INTRODUCTION:
Raltegravir is pharmacologically known as an Anti-HIV drug having IUPAC name N-[2-[4-[(4-fluorophenyl) methylcarbamoyl]-5-hydroxy-1-methyl-6-oxopyrimidin-2-yl] propan-2-yl]-5-methyl-1, 3, 4-oxadiazole-2-carboxamide shown in Fig. 1. As such, it’s a structural analogue of the di-keto acid category of compounds and shares their β-hydroxy-ketone structural motif. Raltegravir inhibit the integrase enzyme, an HIV enzyme that integrates the infectious agent genetic material into human chromosomes, an essential step within the pathological process of HIV which reduces the HIV infection in the human body. The drug is metabolized away via glucuronidation[1]. It is quickly absorbed with a median tmax of 4 hours in fasting condition[2].
Raltegravir shows 32% of bioavailability with rapid absorption and 83% was binded with human plasma protiens. After oral administration, 31% of Raltegravir was excreted through urine[3]. Literature survey discloses some analytical procedures are developed for the estimation of Raltegravir such as high performance liquid chromatography[4-10], high performance thin layered chromatography[11-13], LC-MS[14-27] and spectrophotometric[28-35] methods. After thorough survey, there is no colorimetric method was reported about this drug using MBTH reagent. Thus an effort was created for the estimation of Raltegravir by colorimetric method using MBTH reagent. Within the present study a straightforward, rapid, precise and accurate colorimetric methodology has been developed for the determination of Raltegravir in pharmaceutical formulation, serum and urine samples.
Figure 1: Chemical structure of Raltegravir
MATERIALS AND METHODS:
Apparatus:
Shimadzu UV-1800 model double beam spectro photometer connected to system equipped with spectra manager UV Probe software was utilized in this method. A 1cm thickness glass cuvette is used as sample cell. Contech electronic balance is used for weighing all the samples required for this work. Scientech SE-366 model ultrasonicator is utilized for mixing, preparation and sonication of standard and sample solutions.
Materials and reagents:
The entire chemicals and reagents are used in this work are analytical grade and the solvent used is double distilled water. Raltegravir pure drug was acquired from Hetero Pharma Ltd, Hyderabad, India. Isentress® tablets contain 25 mg Raltegravir were used as a pharmaceutical formulation procured from Merck Pharmaceutical Ltd, India. Methanol, MBTH and Ferric chloride were acquired from Merck chemicals Ltd, India. Hydrochloric acid is obtained from Qualigens. Serum and urine collected from healthy human volunteers.
Method development:
Selection of solvent:
In this work solvent was selected by investigating numerous solvents according to stability, solubility, suitability and economic conditions. Methanol was chosen as a suitable solvent.
Selection of wavelength:
Analytical wavelength was selected by scanning the standard and sample solution each having the concentration of 100 µg/mL of Raltegravir with MBTH reagent and ferric chloride solution followed by oxidative coupling reaction produces a bluish green chromogen shows absorption maxima at 572nm which was depicted in Fig. 2.
Preparation of standard solution:
10 mg of Raltegravir was accurately weighed and transferred into a 10 mL volumetric flask then a small amount of methanol was added to it for dissolving the drug. After the drug is completely dissolved then the volume was made up to the mark with methanol which gives 1000 µg/mL.
Figure 2: Overlain spectra of standard and sample solution of 100 µg/mL of Raltegravir at 572nm
Preparation of sample solution:
Twenty Isentress® tablets each contain 25 mg Raltegravir were taken and make them crushed in mortar and pestle into a fine powder. Powder equivalent to 10 mg of Raltegravir was accurately weighed and transferred into a 10 mL volumetric flask then a small amount of methanol was added to it for dissolving the drug. After that the volume was made up to the mark with methanol and then it was taken into an ultrasonicator for mixing followed by filtration through 0.45 micron Whatmann filter paper No. 41.
