DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR ESTIMATION OF DARUNAVIR

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR ESTIMATION OF DARUNAVIR

Anuruddha R. Chabukswar ¹ and Ayushi S. Gadekar ^{* 2}

Department of Pharmaceutical Chemistry ¹, Dr. Vishwanath Karad MIT World Peace University, School of Pharmacy, Kothrud, Pune - 411038, Maharashtra, India.

Department of Pharmaceutical Chemistry ², MAEERS Maharashtra Institute of Pharmacy, Kothrud, Pune -411038, Maharashtra, India.

ABSTRACT: A simple, precise, and sensitive stability-indicating high-performance thin layer chromatographic (HPTLC) method has been developed and validated for the analysis of Darunavir in bulk and in the tablet dosage form. The separation was performed on pre-coated silica gel 60 GF₂₅₄ plates using Toluene: Methanol: Triethylamine (8.5:1:0.5 v/v/v) as the mobile phase. The retention factor (R_f) was found to be 0.61 ± 0.89. The detection of a band was carried out at 267 nm. The drug was subjected to different stress conditions like acid, base hydrolysis, oxidation, thermal degradation, and photolysis. The method was successfully validated according to ICH Q₂ (R1) guidelines. The linear regression analysis data for the calibration plot showed a good linear relationship with R² = 0.992 in the range of 500-3000 ng band^-1. The method found to be accurate as results of the recovery studies are close to 100%. The developed method can be adopted for routine analysis of Darunavir in bulk and pharmaceutical dosage form.

High-performance thin-layer chromatography (HPTLC), Darunavir, method validation, ICH guidelines,  stability-indicating method

INTRODUCTION: Darunavir is chemically [(3aS,4R,6aR)- 2,3,3a,4,5,6a-hexahydrofuro[2,3-b] furan-4-yl] N- [(2S,3R)-4- [(4-aminophenyl) sulfo-nyl-(2-methylpropyl)amino]- 3- hydroxy- 1- phenyl butan-2-yl] carbamate it is HIV protease inhibitor that is used in the treatment of AIDS and HIV infections. Darunavir is an antiretroviral protease inhibitor that is used in the therapy and prevention of human immunodeficiency virus (HIV) infection and the acquired immunodeficiency syndrome (AIDS). Darunavir can cause transient and usually asymptomatic elevations in serum aminotransferase levels and has been linked to rare instances of clinically apparent, acute liver injury.

In HBV or HCV co-infected patients, highly active antiretroviral therapy with darunavir may result of an exacerbation of the underlying chronic hepatitis B or C. Literature survey reveals that few analytical methods have been reported for the estimation of Darunavir including UV-Vis spectroscopy ^{1, 2}, high-performance liquid chromatography (HPLC) ^{3, 4}, high-performance thin-layer chromatography (HPTLC) ^{5, 6}, Infrared Spectroscopy (IR) ⁷, Capillary Electrophoresis ⁸, LC-MS/MS ^{9, 10}.

FIG. 1: STRUCTURE OF DARUNAVIR

MATERIALS AND METHODS:

Reagents and Chemicals: 20 tablets each containing 300 mg of Darunavir (Darunavir, Cipla Ltd.) was procured from the local market. Methanol (AR grade), Triethylamine (AR grade), toluene (AR grade) were purchased from S.D. Fine Chemical Laboratories, Mumbai. Hydrochloric acid (HCl), acetic acid (CH₃COOH), hydrogen peroxide (H₂O₂), and sodium hydroxide (NaOH); all AR grade were purchased from Loba Chemie Pvt. Ltd., Mumbai.

Chromatographic Conditions: Chromatographic separation of the drug was performed on aluminum plates precoated with silica gel 60 F₂₅₄ (10 cm × 10 cm with 250 μm layer thickness). The sample was applied on the plate as a band of 5 mm width using Camag 100 μl sample syringe (Hamilton, Switzerland) with a linomat 5 applicator (Camag, Switzerland). The mobile phase was composed of Toluene: Methanol: Triehylamine (8.5:1:0.5 v/v/v). 10 cm × 10 cm Camagtwin trough glass chamber was used for linear ascending development of TLC plate under 15 min saturation conditions and 10 ml of mobile phase was used per run.

Migration distance was 80 mm. Densitometric scanning was carried out using Camag TLC scanner at 267 nm, operated by win CATS software (version 1.4.3), slit dimensions were 4.00 × 0.45 mm and Deuterium lamp was used as a radiation source.

Preparation of Standard Stock Solution: The standard stock solution of Darunavir was prepared by dissolving 10 mg of drug in 10 ml of methanol to get a concentration of 1000 µg/ml. From the standard stock solution, the working standard solution was prepared to contain 100 µg/ml of Darunavir.

Selection of Analytical Wavelength: From the standard stock solution further dilutions were made using methanol and scanned over the range of 200 – 800 nm and the spectra were obtained. It was observed that the drug showed considerable absorbance at 267 nm Fig. 2.

