A New Stability Indicating RP-Hplc Method Development and Validation for Simultaneous Estimation of Emtricitabine and Tenofovir with degradation kinetics.

 

K. S. Sumanth1*, A. Srinivasa Rao2, D. Gowri Shankar3

1department of Pharmaceutical Analysis, Sri Vasavi Institute of Pharmaceutical Sciences, Andhra Pradesh, India.

2department of Pharmaceutical Analysis, Shri Vishnu College of Pharmacy, Andhra

Pradesh, India.

3University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra

Pradesh, India.

*CorrespondingAuthorE-mail:sumanth.kamatham222@gmail.com

 

ABSTRACT:

A new, simple, rapid and stability-indicating RP-HPLC method was developed for the estimation of Emtricitabine (EMT) and Tenofovir disoproxil fumarate (TDF) in pharmaceutical dosage forms and validated. The HPLC method was developed on Shiseido C18 column (250 x 4.6 mm i.d, 5) using methanol: 20mM phosphate buffer (pH 5.0) in the ratio of 35:65 v/v at 271 nm. The retention times for EMT and Tdf was found to be 2.79 and 4.91 min respectively. Linearity was established in the range of 0.5-1.5 μg/ml and 0.75-2.0 μg/ml for EMT and Tdf respectively. The method was precise with %RSD < 2 for both intraday and interday precision. The accuracy of the method was performed over three levels of concentration and the recovery was in the range of 98-102%. The drugs were individually subjected to forced degradation (thermal, photolytic, hydrolytic, and oxidative stress conditions) studies for seven days. EMT showed maximum degradation in acidic medium and Tdf showed maximum degradation in basic medium. The method was successfully applied for quantifying the drugs in marketed dosage forms.

 

KEYWORDS:RP-HPLC, Tenofovir disoproxil fumarate, Emtricitabine, Phosphate buffer, Forced degradation.

 

 


1. INTRODUCTION:

Emtricitabine (EMT) is a nucleoside reverse transcriptase inhibitor (NRTI). Chemically it is 5-fluoro-1-(2R, 5S)-[2-(hydroxymethyl)-1, 3-oxathiolan-5-yl] cytosine (Fig. 1). EMT is the (-) enantiomer of thio analog of cytidine which differs from other cytidine analogs, in that it has fluorine in the 5th position. EMT is an antiviral agent used for the prevention of perinatal HIV-1 reverse transcriptase[1].

 

It is also active against Hepatitis B virus[2]. Tenofovir disoproxil fumarate (TDF) is a fumaric acid salt of the bis- isopropoxy carbonyl oxymethyl ester derivative of tenofovir. TDF is a nucleotide reverse transcriptase inhibitor. TDF is converted to Tenofovir, an acyclic nucleoside Phosphonate (Nucleotide) analog of adenosine 51-Monophosphate. Chemically it is 9-[(R)-2[ [bis[ [ (isopropoxycarbonyl) oxy] methoxy] phosphinyl] methoxy] propyl] adenine fumarate[3] (Fig. 2).

 

The present study is to develop a stability indicating RP-HPLC method for EMT and TDF. The objective of the study is to subject the drugs for acid, base, peroxide, light, thermal degradation and estimate its extent of degradation in different days. A Literature survey reveals that several analytical methods for the estimation of EMT and TDF based on UV[4-8], HPLC[9-12], stability indicating HPLC[13-14], UPLC [15], Fluorimetry[16], Chiral chromatography[17], LC-MS[18,19] were reported. The present aim is to develop a more precise, accurate and simple stability indicating method for the estimation of tDF and EMT. The molar absorptivity of TDF and EMT was found maximum at 271nm. The validated method was used for the quantification of marketed formulation containing TDF and EMT.

 

 

Fig.1 Chemical structure of Emtricitabine

 

 

Fig.2 Chemical structure of Tenofovir disoproxil fumerate

 

2. MATERIALS AND METHODS:

2.1 Chemicals and reagents:

Emtricitabine (EMT) and Tenofovir Disoproxil Fumarate (TDF) working standards were procured from Hetero Laboratories Ltd. Commercially available as TENOF-EM tablets were purchased from the local Pharmacy of Tadepalligudem. HPLC grade water was purchased from Thermo Fisher Scientifics Ltd., Mumbai. HPLC grade Methanol, Orthophosphoric acid, hydrochloric acid, sodium hydroxide pellets purified and hydrogen peroxide 30% of AR grade were procured from Merck specialties Pvt. Ltd., Mumbai.