Preparation of 0.3% w/v MBTH reagent:
0.3grams of MBTH reagent was taken in 100mL volumetric flask. To this 30mL of distilled water was added and sonicated for 20 min. Shake the flask and make up the volume to 100mL with distilled water.
Preparation of 0.1N HCl:
Pipette out 0.83mL of concentrated HCl and transferred into a 100mL volumetric flask. To this add 20mL of distilled water and shake it properly. Then make up the volume to 100mL with distilled water.
Preparation of 1% w/v FeCl3 solution:
1gm of FeCl3 was dissolved in 30mL of 0.1N HCl in a 100mL volumetric flask and it was shaked well to dissolve completely and then volume was made up to mark with 0.1N HCl.
Preparation of blank:
For the preparation of blank solution, 1mL of 0.3% w/v MBTH reagent was transferred into a 10mL of volumetric flask. To this 0.8mL of 1% w/v FeCl3 solution was added followed by the addition 0.1N HCl and finally the volume were made up to the mark with methanol. The solution is kept aside for 30min until bluish green color develops.
General procedure:
Aliquots of Raltegravir ranging from 80-220 µg/mL were taken into a series of 10mL volumetric flask. To each volumetric flask, 1mL of 0.3% w/v MBTH reagent, 0.8mL of 1% w/v FeCl3 solution was added followed by the addition 0.1N HCl and finally the volume were made up to the mark with methanol. The solutions are kept aside for 30min until bluish green color develops due to oxidative coupling reaction between Raltegravir and MBTH in presence of ferric chloride and HCl. The above solutions were scanned from 400nm-800nm against blank and the absorption spectrum shows the absorption maximum (λmax) at 572 nm. The drug was estimated by constructing a calibration curve between the concentration of the drug solutions on the x-axis and the corresponding absorbance values on the y-axis.
RESULTS AND DISCUSSION:
Reaction mechanism and absorption spectra:
Raltegravir shows maximum absorbance at 572nm with bluish green colored complex due to oxidative coupling reaction with MBTH in presence of ferric chloride and HCl. In this reaction process, first MBTH undergoes oxidation in presence of ferric chloride and loses two electrons and one proton to produce an electrophilic intermediate. This electrophilic intermediate reacts with Raltegravir in addition with HCl and produced a bluish green chromogen which shows maximum absorbance at 572nm is represented in Fig. 2. The complete reaction scheme is depicted in Fig. 3.
Figure 3: Scheme of oxidative coupling reaction between Raltegravir and MBTH
Optimum reaction for formation of colored complex:
The method was optimized rigorously to achieve complete reaction mechanism with highest sensitivity and absorption maximum. The complete oxidative coupling reaction was established out by investigating the preliminary tests.
Effect of reagent:
100 µg/mL of Raltegravir is added with 1mL of 0.3% w/v MBTH and 0.8mL of 1%w/v ferric chloride solution in presence of HCl undergoes oxidative coupling reaction and produce bluish green chromogen. If the concentration of drug increases in series and reacted with MBTH-ferric chloride reagent in presence of HCl produces increasing in the intensity of the colored complex. So intensity of colored complex increases with increased concentration of drug at particular concentration.
Effect of reaction time and stability of colored complex:
The reaction time was examined in between 1-3 min followed by bluish green color formation at room temperature. If the temperature is increased to 300C there is no effect on the colored complex but more than 300C the colored complex undergoes decay. The stability of the colored complex is about 2 h.
Effect of acid:
It was found that hydrochloric acid was added to the colored mixture to avoid flocculation of colored complex and also it induces the intensity of colored complex between drug and MBTH-ferric chloride reagent.
Effect of series of reagents is added:
The intensity and stability of color with maximum absorbance was achieved by the addition of reagent with drug in this sequence such as Raltegravir-MBTH-ferric chloride-HCl.
Effect of interference:
The drug-excipient interference studies were investigated by examining the interaction between the drug and generally encountered excipients such as microcrystalline cellulose, lactose, eudragit, magnesium stearate and coating agent like opadry II pink. 100 µg/mL of Raltegravir pure drug was added separately with increasing concentration of potential excipients followed by the addition of MBTH-ferric chloride-HCl solution and kept aside for one hour for complete reaction to produce colored complex. These colored complex are stable even after the increasing concentration of potential excipients.
