1. asymmetry were observed in the ratio of 50:50% v/v of acetonitrile and methanol. It was
found to be the optimum mobile phase concentration.
3.2. Choice of internal standard
Several substances were tested as internal standards. Among these, lamotrigine
has beenchosen as the most appropriate in the present analysis because it is
stable. In the present study, itdid not interfere with the matrix of pharmaceutical
samples and it was well separated from TDF. More over, a significant advantage of this
IS was its elution time that was shorter than that of TDFr e s u l t i n g i n s h o r t r u n t i m e ,
l e s s t h a n 5 m i n . A t yp i c a l c h r o m a t o g r a m o f T D F a n d I S u s i n g
t h e proposed method is shown in Fig.2. Sharp and symmetrical peak was obtained with good
baselinefor each compound, thus facilitating the accurate measurements of peak area. The
average retentiontimes for TDF and IS were found to be 4.11 ± 0.03 and 2.27 ±
0.02 min, respectively. Under thedescribed parameters, the respective
compounds were clearly separated and their corresponding peaks were sharply
developed at reasonable retention times.
3.3. Validation of methods3.3.1. Linearity
Five points calibration graphs were constructed covering a concentration range 1-5 µg/
ml(see section 2.3). Three independent determinations were performed at each concentration.
Linear relationships between the ratio of peak area signal of TDF to that of IS
versus the correspondingdrug concentration were observed, as shown by the
results presented in Table 1. The standard deviations of the slope and intercept were
low. The determination coefficient (r
2
) exceeded 0.999.T o d e t e r m i n e w h e t h e r t h e e x p e r i m e n t a l i n t e r c e p t ( a )
o f t h e r e g r e s s i o n e q u a t i o n w a s n o t significantly different from the theoretical
zero value, confidence interval (99%) and student’s t-testwere performed. It concerns the
comparison of t = a/ s
a
where a is the intercept of the regressionequation and s
a
is the standard deviation of a, with tabulated data of the t -distribution.
A s t h e calculated t value (t = 1.4350) does not exceed to (0.001, 14) = 4.140, the
intercept of regressionequation is not significantly different from 0 (point estimation). By
using 99% confidence interval,the value lies between 0.0273 - 0.0452.This shows that the
intercept will fall on this range and thedistance from zero is very short (Interval estimation).
3.3.2. Precision
The repeatability study (n=6) carried out showed a R.S.D. of 1.340 % for the
peak a r e a r a t i o o f T D F o f I S o b t a i n e d , t h u s s h o w i n g t h a t t h e e q u i p m e n t
used for the study worked
correctly for the developed analytical method and being highly repetitive. For
the intermediate precision a study carried out by the same analyst working on 3
consecutive days (n = 3) indicated aR.S.D. of 0.744 and 1.126 %. Both values were
far below 2%, the limit percentage set for the precision and indicated a good method
precision.
3.3.3. Accuracy
The data for accuracy were expressed in terms of percentage recoveries of TDF
inthe real samples. These results are summarized in Table 2. The mean recovery data of TDF
in realsample were within the range of 99.18 and 100.84 % for Forzest and 98.70 and 100.85

2. for Tazzle.Mean % R.S.D. was 1.348 % and 0.880 %, satisfying the acceptance criteria for
the study.
3.3.4. Specificity
The HPLC chromatogram recorded for the mixture of the drug excipients
revealedno peak within a retention time range of 5 min. The results showed that the
developed method wasspecific as none of the excipients interfered with the analytes of
interest (Fig.2).
3.3.5. Stability
The stability of TDF in standard and sample solutions containing IS determined by storingthe
solutions at ambient temperature (20 ± 1°C) protected from light. The solutions were
checkedin triplicate after 3 successive days of storage and the data were
compared with freshly preparedsamples. In each case, it could be noticed that solutions
were stable for 48 hrs, as during this timethe results did not decrease below 97%. This
denotes that TDF is stable in standard and samplesolutions for at least 2 days at
ambient temperature, protected from light and is compatible with IS.
3.3.6. System suitability
The resolution factor between IS and TDF, in the developed method, was above 2.The %
R.S.D. of peak area ratios of TDF to that of IS and retention times for both drug and IS
werewithin 2% indicating the suitability of the system (Table 3). These results indicate the
applicabilityof this method to routine with no problems, its suitability being
proved. The system suitability parameter like capacity factor, asymmetric factor,
tailing factor, HETP and number of theoretical p l a t e s a l s o c a l c u l a t e d . I t w a s
observed that all the values are within the limits. The
s t a t i s t i c a l evaluation of the proposed method revealed its good linearity, reproducibility
and its validation for different parameters and let us to the conclusion that it could
be used for the rapid and reliabledetermination of TDF in tablet formulation.
3.4. Assay of tablets
The validated method was applied for the assay of two
c o m m e r c i a l t a b l e t s containing 20mg of TDF: Tazzle and forzest. Each
s a m p l e w a s a n a l yz e d i n t r i p l i c a t e a f t e r e x t r a c t i n g t h e d r u g a s
mentioned in assay sample preparation of the experimental
s e c t i o n (section 2.4) and injections were carried out in triplicate Fig.2. shows a
HPLC chromatogram of TDF in pharmaceutical tablets. None of the tablet ingredients
interfered with the analyte peak. Ther e s u l t s p r e s e n t e d i n T a b l e 4 a r e i n g o o d
agreement with the labeled content. Assay results, e x p r e s s e d a s t h e
percentage of label claim, were found to be 99.21 ± 1.340 for
F o r z e s t ; 99.27 ± 1.253 for Tazzle showing that the content of TDF in tablet formulations
confirmed to thecontent requirements (95-105 %) of the label claim. Low values of standard
deviation denoted veryg o o d r e p r o d u c i b i l i t y o f t h e m e a s u r e m e n t . T h e a b o v e
r e s u l t s d e m o n s t r a t e d t h a t t h e d e v e l o p e d method achieved rapid and accurate
determination of TDF and could be used for the determinationof TDF drug substance and
pharmaceutical formulations.
4. Conclusion
A validated isocratic HPLC-UV method has been developed for the determination of TDF in
dosage forms Forzest and Tazzle. The proposed method is simple, rapid, accurate,
precise,a n d s p e c i f i c . I t s c h r o m a t o g r a p h i c r u n t i m e o f 5 m i n a l l o w s t h e
a n a l ys i s o f a l a r g e n u m b e r o f s a m p l e s i n a s h o r t p e r i o d o f t i m e .
T h e r e f o r e , i t i s s u i t a b l e f o r t h e r o u t i n e a n a l ys i s o f T D F
i n pharmaceutical dosage forms. The simplicity of the method allows for

3. application in laboratoriesthat lack sophisticated analytical instruments such as
LC-MS that is complicated, costly and time consuming rather than a simple HPLC-UV
method. Hence the proposed method could be useful for the national quality control
laboratories in developing countries