 

2.2 Instrumentation and analytical conditions:

RP-HPLC method was performed on the HPLC system (Shimadzu) consisting of binary gradient pump with UV detector (LC-20AD). rheodyne injector with 20 l fixed loop was used for injecting sample on Shiseido C18 column (250 x 4.6 mm i.d, 5) in the present study.

 

 

2.3    Preparation of solutions:

2.3.1Preparation of standard stock solutions:

Standard stock solution of EMT and TDF were prepared by transferring accurately weighed 10 mg of both drugs into separate 10ml volumetric flask. The compounds are then dissolved in diluent (methanol: water 50: 50 v/v) to obtain a standard solution of EMT (1000 μg/ml) and TDF (1000 μg/ml).

 

2.3.2Preparation of working standard solutions:

From the stock solution, 1 ml of both drugs were diluted to 10 ml with diluent in separate volumetric flasks to get the concentration of 100 g/ml of EMT and 100 g/ml of TDF.

 

2.3.3 Preparation of calibration curve standard solutions:

Calibration curve standards were prepared from working standards at concentrations of 0.5, 0.75, 1.0, 1.25 and 1.5 g/ml for EMT and 0.75, 1.125, 1.5, 1.875 and 2.25 g/ml for TDF.

 

2.4 Preparation of the mobile phase:

The mobile phase is a mixture of phosphate buffer and methanol. Phosphate buffer is prepared by dissolving 2.72 gm in 1000 ml (20 mM concentration) HPLC grade water. pH of the buffer is adjusted to 5.0 using ortho phosphoric acid. The prepared buffer was filtered through 0.45 m membrane filter (Millipore) and sonicated before use. Mobile phase is pumped in the ratio of 35:65 v/v (methanol: buffer).

 

2.5 Method validation:

The developed method was validated according to International Conference on Harmonization guidelines for validation of analytical procedures. The developed method was validated with respect to parameters such as linearity, LOD, LOQ, precision, accuracy and specificity. Forced degradation studies were done according to ICH Harmonized Tripartite Guideline, Stability Testing of New Drug Substances and Products: Q1A (R2) [20]

 

2.5.1 System suitability:

The system suitability of the HPLC method was determined by making six replicate injections from freshly prepared standard solutions and analyzing each solute for their retention time, theoretical plates number (N) and tailing factor (T).

 

2.5.2 Specificity:

It is the ability to assess unequivocally the analyte in the presence of impurities, degradants and matrix. To determine this, 20 l of blank, standard and sample solutions were injected separately in triplicate and respective chromatograms were recorded under the optimized conditions.

2.5.3 Linearity:

The calibration curves were obtained with concentrations of the standard solutions of 0.5-1.5 g/ml (50% to 150%) and 0.75-2.25 g/ml (50% to 150%) for EMT and TDF respectively. Linearity was evaluated by regression analysis, which was calculated by the least square regression method.

 

2.5.4 Accuracy:

To check the degree of accuracy, recovery studies were performed in triplicate by the standard addition method at 50%, 100% and 150% levels.

 

2.5.5 Precision:

Precision was checked by analyzing the samples at different time intervals of the same day (intra-day precision) as well as on different days (inter-day precision).

 

2.2.6 Limit of detection and limit of quantification:

Limit of detection (LOD) and limit of quantification (LOQ) were calculated by using the values of Signal to Noise (S/N) ratio for two drugs. For LOD, S/N ratio should be 3:1 and for LOQ, S/N ratio should be 10:1

 

2.5.7 Robustness:

Robustness was determined by analysis of samples under slight variations in chromatographic conditions. The flow rate of the mobile phase was changed from 0.9 ml/min to 1.1 ml/min. The ratio of the organic phase (methanol) was changed by +2% and -2%. The effect of retention time and peak parameters were studied.