Method validation:
Linearity:
Linearity of the colorimetric method is established by the analysis of aliquots ranging from 80-220 µg/mL were taken into a series of 10mL volumetric flask. To each volumetric flask, 1mL of 0.3% w/v MBTH reagent, 0.8mL of 1% w/v FeCl3 solution was added followed by the addition 0.1N HCl and finally the volume were made up to the mark with methanol. The solutions are kept aside for 30min until bluish green color develops due to oxidative coupling reaction between Raltegravir and MBTH in presence of ferric chloride and HCl. Each concentration of Raltegravir was analyzed at 572nm and the calibration plot between concentration and absorbance was depicted in Fig. 4. The linearity studies were performed and the overlain spectrum of each concentration was shown in Fig. 5. Through linearity studies the optical and regression characteristics was established and shown in Table 1.
Figure 4: Calibration plot of Raltegravir at 572nm
Figure 5: Overlain spectra of different concentrations (80-220 µg/mL) for linearity studies
Table 1: Optical and regression characteristics of Raltegravir
|
Parameters |
Method |
|
λmax (nm) |
572 |
|
Beer’s law limit (µg/mL) |
80-220 |
|
Molar absorptivity (L mol-1 cm-1) |
0.412×10-2 |
|
Sandell’s sensitivity ( μg cm-2/0.001 AU) |
0.242 |
|
Regression equation (Y=mX+C) |
0.004x + 0.007 |
|
Slope (m) |
0.004 |
|
Intercept (C) |
0.007 |
|
Correlation coefficient (r2) |
0.999 |
|
LOD (µg/mL) |
2.3 |
|
LOQ (µg/mL) |
7.1 |
|
Colour |
Bluish green |
Sensitivity:
The LOD and LOQ were calculated based on the standard deviation of the response and the slope of the constructed calibration curve, as described in International Conference on Harmonization guidelines Q2 (R1). The LOD is calculated by the following formula:
The LOQ is calculated by the following formula:
The LOD and LOQ give the information about the sensitivity of the method and it was found to be 2.3µg/mL and 7.1µg/mL respectively.
Specificity:
The analyte was assessed in the presence of the excipients and it was found that there was no interaction with the analyte.
Accuracy:
The accuracy studies were carried out by addition of 80%, 100% and 120% levels of pure Raltegravir with the pre-analyzed formulation of 100mg/mL concentration separately in three replicates of each level. The percentage recoveries were calculated and the results were shown in Table 2.
Table 2: Accuracy data of Raltegravir
|
Spiked level of recovery (%) |
Raltegravir added (µg/mL) |
Raltegravir found (µg/mL) |
Recoverya±RSD% |
|
80 |
80 |
79.88 |
99.85±0.82 |
|
100 |
100 |
100.27 |
100.27±0.61 |
|
120 |
120 |
120.52 |
100.43±0.30 |
a Average of three determinations.
Precision:
Precision studies were carried out by analyzing the six replicates of 100mg/mL Raltegravir to confirm whether method is having the capability of reproducibility and precise. The standard deviation and % relative standard deviation for the precision were calculated statistically which was shown in Tables 3.
Table 3: Precision data of Raltegravir
|
Parameter |
Raltegravir addeda (µg/mL) |
Raltegravir found (µg/mL) |
Recoveryb±RSD% |
|
System precision |
100 |
99.27 |
99.27±1.3 |
|
Method precision |
100 |
99.98 |
99.98±0.02 |
a is the number of six replicates.
b Average of six determinations.
Ruggedness:
Ruggedness was performed by using six replicates of 100mg/mL Raltegravir on different instruments like Lab India double beam UV- spectrophotometer and Shimadzu double beam UV-Visible spectrophotometer and by different days (intra-day and inter-day), analysts and laboratories. The results are statistically analyzed and were depicted in Table 4.