INTRODUCTION:

For bulk drugs and their formulation to remain of excellent quality and integrity, quantitative analysis is most worthy. The quantification and validation of the process utilizing the development of analytical instruments is an important stage in the method development process. Our research team is focuses on the development and validation of bulk drugs and formulations^1-3.

Doravirine is a non-nucleotide reverse transcriptase inhibitor specifically authorized for treating HIV-1 infection⁴.

It works by noncompetitively binding to the virus, thereby preventing the conversion of RNA to DNA and inhibiting viral replication, and is metabolized via the CYP3A pathway⁵. Doravirine does not inhibit human DNA polymerases α, ß, or γ mitochondrial DNA polymerase. In vitro studies show that doravirine is effective against NNRT inhibitor-resistant strains with a single daily dose^6-7. The drug's bioavailability is not affected by its combination with food or other fixed-dose drugs. Chemically, doravirine is 3-chloro-5-({1-[4-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]-2-oxo-4-(trifluoromethyl)-1,2-dihydropyridine-3-yl}oxy)benzonitrile, with a formula of C₁₇H₁₁ClF₃N₅O₃ and a molecular weight of 425.7 g/mol.

Figure 1: Structure of Doravirine

Few methods have been reported for the spectrophotometric and chromatographic separation of doravirine, whether alone or in combination with other drugs^8-14. This study aimed to develop a simple, highly sensitive, accurate, and reproducible analytical method for quantifying doravirine in both its pure form and in tablet formulations. This method was validated according to the International Conference on Harmonization (ICH) Q2(R1) guidelines^15-16.

MATERIAL AND METHODS:

Pure sample:

Doravirine was procured from Mylan Laboratory Hyderabad as a gift sample.

Formulation:

Tablets of doravirine 100 mg were prepared in the laboratory with commonly available excipients.

Table 1: Formulation for Preparation of Laboratory Tablet of Doravirine

Sr No.	Ingredients	Quantity (mg)
1.	Doravirine	100
2.	Microcrystalline cellulose	80
3.	Lactose	20
4.	Talcum powder	2.25
5.	Magnesium stearate	5.75
6.	Starch	42

Chemical and reagents:

Acetonitrile (ACN), water (H₂O), and methanol (HPLC Grade) were all used of pure AR grade purchased from the SRL lab.

Instrumentation:

HPLC system used is of Shimadzu model no. DGU-A5R, A digital analytical balance Shimadzu ATX224 was used for weighing. An ultrasonic bath PCi analytics was used to carry out the assay.

Preparation of standard solution:

Sample preparation for the HPLC method's development to estimate doravirine

Standard stock solution (A1) (1000µg/ml):

To create a 1000µg/ml solution, 10mg of pure Doravirine was accurately weighed and transferred into a clean, dry 10ml volumetric flask. The Doravirine was dissolved in a small amount of methanol and sonicated briefly. The solution was then diluted with methanol up to the 10ml mark on the flask.

Standard stock solution (A2) (100µg/ml):

A volume of 1.0ml volumetric flask, and then the flask was filled to the mark with methanol to achieve a concentration of 100µg/ml.

Standard working solution (A3) (10µg/ml):

A volume of 1.0ml from the solution (A2) was transferred into a 10ml volumetric flask, and then the flask was filled to the mark with methanol to obtain a final concentration of 10µg/ml.

Preparation of mobile phase:

The mobile phase was created by combining acetonitrile and water in a proportion of 60:40%v/v.

Preparation of sample stock solution for HPLC Assay method:

Doravirine tablets were prepared in the laboratory using the necessary excipients, each containing 100mg of Doravirine, and their average weight was determined. The tablets were then crushed into a fine powder. A portion of the tablet powder equivalent to 10mg of Doravirine was accurately weighed and transferred into a 10ml volumetric flask. The powder was dissolved in methanol, and the solution was sonicated for 10minutes. Methanol was added to bring the final volume to 10ml, resulting in a concentration of 1000µg/ml. The solution was filtered through Whatman filter paper no. 41. Finally, appropriate dilutions were performed to obtain a final concentration of 10µg/ml.

Validation of Proposed Method:

System suitability parameters:

As a part of the validation process, system suitability was assessed for each validation parameter by conducting five consecutive injections of the drug standard solution (10µg/ml). Parameters such as tailing factor and plate count were observed and recorded.