 

2.6. Stress testing studies[21]:

Stability studies were performed for EMT and TDF in dilute solutions of acid (0.1 M), base (0.1 M) and peroxide (3%). Stability studies were also done in thermal and photolytic conditions.

 

2.6.1. Acidic degradation :

Stock solutions of 100 g/ml EMT and TDF were prepared using 0.1 M HCl as diluent and stored at room temperature for seven days. Each day an aliquot of these solutions were diluted to 10 ml with mobile phase to get a concentration of 1 g/ml EMT and 1.5 g/ml TDF.

 

2.6.2. Alkaline degradation:

Stock solutions of 100 g/ml EMT and TDF were prepared using 0.1 M NaOH as diluent and stored at room temperature for seven days. Each day an aliquot of these solutions were diluted to 10 ml with mobile phase to get a concentration of 1 g/ml EMT and 1.5 g/ml TDF.

 

2.6.3. Oxidative degradation:

Stock solutions of 100 g/ml EMT and TDF were prepared using 3% v/v H2O2 as diluent and stored at room temperature for seven days. Each day an aliquot of these solutions were diluted to 10 ml with mobile phase to get a concentration of 1 g/ml EMT and 1.5 g/ml TDF.

 

2.6.4. Photolytic degradation:

Stock solutions of 100 g/ml EMT and TDF were prepared using diluent (methanol: water 50:50) and stored under UV radiation (overall illumination ≥ 210 Wh/m2 at room temperature) for seven days. Each day an aliquot of these solutions were diluted to 10 ml with mobile phase to get a concentration of 1 g/ml EMT and 1.5 g/ml TDF.

 

2.6.5. Thermal degradation:

The Stock solutions of 100 g/ml EMT and TDF were prepared using diluent (methanol: water 50:50) and stored at 40C2C/75% RH5% RH for seven days. Each day an aliquot of these solutions were diluted to 10 ml with mobile phase to get a concentration of 1 g/ml EMT and 1.5 g/ml TDF.

 

3. RESULTS AND DISCUSSION:

3.1. Method development and optimization:

The standard solutions of EMT and TDF were scanned in UV spectrophotometer in the range of 200-400 nm. The λmax of both drugs were found to be 280.14 and 261.25 nm for EMT and TDF respectively. The iso-absorptive point for both drugs was observed as 271 nm (Fig. 3) which was selected for detection of drugs. Trails were performed using different columns (Hypersil BDS C18, Symmetry C18, Phenomenex C18 and Shiseido C18), buffers (Acetate, Phosphate, Ortho phosphoric acid), pH (3-6), organic phases (Acetonitrile, Methanol). Shiseido C18 column (250mm X 4.6 mm, 5 μ) produced good separation with efficient resolution and more theoretical plates. The drugs were eluted at a flow rate of 1.0 ml/min using a mobile phase consisting of methanol: 20mM phosphate buffer (pH 5.0) in the ratio of 35:65 v/v respectively. The retention times of the drugs were observed to be 2.79 min for EMT and 4.91 min for TDF.


 

Fig.3 UV Overlay spectrum of EMT and TDF.

 


3.1.1. System suitability:

Under optimized chromatographic conditions 20 l of solution containing 1μg/ml of EMT and 1.5 μg/ml of TDF was injected into the system in six replicates. Chromatograms were recorded and studied for different system suitability parameters like retention time, peak area, number of theoretical plates, tailing factor and resolution. The system suitability parameters were shown in Table 1.

 

Table. 1 System Suitability Parameters of EMT and TDF

Parameter

EMT

TDF

Retention Time (minutes )

2.79

4.91

Peak area

36235

42297

No. of Theoretical plates

6412

5192

Tailing Factor

1.12

1.14

Resolution

--

9.89

 

3.1.2 Specificity:

The HPLC chromatograms were recorded for blank (Fig. 4a) and standard (Fig. 4b-d) under optimized analytical conditions and compared for additional peaks, however found no additional peaks. The two peaks were completely separated in HPLC chromatogram and the resolution was found to be more than 2.

