Development and Validation of Stability-indicating HPLC method for simultaneous estimation of Tenofovir, Emtricitabine and Efavirenz in fixed dose combination drug product

 

P. Sreelatha*, B. Rama Devi

Department of Chemistry, Jawaharlal Nehru Technological University, Hyderabad, Telangana, India-500085.

*Corresponding Author E-mail: bonugulatha@gmail.com

A novel simultaneous determination of stability indicating method was developed for three combination drug product, the commercially available Atripla tablets were used for development, commercially available tablet combination of the Emtricitabine, Efavirenz, and Tenofovir. The developed novel isocratic HPLC method able to separate the three compounds in shorter runtime than reported methods and offering minimum usage of chemical reagents. The separations was achieved on Kromosil C18 100 x 4.6,3.5µ Column, Mobile phase 0.1M ortho phosphoric acid buffer and Acetonitrile in 60:40 v/v ratio, at flow rate of 0.5 mL/min and detection wavelength at 265 nm. The drug product was subjected to various stress conditions as per ICH stress conditions. The developed method was validated with respect to specificity, linearity, accuracy, precision, ruggedness and robustness as per ICH guidelines. The developed method transferred to Quality control for routine testing and stability studies.

 

 

 

1. INTRODUCTION:

Emtricitabine (FTC) is a nucleoside reverse transcriptase inhibitor(NRTI). Chemically it is described as 5-fluoro-1-(2R,5S)-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine (Fig. 1). FTC isthe (^_) enantiomer of thioanalog of cytidine which differs from other cytidine analogs, in that it has fluorine in the 5th position.FTC is an antiviral agent used for the prevention of perinatalHIV-1 reverse transcriptase (Budavari, 2001). It is also active against Hepatitis B virus. [1, 4-7].

 

Efavirenz is a human immune deficiency virus type-I (HIV-I) specific non-nucleoside reverse transcriptase inhibitor (NNRTI). Efavirenz is chemically described as (S)-6-chloro-4-(cyclopropyl ethynyl)-1, 4-dihydro-4-(trifluoro methyl)-2H-3,1-benzoxazin-2-one [2, 4-7]

 

Tenofovir disoproxil fumarate (TDF) belongs to the class of antiretroviral drugs known as nucleotide analogue reverse transcriptase inhibitors (nRTIs), which blocks reverse transcriptase, an enzyme crucial to viral production in HIV-infected people.

 

Chemically TDF is 9[(R)-2-[[bis [[(isopropoxycarbonyl) oxy] methoxy]phosphinyl] methoxy] propyl] adenine fumarate. TDF is the first nucleotideanalog approved for HIV-1 treatment [3, 4-7].

 

A literature survey reveals that analytical methods based on HPLC [8-18] are available for the determination of these drugs individually and in combination with other drugs in different dosage forms.But these reported analytical methodshave longer run times, usage of gradient programs, etc.

 

Hence the aim of the present work is to develop and validate a simple, precise, accurate, and rapid stability indicating method for the determination of Tenofovir, Emtricitabine and Efavirenz in a combined dosage form as per ICH guidelines [19-21].

 

 

 

 

 

Fig. 1: Chemical Structure of (a) Emtricitabine (b) Efavirenz (c) Tenofovir

 

2.MATERIALS AND METHODS:

2.1 Chemicals and reagents:

Emtricitabine, Efavirenz and Tenofovir reference standards were supplied as a gift sample by regional Pharmaceutical Company, Hyderabad, India. Tablets were purchased from local pharmacy, all chemicals and reagents used were of analytical grade and were purchased from Fisher scientific Chemicals, India. High purity water was prepared by using Milli-Q water purification system.

 

2.2 Equipment:

HPLC assay was performed on a Liquid Chromatography Waters HPLC PDA 2996 system used consists of Quaternary solvent manager, sample manager and waters Empower3 software was used to control the equipment and to calculate data and responses from the LC system. Cintex digital water bath was used for hydrolysis studies. Photo stability studies were carried out in a photo stability chamber (Sanyo, Leicestershire, UK). Thermal stability studies were performed in a dry air oven (Cintex, Mumbai, India).

 

2.3 Chromatographic conditions:

The method was developed using Kromosil C18(250 x 4.6 mm, 5μm) column with mobile phase containing a mixture 60:40 v/v ratio of mobile phase A (1 mL of ortho phosphoric acid in 1000mL of milli-Q water in a volumetric flask) and mobile phase B (ACN). The mobile phases were filtered through nylon 0.45μm membrane filters and degassed in sonicator. The flow rate of the mobile phase was 1 mL/min with a run time is 6min.The column temperature was maintained at 30°C and the eluted compounds were monitored at the wavelength of 265 nm (Fig:2) using photodiode array (PDA) detector and the injection volume was 10μL.

