Development and Validation of RP-HPLC Method for Determination of Ritonavir and Lopinavir

 

Fathima Qurratul Ayeen1, Dr. Ruheena Yasmeen2, Humera Badar3

1Assistant Professor, Department of Pharmaceutical Analysis and Quality Assurance, Nizam Institute of Pharmacy, Deshmukhi (V), Pochampally (M), Behind Mount Opera, Yadadri Bhuvanagiri (Dist)-508284, Telangana, India.

2Assistant Professor, Department of Pharm. D (Pharmacy Practice), Nizam Institute of Pharmacy,

Deshmukhi (V), Pochampally (M), Behind Mount Opera, Yadadri Bhuvanagiri (Dist)-508284, Telangana, India.

3Assistant Professor, Department of Pharmaceutical Analysis and Quality Assurance, Nizam Institute of Pharmacy, Deshmukhi (V), Pochampally (M), Behind Mount Opera, Yadadri Bhuvanagiri (Dist)-508284, Telangana, India.

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

 

ABSTRACT:

A novel simple, accurate, precise and selective high performance liquid chromatographic (HPLC) method was developed and validated for simultaneous estimation of Ritonavir and Lopinavir in tablet dosage form. An isocratic, reverse phase HPLC method was developed and validated using Hypersil C18 (250 mm×4.6 mm i.d.) 5µm, column and orthophosphoric acid pH3 and methanol (40:60) as mobile phase and detection is carried out at a wavelength of 273nm.The retention time for RITO and LOPI were 3.3±0.1 min and 4.7±0.1min respectively. The method was validated with respect to linearity, precision, accuracy and robustness.

 

KEYWORDS: Ritonavir, lopinavir, HPLC, UV, orthophosphoric acid (OPA).

 

 

INTRODUCTION:

Ritonavir (RITO),chemically 1,3-thiazol-5-ylmethylN-[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amin}butanamido]-1,6 diphenylhexan-2-yl] carbamate (Fig 1) is an inhibitor of HIV-1 protease used to treat HIV infection and AIDS. Currently rarely used for its own antiviral activity, but remains widely used as a booster of other protease inhibitors.1 It is official in Indian pharmacopeia2.

Lopinavir (LOPI), (2S)-N-[(2S,4S,5S)-5-[2-(2,6-dimethylphenoxy) acetamido]-4-hydroxy-1,6-diphenylhexan-2-yl]-3-methyl-2-(2-oxo-1,3-diazinan-1-yl) butanamid (Fig 2) is an antiretroviral of the protease inhibitor class. Inhibition of HIV-1 protease prevents cleavage of the viral polyprotein precursor and results in the release of immature, noninfectious virions.3

 

 

It is official in united states pharmacopeia4 The literature survey reveals that several UV spectrophotometric estimation in combinations5,6, HPLC7-16 and HPTLC17 have been reported till date for the analysis of RITO and LOPI as a single drug or in combination with other drugs in pharmaceutical dosage form. No reports were found to develop novel analytical method for this drug with low retention time. The existing available literature reports do not show any simple analytical method for the simultaneous estimation of RITO and LOPI in bulk and pharmaceutical dosage form. This paper describes simple, precise, accurate and sensitive HPLC method development and validation as per international conference on harmonization guidelines18.

 

 

Figure1: Structure of Ritonavir

 

Figure 2: Structure of Lopinavir

 

EXPERIMENTAL:

Reagents and chemicals:

Authentic sample of RITO and LOPI were obtained from Rainbow Laboratories Pvt Ltd (Hyderabad), respectively. The brand of tablet LOPIMUNE (manufactured by Cipla Ltd) labeled to contain Ritonavir 50mg, Lopinavir 200mg were procured from local market. Methanol (HPLC grade), HPLC grade water (HPLC/Merck), orthophosphoric acid (LR/ Standard Reagents) was obtained from rainbow laboratory (Hyderabad, India).

