Validation of a Cost-Effective RP-HPLC Method for Quantitative Investigation of Daclatasvir Dihydrochloride in Pharmaceutical Formulations

Abstract

A well-known direct-acting antiviral (DAA) drug called daclatasvir may be used to treat chronic hepatitis C virus (HCV) infection. Herein, we reported a selective, precise, and a cost-effective analytical method for the measurement of an active pharmaceutical ingredient (API) of daclatasvir dihydrochloride in drug substances as well as drug products via the reversed-phase RP-HPLC technique. To obtain greater separation, the majority of the chromatographic conditions were improved. Best separation findings were achieved under chromatographic conditions with an HPLC column of USP L1 (150 × 4.6 mm, 5 μm) by utilizing a combination of acetonitrile and buffer solution of KH₂PO₄ (30 : 70, v/v) as a mobile phase at a stream rate of 1 mL.min⁻¹ with a finding at 300 nm and a column temperature of 40°C. Linearity was examined in the range of 90–210 ppm (R² = 0.999) for daclatasvir dihydrochloride. The new technique has been verified using industry-recognized criteria, including applicability, system precision, accuracy, robustness, specificity, range, linearity, quantification limit, reagent stability, and detection limit. All the measured metrics were determined to be within acceptable limits using the criteria of the Worldwide Council for Harmonisation (ICH). In pharmaceutical labs, daclatasvir dihydrochloride may be analyzed qualitatively and quantitatively using the well-established RP-HPLC technique. Our study also highlights the need to evaluate the greenness of the method developed using a recognized tool ,i.e., Analytical Greenness Metrics (AGREE).

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

A newly licenced direct-acting antiviral (DAA) medication called daclatasvir, sold under the brand name Daklinza®, is used to treat persistent HCV infection [1–3]. It is a novel inhibitor targeting the nonstructural protein 5A (NS5A) of HCV, developed by Bristol-Myers Squibb [4]. Daclatasvir binds to the N-terminus of NS5A to exert its inhibitory effects on viral RNA replication and virion assembly [5]. This interaction leads to structural changes in the protein, ultimately inhibiting its functions [6]. The chemical composition of daclatasvir dihydrochloride and the analytical methods employed to analyse it have been crucial in its development and clinical use.

Daclatasvir (generic name, INN) is a pharmaceutical drug having a molecular formula C₄₀H₅₂Cl₂N₈O₆ with a molecular weight of 811.8 g mol⁻¹ [6]. The structural representation of the daclatasvir dihydrochloride drug is provided in Figure 1. This drug can be obtained by combining one mole of daclatasvir with two equivalent moles of hydrochloric acid. However, the qualitative and quantitative measurements of these types of expensive pharmaceutical drugs require a rapid, selective, precise, and cost-effective analytical method that needs to be developed and validated for their commercial applications [7].

[figure(s) omitted; refer to PDF]

Several analytical (chromatographic and spectroscopic) methods have been reported in the literature to determine the amount of daclatasvir dihydrochloride drug in bulk and dosage forms [8, 9]. These reported chromatographic methods have several drawbacks which make them unsuitable for their commercial application in pharmaceutical industries. In addition, previously reported methods to determine daclatasvir dihydrochloride entail low sensitivity. Mostly, the complications are associated with column dimensions [10] and retention times [11, 12]. In the case of spectroscopic methods, impurities, degradants, and other excipient components can interfere with the absorbance of the desired analyte in the sample, and consequently, the observed results may differ from the true value [13, 14]. However, in the presence of contaminants or excipients that elute at various intervals, chromatographic procedures give an accurate estimate of the desired analyte, making quantification achievable [15, 16]. The comparison indicates that the quantification by HPLC is better than that carried out via UV-Visible spectroscopy [17]. Hence, there is a strong need to develop a standard chromatographic method to measure the actual amount of daclatasvir dihydrochloride in drug substances as well as the product of daclatasvir tablets.

Using RP-HPLC, we developed an effective analytical approach for the detection of daclatasvir dihydrochloride medications to fill this research gap. To achieve better separation, all the parameters, including (i) column temperature, (ii) flow rate, (iii) mobile phase ratio, and (iv) HPLC column length, were tuned. The proposed technique is approved for use in terms of reagent stability, detection limit, precision, accuracy, specificity, robustness, linearity, range, and system appropriateness. Its simplicity, dependability, selectivity, and speed are used to define the originality of this RP-HPLC technology. The results of this research suggest that the suggested approach has a number of benefits over the previously published method because it makes it possible to determine the examined medication in a straightforward and sensitive manner with reliable Greeness results. A short description of the suggested strategy for comparing the results of the present investigation with those previously published is given in Table 1.

