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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access Full Text Article   Research Article

Development and validation of a novel analytical approach based on 2D diffuse light reflection for the quantitative determination of ribavirin in aqueous solutions

Ilaha Vagif Kazymova *, Rahman Khamzatovich Ushaev , Elena Valeryevna Uspenskaya , Anton Vladimirovich Syroeshkin 

Department of Pharmaceutical and Toxicological Chemistry, Peoples’ Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya St, Moscow, 117198, Russian Federation

 

 

INTRODUCTION:

Viral diseases remain a major threat to public health, demonstrating a significant impact on mortality, economics, and healthcare systems ¹. The COVID-19 pandemic serves as an example not only of the large-scale spread of RNA viruses and higlights as evidence of the limitations of existing antiviral strategies ². Among clinically significant viruses, respiratory syncytial virus and hemorrhagic fever viruses are of particularly concern, as they continue to cause outbreaks, high hospitalization rates, and significant mortality in vulnerable population groups ³.

In this context, ribavirin (RBV), a broad-spectrum nucleoside analogue, is of particular interest. Unlike most modern antiviral agents against which RNA viruses exhibit high genetic variability, RBV is characterized by a multi-mechanistic mode of action, including inhibition of inosine monophosphate dehydrogenase, depletion of guanosine triphosphate, and induction of lethal mutagenesis in the viral genome ⁴. The multimodal activity of RBV determines its broad application against RNA viruses and highlights its relevance in the context of emerging or insufficiently characterized pathogens ⁵. However, in current clinical practice, it remains a subject of debate, and its use is limited by an unfavorable safety profile, including the risk of hemolytic anemia ⁶. On the other hand, the lack of universally effective broad-spectrum antiviral drugs maintains and supports interest in RBV-based therapy, including its use in combination regimens ⁷. Given the growing interest in RBV therapy, the development of modern and advanced quality control methods represents one of the key tasks of contemporary pharmaceutical chemistry ⁸. Ribavirin (RBV) is officially included in both the United States Pharmacopeia and the British Pharmacopoeia, which describe analytical methods such as high-performance liquid chromatography (HPLC) for quantitative determination of RBV in powder and inhalation formulations, spectrophotometric methods for its determination in pharmaceutical preparations, as well as other techniques including capillary electrophoresis (CE) and polarimetry ⁹. However, despite their advantages, these methods have several limitations, such as cost, complexity, and selectivity issues, which justify the need for the development of alternative analytical approaches. Modern optical approaches have enabled the development of an innovative method based on the kinetics of diffuse light reflection from a rough surface, followed by chemometric processing of dynamic speckle patterns. Chemometric analysis of speckle structures was performed using 10 mathematical descriptors analogous to QSAR parameters ¹⁰.  Mathematical descriptors enable both quantitative and qualitative characterization of substances, including the determination of the concentration of the active pharmaceutical ingredient. The advantages of the diffuse reflectance light method include high selectivity and sensitivity. Measurements can be performed directly in aqueous solutions, which reduces analysis time and reagent consumption. The method is applicable to solid, liquid, and amorphous pharmaceutical materials, making it useful for both quality control and authenticity verification of pharmaceutical products ¹¹.

In this study, in addition to bidistilled water (BD), deuterium-depleted water (DDW) was used. It is known that DDW is characterized by an altered protium/deuterium (¹H/²H) ratio, which leads to a reduction in isotope effects ¹². A decreased deuterium content results in increased proton mobility and, consequently, changes in the kinetics of reactions involving proton transfer ¹³.

The interaction of DDW with pharmaceutical compounds, including RBV, may lead to variations in solubility, stability, and the kinetics of interactions with biological targets ¹⁴. Changes in isotopic composition may influence physicochemical properties, reduce isotope-related effects, and enhance the analytical sensitivity toward structural features of RBV ¹⁵.
Thus, DDW acts not only as a solvent but also as a factor capable of influencing and directionally modifying physicochemical characteristics, including the biological activity and efficacy of RBV. This makes it a promising tool in pharmaceutical analysis and in the improvement of quality control systems for RBV ¹⁶.

MATERIALS AND METHODS

2.1. Reagents

The study object was ribavirin (RBV), a synthetic nucleoside analogue structurally related to guanine (Figure 1) (manufacturer: Jiangling Benda Pharmaceutical Co., China). The substance complied with pharmacopoeial requirements. Samples were stored in tightly closed containers at a temperature of (25 ± 2) °C. The shelf life of the substance was 24 months under the specified storage conditions according to the manufacturer’s data ¹⁷.

