques [13] have been suggested for the quantification of Gancyclovir (GCV), such as high-performance liquid chromatography (HPLC) [14, 15], liquid chromatography-mass spectrometry (LC-MS) [16], flow injection [17], UV-vis spectrophotometry [18], capillary electrophoresis [19], Surface enhanced Raman spectroscopy (SERS) [13], and spectrophotometric methods [9]. Nevertheless, these methodologies frequently encounter constraints such as costly equipment, intricate specimen processing, and time-intensive protocols, thereby impeding their widespread implementation in resource-constrained laboratory settings [20].

In recent times, there has been a significant focus on oxidation reactions as a viable method for the analysis of pharmaceutical compounds [21]. Colorimetric reagents are commonly employed in the realm of spectroscopic quantitative and qualitative analysis [22]. One of the colorimetric reagents is N-bromosuccinimide (NBS) [23] that is a bromination reagent with high specificity in both free-radical substitutions and electrophilic additions of unsaturated systems due to its ability to liberate small amounts of bromine [24]. The fast reaction between N-Bromosuccinamide and luminol in alkaline medium used for chemiluminescence of uric acid in saliva samples [25]. N-bromosuccinimide (NBS) [26] is a notable oxidant frequently utilized in chemical reactions due to its favorable attributes, particularly its remarkable selectivity towards nucleophilic and aromatic compounds [27]. Consequently, NBS emerges as a highly suitable candidate for the oxidation of pharmaceutical substances such as Gancyclovir (GCV) [28]. The choice of NBS is based on its ability to specifically target the functional groups present in the Gancyclovir, facilitating the formation of an oxidation product that can be easily quantified and characterized using analytical techniques [18].

Oxidative coupling reactions are highly significant organic reactions that find extensive applications, particularly in the field of analytical chemistry [29]. These reactions involve the coupling of two organic substances in the presence of an oxidizing agent, under specific reaction conditions. The oxidation of these substances results in the formation of multiple compounds that interact with each other, ultimately leading to the production of a colored product. This colored product can be quantitatively measured using spectroscopic techniques.

Figure 1: Chemical structure of Ganciclovir [30]

Materials

The NBS oxidizing agent, working standard (GANCICLOVIR), and methylene blue were purchased from Sigma-Aldrich in Mumbai.

Instrumentation

The following equipment and instruments were used:

• A pH meter (Model EQ 621, Equiptronics, India)
• An electronic balance (Model Shimadzu AUX 120)
• A double-beam UV/Visible spectrophotometer (Shimadzu, Japan) equipped with a deuterium lamp
• A thermostatically controlled water bath

Methods

Preparation of Stock Standard Solution

To create the stock solution, 1g of GANCICLOVIR was precisely dissolved in 1000 mL of distilled water (D.W). Different concentrations were prepared by diluting a suitable volume of the stock solution with the same solvent.

Preparation of Reagents

The stock solutions of N-bromosuccinimide and methylene blue, as well as HCl, were prepared by accurately weighing them and dissolving them in 10 mL of distilled water with vigorous stirring for 10 minutes. The solutions were then transferred to 100 mL amber-colored volumetric flasks, and the volume was adjusted.

General Procedure

A series of 10 mL volumetric flasks was used to prepare a range of calibration solutions for Gancyclovir (100 \(\mu\)g/mL). In each flask, the following steps were followed:

• Transfer the required volume of Gancyclovir to cover the calibration range (1 - 35 \(\mu\)g/mL).
• Add 0.5 mL of 1M HCl and 2 mL of 0.001 M N-bromosuccinimide.
• Keep the solution at a temperature of 20\(^\text{o}\)C for 10 minutes.
• After 10 minutes, add 1 mL of methylene blue.
• Dilute the flask contents with distilled water to the mark.
• Measure the absorbance at 610 nm compared to a blank reagent, prepared in the same way but without adding any medication.

Oxidation reaction was used in the process by adding an excess of N-bromosuccinimide. The unreacted N-bromosuccinimide was used to bleach the methylene blue color. The absorption was measured at 610 nm, and it exhibited a linear increase with increasing Ganciclovir concentration.

To determine the optimal conditions for the identification of Ganciclovir in pharmaceutical preparations, the effect of different parameters on color production was tested. One of the parameters considered was the volume of the dye. In order to optimize the effective and optimal dye concentration (methylene blue, malachite green, and crystal violet) that can be calculated spectrophotometrically, experiments were conducted and the results are presented in Table 1.