Table 1

Comparison of performance features of the current work vs published work.

Analytical techniques	Linearity (ppm)	Remarks	Reference
HPLC-UV	0.6–6	Poor sensitivity acetonitrile–10 mM acetate buffer	[18]
HPLC–UV	0.5–101	Poor sensitivity phosphate buffer : acetonitrile (50 : 50) tR = 2.33 min	[19]
HPLC–UV	2–199	Poor sensitivity phosphate buffer : acetonitrile (60 : 40) tR = 7.3 min	[14]
HPLC–UV	10–151	Poor sensitivity water and methanol (20 : 80) tR = 2.0 min	[20]
HPLC–UV	21–80	Poor sensitivity acetonitrile : methanol (70 : 30) tR = 2.7 min	[11]
HPLC–UV	2–100	Poor sensitivity methanol : water : acetonitrile (25 : 30 : 45) tR = 6.9 min	[21]
HPLC–DAD	0.6–60	Poor sensitivity phosphate : acetonitrile (75 : 25) tR = 5.4 min	[5]
Derivatization	6–50	Very poor sensitivity and reagent use	[13]
Spectrophotometry	4–12	Low sensitivity
Proposed HPLC method	90–210	Highly sensitive and simple method with agree (greenness calculator) score 0.68	—

2. Materials and Methods

2.1. Chemicals and Reagents

Acetonitrile (CH₃CN, 99.8%), potassium dihydrogen phosphate (KH₂PO₄, >99%), phosphoric acid (H₃PO₄, 99.99%), and deionized water for chromatography (HPLC grade) were procured from Merck. Reference samples of daclatasvir dihydrochloride and drug products were supplied by the pharmaceutical industry in Lahore.

2.2. Preparation of Solutions

2.2.1. Buffer Preparation

The pH of this solution was raised to 3.0 ± 0.05 using diluted phosphoric acid, and the volume was increased to one litre with deionized water after accurately weighing and dissolving 2.73 g of KH₂PO₄ in about 800 mL of deionized water. After being sonicated for ten minutes, a 0.45 m hydrophobic PTFE membrane filter was used to filter the buffer solution (Millipore, USA).

2.2.2. Mobile Phase Preparation

A 30 : 70 (v/v%) combination of acetonitrile and a buffer solution of potassium dihydrogen phosphate was made, and it was sonicated for 30 minutes to remove gas. In addition, acetonitrile and water were taken as diluents in a proportion of 20 : 80 (v/v%).

2.2.3. Preparation of the Typical Stock Solution

A 50 mL volumetric flask was filled with precisely weighed 41.25 mg of daclatasvir dihydrochloride, which is equivalent to 37.50 mg of daclatasvir. To achieve the desired concentration of 750 ppm, daclatasvir was dissolved into the diluent by sonicating the flask for 10 minutes. A 0.45 m hydrophobic PTFE membrane filter was also used to filter the standard stock solution.

2.2.4. Formation of the Standard Solution

To create the typical solutions of 90, 120, 150, 180, and 210 ppm, correspondingly, 3.0, 4.0, 5.0, 6.0, and 7.0 mL of the standard stock solution (750 ppm) were accurately transferred into each 25 mL volumetric flask, diluted to the proper concentration with dilutant, and thoroughly mixed by sonication.

2.2.5. Preparation of a Sample Solution

Ten daclatasvir tablets were roughly weighed, ground, and added to the sample in a 50 mL volumetric flask. Each tablet contained around 37.50 mg of the medication. The mixture was then sonicated for 25 minutes to thoroughly saturate the active ingredient in the diluent after 30 mL or so of diluent was inserted. After being diluted to the right concentration, the solution was filtered and kept at room temperature. A 25 mL flask was filled to the mark with a diluent after 5.0 mL of the sample solution’s supernatant was transferred there. This created the sample solution, which had an estimated concentration of 150 ppm.

