RP-HPLC Method Development and Validation for the Quantification of a Selected Active Pharmaceutical Ingredient in Bulk and Tablets

The field of pharmaceutical analysis continues to evolve in response to the increasing complexity of drug formulations and the stringent regulatory requirements governing their quality, safety, and efficacy.^1-4 Reliable analytical methods are indispensable for ensuring that pharmaceutical products meet the desired specifications throughout their lifecycle—from raw material testing to finished product evaluation. Among the diverse analytical techniques available, High-Performance Liquid Chromatography (HPLC) has established itself as a benchmark in both qualitative and quantitative drug analysis. Its superior sensitivity, selectivity, reproducibility, and capability to separate structurally related compounds make it the most widely adopted method in pharmaceutical research and quality control laboratories.

Within this analytical framework, Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) has emerged as the most commonly used chromatographic technique due to its versatility and compatibility with a broad range of analytes.^5-8 Unlike normal-phase chromatography, RP-HPLC employs a non-polar stationary phase and a polar mobile phase, facilitating the effective separation of polar, semi-polar, and non-polar molecules. The technique is particularly advantageous for analyzing complex pharmaceutical matrices since it enables the simultaneous separation of the active pharmaceutical ingredient (API), excipients, degradation products, and impurities. Additionally, the relatively simple sample preparation, excellent reproducibility, and adaptability to routine analysis have further contributed to its extensive application in the pharmaceutical industry.^9-12

The present study focuses on Adefovir, an important nucleotide analog antiviral drug used primarily for the management of chronic Hepatitis B Virus (HBV) infection. Adefovir exerts its therapeutic action by inhibiting viral DNA polymerase, thereby suppressing viral replication and disease progression. Clinically, Adefovir is administered in the form of its prodrug, Adefovir Dipivoxil, which enhances oral bioavailability and systemic absorption. Despite its clinical value, Adefovir possesses a narrow therapeutic index and is known to cause dose-dependent nephrotoxicity at higher concentrations. Therefore, the accurate quantification of Adefovir in both its bulk form and finished dosage formulations is critical for maintaining therapeutic efficacy, ensuring patient safety, and complying with pharmacopoeial standards.^13-20

Fig. 1: Structure of Adefovir dipivoxil

However, the analytical quantification of Adefovir poses significant challenges. Its hydrophilic nature, limited UV absorbance, and structural similarity to endogenous nucleotides make its detection and separation demanding. These challenges necessitate the development of a highly specific and optimized chromatographic method capable of resolving Adefovir from excipients, impurities, and possible degradation products. RP-HPLC, with its flexibility in adjusting chromatographic parameters such as mobile phase composition, pH, column selection, flow rate, and detection wavelength, provides an ideal platform to overcome these analytical difficulties. Proper optimization of these parameters ensures sharp, symmetrical peaks, enhanced resolution, and precise quantification of the analyte.^21-23

In addition to method development, the validation of the analytical procedure is an integral part of pharmaceutical quality assurance. As per the International Council for Harmonisation (ICH) guidelines Q2(R1), method validation ensures that the developed analytical technique is suitable for its intended purpose and produces reliable and reproducible results. The major parameters evaluated during method validation include system suitability, linearity, accuracy, precision, specificity, robustness, limit of detection (LOD), and limit of quantitation (LOQ). Each of these parameters provides evidence of the analytical method’s capability to consistently deliver accurate results under varied conditions.^24-25

The objective of the current research is to develop a simple, rapid, precise, and cost-effective RP-HPLC method for the quantification of Adefovir in its bulk form and tablet dosage formulation. The developed method aims to offer enhanced reproducibility and robustness while being suitable for high-throughput analysis in pharmaceutical quality control laboratories. Comprehensive validation will be carried out in accordance with ICH guidelines to confirm the reliability and applicability of the method for routine use. The successful establishment of such a validated method is expected to provide a significant contribution to the field of pharmaceutical analysis, ensuring the consistent quality of Adefovir-containing formulations and supporting regulatory compliance in both industrial and clinical settings.^26-28

MATERIALS AND METHODS:

MATERIALS:

The active pharmaceutical ingredient (API), Adefovir Dipivoxil, utilized in the present research work, was kindly provided as a gift sample by Lupin Pharmaceuticals Ltd., Pune, India. A commercially available tablet formulation, Hepsera® (10 mg), manufactured by Dr. Reddy’s Laboratories Ltd., Hyderabad, India, was selected for the purpose of method validation and assay studies.

