Stability indicating rp-hplc method for the estimation of Emtricitabine and Tenofovir disoproxil fumerate in tablet dosage form

 

Shailaja K.*, Revathi R., Saravanan V. S.

Department of Pharmaceutical Analysis, The Erode College of Pharmacy and Research, Erode- 638112, India

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

 

ABSTRACT:

A simple, accurate and precise reverse phase high performance liquid chromatographic (RP-HPLC) method was developed for the simultaneous quantitative estimation of Emtricitabine (EMT) and Tenofovir disoproxil fumerate (TDF) in tablet dosage form. Quantitation was carried on an ACE (150 mm x 4.6mm, 5µ) column using gradient composition of 0.01 M citric acid monohydrate buffer pH 4.5 as mobile phase A and methanol as mobile phase B at a flow rate of 1.0 mL/min with detection at 260 nm. Determination of the different validation parameters such as precision, linearity, accuracy, specificity, robustness, limit of detection, limit of quantification and filter validation was done. The system suitability parameters such as retention time, theoretical plates and tailing factor were found to be 3.181min, 13438, 0.14 and 6.090min, 48847, 1.05 respectively for EMT and TDF. The detector response is linear from 16 µg/mL to 64 µg/mL for EMT and 24 µg/mL to 96 µg/mL TDF. The limit of detection and limit of quantification was 0.101, 0.189 μg/mLand 0.306, 0.575 μg/mL for EMT and TDF respectively. The percentage recovery for average of three different concentrations (50, 100 and 150%) was 101.1, 100.6, 99.8 and 99.7, 99.4, 99.6% for EMT and TDF respectively.The high percentage of recovery and low % RSD confirms the suitability of the method for the simultaneous estimation of EMT and TDF in tablet dosage form.The method could effectively separate the drugs from their degradation products, it can be regarded as stability indicating method.

 

 

 

INTRODUCTION:

EMT,(–)4-amino-5-fluoro-1-[(2R,5S)-2-(hydroxylmethyl)-1,3-oxathiolan-5-yl]pyrimidin-2(1H)one Fig 1(a), is a nucleoside reverse transcriptase inhibitor for the treatment of HIV infection in adults and children. TDF is chemically[[(1R)-2(6-Amino-9H-purin-9yl) methyl ethoxy] methyl] phosphonate, bis (isopropyloxy carbonyloxy-Methyl ester), Fumarate (1:1) Fig 1(b), belongs to a class of antiretroviral drugs known as nucleotide analogue reverse transcriptase inhibitors¹, which blocks reverse transcriptase, an enzyme crucial to viral production in HIV infected people. Both the drugs are official in Indian Pharmacopoeia.

 

 

Fig 1(a): Chemical structure of EMT

 

Fig 1(b): Chemical structure of TDF

Literature survey reveals that few RP-HPLC^2,3 methods are reported for estimation of EMT and TDF in pharmaceutical formulation.TDF is estimated individually by UV⁴, derivative - HPLC⁵, Plasma RP-HPLC ^{6, 7} and Plasma LC/MS/MS ^{8, 9, 10} methods. Similarly for EMT, HPLC with Fluorometric detection¹¹ in human plasma and stability indicating liquid chromatographic¹² methods were reported. HPLC¹³ and LC-MS/MS¹⁴ method is reported for simultaneous estimation of EMT and TDF in human plasma. HPTLC¹⁵ method is also reported for simultaneous estimation of EMT and TDF in pharmaceutical formulation. It is intended to develop and validate a simple, precise, accurate, economical and stability indicating HPLC method. The developed HPLC method was applied for the forced degradation studies and the method was validated for its parameters as per ICH guidelines.

 

Materials and Methods:

Chemicals and Reagents:

EMT and TDF were obtained as a gift sample from MYLAN LABS Ltd, Hyderabad. HPLC grade methanol (Merck) and analytical reagent grade citric acid monohydrate (Merck) and Milli-Q water was used in mobile phase preparation. Commercially available EMT and TDF tablet dosage forms (Truvada) containing 200mg of EMT and 300mg of TDF were purchased from local market.