Figure 1: Structural formula of RBV

Chemical name: 1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) oxolan-2-yl]-1,2,4-triazole-3-carboxamide.

The study was conducted using model aqueous solutions of ribavirin (RBV) prepared in the concentration range of 0.1–5.0 mg/mL. Bidistilled water (BD, deuterium content D/H ≈ 145 ppm) and deuterium-depleted water (DDW, deuterium oxide content ≤ 1 ppm; Merck Millipore, Burlington, MA, USA) were used as solvents; for DDW, the concentration range was 0.25–10.0 mg/mL. Solutions were prepared at room temperature using analytical balances and laboratory glassware pre-rinsed with the respective solvent.

2.2. Methods

2.2.1. Diffuse Light Reflection
 The study was carried out using an original approach based on the kinetics of diffuse light reflection. The experimental setup is presented in Figure 2.

 

 

 

                

 

 

Figure 2.  Schematic scheme of the diffuse light reflection setup: 1 — compact light source, 2 — laser processing module, 3 — test sample, 4 — collecting lens, 5 — charge-coupled device (CCD), 6 — USB cable, 7 — personal computer.

 

Intense photoexcitation of optical inhomogeneities (scattering centers), caused by density fluctuations in the near-surface layer, leads to the formation of diffuse reflection, the parameters of which are determined by the Rayleigh criterion:

                                                           (1)

where h is the height of density inhomogeneities, λ is the wavelength of the incident light, and φ is the angle of incidence on the surface. The light scattering centers can be described by a transmission matrix (TM), which represents the relationship between the incident light (E_incident) and the transmitted light (E_transmitted) ^18,19.

  ₌                                                       (2)

When laser radiation interacts with surface roughness, each irregular fragment acts as a secondary source of spherical waves. As a result of their mutual superposition, a specific pattern—known as a speckle pattern—is formed. The temporal dynamics of speckle structures manifest as a chaotic alternation of intensity maxima and minima regions (bright and dark fringes), as shown in Figure 3. The analysis of the obtained speckle patterns was performed using a specialized software package, “Vidan”.

 

Figure 3. Scheme of diffuse laser scattering: (a) the incident optical field is scattered by an optical diffuser and recorded by a camera; (b) the captured speckle pattern region is generated by a laser diode.

 

The obtained speckle pattern does not reproduce the studied object; however, it contains comprehensive visual information about the distribution of scattering centers within it ²⁰. The role of scattering centers is played by structural fluctuations of the aqueous medium, including transient density inhomogeneities, nanoscale heterophase fluctuations, and micro- or nanobubble formations, which act as efficient scattering sites contributing to multiple light scattering and speckle field formation. ^21,22. Subsequent chemometric processing is performed using the “Vidan” software package. Each of the ten topological descriptors (analogous to QSAR Wiener (W) and Balaban (J) descriptors (Table 1)) converts the matrix into numerical values through background noise subtraction ²³.

 

 

Table 1. Representation of chemometric descriptors ²⁴.

Descriptor	Mathematical representation	Description
d1		It determines the percentage ratio of elements whose signal level has changed above the defined threshold S_b to the total number of pixels i_t. This parameter allows the spatial extent of changes to be assessed without considering their amplitude.
d2		It is calculated based on the cumulative intensity of differences of all elements relative to the mean signal value of the original frame. This descriptor reflects not only the number of altered pixels but also the magnitude (degree) of these variations.
d3		The method is based on the magnitude of the maximum possible deviations between two states—complete absorption (“black”) and maximum reflection (“white”). It is used to normalize the real dynamics relative to the theoretical limit ΔS_max.

 

 

The descriptor set d1, d2, d3 was extended into three triads to improve statistical reproducibility: sd1, sd2, sd3 (standard deviations); r1, r2, r3 (ri = di/sdi), as well as the composite descriptor R, which integrates all the above parameters. A comprehensive analysis involving ten descriptors enables reliable differentiation of speckle structures and describes the nature of near-surface scattering, thereby characterizing the properties of the studied objects.

Statistics

The results of the analyses are presented as mean ± standard deviation (n=6). All calculations and statistical processing were conducted using OriginPro 21 software (OriginLab, USA).

RESULTS AND DISCUSSION

The procedure for two-dimensional processing of speckle structures for the analysis of ribavirin (RBV) in aqueous solutions with variations in hydrogen isotopic composition consisted, at the first stage, of analyzing graphical representations of descriptor distributions (see Table 1) for bidistilled water (BD) and deuterium-depleted water (DDW) over time, as well as scanning accompanied by high-speed imaging (more than 10 frames per second) over a period of 60 s. All measurements were performed under repeatability conditions (n = 6). Images were captured using a charge-coupled device (CCD) sensor.