Effect of the volume of oxidant

The impact of various volumes of N-bromosuccinimide (0.5-3 mL) of 0.001 M on the color of methylene blue dye was observed. These observations were made without the presence of Ganciclovir. The results are summarized in Table 2.

It was observed that 2 mL of the N-bromosuccinimide solution was sufficient to achieve optimal bleaching of the methylene blue dye pigment. Therefore, in subsequent experiments, this volume was chosen for use.

The Acid Influence

The influence of acid on the oxidation of Ganciclovir was tested. The study revealed that the proximity of acid increased the absorption of the resulting product. Among the acids explored, including HCl, \(CH_3COOH\), \(H_2SO_4\), and \(HNO_3\), it was found that each of them could produce the color product. However, HCl provided better absorption with the highest color stability.

In subsequent experiments, 0.5 mL of 1M HCl was used. The results of this investigation are summarized in Table 3.

Temperature Effect

The influence of temperature on the color intensity of methylene blue was investigated. It was observed that the maximum absorbance was obtained when the color was formed at 20\(^\text{o}\)C. Color loss was observed at both low and high temperatures. Therefore, it is recommended to maintain a temperature of 20\(^\text{o}\)C for subsequent experiments.

Sequence of Addition

The sequence of introduction of the oxidant reagent (NBS) should follow the analytical procedure to achieve the best results. Deviating from the specified sequence resulted in a loss of color intensity and reduced stability. The experimental results are summarized in Table 4.

The Influence of Time on the Dye’s Oxidation and Bleaching

The impact of time on N-bromosuccinimide Ganciclovir oxidation and the time required for maximal methylene blue bleaching color were investigated. The results are presented in Tables 5 and 6.

It was found that a sitting time of 10 minutes was required for the full oxidation of Ganciclovir, and 15 minutes were necessary for the bleaching of methylene blue dyes.

Interference Influence

The influence of certain excipients explicitly labeled in pharmaceutical preparations was evaluated in the determination of GANCICLOVIR in the presence of various excipients, including glucose, starch, tween 80, sucrose, benzoic acid, aspartate, lactose, PVP, and microcrystalline cellulose. The experimental findings revealed that there was no interference with the experimental technique by these excipients, as shown in Table 7.

Absorption Spectra

When a 1 mL diluted aqueous solution of Ganciclovir is mixed with 0.5 mL of HCl and 2 mL of N-bromosuccinimide, followed by the addition of 2 mL of methylene blue and a 15-minute incubation, a deep blue color is spontaneously generated. The maximum absorption at 610 nm is shown in Figure 2.

Figure 2: The maximum absorption for drug B): the
maximum absorption for GANCICLOVIR-NBS

Calibration Graph

A linear relationship between absorption and Ganciclovir concentration within the range of 1-35 \(\mu\)g/mL\(^{-1}\) was established. The determination coefficient value (R\(^2\) = 0.9997) indicates a strong correlation. The calibration graph was obtained following the general procedure, as shown in Figure 3.

It was tested to determine the effect of concentration on the absorption of the color product. The Sandell sensitivity was observed to be (0.169 g/cm\(^{-2}\)). The high molar absorptivity was found to be \(3.39 \times 10^{3}\) L.mol\(^{-1}\).cm\(^{-1}\) for the color product. The equations for the limit of detection (LOD) and the limit of quantitation (LOQ) were verified:

\[\text{LOD} = 3 \times \frac{s}{S},\] \[\text{LOQ} = 10 \times \frac{s}{S},\] where ’s’ is the standard deviation for the intercept, and the slope of the intensity line was 0.0059. All analytical optical characteristics are listed in Table 8.

Precision and Accuracy

The precision and accuracy of the methodology were evaluated by conducting five replicate tests of the pure drug solution at two separate Ganciclovir concentrations. The results are presented in Table 9, demonstrating the professional precision and accuracy achieved.

Application of the Technique

The estimation of Ganciclovir in the pharmaceutical formulation was successfully achieved using NBS in the studied analysis method. The values of %RSD, %Error, and Recovery were calculated and are shown in Table 10. Additionally, a T-test was conducted, which did not reveal any significant change. This suggests that the method is appropriate for the determination of Ganciclovir.

The method employed in this study for determining the composition of Ganciclovir in pharmaceutical products has proven to be simple, rapid, versatile, sensitive, and easily reproducible. It offers several advantages, including the absence of the need for heating or extraction.

