Analytical Method Development and Validation for the Estimation of Sacubitril and Valsartan in Combined Pharmaceutical Dosage Forms by RP-HPLC

 

Y. Phalguna1, Noor Jahan2*, Indraja N3, Satheesh Kumar G2

1Associate Professor, Jyothishmathi College of Pharmacy, Turkapally.

2Assistant Professor, Jyothishmathi College of Pharmacy, Turkapally.

3Assistant Professor, CMR College of Pharmacy, Kandlakoya.

*Corresponding Author E-mail: azher9849022431@gmail.com

 

ABSTRACT:

The Present work was to develop a simple, fast, accurate, precise, reproducible, Reverse Phase High Performance Liquid Chromatographic Method for simultaneous estimation of Valsartan and Sacubitril in pure drug form. Chromatographic separation was done using thermosil C18 column having dimension of (100 mm x 4.6 mm) having particle size of 5.0 µm, with mobile phase consisting of Phosphate buffer (KH2PO4 and K2HPO4) pH 3 ±0.02 pH adjusted with ortho phosphoric acid and Acetonitrile  (25:75 %v/v), flow rate was adjusted to 0.8 ml/min and detection wavelength at 254nm. The retention times of Valsartan and Sacubitril was found to be 2.589 and 3.711mins. The method has been validated for accuracy, precision, linearity, robustness and range. Linearity for Valsartan and Sacubitril was found 0.2µg-0.6µg and 0.1µg-0.3µg and correlation coefficient was 0.999 and 0.999 % RSD for intermediate precision was found to be 0.1 and 0.2, for repeatability was 0.2 and 0.5, % mean recovery for Valsartan and Sacubitril was found to be 100.599% to 101.22% respectively. The method was found to be robust even by change in the mobile phase ±5% and in less flow condition.

 

KEYWORDS: Valsartan, Sacubitril, RP-HPLC, Accuracy, Precision, Linearity, Robustness, Range.

 

 


INTRODUCTION:

High Performance Liquid Chromatography (HPLC) is most widely used analytical technique. Chromatography process can be defined as separation techniques involving mass-transfer between stationary and mobile phases.

 

Principle:

The principle involved in high pressure liquid chromatography is adsorption. The separation of mixture of components is based on their relative affinities towards the stationary phases. The component has more affinity towards the stationary phase travels slower and eluted later2-3. The component has less affinity towards the stationary phases travels faster and eluted first. Since no two components have the same affinity towards the stationary phases so the components were separated4-5.

 

METHOD DEVELOPMENT:

The method development of analysis of any compound is usually based on existing literature, using same or quite similar instrumentation. The development of new or any improved method should be beneficial in any way than the existing method. Method development usually requires selecting the method requirement and deciding the instrumentation to utilize for that purpose6. Method development is done for, new drug products and already existing products. The various chromatographic conditions that include to be optimized during method development are:

·      Mode of separation.

·      stationary phase Selection

·      mobile phase Selection

·      detector  Selection

 

ANALYTICAL METHOD VALIDATION7:

Here the method which is modified to estimate the content, assay, purity or standard of a pharmaceutical product, test for its efficacy and accuracy, that is it is assessed if the method adapted is capable of giving consistent and correct result without any error even if there is a change in change in chemicals, instrument or person this has to be validated by cross checking separated and documented as evidence for efficacy and efficiency of method adapted.


 

HPLC Instrumentation:

The Various parts of HPLC equipment are:

 

1. Reservoir - that holds the mobile phase. ; 2. Pumps - A & B to pump the mobile phase. ; 3. Sample injection - to inject the sample.

4. Columns - that contains the stationary phase. ; 5. Detectors - to detect the results. ; 6. Recorder – to display the results.

 


Hypertension is defined as increase in arterial blood pressure above normal i.e., 120/80 mm Hg. It is itself not a disease but is an indicative of other CVS complications like Myocardial infarction, Stroke, Malignant hypertension, Peripheral vascular diseases etc. Valsartan and Sacubitril are in clinical development as a treatment in both the cases-Hypertension and Heart failure.

 

Sacubitril is a prodrug that is activated to sacubitrilat (LBQ657) by de-ethylation via esterases. Sacubitrilat inhibits the enzyme neprilysin, which is responsible for the degradation of atrial and brain natriuretic peptide, two blood pressure-lowering peptides that work mainly by reducing blood volume.

 

 

Valsartan is an ARB that selectively inhibits the binding of angiotensin II to AT1, which is found in many tissues such as vascular smooth muscle and the adrenal glands. This effectively inhibits the AT1-mediated vasoconstrictive and aldosterone-secreting effects of angiotensin II and results in a decrease in vascular resistance and blood pressure. Valsartan is selective for AT1 and has virtually no affinity for AT2. Inhibition of aldosterone secretion may inhibit sodium and water reabsorption in the kidneys while decreasing potassium excretion8.

 

Present work is aimed to develop a new, simple, fast, rapid, accurate, efficient and reproducible RP-HPLC method for the simultaneous analysis of valsartan and sacubitril .the development method will be validated according to ICH guideline.

