1. INTRODUCTION:

Raltegravir Potassium, [N-[(4-fluorophenyl)methyl]-5-hydroxy-1-methyl-2-{2-[(5-methyl-1,3,4-adiazol-2-yl)formamido]propan-2-yl}-6-oxo-1,6-dihydropyrimidine-4-carboxamide, is involves integrase which is an enzyme necessary for the HIV virus to successfully insert its viral DNA into a human host’s DNA. The virus must be able to carry out this process in order to use the host’s cellular machinery to make copies of its viral DNA in order to successfully spread the HIV infection. Integrase inhibitors, like Raltegravir, block the action of integrase and prevent the HIV virus from successfully inserting its DNA into the host DNA.^1,2

Raltegravir is absorbed with a T max of approximately 3 hours post dose in the fasted state. It is approximately 83% bound to human plasma protein over the concentration range of 2 to 10 μM. The apparent terminal half-life of Raltegravir is approximately 9 hours, with a shorter α-phase half-life (~1 hour) accounting for much of the AUC.^3,4

2. EXPERIMENTAL:

2.1 Instrumentation:

HPLC was carried out using instrument. Densitometric scanning was done using integrating software. Melting points were recorded using Melting Point Apparatus.

2.2 Chemicals and Materials:

Raltegravir Potassium (RAL) was taken for the experiment. The experiments were performed with all analytical-grade reagents.

Hydrochloric acid and sodium hydroxide were prepared in distill water for forced degradation studies. Hydrogen Peroxide (6 %v/v) was used for oxidative study.

2.3 Chromatographic Conditions:

The samples were spotted in the form of bands of width 6 mm with a microlitre syringe on aluminum plate pre-coated with silica gel 60. The mobile phase consisted of Toluene: ethylacetate: methanol: acetic acid (8:1:1:0.1v/v/v/v). The plates were prewashed with methanol and activated at 60^◦C for 15 min prior to chromatography. Samples were applied at 5 mm intervals under a stream of nitrogen. The slit dimensions were 5×0.45 mm. Linear ascending chromatogram development, to a distance of 8 cm was performed in twin trough TLC developing chamber at room temperature (25 ± 2˚C) without saturation with a mobile phase. The detection wavelength of 299 nm was selected for analysis.⁵

2.4 Forced Degradation study of RAL: Stressed Sample Preparation:

Stressed samples were chromatographed along with the non-stressed standard sample.

2.4.1 Hydrolytic Degradation: Acid, Base and Neutral Hydrolysis:

RAL Solutions containing (10mgmL⁻¹) were prepared in 1N HCl, 1NNaOHand water. These were subjected to conditions mentioned in Table 1 and analyzed by TLC for appearance of additional spots. For HPLC, 1 mL of the samples were withdrawn in 10 mL volumetric flasks after specified duration of time, neutralized with respective acid or base and were diluted to the mark with methanol. A fixed volume (5μL) was spotted on TLC plates and the plates were developed and analyzed.⁶

2.4.2 Oxidative Degradation: Hydrogen Peroxide Induced:

Solution of RAL (10 mgmL⁻¹) was prepared in water containing 6 % v/v of H₂O₂, treated in the dark under the conditions shown in Table 1 and periodically analyzed for the appearance of additional spots by TLC. For HPLC analyses, 1.0 mL aliquots of the stressed samples were transferred to 10mL volumetric flasks and diluted to the mark with methanol. A fixed volume (5μL) was spotted on TLC plates and the plates were developed and analyzed.

2.4.3 Thermal Degradation: Dry Heat Induced:

The powdered RAL was spread in a flat-bottomed glass tube to give a homogeneous layer and was subjected to the conditions indicated in Table 1. After the specified time duration, accurately weighed powder (10mg) from the blend was transferred into 10mL volumetric flask and diluted with methanol upto the mark. A fixed volume (5μL) was spotted on TLC plates and the plates were developed and analyzed.⁷

2.4.4 Photolytic Degradation:

Solution of RAL (10 mgmL⁻¹) were prepared in methanol in quartz vessel and exposed to forced irradiation (at 15 cm from source) in a 40cm×30cm×30cm chamber fitted with UV lamp (8W). For chromatographic analysis, 1 mL of the sample was withdrawn at an interval of 12 hours and diluted to10 mL with methanol. A fixed volume (5μL) was spotted on TLC plates and the plates were developed and analyzed.

2.5 Preparation of Solutions:

2.5.1 Preparation of RAL stock solution: Accurately weighed RAL (10mg) was transferred into10 mL volumetric flask, dissolved and diluted with water up to the mark (1mg/mL).

2.5.2 Preparation of RAL working standard solution: RAL stock solution (0.5mL) was transferred into 10 mL volumetric flask and the flask was diluted with methanol upto the mark (50µg/mL RAL).⁸

2.6 Validation of developed HPLC method:

2.6.1 Linearity:

Linearity was established on the basis of trial and error method. The linearity of response for RAL was assessed in the range of 200–700 ng/spot.

