Stability indicating RP-HPLC method for the determination of Tenofovir in pharmaceutical formulation

 

Gurmeet S. Chhabra*, Aayushi Rajora, Dinesh K. Mishra

Indore Institute of Pharmacy, Indore (M.P), India.

 

ABSTRACT:

Stability indicating high performance liquid chromatography (HPLC) method was developed for the assay of Tenofovir in bulk and solid dose formulation. The HPLC separation was achieved on kromasil C18 (100mm × 4.6mm, 5 μm) column using a mobile phase of Methanol: Potassium dihydrogen orthophosphate buffer (30:70,v/v) at a flow rate of 1 ml min^-1 and UV detection at 260 nm. Peak elutes at 7.33 appropriate. The method was validated for linearity, repeatability, accuracy, precision, robustness, limit of detection and limit of quantification. The accuracy was between 99.14 - 99.97%. The highest R.S.D. amongst interday and Intraday precision was found 0.808 and 0.473 respectively.The assay was linear over the concentration range of 10-50 μg/ml (R≈0. 999). The method was robust as no significant change in chromatographic parameters. LOD and LOQ was found to be 0.90 and 2.71 respectively. The stress studies were performed per ICH guidelines to confirm its Stress testing was carried out in presence of acid, base, hydrogen peroxide, heat and light to demonstrate specificity of the method as per ICH guidelines. The developed method could separate the potential degradation products from the Tenofovir peak. It was concluded that   highest degradation occurs in basic condition. This proposed method was suitable and practical for analysis the content of Tenofovir in pharmaceutical products and could be of benefit for the prediction shelf life of Tenofovir in marketed formulations.

 

 

INTRODUCTION:

Tenofovir Alafenamide Fumarate (TAF) belongs to the class of nucleotide reverse transcriptase inhibitor (NRTI). It is a novel ester prodrug of the antiretroviral Tenofovir. It is chemically called as (2E)but-2-enedioicacid; bis(propan-2-yl(2S)-2-{[(S)-({[(2R)-1-(6-amino9H-purin-9-yl)propan-2yl]oxy}methyl)(phenoxy)phosphoryl]amino}propanoate).It has a molecular formula ofC₂₃H₃₁N₆OP and a molecular weight of 476.47 g/mol. It has the following structure (Figure 1) It is Slightly soluble in water, soluble in methanol, very slightly soluble in dichloromethane^.1,2

 

Fig. 1. Structure of Tenofovir

 

Several high-performance liquid chromatography (HPLC) methods have been published for direct analysis of Tenofovir in bulk materials and formulations in pharmaceutical product^3,16. However, the purpose of this work was to develop and validate an economical, simple and stability-indicating HPLC method for Tenofovir bulk materials and pharmaceutical formulations using a C18 column for chromatographic separation followed by UV detection at 260 NM. Stress testing was carried out to demonstrate specificity of the method. ¹⁷ The developed method could be applied for prediction of shelf life of Tenofovir in related pharmaceutical products.

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Tenofovir bulk material was purchased from Mylan Pharmaceutical Ltd.Hyderabad. HPLC grade methanol was purchased from Merck Specialities Pvt. Ltd. with analytical reagent grade and water was obtained from Millipore.The marketed formulation was purchased from market and used for assay purpose.

 

HPLC Apparatus and conditions:

HPLC was selected as analytical technique for estimation of Tenofovir. The analysis of the drug was carried out on Agilent (S.K.) having Gradient System with UV Detector well Equipped with Reverse Phase (Cosmosil) C18 column (4.6 id x 250mm; 5µm), A SP930D pump and a 20µl injection loop with UV730D Absorbance detector and running Chemstation software. The elution was performed on isocratic solvent system using methanol: Potassium dihydrogen orthophosphate buffer (30:70v/v) at a flow rate of 1 ml min^-1 , UV detection at 260 nm and the runtime was set to be 17 minutes with controlled temperature at 27ºC and injection volume was 20 µL for every samples and standard.

 

Stock and working solutions of standard compound:

Stock solution was prepared by accurately weighed standard Tenofovir 10 mg, dissolved in mobile phase, and adjusted to 100 mL with a volumetric flask. Working standard solutions were obtained by appropriate dilution of the stock solution with mobile phase.

