RP-HPLC Method Development and Validation for the Analyisis of ...

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701 

_________________________________________Research Article 

RP-HPLC Method Development and Validation for the 

Analyisis of Telaprevir in Pharmaceutical Dosage Forms 

B. Lakshmi 1 , K.Saraswathi 2 and TV.Reddy 3 

1 

Kallam Haranadha Reddy Institute of Technology,NH-5, Chowdavaram, Guntur, 

Andhra Pradesh, India. 

2 

SV University, Tirupati, Andhra Pradesh, India. 

3 

Department of chemistry, Malla Reddy College of Engineering, Secunderabad, 

Andhra Pradesh, India. 

ABSTRACT 

A simple, selective, linear, precise and accurate RP-HPLC method was developed and validated for rapid assay 

of Telaprevir in tablet dosage form. Isocratic elution at a flow rate of 0.8ml/min was employed on a symmetry 

C18 (250x4.6mm, 5µm in particle size) at ambient temperature. The mobile phase consisted of 

0.1%orthophosphoric acid: Acetonitrile: Methanol 5:15:80 (v/v/b). The UV detection wavelength was 254nm 

and 20µl sample was injected. The retention time for Telaprevir was 4.282 min. The percentage RSD for 

precision and accuracy of the method was found to be less than 2. The method was validated as per the ICH 

guidelines. The method was successfully applied for routine analysis of Telaprevir in tablet dosage form and in 

serum. 

Key Words: Telaprevir, RP-HPLC, UV detection, serum, recovery, precise. 

INTRODUCTION 

Telaprevir is a pharmaceutical drug for the 

treatment of hepatitis C co-developed by Vertex 

and Johnson & Johnson. It is a member of a class 

of antiviral drugs known as protease inhibitors 1 . 

Specifically, telaprevir inhibits the hepatitis C virus 

NS3.4A serine protease 2 . 

IU/mL at treatment weeks 4 or 12, OR and HCV- 

RNA levels detectable at treatment week 24. 

Telaprevir Must be administered in combination 

with peginterferon alfa and ribavirin. If 

peginterferon alfa or ribavirin is discontinued for 

any reason, telaprevir must also be discontinued. 

To prevent treatment failure, dose must not be 

reduced or interrupted. 

EXPERIMENTAL 

Chemicals and reagents 

HPLC grade Actonitrile,Methanol and 

Orthophosphoric Acid was purchased from Merck 

Specialities Pvt. Ltd. 

Fig. 1: Structure of Telaprevir 

Telaprevir is the first hepatitis C drug that has 

demonstrated activity in patients who have failed 

prior therapy 3 . 

Patients with inadequate viral response are unlikely 

to achieve sustained virologic response, and may 

develop treatment-emergent resistance 

substitutions. Discontinuation of therapy is 

recommended in all patients with either of the 

following circumstances: HCV-RNA levels >1000 

Instrumentation and analytical conditions 

The analysis of drug was carried out on a PEAK 

HPLC system equipped with a reverse phase C18 

column (250x4.6mm, 5µm in particle size), a LC- 

P7000 isocratic pump, a 20µl injection loop and a 

LC-UV7000 absorbance detector and running on 

PEAK Chromatographic Software version 1.06. 

Isocratic elution with 0.1% Orthophosphoric Acid: 

Acetonitrile:Methanol 5:15:80 (V/V) (P H -4.8) was 

used at a flow rate of 0.8ml/min and Detector 

wavelength 254nm. The mobile phase was 

prepared freshly and degassed by sonicating for 5 

min before use. 

Vol. 3 (4) Oct – Dec 2012 www.ijrpbsonline.com 1483


Stock and Working standard and sample 

solutions 

Accurately weigh and transfer 10mg of Telaprevir 

working standard into a 10ml volumetric flask add 

diluent and sonicate to dissolve it completely and 

make volume up to the mark with the same solvent. 

Further pipette 1ml of the above stock solution into 

a 10ml volumetric flask and dilute up to the mark 

with diluent. Mix well and filter through 0.45µm 

nylon filter paper. The calibration curve was 

plotted with the five concentrations of the 160-60 

µg/ml working standard solutions. Calibration 

solutions were prepared daily and analyzed 

immediately after preparation. 