 

 

Fig. 4a Chromatogram of blank

 

Fig. 4b Standard chromatogram of EMT

 

 

Fig. 4c Standard chromatogram of TDF

 

 

Fig. 4d Chromatogram of well resolvedpeaks of EMT and TDF

3.1.3. Linearity:

The calibration curves of EMT and TDF were constructed in the concentration range of 0.5-1.5 g/ml, 0.75-2.0 g/ml for EMT and TDF respectively. The plots obtained from linear regression were shown in Fig. 5 and the result analysis was shown in Table 2.

 

 

 

 

 

 

 

 

 

 

Table 2. Linearity of EMT and TDF

S.No

Emt

TDF

Concentration (ug/ml)

Peak area

Concentration (ug/ml)

Peak area

blank

0

0.00

0

0.00

Inj 1

0.5

20578

0.75

23247

Inj 2

0.75

28082

1.125

34034

Inj 3

1.0

36235

1.50

42297

Inj 4

1.25

43191

1.875

48858

Inj 5

1.5

54267

2.25

56133

Regression equation

Y=34981x + 1241

Y=26601x + 579.7

Linearity (μg/ml)

0.5-1.5 μg/ml

0.75-2.25 μg/ml

Correlation coefficient

( R2)

0.995

0.997


Fig. 5 Graph of Linearity of EMT and TDF

 

 

3.1.4. Accuracy:

The accuracy for proposed method was determined, recovery studies were performed in mentioned levels and recorded (Table 3), Obtained results were found to be within the limits of 98-102%, indicating an agreement between the true value and found value.

 


 

 

Table 3. Accuracy of EMT and TDF

 

EMT

TDF

Level

50%

(0.5 g/ml)

100% (1.0 g/ml)

150% (1.5 g/ml)

50% (0.75 g/ml)

100% (1.5 g/ml)

150% (2.25 g/ml)

Amount of drug added

0.5 g/ml

1 g/ml

Amount of drug recovered( g/ml)

1.010.34

1.520.17

1.990.16

1.760.25

2.540.21

3.240.12

% Recovery

101.00

101.33

99.50

100.57

101.60

99.69

%CV

1.07

0.63

0.61

1.27

1.00

0.73

N

6

6

 


3.1.5. Precision:

Precision was calculated as intra-day and inter-day variations for the drugs. Percent relative standard deviations for estimation of EMT and TDF under intra-day and inter-day variations were found to be less than 2. Results were showed in Table 4 and 5


 

Table 4. Intraday Precision of EMT and TDF

Drug

level

Concentration (μg/ml)

Concentration found* (μg/ml)

SD

(%RSD)

%Recovery

EMT

50%

0.50

0.50

0.0039

0.79

100.00

100%

1.00

1.01

0.0046

0.45

101.00

150%

1.50

1.48

0.0059

0.39

98.66

TDF

50%

0.75

0.74

0.0026

0.35

98.66

100%

1.50

1.51

0.0055

0.36

100.66

150%

2.25

2.23

0.0025

0.11

99.11

*Average of 6 determinations

 

Table 5. Interday Precision of EMT and TDF

Drug

level

Concentration (μg/ml)

Concentration found* (μg/ml)

SD

(%RSD)

%Recovery

 

EMT

50%

0.50

0.495

0.0049

0.98

99.00

100%

1.00

0.99

0.0056

0.56

99.00

150%

1.50

1.49

0.0069

0.46

99.33

 

TDF

50%

0.75

0.76

0.0026

0.34

101.33

100%

1.50

1.49

0.0065

0.43

99.33

150%

2.25

2.27

0.0045

0.19

100.88

*Average of 6 determinations

 


3.1.6. Limit of detection (LOD) and Limit of quantitation (LOQ):

The LOD and LOQ were calculated according to the S/N ratio of the respective drugs. The concentration of the drugs were reduced with regular intervals from 10 μg/ml concentration and injected into HPLC. The concentration with S/N 3:1 is taken as LOD and concentration with S/N 10:1 is taken as LOQ.

