INTRODUCTION:

Darunavir, often known as Prezista¹, is a synthetic nonpeptidic protease inhibitor that is effective against wild-type HIV.² It is a crucial component of HAART, one of the biggest developments in HIV treatment.³ There must be variety in the antiretroviral therapy plans used to treat HIV/AIDS⁴. According to the OARAC, Prezista may be preferred by adolescents and those who have never had treatment.⁵ The pharmaceutical business Tibotec developed it. In 2006, the FDA (Food and Drug Administration) approved darunavir.⁶ It is chemically [(1S,2R)-3-[[(4aminophenyl) sulfonyl] (2-methyl propyl) amino]-2-hydroxy-1-(phenylmethyl) propyl] carbamic(3R,3aS,6aR) hexahydrofuro[2,3-b] furan-3-ylester monoethanolamine (Fig 1)⁷

Figure-1: Structure of Darunavir

A survey of the literature indicates that there are many methods documented for the development of analytical methods and validation of darunavir. Therefore, the development of cleaning methods and the validation of the Darunavir API incorporated into oral films will be the primary focus of the present study.^8-11

MATERIALS AND METHODS:

Instruments:

The UV/Vis spectrophotometers used were a UV1800s model from Shimadzu and UV3200 from lab India, Weighing balance, Stainless steel plate, Swab sticks, Hot air oven, UV chamber, Orbital shaker, FTIR

Chemicals and reagents:

The purest version of darunavir was bought from Dr. Reddy’s laboratories in Hyderabad, India.

Selection of solvent:

To determine the best solvent for dissolving the drug, multiple trials were conducted. 0.1NHcl, 0.1NNaoH, Water, ACN, and Methanol were among the solvents that were tested by the drug's solubility. In ACN, it was found that the drug's maximal absorption was 263nm. ACN was therefore chosen as the solvent.

Darunavir Standard Stock Solution:

Weighing approximately 50mg of the pure drug, the drug was transferred to a 50ml volumetric flask creating the darunavir stock solution. To increase the drug solution’s concentration to the required 1000µg/ml, acetonitrile was added.

Darunavir's working standard solution:

1ml of the stock solution is pipetted into a volumetric flask with a capacity of 10ml. The reactor’s capacity was increased with ACN to bring the concentration to 100µg/ml. Multiple dilutions were prepared by using the 100µg/ml concentration solution.

Darunavir test solution:

The synthetic mixture was prepared by 300mg Darunavir, 25mg Polyvinyl pyrrolidone, 5mg Magnesium stearate, and 5mg Talc, A quantity of 10mg of powder was added to a volumetric flask. The drug solution was filtered before being mixed with ACN to make up the volume.

Determination of absorbance maxima (λ max):

A 10μg/ml concentration was prepared by pipetting 1ml of the working standard solution above into a 10ml volumetric flask and adding acetonitrile to bring the volume up to the required level. After that, the sample was scanned in the 200–400 nm range using acetonitrile as a blank in a UV Spectrophotometer. It was found that 263 nm was the wavelength corresponding to maximal absorbance. (Figure 2)

Figure 2: Spectra of Darunavir

Oral Film

The required quantity of Polymer HPMC E15 was taken and dissolved in the solvent ethanol in a beaker and kept aside for soaking for ½ an hour. For 15 minutes, the mixture is stirred to achieve a uniform consistency. Within the time intervals of 10-15 mins the other excipients mentioned in table- 1 were added in order. The solution is kept for stirring on a magnetic stirrer for 1-2hrs. To remove air bubbles the solution is kept aside for a few minutes. Once the mixture is well distributed throughout the petri dish, it can be dried in a hot air oven or at room temperature for 24 hours.

Table 1: Formulation of Oral Film

S. No	Ingredients	Formulation
1	Darunavir	50(mg)
2	HPMC	400(mg)
3	Mannitol	25(mg)
4	Propylene glycol	0.15(ml)
5	Citric acid	5(mg)
6	Ethanol	10(ml)

Figure-3: Oral film

Cleaning method:

The cleaning process is performed by the swab method, which is known as direct surface sampling. It is a primarily used method that involves the usage of a probe. It is performed methodically on the surface area to remove any residue, a 1ml volume of 100µg/ml is pipetted and added on a stainless-steel plate and evaporated to dryness. Swabs are used to swab the sample first horizontally and then vertically. The swabs were put in a 10 ml volumetric flask with the solvent that had been sonicated, and acetonitrile was used to bring the volume up to the required level. The absorbance of the solution was measured using a UV Spectrometer

Figure 4: Steps involved in the swabbing method

METHOD VALIDATION:

The method was validated in terms of linearity, precision, accuracy, robustness, and ruggedness.