 

Chromatographic condition:

HPLC system used was Waters system equipped with model no 2695, Empower Software 2 Sonicator (Citizen, Digital Ultrasonic Cleaner), Rheodyne sample injection (10µl), PDA detector. A chromatographic column Hypersil (C18, 250 mm× 4.6 mm, 5µm) was used. Separation was carried out at flow rate of 1ml/min using orthophosphoric acid pH 3 and methanol (40:60 v/v) and detection at 275nm.

 

Preparation of standard stock solution:

Selection of detection wavelength:

From the standard stock solution further dilutions were done using HPLC grade water and scanned over the range of 200-400nm and the spectra was obtained. It was observed that both the drug showed considerable absorbance at 275nm. (Fig3)

 

 

 

Preparation of sample solution (Tablet formulation analysis):

Twenty Tablets were weighed and powdered. Tablet powder having weight equivalent to 50 mg of Ritonavir and 200 mg Lopinavir was weighed accurately and taken in a 100 mL volumetric flask. To it 10 mL of the methanol was added and sonicated for 15 minutes to dissolve the drugs. The volume was made up to 100 mL with HPLC grade water. The resulting solution was then filtered through a 0.45 µm membrane filter to prepare a stock solution of the tablet sample. From the above prepared test stock solution, 5 mL was taken to 25 mL volumetric flask and the volume was made up with the HPLC grade water. The concentration of Ritonavir and  Lopinavir in the solution was 100 µg/mL, 400 µg/mL respectively.

 

 

 

 

Figure 3: overlaid UV-vis spectra of RITO (100 µg/mL) and LOPI (400 µg/mL)

 

RESULT AND DISCUSSION:

Table1.Optimization of chromatographic conditions

Trial

Mobile Phase

Column

Ratio

Flow Rate

mL/min.

Retention Time

Observation

RITO

LOPI

Trial 1

OPA: Methanol

C8

60:40

1

2.948

_

Only single peak

Trial 2

OPA: Methanol

60:40

1

7.264

14.712

Theoretical plate below limit

Trial 3

OPA: Methanol

 

 

 

C18

  60:40

1

5.434

11.809

High Retention Time

Trial 4

OPA: Methanol

50:50

1

3.978

6.649

High Retention Time

Trial 5

OPA:      Methanol

40:60

1

3.505

4.724

Optimized Trial

Trial 6

OPA:   Methanol

40:60

1.2

4.065

5.171

Tailing Occurred

 

From the results it was observed that the optimized method selected was trial-5.i.e mobile phase 0.1%OPA: methanol (40:60).The resolution was good 5.50, column efficiency is 4854 for Ritonavir and 6904 for Lopinavir and the asymmetry of both Ritonavir and Lopinavir are 1.42 and 1.65 respectively. All parameters are within limits as per ICH guidelines.

 

 

Figure 4: Chromatograms of Method development trial

METHOD VALIDATION:

Linearity:

The linearity of the responses of the drugs was verified at six concentration levels, ranging from 50-150µg/ml for RITO and 200-600 µg/ml for LOPI respectively. The calibration graph was obtained by plotting peak area versus the concentration and data was treated by least squares linear regression analysis. The equation of the calibration curve found for RITO y=41158x+23829 and for LOPI y=12729x+29894 respectively. The calibration graphs were found to be linear in the plotted concentrations. The coefficient of determination was 0.999 for RITO and LOPI

 

 Figure 5: Graph for linearity data of RITO and LOPI

 

Precision:

The precision of the method was demonstrated by intraday studies. In the intraday studies, 6 replicates of RITO (50 µg/ml) and LOPI (200 µg/ml)were analyzed in a day and percentage RSD was calculated and found to be 0.12%

 

Accuracy:

