2Womens Degree College, Guntur-522004, India
3Vagdevi College of Pharmacy, Gurazala, Guntur-522415, India
#Authors are equally contributed
Keywords: Emtricitabine; Tenofovir disoproxil fumarate; RPHPLC; Validation
Tenofovir disoproxil Fumarate (TDF) is fumaric acid salt of the bisisopropoxycarbonyl- oxymethyl ester derivative of tenofovir. Chemically it is 9-[(R)-2-[[(isopropoxycarbonyl) oxy]-methoxy] phosphiny]methoxy]propyl]adenine fumarate (Figure 1). It is used in combination with other antiretroviral for the treatment of HIV infection [2,3]
Literature survey reveals that few RP-HPLC [4-6] methods are reported for estimation of ETB, TDF and efavirenz in pharmaceutical formulation. TDF is estimated individually by UV [7], derivative-HPLC [8], HPTLC [9] in pharmaceutical formulation. ETB, TDF were estimated in biological matrices by HPLC [11,12] and LC/ MS/ MS [13-25], methods. The reported methods [4-6] for estimation of ETB, TDF in pharmaceutical formulations have some drawbacks in terms of sensitivity, ruggedness and robustness. The purpose of this study was to develop simple, rapid, precise and accurate RP-HPLC method for the simultaneous estimation of both the drugs in combined tablet dosage form.
After series of trials with various C8 and C18 columns, the final choice of stationary phase giving satisfactory resolution and run time was the reversed phase Phenomenex-Luna C18 column. 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 use of acetonitrile: 10 mM phosphate buffer (pH 6.8) (60:40, v/ v), with 1.0 mL/ min. From the overlain UV spectra, suitable wavelength considered for monitoring the drugs was 260 nm (Figure 2). Solutions of FTB 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. Under the optimum chromatographic conditions, the retention time obtained for ETB and TDF were 2.81 and 7.42 min respectively. The chromatogram of placebo, standard and test formulation was depicted Figure 2-4.
Linearity: Linearity was found to be 40-240 μg/ mL for ETB and 60-360 μg/ mL for TDF. The linear regression for ETC and TDF were (r2 = 0.9934) and (r2 = 0.9999). The results were depicted in Table 2 and Figure 5,6.
Accuracy: Accuracy of developed method was confirmed by doing recovery study as per ICH norms at three different concentration levels 80%, 100% and 120% by replicate analysis (n = 3). The result of accuracy study was reported in Table 3. From the recovery study it was clear that the method is very accurate for quantitative estimation of ETB and TDF in tablet dosage form as all the statistical results were within the range of acceptance. The results were depicted in Table 3.
Tablet |
Label claimed (mg) |
Conc. found (mg) |
% Assay |
|||
ETOF Tablets |
ETB |
TDF |
ETB |
TDF |
ETB |
TDF |
20.0 |
30.0 |
20.05 |
29.98 |
100.21 |
98.97 |
S.No. |
Concentration µg/ ml |
Area of Emtricitabine |
Concentration µg/ ml |
Area of Tenofovir |
|
1 |
40 |
22988.567 |
60 |
16998.424 |
|
2 |
80 |
47605.237 |
120 |
33602.702 |
|
3 |
120 |
68028.039 |
180 |
50459.14 |
|
4 |
160 |
90704.169 |
240 |
67137.714 |
|
5 |
200 |
102120.617 |
300 |
84014.837 |
|
6 |
240 |
130654.429 |
360 |
101710.108 |
|
Concentration range µg/ ml |
40-240 |
60-360 |
|||
Slope (m)
|
543.85 |
281.04 |
|||
Correlation co-efficient (r2) |
0.993 |
0.999 |
Level of % recovery |
Target Conc. (µg/ ml) |
Amount of drug spiked (µg/ ml) |
Drug recovered (µg/ ml) |
%Recovery |
Mean |
SD |
%RSD |
80 |
100 |
80 |
181.13 |
100.62 |
99.95 |
1.42 |
1.27 |
180.01 |
100.22 |
||||||
179.85 |
99.98 |
||||||
100 |
100 |
100 |
200.10 |
100.21 |
100.22 |
1.23 |
1.12 |
202.21 |
100.23 |
||||||
200.11 |
100.21 |
||||||
120 |
100 |
120 |
221.21 |
101.15 |
100.32 |
1.15 |
1.46 |
220.34 |
100.33 |
||||||
220.41 |
100.42 |
Sample. No |
Intra day |
Inter day |
||
%Label Claim |
%Label Claim |
|||
ETB |
TDF |
ETB |
TDF |
|
1 |
100.31 |
100.21 |
99.67 |
99.541 |
2 |
99.41 |
99.61 |
99.12 |
99.93 |
3 |
99.34 |
100.02 |
100.42 |
100.35 |
4 |
99.22 |
100.05 |
99.98 |
99.76 |
5 |
100.33 |
100.08 |
100.1 |
100.43 |
6 |
100.14 |
99.99 |
100.22 |
99.99 |
Mean |
99.75 |
100.21 |
99.26 |
99.54 |
S.D |
0.2262 |
0.226247 |
0.3742 |
0.3742 |
% R.S.D |
0.2264 |
0.2264 |
0.3742 |
0.3742 |
Limit of detection and Limit of Quantification: LOD is found to be 1.5456 μg/ mL for Emtricitabine and 0.0712 μg/ mL for Tenofovir and LOQ are found to be 4.5924 μg/ mL for Emtricitabine and 13.931μg/ mL for Tenofovir.
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