All solvents and reagents employed throughout the experimental work were of analytical and HPLC grade to ensure accuracy and reproducibility of the results. These included acetonitrile, methanol, ortho-phosphoric acid, and HPLC-grade water, which were procured from Fisher Scientific Pvt. Ltd., Mumbai, India. All other chemicals and reagents used were of the highest purity and were utilized without further purification.

METHODOLOGY:

Identification and Characterization of Drug^30-32

Before initiating the analytical work, it was essential to confirm the identity and purity of the selected active pharmaceutical ingredient to ensure its suitability for method development. A series of preliminary identification and characterization tests were therefore performed to verify the chemical integrity and physicochemical consistency of the drug. These studies provided essential insights into the drug’s solubility behavior, thermal properties, and spectral characteristics, all of which were critical for the development of a precise and reliable RP-HPLC method.

Selection and Procurement of Drug^33-35

In the present research, Adefovir Dipivoxil (ADF) was selected as the model compound for method development and validation due to its therapeutic relevance and analytical challenges. The pure drug sample was kindly gifted by Lupin Pharmaceuticals Ltd., Pune, India, while the marketed tablet formulation Hepsera® (10 mg), manufactured by Dr. Reddy’s Laboratories Ltd., Hyderabad, India, was procured from a local pharmacy.

Upon receipt, the drug sample underwent preliminary physical evaluation to verify its authenticity. Characteristics such as color, odor, and melting point were examined and compared with standard reference data to confirm its identity and compliance with pharmacopeial specifications.

Physicochemical Characterization^36-38

Comprehensive physicochemical characterization was undertaken to gather information regarding the solubility, stability, and structural features of Adefovir Dipivoxil. Analytical techniques such as UV–Visible spectroscopy, FT-IR spectroscopy, and melting point determination were employed to support method optimization and establish method selectivity.

Solubility Studies^39-42

The solubility of Adefovir Dipivoxil was evaluated in various solvents, including methanol, distilled water, and phosphate buffer solutions, to identify a suitable medium for complete dissolution. These studies assisted in selecting an appropriate solvent system compatible with the chromatographic conditions. Methanol was found to provide excellent solubility and compatibility with the mobile phase, ensuring consistent and reproducible chromatographic responses.

Melting Point Determination⁴³

The melting point of Adefovir Dipivoxil was determined using the capillary tube method. A small quantity of the drug was placed in a capillary tube sealed at one end and inserted into a digital melting point apparatus. The temperature at which the drug began to liquefy was recorded. The observed melting point corresponded closely with literature values, confirming the identity and purity of the sample.

Fourier Transform Infrared (FT-IR) Spectroscopy⁴⁴

FT-IR analysis was performed to identify the functional groups and confirm the molecular structure of Adefovir Dipivoxil. The infrared absorption spectrum was recorded using a Shimadzu FTIR-8400S spectrophotometer over a wavelength range of 4000–400 cm?¹. The obtained spectrum displayed characteristic absorption bands corresponding to key functional groups such as ester, carbonyl, hydroxyl, and amine moieties, validating the structural integrity of the compound.

UV–Visible Spectroscopy^45-46

The UV absorption spectrum of Adefovir Dipivoxil was obtained using a Shimadzu UV-1800 spectrophotometer equipped with 1 cm quartz cells. Scanning was carried out in the range of 200–400 nm to determine the maximum absorbance wavelength (λmax) of the drug. The λmax was subsequently used as the detection wavelength in the RP-HPLC method to ensure optimal sensitivity and accurate quantification.

Selection of Mobile Phase and Standard Preparation^47-52

Preparation of Standard Solution

An accurately weighed quantity of 5 mg of Adefovir Dipivoxil was transferred into a 25 mL volumetric flask and dissolved in methanol to prepare a stock solution with a concentration of 200 µg/mL. A 1 mL aliquot of this stock solution was further diluted to 20 mL with methanol to obtain a working solution of 10 µg/mL, which was used for subsequent chromatographic analysis.