Linearity

There were several dilutions prepared, ranging from 2 to 16µg/ml. At 263nm, the absorbance values were taken. Plotting the linearity curve against concentration vs absorbance was done. The value of the correlation coefficient (R²) data in (Table 02 and Fig 05).

Precision:

Studies conducted throughout the day proved it. Six times a day, the identical concentration of the intraday solution was examined, and the % RSD was determined and mentioned in (Table 03).

Accuracy:

In this test, it included three replicates of each percentage at the concentration levels of 50%, 100%, and 150%.0.2ml, 0.4ml, and 0.6ml of darunavir were pipetted at respective concentrations of 2,4,6µg/ml onto a stainless-steel plate with a predefined 10cm²surface area and left to dry. The swabs were soaked in acetonitrile. The entire surface of the plates was cleaned, and swabs were put into 10ml volumetric flasks containing 5 ml of solvent; the volumetric flasks were then sonicated for 15 minutes and diluent was used to bring the volume up to the required level,4 µg/ml of test solution was used for spiking. The absorbance of the solution was measured using a UV-vis spectrophotometer. % Recovery mentioned (Table 04).

Robustness:

This technique was established by analyzing data at two distinct wavelengths (263+/-1) and noting the absorbance. The results were expressed as a percentage of RSD in (Table 05).

Ruggedness:

Two separate analyzers completed the analysis and recorded the absorbance, the method's results were expressed as a percentage of relative standard deviation (%RSD) in (Table 06).

Assay of Oral film:

The oral film was dissolved in the required amount of ACN, the absorbance of the solution was measured at 263nm using UV spectroscopy., and according to beers Lamberts law the concentration was determined to be 3.27µg/ml.

Absorbance of sample Concentration of the standard

%Assay= –––––––––––––––––––– × ––––––––––––––––––––––––– × 100 = (0.236/0.288) × (4/3.27) ×100 =99.9%

Absorbance of standard Concentration of sample

Forced Degradation Studies:

Stress testing, commonly referred to as forced degradation tests, involves exposing drug substances and drug products under a variety of stressful situations to measure the amount and rate of degradation that occurs during storage and after the drugs are administered to the body. Acid, base, oxidative, and thermal degradations are the several types of degradation studies (Table 07).

Acid Degradation:

6 ml of 10 ppm drug solution and 10 ml of 0.1N HCl were taken into the 50 ml volumetric flask and kept in an orbital shaker for 6 or 7 hours. 10 ml of 0.1N NaOH is used to neutralize the solution, and diluent is used to bring the volume up to the required level.

Base Degradation:

6 ml of 10 ppm drug solution and 10 ml of 0.1N NaOH were taken into the 50 ml volumetric flask and kept in an orbital shaker for 6 or 7 hours. 10 ml of 0.1N HCl is used to neutralize the solution, and diluent is used to bring the volume up to the required level.

Oxidative Degradation:

6 ml of 10 ppm drug solution and 10 ml of 3%H₂O₂ were taken into the 50 ml volumetric flask and kept in an orbital shaker for 6 or 7 hours, and the diluent was used to bring the volume up to the required level.

Thermal Degradation:

Thermal degradation was performed in a hot air oven. For this study, 50mg of darunavir was taken in a glass petri dish and placed in an oven for 6 hours. After the specified time, the product was cooled and transferred into a 50ml volumetric flask, and the diluent was used to bring the volume up to the required level.

UV Degradation:

For the UV degradation study, 50mg of darunavir was placed in a glass petri dish and left in the UV chamber for six hours at a wavelength of 256nm. Once the stress was over, the product was cooled and transferred into a 50ml volumetric flask, and the diluent was used to bring the volume up to the required level.