 

Fig. 2: UV Spectrum of Efavirenz, Tenofovir, and Emtricitabine

 

2.4 Preparation of standard solution and system suitability solution:

A standard stock solution was prepared by accurately weighed and transferred 20mg of Emtricitabine,60 mg of Efavirenz and 30mg of Tenofovir of working standards into a 10mL clean dry volumetric flask, added 5mL volume of diluent (methanol: water (50:50 v/v), sonicated for 15 minutes and make up to the final volume with diluent. 1mL from the above standard solutions was taken into a 10mL volumetric flask and made up to the volume with diluents as used as system suitability solution.

 

2.5 Preparation of test solution:

Tablets was weighed and crushed powdered (equivalent to 20mg of Emtricitabine, 60 mg of Efavirenz and 30mg of Tenofovir) was transferred into a 100mL volumetric flask, 50mL of diluent added and sonicated for 25 min, further the volume made up with diluent and the solution was centrifuged at 4000rpm for 10 minutes in order to eliminate insoluble excipients and filtered through a 0.45μm pore size Nylon 66 membrane filtered. From the filtered solution 1mL was pipette out into a 10 ml volumetric flask and made up to 10mL with diluent and injected in to HPLC system as per chromatographic conditions mentioned.

3. RESULTS AND DISCUSSION:

3.1 Method development and optimization:

To develop a simple and robust method for simultaneous determination of Emtricitabine, Efavirenz and Tenofovir in fixed dose combination product by using HPLC, the wavelength selection was performed by injecting the diluted individual standard solution in to PDA detector and collected the PDA spectrum of the all three compounds. The three compounds maximum absorbance was observed between 240 to 290 nm, the peak of the all the three drugs Emtricitabine, Efavirenz and Tenofovir were showed good response at 265 nm. Hence 265 nm was selected for detection wavelength for quantification of Emtricitabine, Efavirenz and Tenofovir.

 

To optimise the chromatographic conditions preliminary development trials performed with C18 column with  mixture of Milliqwater, solvent like methanol and acetonitrile without any buffer, tried several solvent mixture compositions of 80:20 v/v, 70:30 v/v, 60:40 v/v and 50:50 v/v combination at 60:40 mobile phase combination with Milliqwater and Acetonitrile the three component were well separated. To improve the peak shape of and improve the base line 0.1M Ortho phosphoric acid were added in to the mobile phase. The separation of the three components were good in the Kromosil C18 250 mm column. To reduce the runtime of the method, 150 mm and 100 mm length columns with 5µ and 3.5µ columns were tried. In 100 x 4.6, 3.5µ column Emtricitabine, Efavirenz and Tenofovir were well separated and the peak was symmetrical.

 

For selection of diluents, solubility of each analyte was observed as, Emtricitabine is soluble in water, Efavirenz is soluble in Methanol, Tenofovir is soluble in methanol and slightly soluble in water. Based on the solubility data of Emtricitabine, Efavirenz and Tenofovir, water: methanol 50/50 v/v composition was selected for diluent to extract the drugs from the fixed dose combination drug product.

 

The final chromatographic conditions of developed method Kromosil C18 (100 mm × 4.6 mm, 3.5m) isocratic reversed phase LC method consist of 0.1M Ortho phosphoric acid buffer: acetonitrile in the ratio of 60:40 v/v respectively as mobile phase at flow rate 0.8 mL/min. The column temperature was maintained at 30°C and the detection monitored at 265 nm. A typical  chromatogram of Emtricitabine, Efavirenz and Tenofovir injection  obtained  from  a 10 µL  injection  is  provided. The overall chromatographic run time was 6 minutes. The peak shapes of Emtricitabine, Efavirenz and Tenofovir was found symmetrical and retention time is 2.155, 2.654 and 3.137 min. The system suitability is given in Table 1, the chromatogram of standard shown in Fig. 3.

 

 

Fig. 3: Standard Chromatogram of Emtricitabine, Efavirenz and Tenofovir

 

Table 1: System Suitability and System Precision

* RT-Retention time, TF-Tailing factor, TP-Theoretical plates

 

 

Table 2: Results of forced degradation

Stress Conditions	% Degradation			Purity angle			Purity threshold
	Emtricitabine	Efavirenz	Tenofovir	Emtricitabine	Efavirenz	Tenofovir	Emtricitabine	Efavirenz	Tenofovir
Acid stress	97.18	96.76	99.20	0.39	09.15	0.69	0.90	24.41	0.79
Base stress	97.82	97.72	97.92	0.63	13.89	0.66	1.15	24.01	0.84
Peroxide stress	98.51	98.58	98.38	5.39	09.15	0.69	7.06	24.41	0.79
UV stress	99.71	99.43	99.18	NA	24.08	0.70	NA	24.08	0.91
Thermal stress	99.35	99.34	98.59	2.04	12.56	0.86	5.09	24.38	0.89
Water stress	99.65	99.66	99.53	1.38	10.18	0.73	1.53	24.16	0.80

 

3.2 Validation of the method:

3.2.1 System suitability:

System suitability is used to verify that the system is adequate for the analysis to be performed. Our method shows all the values for the system suitability parameters are within limits. The column efficiency is about 5177, 12014and 8165 theoretical plates for Emtricitabine, Efavirenz and Tenofovir respectively. The tailing factors are about1.3, 1.2 and 1.4 for Emtricitabine, Efavirenz and Tenofovir respectively. The results are summarized inTable1.