Accuracy of the method was determined by Recovery studies. To the formulation (pre-analyzed sample), the reference standards of the drugs were added at the level of 50%, 100%, 150%. Formulation product (Tablet) conc. is kept constant (RITO – 100 mcg and LOPI – 400 mcg) and standard drugs were spiked. Three 25mL vol. flasks were taken and 0.25mL of the test stock solution was added to each flask and  2.5mL (50%), 5mL(100%) and 7.5mL(150%) of the standard stock solution (spike) were added to different vol. flask and the volume was made up to 25mL with mobile phase to obtain concentrations of RITO – 50 mcg and LOPI – 200 mcg (50%), RITO – 100 mcg and LOPI – 400 mcg (100%) and RITO  – 150 mcg and LOPI – 600mcg (150%) respectively.

 

The recovery studies were carried out by injecting three times and the percentage recovery and percentage mean recovery were calculated and shown below in table no 2 and table no3

 

Specificity:

The specificity of the method was ascertained by peak purity profiling studies, indicating no interference of any other peak by impurities.

 

 

Figure 5: Chromatogram of blank, Placebo, Standard and Sample

 

Limit of detection (LOD) and Limit of quantification (LOQ):

LOD and LOQ were calculated as 3.3σ/s and 10 σ /s, respectively; where is standard deviation of response and S is the slope of the calibration plot. The LOD of RITO and LOPI were 0.086 µg/mL  and 0.3023µg/mL respectively. The LOQ of RITO and LOPI were 0.287µg/mL 1.0078µg/mL respectively.

 

Robustness studies:

Robustness of the method was determined by carrying out the analysis under conditions during which flow rate and temperature were altered and observed that not much variation in retention time and are within limits. The results are shown in table no 4.

 

Table 2. Results of recovery tests of RITO

% Level

Amount present(µg/mL)

Amount added (µg/mL)

Peak area

Amount found(µg/mL)

% Recovery*

% mean Recovery

50

100

50

2056847

49.70

99

 

99

 

100

100

100

4106526

99.22

99

150

100

150

6148453

148.56

99

*Average of three determinations

 

Table 3.Results of recovery tests of LOPI

% Level

Amount present (µg/mL)

Amount added (µg/mL)

Peak area

 

Amount found(µg/mL)

% Recovery*

%  mean Recovery

50

400

200

2559897

199.51

100

 

100

 

100

400

400

5127798

399.64

100

150

400

600

7667947

597.61

100

*Average of three determinations

 

 

 

 

Table 4: Robustness data for RITO and LOPI

Para-meter

Value

RITO

LOPI

RT

Peak area

RT

Peak area

Flow-rate

(mL/min)

0.8

3.093

3754153

4.265

4751624

1.0

3.505

2800420

4.724

1941208

1.2

3.392

4126505

4.674

5109555

Column Temp. (0C)

25

3.092

3749235

4.258

4708205

30

3.580

2942652

4.750

7984721

35

3.388

4100799

4.656

5095025

 

 

CONCLUSION:

A renovative, simple and rapid RP-HPLC method was successfully developed for simultaneous estimation of Ritonavir and Lopinavir. The proposed method was found to be rapid, accurate, precise, specific, robust and economical. This method is also having an advantage that it has a short chromatographic time course of 7mins, so both the drugs can be assayed within the short time. The retention times for both the drugs were considerably less compared to the retention time obtained in past works done. It also found that the method shows non-interference of formulation excipients in the estimation of pharmaceutical dosage forms. Thus, the method is not time consuming and can be used in laboratories for the routine analysis of combination drugs.

 

REFERENCES:

1.          Drug information of  Ritonavir  http://www.scbt.com/datasheet-208310-ritonavir.html

2.          Indian Pharmacopoeia, 2007, volume 3, 1057-1060.

3.          Drug information of Lopinavir http://www.scbt.com/datasheet-207831-lopinavir.html

4.          United States Pharmacopoeia, 30. National Formulary, 25, 2007, 3143

5.          Jaiprakash N, Sachin B, Baig Salim, Indrajeet G. Spectrophotometric Method for Simultaneous Estimation of Lopinavir And Ritonavir In Bulk And Tablet Dosage Form International Journal Of Chem. Tech Research, 2014, 6(1): 823-827.