Selection of Mobile Phase and Chromatographic Conditions: Chromatographic separation studies were carried out on the working standard solution of Darunavir 100 µg/ml. Initially, trials were carried out using various solvents in various proportions on normal TLC plates to obtain the desired R_f and shape for drug peak. After a few trials, Toluene: Methanol: Triethylamine (8.5:1:0.5 v/v/v) was chosen as the mobile phase, which gave acceptable peak parameters.

Preparation of Sample Solution: 20 tablets each containing 300 mg of Darunavir (Darunavir, Cipla Ltd.) was weighed and powdered. Powder equivalent to 10 mg of drug was transferred to 10 ml volumetric flask and volume was made up with acetonitrile to get concentration (1000 µg/ml) and was sonicated for 10 min. Solution was filtered, from this solution 1 ml of drug was taken in 10 ml volumetric flask and volume was made up with acetonitrile. Further dilution in acetonitrile was done to get concentration 10 µg/ml.

Densitogram and System Suitability Parameter of Drug: Solution of Darunavir (100 µg/ml) was prepared. 10 µl (1000 ng/band) of solution was applied on pre-activated TLC plate with the help of Hamilton syringe (100 μl), using Linomat 5 sample applicator. The development chamber was saturated with the mobile phase for 15 min. The spotted plate was placed in the saturated chamber and developed up to 80 mm distance. The plate was dried and was scanned over 90 mm distance at 267 nm. The retention factor was found to be: 0.61 ± 0.89 Fig. 3, Table 1.

Stress Degradation Studies of Bulk Drug: Stress degradation studies were carried under the condition of acid as well base hydrolysis, oxidation, and dry heat. Dry heat and photolytic degradation were carried out in solid-state.

Preparation of Standard Stock Solution: Accurately weighed 25 mg of Darunavir was transferred to the 5 ml pre-calibrated volumetric flask. Darunavir was dissolved in small quantity of water. Volume was made up to 5 ml with water to achieve a stock solution of 5000 μg/ml (Stock-1).

Preparation of Sample:

Degradation under Acid-Catalyzed Hydrolytic Condition: To 1 ml of 5000 µg/ml solution of Darunavir, 1 ml of 0.5 N HCl was added. The above solution was kept for 4 h at room temperature. After exposure the solution was neutralized with 0.5 N NaOH and volume was made up to 10 ml with methanol 0.5 µl of the resultant solution was then applied at TLC plate and densitogram was developed. 19.77% Darunavir was recovered with no peak of degradant Fig. 4.

Degradation under Alkali Catalyzed Hydrolytic Condition: To 1 ml of 5000 µg/ml solution of Darunavir, 1 ml of 0.5 N NaOH was added. The above solution was kept for 4 h at room temperature. After exposure, the solution was neutralized with 0.5 N HCl and the volume was made up to 10 ml with methanol. 5 µl of the resultant solution was then applied at the TLC plate and densitogram was developed. Average 16.08% of Darunavir was recovered with no peak of degradation Fig. 5.

Degradation under Oxidative Condition: To 1 ml of 5000 µg/ml solution of Darunavir, 1 ml of 10 % H₂O₂ was added. The above solution was kept for 1 h at room temperature. The volume was made up to 10 ml with methanol. 5µl of the resultant solution was then applied at the TLC plate and densitogram was developed. Average 25.44% of Darunavir was recovered with no peak of degradant Fig. 6.

Degradation under Dry Heat: Dry heat studies were performed by keeping drug sample in the oven (80 °C) for a period of 4 h. The sample was withdrawn, dissolved in methanol and diluted to get 500 μg/ml. 5 µl of the resultant solution was then applied at TLC plate and densitogram was developed. Average 76.33% Darunavir was recovered with no peak of degradant Fig. 7.

Photo-Degradation Studies: The photo degradation stability study of the drug was studied by exposing the drug to UV light providing illumination of NLT 200 watt hr/m². After exposure accurately weighed 10 mg of drug was transferred to 10 ml of the volumetric flask; the volume was made up with methanol to obtain 1000 µg/ml solution. 5 ml of the resultant solution was then diluted with methanol to get a concentration of 500 µg/ml. 5µl of the resultant solution was then applied at the TLC plate, and densitogram was developed. Average 50.38% of Darunavir was recovered with no peak of degradant Fig. 8.

Validation of Analytical Method: ¹¹ The method was validated as per ICH Q2 (R1) guidelines.

Specificity: The specificity of the method was ascertained by peak purity profiling studies. The peak purity values were found to be more than 0.998, indicating the non-interference of any other peak of degradation product or impurity.

Linearity: From the standard stock solution (1000µg/ml) of Darunavir, a solution was prepared to contain 100µg/ml. This solution was further used for spotting. Six replicates per concentration were spotted. The linearity (relationship between peak area and concentration) was determined by analyzing six concentrations over the concentration range of 500-3000 ng/band.

The results obtained are shown in Table 2, the peak areas were plotted against the corresponding concentrations to obtain the calibration curve as shown in Fig. 9.

