Raltegraviris Chemically, N-(4-Fluorobenzyl)-5-hydroxy-1-methyl-2-(2-{[(5-methyl-1,3,4-oxadiazol-2-yl) carbonyl]amino}-2-propanyl)-6-oxo-1,6-dihydro-pyrimidine carboxamide^1-2 structure are shown in Figure 1.

Figure1: Structure of Raltegravir

Raltegravir is an antiretroviral drug used to treat HIV infection. The first of a new class of HIV drugs, the integrase inhibitors. Raltegravir targets integrase, an HIV enzyme that integrates the viral genetic material into human chromosomes, a critical step in the pathogenesis of HIV. The drug is metabolized away via glucuronidation. Different doses of Raltegravir oral tablets were 200, 400 and 600 mg. Chewable tablets are of 25 and 50mg.

A thorough literature survey has revealed that UV spectroscopy³, HPLC^3-6 method for Raltegravir with combination of other drugs, for its estimation in bulk, pharmaceutical dosage forms and biological samples.

The aim of present research work for the development and validation of HPLC method, an attempt was made to develop a simple, accurate, precise and rapid method for the estimation of Raltegravir in bulk and pharmaceutical dosage form.

EXPERIMENTAL:

MATERIALS AND REAGENTS:

Raltegravir was gift sample from Hetero drugs. Acetonitrile HPLC Grade was purchased from Merck Chemical Company. HPLC Grade water was purchased from Avantor performance materials limited. The 0.45 μm pump Nylon filter was obtained from Advanced Micro Devices (Ambala Cantt, India) and Whatman no 5 filter paper was obtained from Modern Science lab, (Nashik, India).Glasswares used were Class A grade.

HPLC instrumentation and conditions:

Chromatographic separation was achieved by using maintained at 25 ºC. Isocratic elution was performed using methanol: water (80:20) (v/v) at a flow rate 0.80 mL min^-1 with UV detection at 240 nm. The overall run time was 5 min and 10μL of sample was injected into the HPLC system.

Preparation of stock solution:

Raltegravir stock solution (1000 μg mL^-1) was prepared by accurately weighing 25 mg of Raltegravir in a 25 mL volumetric flask and making up to volume with mobile phase. Working solutions for HPLC injections were prepared on a daily basis from the stock solution in a solvent mixture of methanol: water (80:20) (mobile phase). Solutions were filtered through a 0.45 μm membrane filter prior to injection.

Development of calibration curve:

Appropriate aliquots of standard Raltegravir stock solution were taken in different volumetric flasks and the resultant solution was diluted up to the mark with the mobile phase to obtain a final concentration of 25, 50,100,150 and 200μg mL^-1. Each of the solutions was injected using isocratic mode with UV detection at 240nm and flow rate of 0.8mLmin^-1. From the resultant chromatogram obtained, peak area was used to plot the calibration curve.

Method Validation:

The method was validated for the following parameters: linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, system suitability androbustness.^{7, 8}

Linearity:

Raltegravir appropriate aliquots were pipette out from the standard stock solution into a series of 10ml volumetric flasks. The volume was made up to the mark with mobile phase (Acetonitrile, water in 80:20) ranging from 25-200 μg mL^-1 of Raltegravir. 20 μL of each solution was injected in to the HPLC system and the peak area of the chromatogram obtained was noted. Correlation coefficient (r) of the line, constructed by plotting mean of peak areas against corresponding concentration, was found to be 0.9997.

Accuracy:

To ensure accuracy of the method, recovery studies were performed by standard addition method at 50%, 100% and 150% level to pre-analyzed samples and subsequent solutions were re-analyzed. At each level three determinations were performed.

Precision:

Precision of the method was determined in terms of repeatability and intra-day and inter-day precisions.

Repeatability of the method was determined by analyzing six samples of same concentrations of drug. Chromatographs were recorded and area of each chromatograph was measured. Results of this determination are reported in table 4.The % RSD of three obtained assay values at three different concentration levels were calculated. The inter-day precision and intra-day precision studies were performed. The % RSD was calculated.