The authors extend their sincere appreciation for the provision of facilities and instruments at the DNA Research Center, University of Babylon, Iraq, and the Department of Chemistry, College of Sciences, University of Kufa, Iraq.

This research paper received no external funding.

The authors declare no conflicts of interest.

All authors contributed equally to this paper. They have all read and approved the final version.

1. Kumar, T. A., Gurupadayya, B. M., & Rahul Reddy, M. B. (2012). Selective and Validated Spectrophotometric Assay for Microgram Determination of Ganciclovir with 1-fluoro-2, 4-dinitrobenzene and N-Bromosuccinimide Reagents, 27, 14-27.
2. Greeley, Z. W., Giannasca, N. J., Porter, M. J., & Margulies, B. J. (2020). Acyclovir, cidofovir, and amenamevir have additive antiviral effects on herpes simplex virus TYPE 1. Antiviral Research, 176, 104754.
3. Naguib, M. J., Hassan, Y. R., & Abd-Elsalam, W. H. (2021). 3D printed ocusert laden with ultra-fluidic glycerosomes of ganciclovir for the management of ocular cytomegalovirus retinitis. International Journal of Pharmaceutics, 607, 121010.
4. Razonable, R. R. A. H. (2019). Cytomegalovirus in solid organ transplant recipients Guidelines of the American Society of Transplantation Infectious Diseases Community of Practice. Transplant Infectious Diseases, 33(special), 1-23.
5. Heikkinen, E. M., Ruponen, M., Jasper, L. M., Leppanen, J., Hellinen, L., Urtti, A., ... Vellonen, K. S. (2020). Prodrug Approach for Posterior Eye Drug Delivery: Synthesis of Novel Ganciclovir Prodrugs and in Vitro Screening with Cassette Dosing. Molecular Pharmaceutics, 17(6), 1945-1953.
6. Chuchkov, K., Chayrov, R., Hinkov, A., Todorov, D., Shishkova, K., & Stankova, I. G. (2020). Modifications on the heterocyclic base of ganciclovir, penciclovir, acyclovir - syntheses and antiviral properties. Nucleosides, Nucleotides and Nucleic Acids, 39(7), 979-990.
7. Martson, A. G., Edwina, A. E., Kim, H. Y., Knoester, M., Touw, D. J., Sturkenboom, M. G. G., & Alffenaar, J. W. C. (2022). Therapeutic Drug Monitoring of Ganciclovir: Where Are We? Therapeutic Drug Monitoring, 44(1), 138-147.
8. Alinejad, T., Chen, C. S., Shamsipur, M., Gholivand, M. B., & Paimard, G. (2023). Electrochemical evaluation and determination of antiretroviral drug ganciclovir based on Fe-Cu/TiO2/multi-walled carbon nanotubes sensor. Measurement, 214, 112846.
9. Li, D., Zhang, Q., Deng, B., Chen, Y., & Ye, L. (2021). Rapid, sensitive detection of ganciclovir, penciclovir, and valacyclovir-hydrochloride by artificial neural network and partial least squares combined with surface enhanced Raman spectroscopy. Applied Surface Science, 539(2), 148224.
10. Yari, A., & Shams, A. (2018). A Sensitive Electrochemical Sensor for Voltammetric Determination of Ganciclovir Based on Au-ZnS Nanocomposite. Electroanalysis, 30(5), 803-809.
11. Cui, P., & Wang, S. (2019). Application of microfluidic chip technology in pharmaceutical analysis: A review. Journal of Pharmaceutical Analysis, 9(4), 238-247.
12. Selby, P. R., Shakib, S., Peake, S. L., Warner, M. S., Yeung, D., Hahn, U., & Roberts, J. A. (2021). A Systematic Review of the Clinical Pharmacokinetics, Pharmacodynamics and Toxicodynamics of Ganciclovir/Valganciclovir in Allogeneic Haematopoietic Stem Cell Transplant Patients. Clinical Pharmacokinetics, 60(6), 727-739.
13. Ganduh, S. H., Aljeboree, A. M., Mahdi, M. A., & Jasim, L. S. (2021). Spectrophotometric Determination of Metoclopramide-HCL in the Standard Raw and it Compared with Pharmaceuticals. Journal of Pharmaceutical Negative Results, 12(2), 44-48.