A composite of 20 tablets was prepared by grinding 

them to a fine, uniform size powder. 10 mg of 

Telaprevir was accurately weighted and 

quantitatively transferred into a 100 ml volumetric 

flask. Approximately 25 ml mobile phase were 

added and the solution was sonicated for 15 min. 

The flask was filled to volume with mobile phase, 

and mixed. After filtration, an amount of the 

solution was diluted with mobile phase to a 

concentration of 100μg/ml. 

Validation procedure 

The objective of the method validation is to 

demonstrate that the method is suitable for its 

intended purpose as it is stated in ICH guidelines. 

The method was validated for linearity, precision 

(repeatability and intermediate precision), 

accuracy, specificity, stability and system 

suitability. Standard plots were constructed with 

five concentrations in the range of 160µg/ml to 

60µg/ml prepared in triplicates to test linearity. The 

peak area of Telaprevir was plotted against the 

concentration to obtain the calibration graph. The 

linearity was evaluated by linear regression 

analysis that was calculated by the least square 

regression method. The precision of the assay was 

studied with respect to both repeatability and 

intermediate precision. Repeatability was 

calculated from five replicate injections of freshly 

prepared Telaprevir test solution in the same 

equipment at a concentration value of 100% 

(100µg/ml) of the intended test concentration value 

on the same day. The experiment was repeated by 

assaying freshly prepared solution at the same 

concentration additionally on two consecutive days 

to determine intermediate precision. Peak area of 

the Telaprevir was determined and precision was 

reported as %RSD. 

Method accuracy was tested (% recovery and 

%RSD of individual measurements) by analyzing 

sample of Telaprevir at three different levels in 

pure solutions using three preparations for each 

level. The results were expressed as the percentage 

of Telaprevir recovered in the samples. Sample 

solution short term stability was tested at ambient 

temperature (20±10 0 C) for three days. In order to 

confirm the stability of both standard solutions at 

100% level and tablet sample solutions, both 

solutions protected from light were re-injected after 

24 and 48 hours at ambient temperature and 

compared with freshly prepared solutions. 

RESULT AND DISCUSSION 

Optimization of the chromatographic conditions 

Proper selection of the stationary phase depends up 

on the nature of the sample, molecular weight and 

solubility. The drug Telaprevir is non-polar. Nonpolar 

compounds preferably analyzed by reverse 

phase columns. Among C8 and C18, C18 column 

was selected. Non-polar compound is very 

attractive with reverse phase columns. So the 

elution of the compound from the column was 

influenced by polar mobile phase. Mixture of ortho 

phosphoric acid, methanol and acetonitrile was 

selected as mobile phase and the effect of 

composition of mobile phase on the retention time 

of Telaprevir was thoroughly investigated. The 

concentration of the ortho phosphoric acid, 

methanol and acetonitrile were optimized to give 

symmetric peak with short run time (Fig.2). 

Fig. 3: Typical chromatogram of Telaprevir 



Validation of method 

Linearity 

Five points graph was constructed covering a 

concentration range 10-50µg/ml (Three 

independent determinations were performed at each 

concentration). Linear relationships between the 

peak area signal of Telaprevir the corresponding 

drug concentraton was observed. The standard 

deviation of the slope and intercept were low. The 

statistical analysis of calibration is shown in Table. 

Table: Result of Linearity 

S.No 

Sample 

Concentration 

(µg/ml) 

Area 

1 60 335879 

2 80 432879 

3 100 544728 

4 120 648723 

5 140 734892 

6 160 832567 

Result 

Slope: 5203.9 

Intercept: - 

4834.35 

C.C: 0.9991 

Interday Precision 

Table: Results of Interday Precision 

Sample 

Area 

(100µg/ml) 

1 542560 

2 544882 

3 546585 

4 535588 

5 539566 

6 540256 

Result 

R.S.D= 0.732 

Accuracy 

The accuracy of the method was determined by 

calculating recovery of telaprevir (60,90,120ppm) 

by the method of standard addition. Known amount 

of telaprevirwas added to a pre quantified sample 

solution and the amount of telaprevir was estimated 

by measuring the peak area ratios and by fitting 

these values to the straight line equation of 

calibration curve. The recovery studies were 

carried out three times over the specified 

concentration range and amount of telaprevir was 

estimated by measuring the peak area ratios by 

fitting these values to the straight line equation of 

calibration curve. From the above determination, 

percentage recovery and standard deviation of 

percentage recovery were calculated. 