System Suitability parameters:

To ensure the validity of the analytical procedure, a system suitability test was established. System suitability parameters for the developed HPLC method were determined by injecting six replicates of the standard solution (50μg/mL). Parameters such as a number of theoretical plates (N), tailing factor and retention time were calculated.

Robustness:

The drug solution was subjected to small, deliberate changes like flow rate, wavelength and change in mobile phase ratio. The results obtained were not affected by varying the conditions and were in accordance with the results in original conditions.

Ruggedness:

To determine ruggedness, two different analysts performed in similar operational and environmental conditions using developed method.

Limit of quantification (LOQ) and limit of detection (LOD):

The limit of quantification (LOQ) and limit of detection (LOD) were based on the residual standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in International Conference on Harmonization guidelines Q2 (R1).

Analysis of Pharmaceutical formulations:

Twenty tablets were weighed accurately and finely powdered. A powder equivalent to 25mg of Raltegravir was transferred carefully to 25mL volumetric flask and about 15mL diluent was added. The mixture was sonicated for 10 minutes. The volume was made up to 25mL with diluent, filtered through Whatman no. 5 filter paper. From the filtrate further dilutions were made to obtain 25μg/mL. The final solution was injected in HPLC, chromatogram was recorded and area was measured.

RESULTS AND DISCUSSION:

Several mobile phases were tried for the analysis. The mobile phase acetonitrile: water 80:20 (v/v) showed good separation and good peak symmetry. By the proposed method, the retention time of was found to be 1.3 minutes. The resulting chromatogram obtained is shown in Figure 2.

Figure 2: Chromatogram of Raltegravir

Linearity:

Linearity was obeyed in the concentration range of 25-200μg/mL and the correlation 0.998. The regression equation of Raltegravir concentration over its peak area ratio was found to be y =45127x +2E+06,where y is the mean peak area and x is the concentration of Raltegravir (μg/ml). The calibration curve obtained is shown in figure 3.

Figure 3:Calibration curve of Raltegravir

Accuracy:

Accuracy is determined by performing recovery studies at 3 levels in which known amount of analyte shall be added and recovery shall be carried out in three replicates of each concentration level and the % recovery was calculated. The mean recovery was found between 99.7-100.7 % and %RSD between 0.5-0.9. The accuracy results are shown in Table 1.

Results of recovery studies:

Repeatability was determined by analysing 50 μg/ml concentration of Raltegravir for six times and % RSD was found to be < 2 which shown in Table no 2.

Table 1: Accuracy results

Spiked level (%)	Formulation Conc (µg/ml)	Pure Drug Conc (µg/ml)	Amount found	% Recovery	% Mean recovery ±SD	%RSD
50	25	12.5	37.5	100.4	100.1±0.305	0.3055
	25	12.5	37.5	99.8
	25	12.5	37.5	100.2
100	25	25	50	100.1	100.2±0.208	0.2081
	25	25	50	100.5
	25	25	50	100.2
150	25	50	75	99.5	100.3±0.472	0.4724
	25	50	75	100.2
	25	50	75	100.4

Intermediate Precision (reproducibility):

The intraday and interday precision studies were carried out and the mean percent relative standard deviation (% RSD) was calculated and it was found to be 0.25 and 0.89respectively, which is within the acceptable criteria of not more than 2.0. The results are shown in Table 3.

Table 3: Results of intra-day and inter-day precision

Concentration

(µg/ml)

Intra-day precision

Inter-day precision

Area mean ±S.D

(n=3)

RSD

Area mean

±S.D

(n=3)

%RSD

26743802

±95572

0.015279

26743802

±159028.0

0.005

System Suitability parameters:

The System suitability parameters like number of theoretical plates (N) was found to be3258, tailing factor 0.6, which indicates efficient performance of column. The results are shown in Table 4.

Table 4: System suitability parameters

Parameter	Results
Retention time	1.380
Tailing factor	0.6
Number of Theoretical plates	3258
HETP	0.027

Ruggedness:

Using developed method two different analysts performed on 50µg/ml in similar operational and environmental conditions. The results obtained were not affected and were in accordance with the results in original conditions. This shows the method was rugged and results are shown in Table no.5.