2.3. Characterization Techniques

A Shimadzu HPLC system (20AT, Kyoto, Japan) coupled with a UV-visible detector having dual-wavelength was used in this study. The system also has gradient elution capability and an autosampler associated with the Agilent HPLC column of USP L1 (150 × 4.6 mm, 5 μm) containing octadecylsilyl silica gel as a stationary phase [22]. For this investigation, a pH metre (S220, Mettler Toledo), an ultrasonic water bath (8510, Branson), and an analytical balance (PB210S, Sartorius) were also used.

2.4. Chromatographic Conditions

By using chromatographic conditions on reversed-phase octadecylsilyl silica gel (stationary phase) with a pore size of 5 m and a stainless steel HPLC column with dimensions (150 × 4.6 mm) coupled with a UV-visible detector set at 300 nm, analytical separation was achieved. The mobile phase was isocratically eluted at a flow rate of 1 mL.min⁻¹. The system was equilibrated for 30 minutes before the solution was injected into the column.

3. Results and Discussion

3.1. Method Validation

To verify adherence to ICH standards and regulatory compliance Q2(R1) [23, 24], most of the analytical parameters were investigated which define the system’s suitability by measuring relative standard deviation (RSD), standard deviation, accuracy in terms of average recovery for various spiked levels and tailing factor. Moreover, the precision, limit of detection (LOD), specificity, limit of quantification (LOQ), robustness, and correlation coefficient (R²) in linearity were the basis of method validation.

3.1.1. System Suitability

A standard solution of daclatasvir dihydrochloride (150 ppm) was prepared as described above at 100% level and six replicate samples into the HPLC system were injected. The observed chromatogram representing system suitability for the standard solution of daclatasvir dihydrochloride is provided in Figure 2. The system suitability parameters were evaluated in terms of RSD value for retention time (0.06%), peak response (0.07%), tailing factor (∼1.33), and theoretical plates (∼2793) which were found within the acceptance criteria, as expressed in Table 2.

[figure(s) omitted; refer to PDF]

Table 2

System suitability studies for daclatasvir dihydrochloride.

Sr. no.	Parameters	Observed value	Acceptance criteria
1	RSD (retention time)	0.06%	<2.0%
2	RSD (peak response)	0.07%	<2.0%
3	Tailing factor	∼1.33	0.8–2.0
4	Theoretical plates	∼2793	>2000

3.1.2. Linearity

Five concentrations, 90, 120, 150, 180, and 210 ppm, were produced (Table 3) and inserted into the HPLC system under chromatographic conditions for reference solutions that were 60 to 140 percent spiked. By creating a graph between different daclatasvir concentrations and their peak response, the linearity was examined. The correlation coefficient (R²) was then calculated, as shown in Figure 3. The observed linear equation (Y = 72744 X + 385027) shows the values of slope (72744), intercept (385027), and R² = 0.9992. The value of R² indicates excellent linearity [25].

Table 3

Various concentration of standard solution for linearity measurements.

Sample name	Volume of stock solution(mL)	Total volume(mL)	Final concentration(μg.mL−1, ppm)
Linearity–S1	3.0	25	90
Linearity–S2	4.0	25	120
Linearity–S3	5.0	25	150
Linearity–S4	6.0	25	180
Linearity–S5	7.0	25	210

[figure(s) omitted; refer to PDF]

3.1.3. Accuracy

A placebo of daclatasvir tablets was spiked with the standard solution in triplicate at 60%, 100%, and 140% concentration spiked levels. Figure 4 represents chromatogram showing no interference for the placebo sample of daclatasvir tablets. The estimate of the mean recovery of three levels for those three levels is shown in Table 4 because of the injection of these solutions into the HPLC device under predefined chromatographic conditions. The average recovery was calculated as 100.60 percent, which is within the acceptable range of accuracy (98.0 to 102.0 percent), with the mean recovery being determined to be 100.79, 101.01, and 99.99 percent, respectively.

[figure(s) omitted; refer to PDF]

Table 4

Various concentrations of daclatasvir and % average recovery data.

Analyte level	Theoretical concentration(ppm)	Measured concentration(ppm)	Individual recovery(%)	Mean recovery(%)
Level-1	90	90.22	100.25	100.79
	90	92.07	102.30
	90	89.85	99.83
Level-2	150	151.42	100.95	101.01
	150	151.35	100.90
	150	151.77	101.18
Level-3	210	211.43	100.68	99.99
	210	208.42	99.25
	210	210.11	100.05

3.1.4. Range

The lowest and highest concentrations (amounts) of analyte in the sample at which the analytical technique may be used with an acceptable degree of accuracy, precision, and linearity are referred to as the limits of a method. Each of these fundamental qualities has had the validity of the developed analytical approach evaluated, and the results produced satisfy the criteria for approval, as displayed in Table 5.