Optimization of Mobile Phase

To achieve optimal resolution, sharp peak shape, and reproducibility, several mobile phase compositions were systematically evaluated. Combinations of methanol with water and methanol with phosphate buffer at different pH levels (3.5–5.5) were tested. Among the various systems studied, a mobile phase comprising Methanol: Phosphate Buffer (70:30, v/v) adjusted to pH 3.5 produced the best chromatographic performance, yielding well-resolved and symmetrical peaks for Adefovir Dipivoxil with minimal tailing.

Chromatographic Conditions

Chromatographic separation was achieved on a C18 Inertsil column (250 mm × 4.6 mm, 5 µm particle size) under isocratic conditions. The optimized mobile phase consisted of Methanol: Phosphate Buffer (70:30, v/v) at pH 3.5, delivered at a flow rate of 1.0 mL/min. The detection wavelength was set at 262 nm, and the injection volume was 20 µL. All analyses were conducted at ambient temperature. These optimized parameters ensured a stable baseline, high resolution, and reproducible retention times suitable for routine quantification of Adefovir Dipivoxil.

Preparation of Calibration Curve^53-55

To establish the linearity and working concentration range, a stock solution of Adefovir Dipivoxil (100 µg/mL) was prepared by dissolving 10 mg of the drug in methanol and diluting to 100 mL with the same solvent. Aliquots of this solution were diluted with the mobile phase to obtain standard concentrations ranging from 2 to 20 µg/mL. Each concentration was injected into the HPLC system, and the corresponding peak areas were recorded. A calibration curve was plotted between concentration and peak area, exhibiting a linear relationship that confirmed the method’s suitability for quantitative analysis.

System Suitability Testing^56-60

Prior to analysis, system suitability testing (SST) was performed to ensure that the chromatographic system was functioning effectively and provided consistent results. A standard solution of Adefovir Dipivoxil (10 µg/mL) was prepared in the mobile phase and injected five times consecutively. Critical parameters such as retention time, theoretical plate count, tailing factor, and peak symmetry were evaluated. The results were within acceptable limits, confirming the system’s efficiency, reproducibility, and readiness for routine analysis.

Application of the Proposed Method to Laboratory and Marketed Samples^61-65

Analysis of Laboratory-Prepared Samples

To evaluate the practical applicability of the developed RP-HPLC method, laboratory-prepared samples containing Adefovir Dipivoxil (ADF) were analyzed under the optimized chromatographic conditions. A laboratory mixture equivalent to 10 mg of ADF was accurately weighed, dissolved in 100 mL of the mobile phase, and thoroughly mixed using a mechanical shaker. The resulting solution was further diluted to obtain a working concentration of 10 µg/mL.

In order to simulate potential formulation variations, five different laboratory-prepared mixtures were analyzed. The chromatographic peak areas of both standard and sample solutions were recorded, and the concentration of ADF in each sample was determined by comparing the peak area of the sample with that of the standard. The percentage of drug estimated in each sample was calculated using the following equation:

% Estimation = (At / As) × (Ds / Dt) × (Ws / Wt) × 100

Where:
At = Area of sample,

As = Area of standard,

Ds = Dilution factor of standard,

Dt = Dilution factor of sample,

Ws = Weight of standard,

Wt = Weight of sample.

The results confirmed that the developed method accurately quantified Adefovir Dipivoxil in laboratory-prepared mixtures, demonstrating its reliability and suitability for analytical applications.

Application to Marketed Formulation^66-68

The validated RP-HPLC method was further applied to determine the ADF content in a commercially available tablet formulation (Hepsera® 10 mg). Twenty tablets were accurately weighed, finely powdered, and the average tablet weight was determined. A portion of powder equivalent to 10 mg of Adefovir Dipivoxil was transferred to a 100 mL volumetric flask, dissolved in 50 mL of mobile phase, and sonicated for 10 minutes to ensure complete extraction. The volume was then made up to 100 mL with the mobile phase, followed by filtration through a 0.45 μm membrane filter.

The filtrate was appropriately diluted to obtain a 10 µg/mL working solution. Equal volumes (20 µL) of both standard and sample solutions were injected into the HPLC system. The percentage of Adefovir Dipivoxil in the marketed formulation was determined by comparing the peak areas of the sample and standard, confirming the method’s applicability for routine quality control of pharmaceutical dosage forms.