Range: Darunavir = 500-3000 ng/band

Precision: The precision of the method was demonstrated by intra-day and inter-day variation studies. In the Intra-day studies, 3 replicates of 3 concentrations were analyzed on the same day, and percentage RSD was calculated. For the inter-day variation studies, 3 replicates of 3 concentrations were analyzed on 3 consecutive days and percentage RSD was calculated. For intraday precision and inter-day precision results obtained are shown in Tables 3 and 4.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

LOD and LOQ are calculated from the formula: -

LOD = 3.3 σ / S

LOQ = 10 σ / S

Where, σ = standard deviation of Y intercept, S   = slope of the calibration curve Table 5.

Assay: Darunavir tablet formulation analysis was carried out as mentioned under section preparation of sample solution. Procedure was repeated for six times. 2 μl volume of sample solution was applied and area was recorded.

Concentration and % recovery was determined from linear equation. The results obtained are shown in Table 6.

Accuracy: To check the accuracy of the method, recovery studies were carried by spiking the standard drug to the blend, at three different levels 50, 100 and 150%. The basic concentration of the sample chosen was 1000 ng/band.

% recovery was determined from the linearity equation. The results obtained are shown in Table 7.

Robustness: Robustness of the method was determined by carrying out the analysis under conditions during which detection wavelength, chamber saturation time were altered, time was also changed from spotting to development and development to scanning and the effects on the area were noted.

It was found that the method is robust. The results obtained are shown in Table 8.

Summary of Validation Study: The summary of validation parameters are summarized in Table 9.

RESULTS AND DISCUSSION:

Selection of Analytical Wavelength: Detection wavelength was found to be 267 nm.

FIG. 2: UV SPECTRUM OF DARUNAVIR (10 µg/ml)

Typical Densitogram of Darunavir:

FIG. 3: A TYPICAL DENSITOGRAM OF STANDARD SOLUTION OF DARUNAVIR (1000 ng/band)

TABLE 1: SYSTEM SUITABILITY PARAMETERS FOR DARUNAVIR

Stress Degradation Studies of Bulk Drug:

Degradation under Acid-Catalyzed Hydrolytic Condition:

FIG. 4: REPRESENTATIVE DENSITOGRAM OF ACID INDUCED DEGRADATION OF DARUNAVIR (2500 ng/band)

Degradation under Alkali Catalyzed Hydrolytic Condition:

FIG. 5: REPRESENTATIVE DENSITOGRAM OF BASED INDUCED DEGRADATION OF DARUNAVIR (2500 ng/band)

Degradation under Oxidative Condition:

FIG. 6: REPRESENTATIVE DENSITOGRAM OF OXIDATIVE DEGRADATION OF DARUNAVIR (2500 ng/band)

Degradation under Dry Heat:

FIG. 7: REPRESENTATIVE DENSITOGRAM OF DRY HEAT DEGRADATION OF DARUNAVIR (2500ng/band)

Photo-Degradation Studies:

FIG. 8: REPRESENTATIVE DENSITOGRAM OF DARUNAVIR AFTER PHOTOLYSIS (2500 ng/band)

Validation of Analytical Method: Linearity:

TABLE 2: LINEARITY STUDY OF DARUNAVIR

FIG. 9: CALIBRATION CURVE FOR DARUNAVIR

Range: Darunavir = 500-3000 ng/band

Precision:

TABLE 3: INTRA-DAY PRECISION OF DARUNAVIR

TABLE 4: INTER-DAY PRECISION OF DARUNAVIR

Limit of Detection (LOD) and Limit of Quantification (LOQ):

TABLE 5: LOD AND LOQ OF DARUNAVIR

Assay:

TABLE 6: ASSAY OF MARKETED FORMULATION

Accuracy:

TABLE 7: RECOVERY STUDIES OF DARUNAVIR

Robustness:

TABLE 8: ROBUSTNESS STUDY

Summary of Validation Study:

TABLE 9: SUMMARY OF VALIDATION PARAMETERS

CONCLUSION: An accurate, rapid, specific and stability-indicating HPTLC method was developed for the estimation of Darunavir Ethanoate in the bulk and pharmaceutical dosage form.

In the developed method, no interferences were observed from blank or excipients at Darunavir peak retention. The primary advantage of this method is that it is simple and accurate with a shorter run time.

Hence, the proposed method can be conveniently adopted for the routine quality control analysis in bulk and formulation. The developed method was validated according to ICH guidelines.

ACKNOWLEDGEMENT: The authors are thankful to Cipla Ltd., Kurkumbh for providing the gift sample of Darunavir for studies.

CONFLICTS OF INTEREST: Nil

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    How to cite this article:

    Chabukswar AR and Gadekar AS: Development and validation of stability indicating HPTLC method for estimation of darunavir. Int J Pharm Sci & Res 2020; 11(7): 3326-32. doi: 10.13040/IJPSR.0975-8232.11(7).3326-32.

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