Recovery 

Table: Recovery Results 

Sample 

concentration 

(µg/ml) 

Recovery 

(µg/ml) 

% of Recovery 

50% 60 59.48 99.73 

75% 90 89.63 99.58 

100% 120 120.02 100.01 

Graph. 1: Linearity Graph of Telaprevir 

Precision 

The validated method was applied for the assay of 

commercial tablets containing Telaprevir. Sample 

was analyzed for six times after extracting the drug 

as mentioned in assay sample preparation of the 

experimental section. The results presented good 

agreement with the labeled content. Low values of 

standard deviation denoted very precision. The 

result is given in Tables. 

Table: Results of Intraday Precision 

Sample 

Area 

(100µg/ml) 

1 544622 

2 538562 

3 533565 

4 545412 

5 544008 

6 541258 

Result 

R.S.D= 1.81 

Specificity 

The specificity of the method was determined by 

comparing test results obtained from analysis of 

sample solution containing excipients with that of 

test results those obtained from standard drug. 

LOD and LOQ 

Limit of detection (LOD) and limit of 

quantification (LOQ) were calculated as 

5microgram/ml and 15 microgram/ml respectively 

as per ICH guide-lines. 

LOD and LOQ 

Table: LOD and LOQ Results 

LOD 

LOQ 

5(µg/ml) 

15(µg/ml) 

Robustness 

To determine the robustness of the method, three 

parameters from the optimized chromatographic 

conditions were varied. 



Table: Robustness Results 

Sample condition (100µg/ml) Area % of Recovery 

Flow rate change 

0.7ml/min 

0.9ml/min 

Wavelength Change 

256nm 

252nm 

Mobile phase ratio change 

MeOH: ACN: 5%OP 

78 17 5 (v/v/v) 

82 13 5 (v/v/v) 

548253 

543251 

543220 

542545 

544569 

547140 

100.64 

99.72 

99.72 

99.53 

99.97 

100.44 

Table: System suitability parameters 

Parameter 

Values 

λ max (nm) 254 

Beer’s law limit (µg/ml) 60-160 

Correlation coefficient 0.999 

Retention time 

4.282min 

Theoretical plates 9796 

Tailing factor 1.36 

Limit of detection (µg/ml) 5 

Limit of quantification (µg/ml) 15 

Ruggedness 

Inter day variations were performed by using six 

replicate injections of standard and sample 

solutions of concentrations which were prepared 

and analyzed by different analyst on three different 

days over a period of one week. Ruggedness also 

expressed in terms of percentage relative standard 

deviation. 

Ruggedness 

Table: Ruggedness results 

Sample 

(100µg/ml) 

Area 

1 549585 

2 546858 

3 544990 

4 546202 

5 546623 

6 550021 

Result 

R.S.D= 0.363 

System suitability 

The system suitability parameter like capacity 

factor, asymmetry factor, tailing factor and number 

of theoretical plates were also calculated. It was 

observed that all the values are within the limits 

(Table.8). The statistical evaluation of the proposed 

method was 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 reliable determination of 

Telaprevir in tablet formulation. The results are 

furnished in Table. 

Assay of Telaprevir tablets 

Weigh 20 Telaprevir (Gluconil-5mg) tablets and 

calculate the average weight. Accurately weigh and 

transfer the sample equivalent to 10mg of 

Telaprevir in to a 10ml volumetric flask. Add 

diluent and sonicate to dissolve it completely and 

make volume up to the mark with diluents. Mix 

well and filter through 0.45um filter. Further 

pipette 1ml of the above stock solution into a 10ml 

volumetric flask and dilute up to mark with diluent. 

Mix well and filter through 0.45um filter. An 

aliquot of this solution was injected into HPLC 

system. Peak area of Telaprevir was measured for 

the determination. The results are furnished in 

Table. 

Table: Formulation results of Telaprevir 

Formulation Dosage Concentrati 

Incovek 

(Tablet) 

on 

Amount 

found 

375mg 100ppm 99.91pp 

m 

% 

Estimated 

99.91 

CONCLUSION 

A validated RP-HPLC method has been developed 

for the determination of Telaprevir in tablet dosage 

form. The proposed method is simple, rapid, 

accurate, precise and specific. Its chromatographic 

run time of 10 min allows the analysis of a large 

number of samples in short period of time. 

Therefore, it is suitable for the routine analysis of 

Telaprevir in pharmaceutical dosage form. 



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