 

 

Figure 4. Visualization of the chemometric analysis methodology for dynamic speckle images, presented as the dependence of descriptors d1, d2, and d3 on the data acquisition time DSpc for BD (a) and for DDW (b)

 

 

The results were considered reliable when stable data accumulation pattern was observed in the coordinates di–t (sec), accompanied by the absence of significant data scatter in the coordinates sd(di)–di_average (t, sec) (Figure 4).

3.1 Development and validation of a novel analytical approach based on 2D diffuse reflection for the quantitative determination of RBV in BD (RBV/BD).

Figure 5 presents 2D diagrams (di–C, mg/mL) shown as horizontal bands of aqueous RBV/BD.

 

 

Figure 5. Two-dimensional diagrams of the diffuse laser scattering method (n=6) for aqueous solutions of RBV/BD.

 

The 2D diagrams (di–C, µg/mL), presented as horizontal bands, demonstrate value stability for RBV/BD samples, indicating the stability of the entire set of descriptors for a specific RBV/BD sample under intra-laboratory reproducibility conditions (Table 2) ²⁵.

 

3.1.1 Linearity

Among the ten proposed descriptors, the most pronounced and statistically significant performance was observed for the topological descriptor R in RBV/BD solutions, which made it possible to construct a linear calibration curve “R–C, mg/mL” (Figure 6) ²⁶.

 

Figure 6. Linear (calibration) dependence "R-C, mg/ml" RBV/BD

 

Table 2. Validation parameters of the linearity of the diffuse light reflectance method in the analytical range of RBV/BD determination.

 

 

The analytical range (AR) was established, within which a linear relationship (r = 0.982) was observed, covering the interval between the upper (0.25 mg/mL) and lower (5.0 mg/mL) limits of quantification for RBV/BD. The Pearson correlation coefficient demonstrates a strong linear relationship between paired data and serves as a measure for the qualification of linear regression and quantitative evaluation ^27,28.

3.1.2 Precision

The dispersion of the i-th results relative to the mean value (x̄) was evaluated under repeatability conditions (same laboratory, same operator, same equipment within a short time period) and intermediate precision conditions (same laboratory, different operators, over an extended time period). Precision was assessed by determining the standard deviation (SD, S), relative standard deviation (RSD, coefficient of variation), confidence interval (x̄ ± Δx), and percentage mean error (ε%). (Table 3,4) ^29,30.

 

 

Table 3: Repeatability (intra-method precision)

С, mg/ml		, с-1	SD		RSD, %	, с-1 tp, f = 2,57 ; P=0,95, n=6, f=5
1.0	208,6; 218,9; 214,6; 236,4; 247,2; 208,1	222,3	16,0	255.9	7.2	222,316.8

 

Table 4: Intralaboratory precision (intermediate) (n=18, P=0,95, f=17)  

, с-1	, с-1	SD		RSD, %	, с-1 tp, f = 2,101	, %
248,6; 259,7; 219,9; 213,2; 274,8; 233,3; 208,6; 310,6; 259,4; 244,4; 247,2; 205,4; 284,5; 214,6; 240,2; 307,6; 247,2;     205,4	245,8	32,8	1075	13.3	245.816.2	6,6

3.2 Development and validation of a novel analytical approach based on 2D diffuse reflection for the quantitative determination of RBV in DDW (RBV/DDW)

Figure 6 presents 2D diagrams (di–C, mg/mL) shown as horizontal bands of aqueous RBV/DDW.

Figure 7. Two-dimensional diagrams of the diffuse laser scattering method (n=6) for aqueous solutions of RBV/DDW.

 

 

The 2D diagrams (di–C, mg/mL), presented as horizontal bands, demonstrate the stability of values for RBV/DDW samples, indicating the stability of the entire descriptor set for a specific sample under intra-laboratory reproducibility conditions (Table 5).

3.2.1 Linearity

For RBV/DDW, the most pronounced and statistically significant behavior was observed for the topological descriptor R. A linear calibration relationship “R–C, mg/mL” was constructed (Figure 7).

 

Figure 8. Linear (calibration) dependence "R-C, mg/ml" RBV/DDW.

 

Table 5. Validation parameters of the linearity of the diffuse light reflectance method in the analytical range of RBV/DDW determination.