Table 5

Precision, accuracy, and linearity data of RP-HPLC method of daclatasvir.

Sr. no.	Parameter	Results
1	% RSD (area)	0.07
2	% RSD (assay)	0.48
3	% RSD assay (cumulative)	0.53
4	Individual recovery	99.25–102.30%
5	Average recovery	100.6%
6	Correlation coefficient (R2)	0.9992
7	Regression equation	Y = 72744 X + 385027

3.1.5. Specificity

The specificity parameter was also estimated under the optimized chromatographic conditions by injecting various solutions of placebo material (without active substance) for both products of daclatasvir (30 mg, 60 mg) tablets in triplicate preparations against the standard solution [25–27]. It was found that there is no interference in the sample solutions for daclatasvir at the retention period of 5 min when compared to the reference solution, demonstrating the proposed method’s outstanding specificity.

3.1.6. Precision

Using the standard method under normal operating conditions, six determinations of the assay were carried out on both products (30 mg, 60 mg) on the same day (intraday) by a single operator (precision-repeatability) as well as on a different day (interday) by another operator on different equipment (precision-intermediate) against the standard solution. The obtained results of the mean assay and % RSD are presented in Table 6. The calculated precision has average RSD values of 0.51% and 0.44% for 30 mg and 60 mg daclatasvir tablets, respectively. It was observed that the % RSD values for both products (30 mg and 60 mg) lie within the specified limit, i.e., <2%, which showed excellent precision of the developed method [28].

Table 6

Precision data for 30 mg and 60 mg daclatasvir tablets.

Parameter	Operator-1 (day 1)	Operator-2 (day 2)	Average value
30 mg daclatasvir tablets
Mean assay (%)	99.35	99.30	99.33
RSD (%)	0.53	0.48	0.51
60 mg daclatasvir tablets
Mean assay (%)	99.20	99.46	99.33
RSD (%)	0.52	0.36	0.44

3.1.7. Robustness

Intentionally changing a single aspect of the method’s operational circumstances also served to verify it. Six identical samples of a standard daclatasvir solution (150 ppm) were injected into the HPLC system in accordance with usual practise, with the flow rate, wavelength, and column temperature being adjusted. The peak responses were noted in order to evaluate the robustness of the suggested technique in terms of standard deviation and percent RSD. Figures 5–9 indicate chromatograms representing the robustness of daclatasvir dihydrochloride standard solution at different values of flow rates (0.8 mL.min⁻¹ and 1.2 mL.min⁻¹), wavelengths (298 nm and 302 nm), and column temperature (38°C), respectively. As shown in Table 7, the observed findings varied within the predetermined bounds, suggesting that the analytical procedure was unaffected by purposefully adjusting the wavelength, column temperature, and flow rate parameters.

[figure(s) omitted; refer to PDF]

Table 7

By adjusting the wavelength, flow velocity, and column temperature, robustness data may be calculated in terms of standard deviation and percent RSD.

Parameter	Flow rate		Wavelength (λmax)		Temperature
	0.8 mL.min−1	1.2 mL.min−1	298 nm	302 nm	@ 38°C	@ 42°C
Standard deviation	0.02	0.01	0.01	0.00	0.05	0.03
% RSD	0.26	0.29	0.16	0.02	1.02	0.62

3.1.8. Detection Limit

The LOD is a characteristic of a limit test and refers to the smallest quantity of analyte in a sample that can be detected but not always quantitated under certain experimental circumstances. The assessed value (1.40 ppm) of this parameter, which was calculated using the following formula under linearity, is shown in Table 7.$$\begin{array}{}\text{(1)}& \mathrm{L}\mathrm{O}\mathrm{D}=3.3\mathrm{x}\frac{\mathrm{S}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d}\mathrm{d}\mathrm{e}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}}{\mathrm{S}\mathrm{l}\mathrm{o}\mathrm{p}\mathrm{e}}\text{.}\end{array}$$