14. Mulabagal, V., Annaji, M., Kurapati, S., Dash, R. P., Srinivas, N. R., Tiwari, A. K., & Babu, R. J. (2020). Stability-indicating HPLC method for acyclovir and lidocaine in topical formulations. Biomedical Chromatography, 34(3), e4751.
15. Soliman, M., Saad, A. S., Ismail, N. S., & Zaazaa, H. E. S. (2021). A validated RP-HPLC method for determination of nitroxinil and investigation of its intrinsic stability. Journal of the Iranian Chemical Society, 18(2), 351-361.
16. Chen, Y., Wu, S., & Yang, Q. (2020). Development and Validation of LC-MS/MS for Analyzing Potential Genotoxic Impurities in Pantoprazole Starting Materials. Journal of Analytical Methods in Chemistry, 2020.
17. Long, X., & Chen, F. (2012). Flow injection-chemiluminescence determination of acyclovir. Bll, 478-481 in Luminescence, Vol 27.
18. Lasure, A., Ansari, A., & Kalshetti, M. (2020). Uv Spectrophotometric Analysis and Validation of Acyclovir in Solid Dosage Form. International Journal of Current Pharmaceutical Research, 12(2), 100-103.
19. Saleh, S., & Hempel, G. (2006). Quantification of ganciclovir in human plasma using capillary electrophoresis. Electrophoresis, 27(12), 2439-2443.
20. Ansari, S. (2017). Combination of molecularly imprinted polymers and carbon nanomaterials as a versatile biosensing tool in sample analysis: Recent applications and challenges. TrAC - Trends in Analytical Chemistry, 93(8), 134-51.
21. Wei, Y. P., Yao, L. Y., Wu, Y. Y., Liu, X., Peng, L. H., Tian, Y. L., ... He, Q. G. (2021). Critical review of synthesis, toxicology, and detection of acyclovir. Molecules, 26(21), 6566.
22. Ravisankar, P., Sulthana, M. S., Babu, P. S., Basha, S. A., Aswini, R., Swathi, V., ... & Thanuja, I. M. (2017). Comprehensive review of important analytical reagents used in spectrophotometry. Indo Am. J. Pharm. Res, 7(5), 8716-8744.
23. Al-Majed, A. A., Bakheit, A. H. H., Abdel Aziz, H. A., Alajmi, F. M., & AlRabiah, H. (2017). Propranolol. Profiles of Drug Substances, Excipients and Related Methodology, 42(4), 287-338.
24. Pall, B., Kapui, I., Kormany, R., & Horvath, K. (2023). Development of Analytical Methods for the Determination of N-Bromosuccinimide in Different Active Pharmaceutical Ingredients by High-Performance Ion Chromatography with Suppressed Conductivity Detection. Separations, 10(1), 15.
25. Vakh, C., Koronkiewicz, S., Kalinowski, S., Moskvin, L., & Bulatov, A. (2017). An automatic chemiluminescence method based on the multi-pumping flow system coupled with the fluidized reactor and direct-injection detector: Determination of uric acid in saliva samples. Talanta, 167, 725-732.
26. Hassan, A. I. (2019). Utility of N-Bromosuccinimide as a Green Chemical Reagent for Determination of H 2 -Receptor Antagonists in their Pharmaceutical Dosage Forms. Acta Chemica Iasi, 27(1), 47-64.
27. Di Carmine, G., Abbott, A. P., & D'Agostino, C. (2021). Deep eutectic solvents: Alternative reaction media for organic oxidation reactions. Reaction Chemistry and Engineering, 6(4), 582-598.
28. Rimmele, M., Glocklhofer, F., & Heeney, M. (2022). Post-polymerisation approaches for the rapid modification of conjugated polymer properties. Materials Horizons, 9(11), 2678-2697.
29. Bakr, M. H., & Hassan, A. N. (2022). Spectrophotometric Determination of Allopurinol by Oxidative Coupling Reaction Using 2-Nitrophenol Reagent in the Presence of N-Bromosuccinimide. Pakistan Journal of Medical and Health Sciences, 16(6), 814-818.
30. Gaber, D. A., Alnwiser, M. A., Alotaibi, N. L., Almutairi, R. A., Alsaeed, S. S., Abdoun, S. A., & Alsubaiyel, A. M. (2022). Design and optimization of ganciclovir solid dispersion for improving its bioavailability. Drug Delivery, 29(1), 1836-1847.