 

Preparation of Standard Stock Solution:                                                       

The stock solutions of EMT (400µg/mL) and TDF (600µg/mL) working standard were prepared in 0.01M HCl. The standard working concentration of mixed EMT (40µg/mL) and TDF (60 µg/mL) were prepared by diluting the stock solution in 0.01M HCl. This solution was subjected to LC analysis.

 

Test Preparation:

Ten tablets (Truvada) were accurately weighed and crushed into a fine powder. The powder equivalent to one tablet (200 mg of EMT and 300 mg of TDF) was taken in 500 mL volumetric flask. About 400mL diluent was added, then sonicated for 45mins with intermediate shaking. Then the volume was finally made up to the mark with diluent (500mL). Sample solution was centrifuged at 5000 rpm for 10 min to get a clear solution. Then 5mL of supernatant solution was pipetted into 50mL volumetric flask and diluted to volume with diluent and mixed well. The solution was filtered through 0.45µ PVDF (Millipore make). This solution was subjected to LC analysis.

 

Instruments and Chromatographic Conditions:

RP-HPLC was performed with waters HPLC 2695 with PDA Detector and Waters HPLC 2695 with UV detector. The monitoring software was Empower. The equipment was controlled by a PC workstation. The 0.01M HCl is used as diluent. The mobile phase A consisting of 0.01M citrate buffer (pH4.5) and mobile phase B is methanol, prepared and degassed with ultrasonicator. Compounds were separated on ACE  (150 mm × 4.6 mm i.d, 5-μm particle) under reversed-phase partition chromatographic conditions. Analysis was performed under gradient conditions (Table 1) at a flow rate of 1 mL/ min and the analytes were monitored at 260 nm.

 

Table 1: Gradient Time Program:

 

ResultS and Discussion:

HPLC Method Development and Optimization:

Column chemistry, solvent type, solvent strength, wavelength and flow rate were varied to determine the chromatographic conditions giving the best separation. The mobile phase conditions were optimized so that the components were not interfered from the solvent and excipients. After trying with different C8 and C18, the final choice of stationary phase giving satisfactory resolution and run time with reversed phase column ACE C18. Mobile phase and flow rate selection was based on peak parameters (height, area, tailing, theoretical plates, capacity factor and resolution) and run time. The best result was obtained by using 0.01M citrate buffer as mobile phase A and methanol as mobile phase B with 1.0 mL/ min. From the overlain UV spectra suitable wavelength considered for monitoring the drugs was 260 nm. Solutions of EMT and TDF in diluents were also injected directly for HPLC analysis and the responses (peak area) were recorded. It was observed that there was no interference from the mobile phase or baseline disturbances and both the analytes absorbed well at 260 nm. The chromatogram of standard and placebo were shown in Fig 2 and 3 respectively.

 

Fig 2: Chromatogram for standard

 

Method Validation:

Linearity:

The linearity of the method was evaluated by processing five point calibration curves. A series of solutions EMT and TDF standard was diluted in the concentration ranging from 16 µg/mL to 64 µg/mL for EMT    [Fig 4 (a)] and 24 µg/mL to 96 µg/mL for TDF [Fig 4 (b)] and analyzed as per test method. A graph was plotted with concentration in µg/mL on X- axis versus response (area) on Y-axis and determined the correlation coefficient. The linearity analysis data was summarized in Table 2.

 

Fig3: Chromatogram for placebo

 

 

Fig 4(a):  Linearity for EMT

 

 

Fig 4(b): Linearity for TDF

Accuracy

The accuracy was confirmed by recovery studies by adding known amount of placebo to the pure API of EMT and TDF from about 50% to 150% of the initial assay concentration. Sample solutions were prepared in triplicate for each level and analyzed as per test method. The results were shown in Table 3.

 

Table 2: Results of Linearity:

 

 

Table 3: Results of Accuracy:

(*n =3)

 

 

Method Precision:

The precision of test method was evaluated by performing assay for six individual test preparations of 200/300mg strength as per test method. The results were shown in Table 4.

 

 

Table 4: Results of Method Precision:

 

Intermediate Precision (Ruggedness):

The ruggedness of method was demonstrated by conducting the precision study using different HPLC system (ADS/LCC/79 and ADS/LCC/200) and different column of same manufacturer at different day. Assay was performed for six individual test preparations of 200/300mg strengths as per test method. The results were shown in Table 5.