3.1.9. Quantification Limit

The lowest quantity of the analyte in the sample that can be accurately and precisely identified under the experimental circumstances is referred to as the LOQ, and it is used to describe low levels of the substance in sample matrices. This parameter was also evaluated under linearity by the following formula, and the obtained value (4.25 ppm) is specified in Table 8.$$\begin{array}{}\text{(2)}& \mathrm{L}\mathrm{O}\mathrm{Q}=10\mathrm{x}\frac{\mathrm{S}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d}\mathrm{d}\mathrm{e}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}}{\mathrm{S}\mathrm{l}\mathrm{o}\mathrm{p}\mathrm{e}}\text{.}\end{array}$$

Table 8

LOD and LOQ values for daclatasvir dihydrochloride.

Drug	Detection limit (ppm)	Quantification limit (ppm)
Daclatasvir dihydrochloride	1.40	4.25

3.1.10. Solution Reagent Stability

As per the standard analytical method, the sample as well as standard solutions of both products, i.e., daclatasvir 30 mg and 60 mg tablets, were analyzed under optimized chromatographic conditions. Then, these solutions were kept at ambient conditions for 12 hours and tested again under the same chromatographic conditions by recording their peak responses. Finally, the calculated % RSD values of standard and sample solutions are expressed in Table 9. It was observed that the % RSD values vary within the specified limits for both daclatasvir 30 and 60 mg tablets. Therefore, the solution reagent stability test has been found acceptable because the cumulative RSD values for both standard and sample solutions are less than 2%.

Table 9

Solution stability data for 30 and 60 mg daclatasvir tablets.

Parameter	30 and 60 mg daclatasvir tablets
	Standard	30 mg tablets	60 mg tablets
% RSD (initial)	0.05	0.57	0.46
% RSD (after 12 hrs)	0.90	1.24	0.79

The findings of the present study were evaluated with those reported in the literature using chromatographic manner [19]. There was no discernible difference between the findings of the suggested approach and the stated HPLC method, according to statistical analysis of the acquired values done using Student’s t-test (0.46) and variance F-test (1.81) [19].

3.2. Greenness of the Method Developed

In a range of settings, including government policy, industrial management, educational practise, and technological development, the ideas of green chemistry are increasingly extensively employed. A brand-new system for assessing greenness, the Analytical Greenness Metric (AGREE), is based on the 12 green analytical chemistry principles. AGREE is a thorough, adaptable, and simple evaluation approach that yields an understandable and instructive result. The factors that are taken into consideration in AGREE are drawn from the 12 GAC principles and converted into a common 0–1 scale. The availability of freeware software available at (git.pg.edu.pl/p174235/AGREE), which simplifies the applications for this metric, is one of its benefits. The final assessment result is the total of the scores for each of the 12 input variables, which are translated into scores on a 0–1 scale [18, 29–31]. The end result is a clock-like circle with the overall score and colour image in the centre, as shown in Figure 2 [21, 32, 33]. According to findings given in Figure 10, the AGREE score is 0.62. Based on the findings, our proposed approach is found to be greener and is thus recommended for the analysis of daclatasvir dihydrogen chloride in pharmaceutical products.

[figure(s) omitted; refer to PDF]

4. Conclusion

The antiviral compound daclatasvir dihydrochloride has been tested in both drug substance and drug product (tablet dose) forms using a straightforward, dependable, affordable, and quick RP-HPLC approach. The developed method has been found to be linear, selective, precise, accurate, robust, and reproducible, as per ICH guidelines. The acquired findings were found to be well within the predetermined limits after statistical analysis of all the parameters using standard deviation and RSD revealed little variance. The developed RP-HPLC method has been found more applicable compared to other analytical methods reported in the literature [8, 31]. Therefore, this RP-HPLC technique can be applied for qualitative and quantitative evaluation of daclatasvir dihydrochloride in pharmaceutical laboratories for routine quality control measurements in a very short time interval. In addition, the results of the greenness evaluation of the proposed method showcase an exceptional commitment to environmental sustainability and a promotion of a greener future.

Authors’ Contributions

The manuscript was written with the contributions of all authors. All authors have approved the final version of the manuscript.

Acknowledgments

The authors are thankful to the Deanship of Scientific Research at the University of Bisha for supporting this work through the Fast-Track Research Support Program. This research was funded by the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R134), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. The authors extend their appreciation to the Research Center at AlMaarefa University for funding this work.

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