 

 

 

 

 

 

 

 

 

 

Table 6. LOD and LOQ of EMT and TDF

Drug

LOD (μg/ml)

LOQ (μg/ml)

EMT

0.01

0.05

TDF

0.1

0.5

 

3.1.7. Robustness:

For robustness studies, conditions like flow rate and concentration of organic phase were changed and method was performed. In all deliberately varied conditions, percent relative standard deviations for peak areas, retention times, theoretical plates and tailing factor were found to be less than 2% (Table 7).

 

 

 

 


 

Table 7. Robustness Parameters of EMT and TDF

S.No.

Parameter

Retention time* (min)

Peak area*

No of theoretical plates*

Tailing factor*

EMT

TDF

EMT

TDF

EMT

TDF

EMT

TDF

1

Initial conditions

2.79

4.91

32423

38568

6356

2542

1.0

1.0

2

Flow 0.9 ml/min

2.82

5.23

31654

42134

6412

2426

1.1

1.2

3

Flow 1.1 ml/min

2.64

4.74

32521

38534

6371

2416

1.0

1.1

5

Organic phase, 2% less (33%)

2.91

5.34

35269

42630

6459

5186

1.1

1.2

6

Organic phase, 2% more (37%)

2.54

4.63

37651

42123

6300

5031

1.0

1.1

7

Less pH (4.8)

2.75

4.89

35269

42741

6169

5162

1.2

1.1

9

More pH (5.2)

2.80

4.92

37651

42410

6135

5131

1.2

1.0

*Average of 3 determinations

 


3.2. Assay:

20 tablets were taken and their average weight was calculated, tablets were crushed to fine powder. Quantity of powder equivalent to 10 mg of EMT and 15 mg TDF was taken and dissolved using diluent in a 10 ml volumetric flask to obtain a concentration of EMT (1000 μg/ml), TDF (1500μg/ml). 1 ml of the above solution was diluted to 10ml with diluent to obtain a concentration of 100 g/ml of EMT and 150 g/ml TDF. 0.1 ml of the above solution was diluted to 10ml with mobile phase to obtain a concentration of 1g/ml of EMT and 1.5 g/ml of TDF. 20 l of the above solution was injected in to HPLC and the percent of assay was calculated using peak areas of standard and sample. The experimental values obtained for the determination of EMT and TDF in pharmaceutical formulation was within the claimed limits (Table 8).

 

Table 8. Assay data of marketed formulation

Drug

Amount labeled (mg)

Amount found*

% Assay

EMT

200

198.720.51 mg

99.36%

TDF

300

299.150.69mg

99.71%

*Values are expressed as mean SD (n= 3)

 

3.3. Stress testing studies:

The following stress testing studies were performed for EMT and TDF under acidic (0.1% HCl), alkaline (0.1% NaOH), oxidative (3% H2O2), photolytic (overall illumination ≥ 210 Wh/m2 at room temperature) and thermal (40⁰C2⁰C/75% RH5% RH) conditions to evaluate the ability of the method to separate EMT and TDF from their degradation products.

 

EMT and TDF showed high degradation in acidic and basic conditions when compared with oxidative, photolytic and thermal conditions. TDF showed high degradation in acidic and basic conditions when compared with EMT. TDF showed high degradation in Oxidative condition when compared with EMT. both drugs showed good stability under photolytic and thermal conditions with very less degradation.

 

The percent amount of drug degraded after degradation studies were given in Table 9. The chromatograms of EMT and TDF in different conditions were showed in table 10 and11. The pattern of degradation of the drugs individually in all the conditions and in different days was well portrayed in the Fig. 6 and 7. From the graphs it was evident that TDF showed high degradation in acidic and basic conditions when compared with EMT. EMT showed degradation when compared with TDF in UV light. In thermal condition both drugs showed equal degradation.