 

Stress testing:

The stress conditions employed for the degradation study included base hydrolysis, acid hydrolysis, and oxidative condition. Stress testing was done by adding 50 µL of reagent to 1 mL of Tenofovir sample, 1N HCl, 1N sodium hydroxide, and hydrogen peroxide (3% v/v) were used as reagent for acid hydrolysis, base hydrolysis, and oxidative stress, respectively. Deionized water was used as solvent. Each sample was then analyzed with the proposed HPLC method after 1 and 2 hours.The peak purity of stressed samples was monitored by the diode array detector in the wavelength range of 200–400 nm¹⁷

 

Method validation:

Validation of the method was done according to the International Conference on Harmonization guideline (ICH, 1996/2005). The method was validated for linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ).^18,19,20

 

System precision/system suitability:

System precision was determined by measuring the peak area of standard solution containing 100% working concentration for six times and calculates the % RSD. The % RSD should be less than 2.0%. The relative standard deviation of six replicate measurement of standard solution found 0.193 % (limit NMT 2.0%), which indicates that the system is precise to analyze the sample.

 

Linearity:

From Tenofovir standard stock solution, different working standard solution (10-50μg/ml) were prepared in mobile phase. 20μl of sample solution was injected into the chromatographic system using mixed volume loop injector chromatogram were recorded.

 

The area for each concentration were recorded

 

Repeatability:

Repeatability is the result of the method operating over short time interval (within a day) under the same conditions. The peak area of 20µg/mldrug solution was analyzed six times on the same day. The %RSD was calculated for the resultant peak area and retention time.

 

Precision:

The intra-day precision was determined by analyzing the six-time injection within one day of Tenofovir, while the inter-day precision was examined for five consecutive days by the proposed method. The precision was expressed as percentage of relative standard deviation (%RSD).

 

Accuracy/Recovery:

Recovery studies were performed to validate the accuracy of developed method. Standard addition was performed with pre-analyzed standard solution. To pre- analyzed formulation, a definite concentration of standard drug (50%, 100%, and 150%) was added and then its recovery was analyzed. The recovery was calculated as follows: recovery (%) = 100 × (detected amount – original amount)/spiked amount. The % recovery was found to be within 99-100%

 

Robustness:

Robustness was performed by variation of flow rate, wavelength, mobile phase ratio and then the analysis was done.

 

Limit Detection and Limit Quantification:

Determination of signal-to-noise ratio was calculated under the proposed chromatographic condition. LOD was considered as 3:1 and LOQ as 10:1.

 

RESULTS AND DISCUSSION:

Selection of Wavelength:

From the figure 2, UV spectrum of Tenofovir, wavelength 260 nm was selected which showed the maximum absorbance in the wavelength range.

 

Figure-2: UV spectrum of Tenofovir

 

Figure-3: Optimised Chromatogram of Tenofovir

 

Development and optimisation of HPLC method:

A fast stability-indicating HPLC technique was developed for the analysis of Tenofovir in tablet formulation. From several trials, the mobile phase  consisting of Methanol: Potassium dihydrogen orthophosphate buffer in the ratio 30:70 (v/v) was found to be an appropriate mobile phase allowing adequate separation of all the compounds using a symmetry C18 (100 mm×4.6 mm, 5 μm) column at a flow rate of 1.0 ml/min. It provided symmetrical peaks and has the most efficient separation and speed. The maximum absorbance 260 nm was used for wavelength detection. A typical chromatogram of separation of Tenofovir is shown in fig. 3.

 

Specificity:

There was no interferences observed at retention time of parent peak, so method was specific as shown in figure-4.

 

Figure (4a,b): Chromatogram of Blank Solution Chromatogram of optimized method

 

Validation Parameters:

The calibration curve yielded correlation coefficient (r²) 0.9966 for Tenofovir respectively as shown in Table-1 which showed the method was linear. For repeatability studies, based on Peak area and retention time the %RSD was found to be 1.26 and 0.87 respectively as shown in Table-2. Based on recovery studies, the accuracy was between 99.14 - 99.97%. as shown in Table-3. The %RSD for intraday precision and interday precision was found to be between 0.29-0.34 and 0.45 and 0.80 respectively (table-5).By changing the mobile phase composition, flow rate and wavelength change, there was no significant change in chromatographic parameters. There was no effect of mobile phase composition on retention time (table 6).The LOD is the lowest limit that can be detected. Based on the S.D. deviation of the response and the slope the limit of detection (LOD) may be expressed as:

LOD = 3.3 (SD)/S = 3 = 0.90μg/ml Where, SD = Standard deviation of Y intercept S = Slope

The LOQ is the lowest concentration that can be quantitatively measured. Based on the S.D. deviation of the response and the slope, the quantitation limit (LOQ) may be expressed as LOQ = 10 (SD)/ S = 2.71μg/mlWhere, SD = Standard deviation Y intercept S = Slope

 

The LOD of Tenofovir was found to be 0.90 (μg/mL).The LOQ of Tenofovir was found to be 2.71 (μg/mL).