Table 5: Results of ruggedness studies

Parameters

Area

R_T

Mean± S.D

(n=3)

%RSD

Analyst-1

27445976

1.380

27751313±

424012.05

0.015

Analyst-2

27336098

1.380

27751313±

424012.05

0.015

Robustness:

The drug solution was subjected to small, deliberate changes like flow rate and wavelength. The results obtained were not affected by varying the conditions and were in accordance with the results in original conditions. This shows the method was robust and results are shown in Table no.6.

Assay of Pharmaceutical Formulation:

Estimation of Raltegravir in tablet dosage forms were carried out 5.002 as assay value. The result of assay obtained was found to be in good agreement with the labeled claim, indicating the absence of interference of the excipients and results are shown in Table no.7 and chromatogram was recorded and area was measured which was shown in figure 4.

Table 6: Results of Robustness study

Parameter	RT	Area mean ± S.D (n=3)	%RSD
Flow rate 0.7ml/min	1.577	30811307 ± 338908.2	0.010999
Flow rate 0.9ml/min	1.227	24495670 ± 317581	0.700531
Wave length 239nm	1.383	26734315 ± 43445.34	0.001625
Wave length 241nm	1.380	25305139 ± 354497.7	0.014009

Table 7: Assay of Raltegravir in tablets

Formulation	Label claim	Amount found	% Purity	%RSD
Isentress (25mg)	25mg	26.4mg	105.6%	0.771

Figure: 4 Chromatogram of Raltegravir Formulation

CONCLUSION:

A simple, rapid precise, accurate and robust RP-HPLC-UV method was developed, validated and applied for the determination of Raltegravir in pharmaceutical dosage forms. No interference from any components of pharmaceutical dosage form and the method has been successfully used to perform long-term and accelerate stability studies of Raltegravir formulations.

ACKNOWLEDGEMENTS:

The authors are grateful to Chairman, Malla Reddy College of Pharmacy for providing necessary research facilities to carry out the research work and Hetero Labs, Hyderabad for providing the gift sample of the drug.

REFERENCES:

1. "FDA approval of Isentress (raltegravir)". U.S. Food and Drug Administration (FDA). June 25, 2009. Retrieved 2009-11-15.

2. "WHO Model List of Essential Medicines". World Health Organization. October 2013. Retrieved 22 April 2014.

3. Jean Marie Poirier, Pascal Robidou, Patrice Jaillon. Quantification of the HIV-integrase inhibitor Raltegravir in human plasma by high performance liquid chromatography with fluorescence reduction. J chromatography B 2008; 867:277-281.

4. Girija B. Bhavar, Sanjay S. Pekamwar, Kiran B. Aher, Sanjay R. Chaudhari, Simple Spectrophotometric Method for Estimation of Raltegravir Potassium in Bulk and Pharmaceutical Formulations Journal of Applied Pharmaceutical Science, pp. 147-150, October, 2013.

5. Jasmine A Talameh, Naser L Rezk, Angela DM Kashuba. Quantifying the HIV-1 integrase inhibitor raltegravir in female genital tract secretions using High Performance Liquid Chromatography with Ultraviolet detection. J Chromatography B 2010; 878: 92-96

6. Notari.S, Tommasi C, Nicastri E, Bellagamba R, Tempestilli M, Percillo LP et al. Simultaneous determination of Maraviroc and Raltegravir in human plasma by HPLC-UV method. Inter Union BiochemMol Biology life 2009; 61(Suppl 4): 470-475.

7. Michael E., Schartz S., Krull. “Analytical Method Development and Validation.”,2004, 25-46

8. ICH Harmonized Tripartite Guideline, ICH Q2B (May 1997), Validation of analytical procedures: methodology.

Received on 18.03.2015 Modified on 08.04.2015

Asian J. Research Chem 8(5): May 2015; Page 335-339

DOI: 10.5958/0974-4150.2015.00055.3

Concentration [µg/ml]	AUC	RT	AUC Mean ±SD	%RSD
50	27445976	1.380	27482459 ± 132301.58
50	27345268	1.383
50	27668117	1.380		0.004814
50	27336098	1.380
50	27584628	1.382
50	27514665	1.383