Validation of the Developed Method^69-73

The developed RP-HPLC method was validated in accordance with the International Council for Harmonisation (ICH) Q2(R1) guidelines to confirm its suitability for the intended purpose. Parameters including accuracy, precision, ruggedness, specificity, linearity, robustness, LOD, and LOQ were systematically evaluated.

Accuracy (Recovery Studies)

Accuracy was determined by the standard addition method. Known quantities of standard Adefovir Dipivoxil were added to pre-analyzed tablet samples at three concentration levels (80%, 100%, and 120%). The mixtures were diluted appropriately with the mobile phase and filtered before analysis. The recovery percentage was calculated based on the difference between the total and original drug content. The results showed recovery values within 98–102%, indicating the high accuracy and reliability of the developed method.

Precision

The precision of the method was assessed through intra-day and inter-day studies. For intra-day precision, three replicate analyses were performed at different time intervals within a single day, while for inter-day precision, the procedure was repeated over three consecutive days. The relative standard deviation (RSD) values were within acceptable limits (≤2%), confirming that the method is precise and reproducible under normal operating conditions.

Ruggedness

Ruggedness was evaluated by analyzing the same sample under varying conditions, including different analysts, days, and instruments. The analysis was carried out by two independent analysts using the same procedure on different days. The consistency of the results demonstrated that minor variations in experimental conditions did not significantly affect the analytical performance, confirming the rugged nature of the developed method.

Specificity

Specificity was established to ensure that the chromatographic peak corresponding to Adefovir Dipivoxil was free from interference by formulation excipients, impurities, or degradation products. The retention time of the drug in standard and sample chromatograms was identical, and no co-eluting or overlapping peaks were observed. These findings confirmed that the method was highly specific for the detection and quantification of Adefovir Dipivoxil.

Linearity and Range

Linearity was assessed over the concentration range of 2–20 µg/mL, covering 80–120% of the expected working concentration. Standard solutions were prepared at different concentrations within this range, and peak areas were plotted against concentration. The method exhibited excellent linearity, with a correlation coefficient (R²) greater than 0.999, indicating a strong linear relationship between peak area and analyte concentration.

Robustness

Robustness was determined by introducing small deliberate variations in analytical parameters, such as mobile phase composition (±2%), flow rate (±0.1 mL/min), and detection wavelength (±2 nm). These minor changes did not significantly affect the retention time, theoretical plate count, or peak symmetry. Thus, the developed method demonstrated robustness and reliability, even under slightly altered chromatographic conditions.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

The LOD and LOQ were determined to establish the sensitivity of the method. LOD represents the lowest detectable concentration, while LOQ is the lowest concentration that can be quantified with acceptable precision and accuracy. These values ensure that the method is capable of detecting and quantifying low levels of Adefovir Dipivoxil in pharmaceutical samples.

Forced Degradation Studies^62-72

Acid-Induced Degradation

To assess acid stability, the drug was treated with 0.5 N hydrochloric acid and heated at 60°C for one hour. The solution was then neutralized and diluted to obtain a 10 µg/mL concentration and injected into the HPLC system. The degradation was quantified by comparing peak areas with those of untreated samples.

Alkali-Induced Degradation

For alkaline degradation, the drug was treated with 1 N sodium hydroxide under the same conditions. After heating and neutralization, the solution was diluted to the required concentration and analyzed. The percent degradation was calculated based on the decrease in peak area.

Oxidative (Peroxide) Degradation

The drug was exposed to oxidative stress using 30% hydrogen peroxide and heated at 60°C for one hour. After appropriate dilution, the solution was analyzed by HPLC to evaluate degradation under oxidative conditions.

Thermal Degradation

Solid and liquid forms of the drug were subjected to thermal stress by placing them in a hot air oven at 100°C for 24 hours. The degraded sample was then diluted and analyzed using the developed HPLC method to determine thermal stability.

Photodegradation

Photostability was assessed by exposing solid drug samples to sunlight for one full day and also to UV light for three days. Solutions prepared at concentrations of 30 and 50 µg/mL were analyzed for degradation. Chromatograms were evaluated for changes in retention time and peak shape to assess the impact of light exposure.