Linear Dependence Parameters                                                y = ax + b
Constant (Free)Term, b ± SD		Slope, a ± SD	Pearson’s Coefficient, r
РБВ/DDW	159,3±4,2	112,2±3,7	0,997

 

 

The analytical range (AR) was established, within which a linear relationship (r = 0.997) was observed, covering the interval between the upper (0.05 mg/mL) and lower (1.0 mg/mL) limits of quantification for RBV in DDW.

3.2.2 Precision

The dispersion of the i-th results relative to the mean value (x̄) was evaluated under two conditions: repeatability and intermediate (intra-laboratory) precision. Precision was assessed by determining the standard deviation (SD, S), relative standard deviation (RSD, coefficient of variation), confidence interval (Table 6,7).

 

 

Table 6. Repeatability (intra-method precision)

С, mg/ml	, · 10-5	·10-5, с-1	SD·10-5	·10-5	RSD, %	·10-5, с-1 tp, f = 2,57 ; P=0,95, n=6, f=5
1.0	206; 235; 242; 209; 225; 205	220	16	0.00256	7.3	220±17

 

Table 7. Intralaboratory precision (intermediate) (n=18, P=0,95, f=17)  

, · 10-5	·10-5, с-1	SD·10-5	·10-5	RSD, %	·10-5, с-1 tp, f = 2,101	, %
206;235;242;209;225;205; 413;439; 431; 427; 441; 413; 411;393; 370; 382; 410; 395	347	78	0.0608	22.5	347±39	11.24

 

 

The increased linearity of the analytical signal in deuterium-depleted water (DDW) may be attributed to isotope-dependent changes in the structure of water and the dynamics of hydrogen bonding. A reduced deuterium content leads to enhanced proton mobility, alterations in the microheterogeneity of the solution, and the emergence of spatiotemporal fluctuations of the refractive index (Δn), which affect light scattering characteristics and improve the sensitivity of the method. This effect is likely associated with changes in the dynamics of density fluctuations and the properties of scattering centers; however, it requires further investigation and highlights the potential of DDW as an analytical medium for improving the performance of optical and chemometric approaches.

3.3 Comparative Analysis of Analytical Methods

Traditional methods for the quantitative determination of ribavirin, such as high-performance liquid chromatography (HPLC), UV spectrophotometry, and capillary electrophoresis, occupy a leading position in pharmaceutical analysis due to their reliability and sensitivity. However, complex sample preparation, expensive equipment, and significant analytical time limit their use for routine quality control. The advantages of the proposed incorporation, based on two-dimensional light diffusion, are that, compared to HPLC, it does not require chromatographic separation or the use of specified solvents and can be performed in an aqueous environment. The recording of speckle patterns significantly reduces the initial measurement time. The experimental setup is simple, making it feasible in relatively small laboratory settings. The method is nondestructive and requires minimal processing. These advantages make it suitable as a complementary or alternative tool to existing c traditional analytical method

CONCLUSION

In the present study, an original analytical approach based on two-dimensional diffuse light reflection (2D) was proposed and validated for the quantitative determination of ribavirin in aqueous solutions with different hydrogen isotope compositions. A strong linear relationship was established between the topological descriptor (R) and ribavirin concentration both in BD (r = 0.982) and in DDW (r = 0.997). The influence of isotopic composition on the structural organization and dynamics of the aqueous medium was demonstrated. It was shown that the use of DDW as a solvent not only alters the physicochemical properties of the system but also enhances analytical sensitivity, as confirmed by the higher correlation coefficient obtained in DDW. These findings open up opportunities for the targeted optimization of analytical performance through variation of the medium properties. Validation performed in accordance with ICH Q2(R1) guidelines confirmed the precision and repeatability of the method, with relative standard deviation values of approximately 7% for both BD and DDW under repeatability conditions. The proposed approach offers significant advantages, including minimal sample preparation and high analysis speed, enabling real-time measurements without the need for complex instrumentation or expensive reagents. The developed method provides a rapid non-destructive analysis using relatively simple optical equipment, presented in the form of an original hardware-software complex based on two-dimensional diffuse light reflection (2D), which makes it a reliable approach for the systematic determination of ribavirin concentration in aqueous solutions, including under various D/H conditions and can be used for routine quality control.

Conflicts of Interest: The authors declare that they have no conflicts of interest.

Contributors: All authors have read and approved the final manuscript.

Acknowledgement: The authors would like to express their deepest gratitude to the staff of the Department of Pharmaceutical and Toxicological Chemistry, RUDN University.

Source of Support: This paper has been supported by the RUDN University Strategic Academic Leadership program.

Funding: This publication has been supported by the RUDN University Scientific Projects Grant System, project № 033323-2-000

Data Availability Statement: The data presented in this study are available on request from the corresponding author.

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