Table -1- Linearity data

Conc μg/ml	Peak area	Statistical analysis
0	0	Slope	37363
20	887009.01
40	1548374.76	Intercept	80923
60	2425678.67
80	3114533.14	Correlation coefficient	0.9966
100	3718846.09

 

Table-2 Repeatability study (n=6)

Concentration	% RSDa	% RSDb
20 µg ml-1	1.26	0.87

^aBased on peak area ^b Based on retention time

 

Table-3 Recovery studies for Tenofovir

Level of recovery	Drug	Mean  % Recovery	Standard Deviation*	% RSD
50%	Tenofovir	99.14	0.47	0.48
100%	Tenofovir	97.65	2.76	2.82
150%	Tenofovir	99.97	0.66	0.66

 

Table-4 Precision Study (n=3)

Concentration	Mean Peak area	SD	%RSD
Interday
20 µg ml-1	1692255.11	1369.44	0.808
30 µg ml-1	2171945.86	11621.64	0.535
40 µg ml-1	3183764.76	14411.54	0.453
Intraday
20 µg ml-1	1711240.66	4980.75	0.291
30 µg ml-1	2201504.68	10532.61	0.473
40 µg ml-1	3110431.42	10778.43	0.346

 

Table. 5. Robustness study (n = 3)

Concentration	Conditions changed	% RSD	Mean RT
50 µg ml-1	Mobile phase composition
	80:20	1.13	2.17
	90:10	0.56	2.16
	Flow rate
	0.8 ml min-1	0.71	3.75
	1.2 ml min-1	1.08	2.42

 

Figure-5ADegradation study with 1N HCl After 30 minute at room temperature

 

Figure-5B:Degradation study with 1N NaOH After 30 min at 80°C

 

Figure-5CDegradation study with 0.01N NaOH initial at room temperature

 

Figure-5DChromatogram of 3% H₂O₂ treated Tenofovir at 80°C for 30 min

Figure-5 A, B, C, D. Elution profile of Tenofovir obtained after different stress conditions: Figure 5A (1N HCl After 30 min at room temperature) Figure 5B (1N NaOH After 30 min at 80°C), Figure-5C (0.01N NaOH initial at room temperature) Figure-5D (3% H₂O₂ treated Tenofovir at 80°C for 30 min)

 

Forced degradation studies:

The experiment showed that the maximum degradation of Tenofovir occurred under basic conditions. The results of% degradation during forced degradation was shown in Table-6.

 

Figure 5(A, B, C,D) showed chromatograms of acidic, oxidative and basic stressed bulk drug after after 30 minutes by changing the temperature conditions. Baseline resolution between Tenofovir and degradation product peaks was achieved. The elevated peak at beginning of chromatogram showed degradation of Tenofovir under various stressed condition

 

Table-6 Degradation Data

Strength used	Temperature (°C)	Time (min)	% Degradation
Acid
1 N HCl	80	30	48.04
0.1 N HCl	40	10	3.96
1 N HCl	40	60	41.96
Base
1 N NaOH	80	30	100
1 N NaOH	80	5	100
0.1NaOH	40	5	100
0.01 N NaOH	40	5	91.1
Oxidative
3 % H2O2	80	30	47.44

 

CONCLUSION:

A rapid and reliable isocratic RP-HPLC method for determination of Tenofovir has been developed and validated. This chromatographic assay fulfilled all the requirements to be identified as a reliable and feasible method, including accuracy, linearity, recovery, robustness and precision data. It is a highly specific and precise analytical procedure and having narrow range of chromatographic run time allows the analysis of a large number of samples in a short period of time. It was subjected to different stress conditions and the developed stability indicating method can able to detect and quantify the degraded productsTherefore, this economical and simple HPLC-UV method can be used as a routine sample analysis. The developed method was applied for the determination of Tenofovir content in marketed formulation. The result showed the method was suitable for stability-indicating analysis and assay for commercial products.

 

ACKNOWLEDGEMENTS:

We gratefully thank to Glenmark Nashik for providing gift sample of Tenofovir bulk drug.

 

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Accepted on 04.05.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2021; 14(12):6335-6339.