RESULTS AND DISCUSSIONS:

Physicochemical Characterization of Adefovir Dipivoxil

Adefovir Dipivoxil (ADF) is a nucleotide analog antiviral agent that is commercially available as a fixed-dose oral tablet formulation. The active pharmaceutical ingredient used in this study was obtained from a certified source, with a reported purity of 99.7%, which was accepted as the reference standard for all analytical evaluations. Independent purity verification was not performed, as the supplied certificate of analysis met pharmacopoeial specifications.

Physicochemical characterization is a critical preliminary step in ensuring the identity, purity, and suitability of the drug for subsequent analytical method development and validation. In the present study, the solubility behavior, melting point, and UV absorption characteristics of Adefovir Dipivoxil were systematically evaluated.

The melting point of Adefovir Dipivoxil was determined using the open capillary method. A precisely weighed amount of the sample was packed into a capillary tube and heated in a calibrated melting point apparatus until complete liquefaction was observed. The melting point was recorded at 101 ± 1°C, which aligns well with both literature data and manufacturer specifications, confirming the thermal stability and purity of the compound.

The drug exhibited good solubility in polar solvents such as methanol, ethanol, and water, while it was sparingly soluble in non-polar solvents, consistent with its amphiphilic chemical structure. Furthermore, UV spectrophotometric analysis revealed a characteristic absorption maximum (λmax) at approximately 259 nm, which was in close agreement with reported data, thereby validating the spectral identity of ADF.

Overall, the physicochemical characterization confirmed that the obtained Adefovir Dipivoxil sample was of high purity and consistent quality, suitable for use in the development and validation of the proposed HPLC analytical method.

Table 1: Physicochemical Characterization of Adefovir Dipivoxil

FT-IR analysis:

a) FT-IR of Adefovir Dipivoxil:

The IR absorbance spectrum of Adefovir Dipivoxil was recorded using FTIR 8400S spectrometer (Shimadzu) over range of 4000 to 400 cm-1.

Fig. 2: FT-IR Spectra of Adefovir Dipivoxil (ADF)

The IR spectroscopy theory utilizes the concept that molecules tend to absorb specific frequencies of light that are characteristic of the corresponding structure of the molecules. The energies are reliant on the shape of the molecular surfaces, the associated vibronic coupling, and the mass corresponding to the atoms.

UV Spectroscopy Analysis

The ultraviolet absorption spectrum of ADF was obtained using Shimadzu1800- UV visible spectrophotometer and 1cm quartz cells, over a wavelength range of 400 to 200 nm. The wavelength maxima (λ max) were analyzed showed in table no. 2.

Table 2: Drug wavelength maxima (λ max)

Fig. 3:  UV Spectra of Adefovir Dipivoxil (ADF)

Selection of mobile phase:

Each mobile phase was filtered through Whatman filter paper No. 42. Peak, well resolved peaks with symmetry within limits and significant Based on sample solubility & stability, various mobile phase compositions were evaluated to achieve acceptable separation using selected chromatographic conditions. The mobile phases tried are as follows:

Table 3: List of mobile phase tried

From various mobile phases tried, mobile phase containing Methanol: Phosphate Buffer (70:30) pH 3.5 was selected, since it gives sharp reproducible retention time for ADF.

Fig. 4: Trial Chromatogram obtained by using Methanol: Water (90:10) as mobile phase.

Fig.  5: Trial Chromatogram obtained by using Methanol: water (80:20) as mobile phase.

Fig. 6: Trial Chromatogram obtained by using Methanol: Phosphate Buffer (80:20) pH 5 as mobile phase.

Fig. 7: Final Chromatogram obtained by using Methanol: Phosphate Buffer (70:30) pH 3.5 as mobile phase of ADF.

Fig. 8: Blank Chromatogram obtained by using Methanol: Phosphate Buffer (70:30) pH 3.5 as mobile phase of ADF.

The following chromatographic conditions were established by trial and error and were kept constant throughout method.

Column   : Inertsil 4.6 (id) x 250 mm

Particle size packing : 5 ?m

Stationary phases      : C18 Inertsil

Mobile phase : Methanol: Phosphate Buffer (70:30) pH 3.5

Detection wavelength           : 262 nm

Flow rate : 1 ml/min.