 

Table 9. Degradation data of EMT and TDF in different conditions

Day

Condition

% Degraded SD

of EMT

% Degraded SD

of TDF

 

 

 

1st Day

Acid

15.372.10

3.722.01

Base

8.962.66

15.661.83

Oxidative

2.992.51

3.352.58

Photolytic

1.551.40

0.882.01

Thermal

0.992.06

4.191.83

 

 

3rd Day

Acid

50.502.06

25.621.87

Base

21.451.49

34.922.07

Oxidative

13.922.51

14.892.04

Photolytic

1.982.01

2.531.69

Thermal

14.712.02

8.512.02

 

 

5th Day

Acid

84.192.13

55.832.04

Base

30.651.99

54.091.99

Oxidative

25.471.94

36.742.59

Photolytic

2.821.90

4.370.99

Thermal

18.392.52

13.912.11

7th Day

Acid

91.321.91

82.852.35

Base

82.620.53

95.251.25

Oxidative

46.142.36

59.390.29

Photolytic

4.831.89

6.032.45

Thermal

31.692.52

19.281.52

 


Table 10. Degradation chromatograms of EMT in different conditions

Condition

Day 1

Day 7

0.1M HCl

 

 

0.1M NaOH

 

 

Oxidative

 

 

Photolytic

 

 

Thermal

 

 

 

Table 11. Degradation chromatograms of TDF in different conditions

Condition

Day 1

Day 7

0.1M HCl

 

 

0.1M NaOH

 

 

Oxidative

 

 

Photolytic

 

 

Thermal

 

 

 


Fig. 6 showing degradation pattern of EMT

 


Fig. 7 showing degradation pattern of TDF


 

3.4    Kinetic investigation:

Treatment of EMT and TDF under specified stress conditions resulted in a gradual decomposition of both drugs in all conditions. The degradation of both drugs followed pseudo first-order kinetics as a linear relationship between log percentage remaining and time was established, having good correlation coefficients (Fig. 8 and 9). The Rate constant (K), time left for 50% potency (t1/2), and time left for 90% potency (t90) for each stress condition were calculated using eqs (1), (2) and (3) respectively [22].

 

(1)

 

(2)

 

(3)

 

Where

K is the rate constant C0 is the concentration at t=0, and Ct is the concentration at time t.


 

Fig. 8 First order plots for the degradation of EMT under different stress conditions.

 

Fig. 9 First order plots for the degradation of TDF under different stress conditions.

Table 11. Summary of kinetics

Stress condition

EMT

TDF

K (1/day)*

t1/2 (days)**

T90 (days)^

K (1/day)*

t1/2 (days)**

T90 (days)^

Acid

2.75 x 10-1

2.44

0.37

1.19 x10-1

2.01

0.30

Base

6.99 x 10-1

1.88

0.28

7.66 x10-1

1.91

0.28

peroxide

2.83 x 10-2

25.15

3.81

2.45 x10-2

90.36

13.69

Photolytic

3.67 x 10-2

7.17

1.32

3.62 x10-2

4.82

0.42

Thermal

3.19 x 10-2

9.90

1.50

3.43 x 10-2

5.77

0.87

* Rate constant per day; **Half life; ^Time left for 90% potency

 


Extensive degradation was found in basic condition, where K value was highest among all tested conditions. Both t1/2 and T90 were found to be lowest for Photolytic and highest for basic condition.The kinetic values were showed in table 11.

 

4. CONCLUSION:

In the developed HPLC method, different proportions of methanol and phosphate buffer were tried for selection of the mobile phase. Ultimately, 20 mM phosphate buffer (pH 5) in water and methanol in a proportion of 65:35 v/v respectively was finalized as the mobile phase. The elution order was EMT (Rt=2.79 min) and TDF (Rt=4.91 min), at a flow rate of 1.0 ml/min. The chromatogram was recorded at 271 nm. The developed method was validated as per ICH guidelines. Parameters like precision, accuracy, specificity, ruggedness, robustness were done and found to be within the acceptance criteria. The stability of the drugs was examined under different stress conditions such as acidic, basic, peroxide, photolytic and thermal conditions. The t1/2 values under different stress conditions were found to decrease in the following order; peroxide> thermal> photolytic> acidic> basic. Hence the RP-HPLC method could selectively quantify EMT and TDF in presence of its degradation products hence; it can be employed as a stability indicating method.

 

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Received on 20.01.2018 Modified on 01.03.2018

Accepted on 28.03.2018 AJRC All right reserved

Asian J. Research Chem. 2018; 11(3):569-579.

DOI:10.5958/0974-4150.2018.00102.5