Linearity and Range:

The method’s linearity was evaluated using linear regression analysis with the least squares method by preparing standard Doravirine solutions at various concentrations. Linearity indicates the method’s capacity to produce results directly proportional to the concentration of the analyte. For HPLC analysis, different volumes (2, 4, 6, 8, and 10ml) of the standard stock solution were transferred into separate 10ml volumetric flasks, which were then filled with methanol to achieve concentrations of 20, 40, 50, 80, and 100 µg/ml. The areas and retention times of these solutions were measured at a wavelength of 324nm against a blank. Similarly, concentrations from 20 to 100µg/ml were prepared from the working standard solution and automatically injected under optimized chromatographic conditions.

Accuracy:

Accuracy refers to the evaluation of the disparity between the obtained value and the actual value. To accuracy, standard Doravirine solutions (0.8, 1.0, 1.2 µg/ml) were added to the sample solution to achieve measurement levels of 80%, 100%, and 120%. The percentage recoveries were then computed.

Precision:

Precision measures how closely a series of measurements from multiple samples of the same substance match under set conditions. The method's precision was evaluated by conducting studies on variations both between different days (inter-day) and within the same day (intra-day). Five sample solutions were prepared, and the percentage relative standard deviation (% RSD) was calculated.

Intraday and Inter day variation:

Using the HPLC method, five standard solutions of Doravrine with a concentration of 20µg/ml were injected into the HPLC system at different time points: immediately (0hr), after 6hrs, after 1 day, and after 2 days. Subsequently, the chromatograms were recorded.

Ruggedness:

The ruggedness of the proposed method was assessed to investigate the impact of factories unrelated to the procedure, such as variations in instruments and analysts. In this study, Doravirine was analyzed using the proposed methods by two different analysts, both adhering to the same operational and environmental conditions.

Robustness:

Robustness indicates a method’s capacity to remain unaffected by minor, deliberate changes in parameters. In this analysis, robustness was tested by slightly adjusting the detection wavelength to 323nm and 325 nm (from the actual 324nm), modifying the mobile phase composition, and varying the flow rate. The mobile phase flow rate was altered to 0.8mL/min and 1.5mL/min, while the actual flow rate was 1.0mL/min. Additionally, the effect of changing the mobile phase composition to ACN: Water ratios of 65:35 and 55:45 was assessed.

Sensitivity:

The limit of detection (LOD) denotes the smallest amount of analyte in a sample that can be detected. On the other hand, the limit of quantification (LOQ) represents the minimum amount of analyte that can be accurately measured. These values were calculated using the following formulas:

LOD = 3.3 σ/S

LOQ = 10 σ/S

where σ is the standard deviation of the analyte's response, and S is the slope from the linearity plot of the analyte.

RESULTS AND DISCUSSION:

Table 2: Optimized chromatographic parameters

Column	Shim pack C18 (4.6mm x 250mm x 5µm)
Mobile phase	Acetonitrile: water (60:40)% v/v
Flow rate	1 ml/min
Wavelength	324nm
Injection volume	10µl
Column oven temperature	30°C
Retention time	4.920 min
Total run time	10 min

A 10µl volume of the solution was automatically injected into the HPLC system, and the resulting chromatogram was recorded. The mobile phase, consisting of a 60:40% v/v mixture of acetonitrile (ACN) and water (H2O), produced distinct and well-separated peaks with an acceptable retention time.

Figure 2: Chromatogram of Doravirine

Figure 3: 3D Image of Doravirine

After equilibrating the column with the mobile phase, a 1.0ml aliquot from the working standard solution was diluted to 10ml to achieve a concentration of about 10 µg/ml. This solution was auto-injected five times, and the chromatogram was recorded, with the peak areas measured afterward. The results for the system suitability parameters peak area tailing factor and theoretical plates are shown in Table 3 below:

Table 3: Observation of system suitability parameters

Sr no.	Weight of Std. drug taken (mg)	Peak area (mV)	Tailing factor	Theoretical plates
1.	10.0	11518	1.141	5349
2.		11517	1.142	5342
3.		11520	1.141	5346
4.		11519	1.140	5345
5.		11520	1.142	5344
%RSD		0.33	0.42	0.63
Limit		NMT 2%	NMT 2	NTL 2000

Linearity and range:

The HPLC method demonstrated linearity within the concentration range of 20 to 100µg/ml. the regression equation derived from this analysis was y = 2691.41x – 11498.1, with a correlation coefficient value of 0.9997. The results for the Linearity and range parameter are shown in Table 4.

Table 4: Calibration curve HPLC method

Conc. (µg/ml)	Area(mV)
20	41535
40	99065
60	148757
80	200734
100	259841
R²	0.99957

Figure 4: Calibration curve of Doravirine

Accuracy:

The percentage recovery of doravirine at various spiked levels (80%, 100%, and 120%) ranged from 98.50% to 101.6%. The %RSD was below 2, as detailed in Table 5, indicating no interference from the excipients in the formulation. Therefore, the method was determined to be accurate.