Temperature : Ambient

Sample size    : 20 ML

Preparation of calibration curve:

The mobile phase was allowed to equilibrate with the stationary phase until steady baseline was obtained. The series of concentration from 2-20 mcg/ml for ADF drug solutions was injected and peak area was recorded. The graph plotted as the concentration of the drug Vs peak area depicted in following figure.

Table 4: Observation of standard curve of ADF

Fig. 9: Standard calibration curve for ADF

System suitability test:

System suitability is a pharmacopoeial requirement and is used to verify, whether the resolution and reproducibility of the chromatographic system are adequate for analysis to be done. The tests were performed by collecting data from five replicate injections of standard solutions.

Application of proposed method for estimation of ADF Laboratory Sample:

The standard and Sample solution of ADF was prepared and inject. The peak area of standard and sample laboratory was compared to obtain the concentration.

Application of proposed method for estimation of ADF in formulation:

Equal volume (20?L) of standard and sample solution were injected separately after equilibrium of stationary phase. The chromatograms were recorded and the response i.e. peak area of major peaks were measured. The content ADF was calculated by comparing a sample peak with that of standard.

Table 6: Results and statistical data for estimation of ADF in marketed formulation.

Brand name:  Adesera Avarg wt.: 110 mg

*Results are mean of three replicates

Validation Parameters

The developed RP-HPLC method for the estimation of Adefovir Dipivoxil (ADF) was validated in accordance with ICH Q2(R1) guidelines for analytical method validation. The parameters evaluated included accuracy, precision, and ruggedness, and the results demonstrated the reliability and reproducibility of the proposed method.

1. Accuracy (Recovery Studies)

Accuracy was determined through recovery studies using the standard addition method at three concentration levels (80%, 100%, and 120%). Pre-analyzed samples were spiked with known quantities of standard ADF, and the percentage recovery was calculated. The results are summarized in Table 7.

The mean percentage recovery of ADF was found to be 99.90% ± 0.36, with a %RSD value below 2%, confirming the high accuracy and reliability of the method. The close agreement between the theoretical and recovered concentrations indicates that the excipients present in the formulation did not interfere with the assay.

2. Precision

Precision of the method was assessed in terms of repeatability (intra-day precision) by performing replicate estimations of ADF in the marketed formulation (Adesera 10 mg). The statistical data are summarized in Table 8.

The mean assay value for precision was 100.93% ± 0.67 with an RSD of 0.007, which is within the acceptable limit (<2%), indicating excellent method precision and repeatability.

3. Ruggedness

Ruggedness of the developed method was established by performing the assay under variable conditions different days (inter-day) and different analysts. The inter-day precision results are presented in Table 9.

Table 9: Results and Statistical Data for Interday Precision Study (Brand: Adesera)

The mean percentage label claim was 100.10% ± 0.44 with a %RSD of 0.004, confirming that the developed RP-HPLC method is rugged, reproducible, and unaffected by small variations in analytical conditions or operators.

Table 10: Summary of Validation Parameters

4. Linearity and Range

Linearity was evaluated by preparing standard solutions of Adefovir Dipivoxil in the concentration range of 2–20 µg/mL. Each concentration was injected in triplicate, and a calibration curve was constructed by plotting peak area versus concentration. The regression equation and correlation coefficient (R²) were calculated to assess linearity.

Table 11: Linearity Data for Adefovir Dipivoxil

Regression Equation: ( y = 27628x + 328.9 )

Correlation Coefficient (R²): 0.9996

The correlation coefficient close to 1.0 confirms an excellent linear relationship between peak area and concentration across the tested range. The low %RSD (<1%) demonstrates high repeatability and consistent detector response, confirming method linearity and suitability for quantitative analysis.

5. Specificity

Specificity was evaluated by comparing chromatograms of the standard, sample, and blank (mobile phase). No interference was observed at the retention time of Adefovir Dipivoxil (~4.72 min).

Table 12: Specificity Results for Adefovir Dipivoxil

The results indicate that the method is highly specific, as no interfering peaks were observed at the retention time of ADF, confirming the method’s ability to selectively estimate ADF in the presence of excipients and other components.