2.6.2 Precision:

Method precision: The intraday precision of the proposed method was determined by estimating the corresponding responses three times on the same day for three different concentrations of RAL (200, 500, 700 ng/spot). The results are reported in terms of percentage relative standard deviation (%RSD).

Intermediate precision: The interday precision of the proposed method was determined by estimating the corresponding responses on three different days over a period of 1 week for three different concentrations^[9] of RAL (200, 500,700 ng/spot). The results are reported in terms of %RSD.

2.6.3 Repeatability:

Repeatability of measurement of peak area:

Working standard solution of RAL i.e. 12μL was spotted on pre-coated TLC plate. The plate was then developed, dried and analyzed as described previously. The area of spot was measured six times without changing the position of plate and %RSD of obtained data was calculated.

Repeatability of sample application:

Working standard solution of RAL i.e. 12μL was spotted on pre-coated TLC plate six times. The plate was developed, dried, sprayed and analyzed as described. The areas of all six spots were measured and %RSD was calculated.

2.6.4 Limits of detection and quantification:

The limits of detection (LOD) and quantitation (LOQ) of the drugs were calculated using the following equations as per the ICH guidelines.

LOD=3.3×σ/SLOQ=10 ×σ/S

Where σ is the standard deviation of the response, and S is the standard deviation of y-intercept of regression lines.¹⁰

2.6.5 Specificity:

The specificity of the method was ascertained by analyzing standard drug and sample. The spot for RAL in the sample was confirmed by comparing the RAL and spectra of the spot with those of standard. The peak purity of the sample was assessed by comparing the spectra at peak start, peak apex and peak end positions of the spot.

2.7 Degradation Kinetic Study of RAL:

In Alkaline Condition (1N sodium hydroxide):

Accurately weighed RAL (50mg) was transferred in 25mL volumetric flask, dissolved and diluted with 0.5N Sodium hydroxide upto the mark. The volumetric flask was maintained at specified temperature in temperature controlled water bath. Sample (1mL) was transferred into a series of 10mL volumetric flask at (0, 15, 30, 45, 60, 90, 120, 150 and 180 min) time intervals. Each sample was neutralized with 0.5N hydrochloric acid and was diluted upto the mark with methanol. Samples were stored in freezer till analyzed. At the time of analysis, samples were allowed to attain room temperature and 5µL of each was spotted on pre-coated TLC plate along with standard RAL. The plates were developed and analyzed as stated.

The study was carried out in triplicate in three sets viz. 60, 70 and 80˚C temperature.

3. RESULTS AND DISCUSSION:

3.1 Stress testing of RAL:

Stress testing provides evidence on how the quality of a drug may be affected under the influence of different stress conditions. Drug decomposition may result in loss of potency and advent of possible adverse effects due to the formation of degradation products. The tests were performed on 10 mg mL⁻¹solutions of RAL using various conditions, including hydrolytic, photolytic, oxidizing and thermal setups.

RAL undergoes extreme degradation in alkaline conditions to form several degradation products. RAL is found to be stable under other stress conditions, concluding its overall stability except alkaline conditions.

3.2 Development of Stability Indicating Assay Method for RAL:

With standards of the synthetic impurities in hand, a chromatographic method for their separation and quantification on a TLC plates was developed, rationally selecting the detection wavelength and the composition of the mobile phase.

3.2.1 Selection of detection wavelength:

UV Spectrum of all the degradation products along with that of standard RAL were recorded in range of 200-400 nm wavelength using HPLC. The wavelength maxima for all varied; overlay spectra of all, established 299 nm as the wavelength for concurrent determination with higher sensitivity for RAL and other degradation products.

3.2.2 Selection of Mobile Phase Composition:

Several mobile phases with different solvents were tried and tested in varying compositions and the resolution amongst RAL and its degradation products were noted. Mobile phases like hexane: ethyl acetate (8:2, 2:8 v/v), chloroform: methanol (9:1, 7:3), chloroform: methanol: acetic acid (9:1:0.1, 8:2:0.5 v/v/v), toluene: ethyl acetate: methanol: acetic acid (7:2:1:0.1,7:1:2:0.5 v/v/v/v), n-butanol: methanol: acetic acid (8:1:1 v/v/v) and Toluene: ethylacetate: methanol: acetic acid (8:1:1:0.1 v/v/v/v) were tried. Amongst all tried, it was found that Toluene: ethylacetate: methanol: acetic acid (8:1:1:0.1 v/v/v/v) gave clear, compact and well resolved spots for RAL and its degradation products.