Table 5: Results of accuracy (Recovery) studies

Conc. (%)	Tablet (µg/ml)	Pure drug (µg/ml)	Total amount (µg/ml)	Actual amount found (µg/ml)	Recovery %	Mean recovery %
80	10	8	18	17.86	98.81	98.96
80	10	8	18	17.86	98.82
80	10	8	18	17.85	98.32
100	10	10	20	19.88	99.08
100	10	10	20	19.87	99.05
100	10	10	20	19.89	99.04
120	10	12	22	22.0	100
120	10	12	22	22.1	101
120	10	12	22	21.9	99.69

Precision:

The precision of the analytical method was assessed by conducting multiple samplings of a uniform sample. The results for both intraday analyses are presented in Table 6. It is noteworthy that the %RSD for Doravirine in the standard solution was below 2% signifying a high level of precision in the proposed method.

Table 6: Intra–day and Inter-day results

Concentration (10 µg/ml)	Intra-day study (n=6)		Inter – day study (n=6)
Concentration (10 µg/ml)	0 hour	6 hours	After Day 1	After Day 2
*Mean area	41530	41511	4189	41256
Standard deviation	6263.2	6290.4	6271.9	6245.1
%RSD	0.81	0.87	0.89	0.85

n= number of measurements, % RSD= percentage relative standard deviation,*Mean of five observation

Ruggedness:

The method’s ruggedness was assessed by applying it to the same procedure with different analyses, and two distinct HPLC instruments were employed for this ruggedness study. The %RSD fell within the specified limits, affecting the robustness of the methods. The outcomes of this study can be found in Table 7.

Table 7: Ruggedness study result (Analysis performed by different analysts)

Conc. (10µg/ml)	Analyst 1	Analyst 2
*Mean area	41520	41540
Standard deviation	6253.2	6243.2
%RSD	0.84	0.81

%RSD= Percentage Relative standard deviation,

*Mean of Five observation

Robustness:

To assess the robustness of the HPLC method, intentional minor adjustments were made to the chromatographic conditions. These adjustments included variations in the acetonitrile and water percentages in the mobile phase (±5%) and changes in the flow rate (±0.2ml/min). The results are shown in Table 8. The % RSD stayed below 2%, which strongly demonstrates the method's robustness.

Table 8: Robustness study for HPLC

Parameters	*Mean Peak Area (mV)	%RSD
ACN: Water (55:45)	11518	0.86
ACN: water (65:35)	11506	0.95
Flow rate (0.8ml/min)	11515	0.99
Flow rate (1.2ml/min)	11517	0.98

Sensitivity:

Table 9 reported the LOD and LOQ values that were calculated using the given equations.

Table 9: Sensitivity

LOD (µg/ml)	0.71
LOQ(µg/ml)	0.12

Assay of Doravirine by HPLC method:

A standard working solution of doravirine (10µg/ml) and a sample solution (10µg/ml) were auto-injected into the HPLC system. The doravirine content in the laboratory-prepared tablet was measured using HPLC. The results for the % labeled claim obtained from HPLC are presented in Table 10. The percentage purity of the doravirine formulation was determined to be 98.56% by HPLC, indicating that the assay value of doravirine complies with ICH guidelines.

Table 10: Result of analysis of tablet formulation

Methods

Sample

Label claim

(100mg/tablet)

% label claim estimated (n=6)

% RSD

HPLC

laboratory prepared tablet

100

98.56

0.89

n= number of measurements; %RSD = percentage relative standard deviation

CONCLUSION:

Based on the analytical results, the method demonstrated accuracy, precision, and sensitivity. It requires minimal solvent use and allows for quick adjustments to experimental conditions. This method is highly effective for the routine analysis of doravirine in both bulk and tablet forms and is not influenced by common excipients in pharmaceutical preparations. Validated according to ICH guidelines, it covers Accuracy, Precision, Specificity, Linearity, LOD, LOQ, Robustness, and system suitability. Thus, this RP-HPLC method is well-suited for quality control analysis and routine assessment of doravirine.

ACKNOWLEDGMENT:

The authors are grateful to Mylan Labs. Ltd. (India) for providing the gift samples of doravirine and also thankful to colleague Pranita Kanojiya for helping in the preparation of tablets in the laboratory.

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Received on 03.04.2024 Revised on 13.08.2024

Accepted on 16.10.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1486-1490.

DOI: 10.52711/0974-360X.2025.00213

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