6.          Vaishali P, Nagulwar. Simultaneous estimation of Ritonavir and Lopinavir by Absorbtion ratio (Q-analysis) UV spectrophotometric method in combined tablet dosage form. Scholars Research Library.2010, 2(1): 196-200.

7.          An and Babu K, Jayakar B, Vinoth Kumar. G. Method Development And Validation Of Simultaneous Determination Of Ritonavir And Lopinavir In Pharmaceutical Dosage Forms by Rp-Hplc. Journal Of Pharmacy Research.2011, 4(9): 2985.

8.          Nagaraju P,  Indira Priyadarshini G and Appaji. Development and Validation of Reverse Phase HPLC Method for the Simultaneous Estimation of Lopinavir and Ritonavir in Pharmaceutical Dosage Forms. International Journal Of Research In Pharmaceutical And Biomedical Sciences.2012, 3(3): 1119-1124.

9.          Dasari V, Bahlul A, Chandu BabuRao, Khagga Mukkanti, Pappula Nagaraju. A Validated Reverse Phase HPLC method for the Simultaneous Estimation of Ritonavir and Lopinavir in Pharmaceutical Dosage Forms. Asian Journal of Research in Chemistry.2012, 3(3): 805-808.

10.        Kumar P, Murthy, Jithendra, Ryali, Nagaraju P.A Validated Reverse Phase HPLC Method for The Simultaneous Estimation of Lopinavir And Ritonavir In Pharmaceutical Dosage Forms. Academic Journal .2011, 4(8): 2819.

11.        Sarika R, Hemant P, Joshi S. Development of New RP HPLC Method for the Simultaneous Estimation of Lopinavir and Ritonavir in API and in Tablet Dosage Form. Asian Journal Of Research In Chemistry .2013, 6(6): 555–560.

12.        Vara Prasad L, Harinadha Baba k. Development method validation of RP-HPLC method for simultaneous determination of Lopinavir and Ritonavir in bulk and formulation dosage. International Research Journal Of Pharmaceutical And Applied Sciences.2012, 2(4):84-90.

13.        Jyothi M. Salunke, Vinit D. Chavhan, Sawant. S. A validated RP-HPLC method for simultaneous estimation of lopinavir and ritonavir in combined dosage form. Scholars Research Library.2013, 5(4):1-6.

14.        Uneetha A, Kathirvel S.A validated RP-HPLC method for simultaneous estimation of lopinavir and ritonavir in combined dosage form. International Journal Of Pharmacy And Pharmaceutical Sciences.2011, 3(1):50-51.

15.        Jagadeeswaran M, Gopal N, Pavan Kumar K and Siva kumar T. Quantitative Estimation of Lopinavir and Ritonavir in Tablets by RP-HPLC Method. Pharmaceutica Analytica Acta.2012, 3(5): 1-3.

16.        Vinodkumar K, Sudhakar M, Padmanabha Reddy Y, Hafeez. RP-HPLC Method Development And Validation For Simultaneous Estimation Of Lopinavir And Ritonavir In Dosage Form And In Plasma. International Journal Of Pharma Research and Review.2014, 3(9):1-8.

17.        Dharaj. Patel, Sapna D. Simultaneous HPTLC Determination Of Lopinavir And Ritonavir In Combined Dosage Form. Asian Journal of Pharmaceutical And Clinical Research.2011, 4(1).

18.        ICH, International Conference on harmonization of technical requirements for registration of pharmaceuticals for human use, Q2(R1),Validation of Analytical Procedures, Methodology, 19996, London, 2005.

 

 

 

 

 

 

 

 

Received on 20.12.2018           Modified on 21.01.2019

Accepted on 26.02.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(7):3413-3417.

DOI: 10.5958/0974-360X.2019.00577.8