RESULTS:

Table-2. Linearity study for darunavir

S. No	Concentration	Absorbance
1	2	0.2
2	4	0.288
3	6	0.377
4	8	0.449
5	10	0.522
6	12	0.603
7	14	0.679
8	16	0.773

Figure 5: Linearity curve of Darunavir

Table- 3. Precision study for Darunavir

S. No	Absorbance
1	0.548
2	0.549
3	0.549
4	0.550
5	0.538
6	0.553
AVERAGE	0.547833
SD	0.005115
%RSD	0.93374

Table-4. Accuracy study for Darunavir

Concentration	Drug Sample	Drug Product	% Recovery	Mean Recovery	% RSD
50%	2ppm	4ppm	99.3	99.8	0.569954
50%	2ppm	4ppm	99.6
50%	2ppm	4ppm	100.4
100%	4ppm	4ppm	100.6	100.7	0.30449
100%	4ppm	4ppm	100.0
100%	4ppm	4ppm	100.4
150%	6ppm	4ppm	99.5	99.4	0.353072
150%	6ppm	4ppm	99.8
150%	6ppm	4ppm	99.1

Table-5. Robustness study for Darunavir

S. No	Absorbance at 264nm	Absorbance at 262nm
1	0.565	0.547
2	0.564	0.548
3	0.565	0.548
4	0.565	0.548
5	0.565	0.522
6	0.566	0.549
Average	0.565	0.548
SD	0.000632	0.010
%RSD	0.11	1.95

Table-6. Ruggedness study for Darunavir

Instrument name- Shimadzu			Instrument name- Lab India
Day-1			Day-2
Analyst-1		Analyst-2	Analyst-3	Analyst-4
Concentration	Absorbance	Absorbance	Absorbance	Absorbance
10PPM	0.523	0.520	0.401	0.395
10PPM	0.522	0.522	0.404	0.394
10PPM	0.521	0.518	0.403	0.393
10PPM	0.519	0.515	0.405	0.399
10PPM	0.520	0.518	0.402	0.396
10PPM	0.522	0.521	0.403	0.395
Average	0.5211	0.519	0.403	0.3953
SD	0.0014	0.0025	0.0014	0.0020
%RSD	0.28	0.48	0.35	0.52

Table 7: Forced degradation studies

Nature of stress	% Degradation of API	% Degradation of Synthetic Mixture
Acid degradation	88.12	85.6
Base degradation	90.6	91.5
Oxidative degradation	91.9	86.3
Thermal degradation	93.1	92.3
Light degradation	92.5	94.2

Figure-6: Stress studies

FTIR Studies

Figure 7 shows the FTIR spectra of pure Darunavir, Oral film, and Synthetic mixture. The characteristics of OH,N-H, O=C=O, C=C=C, and C=O stretching’s of the Darunavir were found at 3361 cm^-1,2909 cm^-1, 2312 cm^-1, 1947 cm^-1, 1701 cm^-1.

Figure-7: Over lay FTIR spectra of Darunavir, Oral film, Synthetic Mixture

DISCUSSION:

In this investigation, the concentration of remaining residual chemicals is measured using a linear correlation coefficient of 0.999. To identify residues on the stainless-steel plate, a swab sample methodology was applied. An absorbance maximum of 263nm for darunavir was used for this study. The standard curve between 2 and 16 µg/ml was found to be linear, with an outstanding R² value of 0.998(Table 2 & Fig 05). It was found that the precision's %RSD was less than 2(Table 3). Recovery studies were used to examine the method's accuracy; the findings were reported as a percentage of recovery between 98 to 100% (Table 04). By altering the wavelength, using a different analyst, and analyzing the samples on different days, the robustness and ruggedness were assessed (Table 05& Table 06). The analysis remained unchanged after the study's adjustments. Therefore, it was discovered that the darunavir approach was reliable. By ICH requirements, the forced degradation studies were carried out in a variety of conditions (Table 06). 99.9% was the assay percentage.

As Per ICH requirements, the designed and verified Ultraviolet spectrophotometric approach was found to be quick, easy to use, accurate, and economical. It was established that every parameter fell inside the permissible range. The suggested technique for estimating darunavir residue can be used in industries such as manufacturing. Thus, regular analysis can effectively use this proven technique.

ACKNOWLEDGEMENT:

I would like to thank Sarojini Naidu Vanitha Pharmacy Maha Vidyalaya for continuous support, and Dr. Reddy’s for the gift sample.

CONFLICT OF INTEREST:

The author declares is no conflict of interest.

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Received on 14.03.2024 Modified on 29.03.2024

Research J. Science and Tech. 2024; 16(2):107-114.

DOI: 10.52711/2349-2988.2024.00016