 

3.2.2 Specificity:

A study to establish the interference of degradation product was conducted. Forced degradation was performed on Emtricitabine, Efavirenz and Tenofovir ablets and on placebo individually. Degradation was conducted by 2N hydrochloric acid refluxed at 60°C (Fig.4), 2N Sodium Hydroxide refluxed at 60°C (Fig.5), 20% Peroxide oxidation at RT (Fig. 6), degradation by UV radiations (Fig. 7), degradation by heat at 105°C for 6 h (Fig. 8), Water (Fig. 9) conditions.

 

All the samples were evaluated for peak purity using PDA detector (Waters Empower software3) and are found to be pure. Emtricitabine, Efavirenzand Tenofovirpeak did not have any flag in the purity results table. This indicates that all the degradation products are well separated from the main peak and the test method is stability indicating. The results are summarized in Table 2.

 

Fig. 4: HPLC Chromatogram and Purity Plots of acid stress sample

 

 

Fig. 5: HPLC Chromatogram and Purity Plots of base stress sample

 

 

Fig. 6: HPLC Chromatogram and Purity Plots of oxidation stress sample

 

Fig. 7: HPLC Chromatogram and Purity Plots of UV light stress sample

 

Fig. 8: HPLC Chromatogram and Purity Plots of thermal stress sample

 

 

3.2.3 Precision

The precision (repeatability) of an analytical method refers to the use of the analytical procedure within a laboratory over a short period of time using the same analyst with the same equipment and is expressed as the %RSD. The test preparation of 20 µg/mL of Emtricitabine, 60 µg/mL of Efavirenz and 30 µg/mL of Tenofovir were injected in HPLC for precision study, the precision study (Table 3) showed that method has a good reproducibility which was approved by the analysis of six replicate injections of the test solution.

 

 

Table 3: Precision results of Emtricitabine, Efavirenz and Tenofovir

% Assay
Sample No.	Emtricitabine	Efavirenz	Tenofovir
01	98.67	99.40	101.70
02	99.51	99.36	99.87
03	99.31	98.86	100.71
04	99.85	100.62	98.43
05	99.28	100.81	101.99
06	98.67	101.04	98.48
Mean	99.22	100.02	100.20
Std. Dev.	0.469	0.915	1.544
%RSD	0.472	0.915	1.541

 

 

Fig. 9: HPLC Chromatogram and Purity Plots of water stress sample

 

Table 4: Linearity data of Emtricitabine, Efavirenz and Tenofovir

 

Table 5: Accuracy data of Emtricitabine

 

Table 6: Accuracy data of Efavirenz

Table 7: Accuracy data of Tenofovir

 

3.2.4 Linearity:

A study to establish the linearity of detector response of Emtricitabine, Efavirenz and Tenofovir was conducted. The calibration curve was plotted over the concentration range of 50-300µg/mL, 30-180µg/mLand 75-450µg/mL (Table4) for Emtricitabine, Efavirenz and Tenofovir. Each of this drug solution (10 µL) was injected under the operating chromatographic conditions as described above.

 

A linearity graph of Concentration in ppm versus peak area of theEmtricitabine, Efavirenz and Tenofovir at each level were studied and the correlation coefficient was found to be 0.999.These results show there was an excellent correlation between the peak area and concentration for the drug (Fig. 10).

 

 

Fig. 10: Linearity plot of Emtricitabine, Efavirenz and Tenofovir

 

3.2.5 Accuracy:

A study of Accuracy was conducted by preparing the test concentration at a level of 50%, 100% and 150% for Emtricitabine, Efavirenz and Tenofovir. Accuracy was performed by transferring the tablets content proportionately for each level to get the concentration of Emtricitabine, Efavirenz and Tenofovir equivalent to 50%, 100% and 150% test preparation as per the test method. The mean % recovery of Emtricitabine, Efavirenz and Tenofovir at all the levels of Emtricitabine, Efavirenz and Tenofovir at higher and lower levels was found to be within the limits (Table 5-7).

 

 

 

3.2.6 Robustness:

The robustness of the proposed method was evaluated by slight modification in the organic composition and pH values of aqueous phase of the mobile phase and flow rate. During these studies it was found that there was not much change retention time, area and symmetry of peak.  The developed method was used for the assay of commercially available tablets and six replicate determinations were performed.

 

4. CONCLUSION:

The stability indicating HPLC method was successfully developed and it showed several advantages over other known methods, for the analysis of these compounds this method is an economical, well resolved peaks in single method that can be used for assay of the three active ingredients. The method was validated in accordance to the ICH guidelines shown linearity, accuracy, precision, selectivity, stability and system suitability. The method can also be used for purity and degradation evaluation. 

 

5. ACKNOWLEDGEMENT:

The Authors are thankful to the authorities of Jawaharlal Nehru Technological University, Hyderabad, India for providing the laboratory facilities.

 

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Received on 27.08.2017         Modified on 13.10.2017

Accepted on 24.11.2017         © AJRC All right reserved