6. Limit of Detection (LOD) and Limit of Quantitation (LOQ)

LOD and LOQ were calculated based on the standard deviation of the response (σ) and the slope (S) of the calibration curve.

Table 13: LOD and LOQ for Adefovir Dipivoxil

The low values of LOD (0.15 µg/mL) and LOQ (0.45 µg/mL) indicate the high sensitivity of the developed RP-HPLC method, allowing for accurate detection and quantification of even small quantities of ADF in pharmaceutical preparations.

7. Robustness

Robustness was determined by making small, deliberate variations in chromatographic conditions, including flow rate (±0.1 mL/min), mobile phase composition (±2%), and detection wavelength (±2 nm). The results are presented in Table 14.

Table 14: Robustness Study for Adefovir Dipivoxil

The chromatographic parameters remained consistent with minor changes in experimental conditions, showing %RSD < 1% in all cases. This confirms the robustness and stability of the method under small deliberate variations, making it suitable for routine analysis.

Table 15: Summary of Method Validation Results

The developed RP-HPLC method for Adefovir Dipivoxil was found to be simple, precise, accurate, linear, specific, robust, and sensitive. All validation parameters complied with ICH Q2(R1) requirements, establishing the method’s suitability for routine quality control analysis of ADF in both bulk and marketed formulations.

Intraday:

It was performed by using same procedure as under marketed formulation analysis and absorbance recorded at 3.0 hrs. Interval within a day.

Forced degradation study

Forced degradation studies were carried out to evaluate the stability-indicating capability of the developed RP-HPLC method for Adefovir Dipivoxil (ADF). The drug was subjected to various stress conditions including acid, alkaline, oxidative, thermal, and photolytic degradation. These studies help in determining the drug’s intrinsic stability and identifying possible degradation pathways.

The chromatograms obtained under each condition were evaluated for peak purity, area reduction, and percentage degradation to confirm that the method could distinctly resolve ADF from its degradation products. The purity flag “No” in each case indicates that the peak of ADF was spectrally pure and no co-eluting impurities were detected.

1. Acid Stress Degradation

Adefovir Dipivoxil was subjected to acidic hydrolysis using 0.5 N HCl and kept at 60°C for one hour. The sample was neutralized and analyzed under optimized chromatographic conditions.

Table 16: Acid-Stressed Degradation of ADF

The drug showed approximately 8.1% degradation under acidic conditions, suggesting moderate susceptibility to acid hydrolysis. No additional impurity peaks were observed, confirming method specificity.

2. Base Stress Degradation

Alkaline degradation was induced by treating ADF with 0.5 N NaOH and maintaining the solution at 60°C for one hour before neutralization and analysis.

Table 17: Alkali-Stressed Degradation of ADF

Under alkaline conditions, the drug underwent mild degradation (4.5%), indicating relative stability in basic media compared to acidic conditions.

3. Oxidative (Peroxide) Degradation

Oxidative degradation was performed using 30% hydrogen peroxide and heating the solution at 60°C for one hour.

Table 18: Peroxide-Stressed Degradation of ADF

ADF was found to be highly stable under oxidative conditions, exhibiting only 1.1% degradation, which suggests limited oxidation sensitivity.

4. Thermal Stress Degradation

The thermal degradation study was carried out by keeping the drug sample at 100°C for 24 hours in a hot air oven before analysis.

Table 19: Thermal-Stressed Degradation of ADF

ADF showed minimal degradation (0.7%) under elevated temperature, confirming its thermal stability.

5. Photodegradation Study

Photostability testing was performed by exposing ADF samples (30 and 50 µg/mL) to UV light and direct sunlight for 24 hours.

Table 20: Photo-Stressed Degradation of ADF

Exposure to UV and visible light caused minor degradation (1.5%), confirming that Adefovir Dipivoxil is photostable under ordinary laboratory conditions.

6. Summary of Forced Degradation Studies

The forced degradation experiments collectively demonstrated that Adefovir Dipivoxil exhibits good stability under most stress conditions, with acidic medium causing the highest degradation (8.1%). All degradation samples showed purity angles lower than purity thresholds, and no purity flag, confirming the stability-indicating nature of the developed RP-HPLC method.