3.3 Forced degradation study of RAL:

RAL was subjected to various conditions as depicted in Table 1 and the percentage degradation occurred of RAL was calculated using linearity equation nf RAL. The percentage of degradation products formed was also calculated.

The chromatograms for different stressed conditions are depicted in Figure 1-4.

Table1 Stress conditions of RAL

Condition	Solvent	Temperature (˚C)	Time (h)	% Degradation
Hydrolytic
Acid	1NHCl	100	1	89.33
Base	1N NaOH	100	4	99.75
Neutral	Water	100	12	72.35
Oxidation	^{6% H}2^O2	Room Temperature	48	49.21
Thermal	-	100	12	2.98

Figure 1 HPLC chromatogram of acidic degradation of RAL

Figure 2 HPLC chromatogram of alkaline degradation of RAL

Figure 3 HPLC Chromatogram of neutral hydrolytic degradation of RAL

Figure 4 HPLC chromatogram of oxidative degradation of RAL

3.4 Validation of the developed HPLC method:

The optimized HPLC method was validated as per ICH guidelines, with regards to linearity, range, precision (repeatability and intermediate precision levels), limit of detection, limit of quantification and specificity.

3.4.1 Linearity and Range:

Linearity was accessed in range of 200-700 ng/spot for RAL. The plots of area under the curves (AUC) of the peak responses of the analyte against their corresponding concentrations, they fitted straight lines responding to equation¹.

Y=9.198X +6936, r²0.9923 [1]

Where,

Y =Area under curve for RALX_,= Concentration of RAL

3.4.2 Precision:

The data for intraday and interday precision was in range of 0.64-1.04 and 1.44-1.78 respectively.

3.4.3 Limit of detection, limit of quantification:

Limit of detection and quantification were computed using equations suggested by ICH guidelines and were 33.99 ng/spot and 103.01 ng/spot respectively.

3.4.4 Specificity:

The peak purity indices of the analytes in stressed solutions, determined with a deuterium detect or under the optimized chromatographic conditions, were found to be more than 0.9997, indicating that no additional peaks were co-adsorbing with each of the analytes and evidencing the ability of the method to assess unequivocally the analytes of interest in the presence of potential interferences. Baseline resolution was achieved for all investigated compounds.

Table 2 Summary of Validation Parameters of developed HPLC method

Parameter	RAL
Linearity(μg/mL)	1-5
r2	0.999
Intraday Precision (CV)	0.52-0.83
Interday Precision (CV)	0.77-1.28
LOD(μg/mL)	0.29
LOQ(μg/mL)	0.95
Robustness	Robust

4. CONCLUSION:

A stability indicating HPLC method for determination of RAL was developed and validated as per ICH guidelines. The method could resolve all the degradation products generated during stressed conditions. The developed HPLC method is simple, precise, accurate and cost-effective and can be adopted for routine analysis of RAL in bulk and its available marketed formulations without any interference from the excipients or degradation impurities.

The developed and validated analytical method was capable of resolving all degradation products from stressed samples of RAL. Due to these characteristics, the method has stability-indicating properties for RAL and, being fit for its intended purpose; it may find application for the routine analysis of this active pharmaceutical ingredient and its degradation impurities.

5. REFERENCES:

1. White DE, Frank J.Medical Virology. 4^th ed. California USA: Academic Press. 1994; 235-296.

2. HIV AIDS. Treatment and Prevention in India: Modeling the costs and consequence; The World Bank: Washington D.C, India Art Report. 2004.

3. Erik Declercq. Antiviral durgs in current clinical use. Journal of Clinical Virology. 2004; 30: 115-133.

4. Sethi PD. HPTLC Quantitative Analysis of Pharmaceutical Formulations.2nd ed. New Delhi: CBS Publication and Distributors. 2001;48-55.

5. Hamilton RJ, Sevell PA. Introduction to HPLC. 2nd ed. London: Chapman and Hall. 1982.

6. Munson JW. High-Performance Liquid Chromatography: Theory, Instrumentation, and Pharmaceutical Applications. New York: Marcel Dekker Inc. 2001; 665-668.

7. Sharma J, Fried B. Hand Book of Thin Layer Chromatography. 3^rd ed. New York: Marcel Dekker Inc. 2003; 767-805.

8. Sethi PD. HPTLC Quantitative Analysis of Pharmaceutical Formulations. 1st ed. New Delhi: CBS Publication and Distributors. 1996; 4-28.

9. Cartenson JT, Swarbirck J. Solution Kinetics- Drug Stability Principles and Practice. 2^nd ed. New York: Marcel Dekker Inc. 1995.

10. Q1A (R2): Stability Testing of New Drugs and Products. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonized Tripartite Guideline. 2003.

Received on 18.12.2023 Modified on 14.01.2024

Asian J. Pharm. Ana. 2024; 14(1):21-25.

DOI: 10.52711/2231-5675.2024.00005