 

 

 

Table 5: Results of Intermediate Precision:

 (*n =6)

I = Using column I and system I

II = Using column II and system II

 

Specificity (Forced Degradation Study):

A study was conducted to demonstrate the effective separation of degradants from EMT and TDF in assay method. Separate portions of drug product and placebo were exposed to the following stress conditions to induce degradation.

 

The sample solution was treated with 0.1N HCL, 0.1N NaOH, 3% H₂O₂ for about 5 minutes on bench top, exposed to heat for about 24 hrs at 105ºC, exposed to humidity at 25ºC/ 90 % RH for about 48 hrs.

 

Stressed samples were analyzed as per test method with photo diode array detector and the chromatograms of the stressed samples were evaluated for peak purity of emt and tdf peaks using waters system. The results were shown in Table 6 and the degradation chromatograms were shown in Fig 5 to 9.

 

Acceptance criteria:

The Purity Angle (PA) should be less than Purity Threshold (PT) and no purity flag for EMT and TDF peak

 

 

Fig 5: Chromatogram for acid degradation

 

              

Fig 6: Chromatogram for base degradation

 

Robustness:

The robustness was performed by making deliberate changes in flow rate, column temperature and pH of the buffer solution (mobile phase A). It shows that there is no change in the retention time even after making deliberate change in the analytical procedure. Then the method was found to be robust. The results were shown in Table 7

 

 

Fig 7: Chromatogram for peroxide degradation

 

      

Fig 8: Chromatogram for thermal degradation

 

Fig 9: Chromatogram for humidity degradation

 

Stability of Solutions:

The stability of solutions were studied by keeping the standard and test preparations on bench top at room temperature for 24 hrs and mobile phase for 5 days.  No significant difference in the assay results at initial time and after the specified time.

 

Filter Validation:

The filter validation was performed by using two different filters namely, 0.45µm PVDF filter (Mfg. by: M/s. Millipore) and Nylon 0.45 µm filters (Mfg. by: M/s.  Millipore).The difference in assay of EMT and TDF in filtered standard solutions against centrifuged standard solution was found to be within the limit. From the above study, it was concluded that both the (PVDF and Nylon) filters are suitable for standard and test solution filtration. The results were summarized in Table 8.

 

Conclusion:

The developed RP-HPLC method is precise, specific, accurate and stability indicating. This method is able to discriminate between EMT, TDF and its degradation products. The same solvent was used throughout the experimental work and no interference from any excipient was observed. Statistical analysis proved that the method is suitable for the analysis of EMT and TDF in bulk drug and in tablet dosage form within a short analysis period of time.

 

 

 

 

 

Table 6: Peak Purity Results from Forced Degradation Studies:

 

 

 

 

 

 

Table 7: Results for Robustness:

* (n = 6)

 

 

 

 

 

Table 8: Results for Filter Validation:

List of Abrreviations:

ºC                          : Degree Celsius

EMT                      : Emtricitabine

H₂O₂                      : Hydrogen peroxide

HCl                       : Hydrochloric acid

HIV                       : Human immuno deficiency virus

HPTLC                 : High Performance thin Layer                                           Chromatography

Hrs                        : Hours

i.d                          : Internal diameter

ICH                       :International Conference on                                             Harmonization

LC                         : Liquid chromatography

LC/MS/MS           : Layer Chromatography/mass                                            spectroscopy/ mass spectroscopy

M                           : Molar

µ                            : Micron

Mg                         : Milligram

min                        : Minute

mL                         : Milliliter

μg/mL                   : Microgram per milliliter

μm                         : Micro meter

mL/min                  : Milliliter per minute

Mm                        : Mili molar

N                           : Normality

nm                         : Nanometer

pH                         : Negative Logarithm of Hydrogen Ion

PDA                      : Photo Diode Array

% RH                    : Percentage relative humidity

% RSD                  : Percentage Relative Standard Deviation

%                           : Percentage

RP-HPLC             :Reverse Phase -High Performance                                    Liquid Chromatography

rpm                        : Rotations per minute

NaOH                   : Sodium hydroxide

TDF                       : Tenofovir disoproxil fumerate

UV                        : Ultraviolet

 

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Received on 28.09.2012       Modified on 09.10.2012

Accepted on 27.10.2012      © RJPT All right reserved