 

The optimized method uses Column Thermosil C18 (100 mm x 4.6 mm) 5µg, mobile phase composition of ortho phosphoric acid and Acetonitrile in the ratio (25:75% V/V). The Flow rate was set to 0.8ml min-1 with UV detection was carried out at 254 nm.

 

The developed method was validated for various parameters such as accuracy, precision, ruggedness, linearity, robustness, system suitability, specificity as per ICH guidelines. All the validation parameters were found to be within the Acceptance criteria.

 

MATERIALS AND METHODS:

The materials employed in the study are Methanol, Acetonitrile, Ortho phosphoric acid, potassium dihydrogen phosphate and distilled water which are purchased from MERCK manufacturer. The equipments utilized in the work are U. V. Spectrometer (Lab India), HPLC-auto sampler – UV Detector (Waters), Digital weighing balance (Ascoset), pH meter (ADWA) and Sonicator (Enertech).

 

From the solubility analysis of the drugs Valsartan and Sacubitril it was found that they are soluble in water and methanol whereas insoluble in Acetonitrile. This was followed by method development procedure wherein suitable mobile phases are selected and the flow rate of mobile phase is set. Different trials were performed as follows by changing the mobile phases and columns employed in RP-HPLC.

 

Buffer preparation:

About 7.0g of potassium di hydrogen ortho phosphate was dissolved in 1000ml of HPLC grade water and PH 2.5 was adjusted with ortho phosphoric acid. It was filtered through 0.45µm nylon membrane filter and degassed with sonicator. It was used as a diluent for the preparation of sample and standard solution.

 

TRIAL-1

Preparation of mobile phase:

Mobile phase consist of water: methanol HPLC of PH 2.5 (30:70) was taken, sonicated and degassed for 10 min and filtered through 0.45 µm nylon membrane filter9-10.

 

Standard Preparation:

Weigh accurately 10mg Valsartan Working Reference Standard and 15mg of Sacubitril Working Reference Standard is taken in to 100ml volumetric flask and then it was dissolved and diluted to volume with mobile phase up to the mark. After that 50ml of the above solution was taken into 100ml standard flask and made up with mobile phase (Stock solution). Further pipette 0.5ml of the above stock solution in to a 10ml volumetric flask and dilute up to the mark with diluent.

 

Observation:

The separation of two analytical peaks was not proper, So the mobile phase ratio has been changed for next trial.

 

TRIAL-2

Preparation of mobile phase:

Mobile phase consist of water: acetonitrile of PH 2.5 (30:70) was taken sonicated and degassed for 10 min and filtered through 0.45µm nylon membrane filter.

 

Observation:

The separation of two analytical peaks was not proper, So the mobile phase ratio has been changed for next trial.

 

TRIAL-3

Preparation of mobile phase:

Mobile phase consist of buffer: Methanol of PH 2.5 (20:80) was taken sonicated and degassed for 10min and filtered through 0.45µm nylon membrane filter11-13.

 

Observation:

The separation of two analytical peaks was not proper, so the mobile phase ratio has been changed for next trial.

 

TRIAL-4

Preparation of mobile phase:

Mobile phase consist of buffer: Methanol of PH 2.5 (30:70) was taken sonicated and degassed for 10min and filtered through 0.45µm nylon membrane filter14.

 

Observation:

The separation of two analytical peaks is occurred but fronting occurs in Valsartan peak.

 

TRIAL-5

Preparation of mobile phase:

Mobile phase consist of buffer: Methanol of PH 2.5 (30:70) was taken sonicated and degassed for 10min and filtered through 0.45µm nylon membrane filter15.

 

Observation:

The separation of two analytical peaks was good but base line noise is occurred. So the mobile phase ratio has been changed for next trial.

TRIAL-6 (OPTIMIZED METHOD):

Preparation of mobile phase:

Mobile phase consisting Orthophosphoric acid: Acetonitrile (25: 75% v/v) pH 3 +/- 0.021 was taken, sonicated and degassed for 10min and filtered through 0.45 µm nylon membrane filter.

 

Observation:

The separation of two analytical peaks was good. The plate count also above 2000, tailing factor below 2, and the resolution is above 2. Hence this condition is taken as optimized method.

 

METHOD VALIDATION:

The chromatographic conditions were validated by evaluating linearity, accuracy, method precision, limit of detection (LOD), limit of quantization (LOQ), ruggedness and robustness in accordance with ICH guidelines.

 

1.    LINEARITY AND RANGE:

Preparation of stock solution:  

Weigh accurately 10mg Valsartan Working Reference Standard and 15mg of Sacubitril Working Reference Standard is taken in to 100ml volumetric flask and then it was dissolved and diluted to volume with mobile phase up to the mark. After that 50ml of the above solution was taken into 100ml standard flask and made up with mobile phase. (Stock solution)

 

Further pipette 0.5ml of the above stock solution in to a 10ml volumetric flask and dilute up to the mark with diluent. The solution was mixed well and used for chromatographic injection.

 

Valsartan:

20%, 30%, 40%, 50%, 60% dilutions are prepared using the stock solution.

 

Sacubitril:

10%, 15%, 20%, 25%, 30% dilutions of Sacubitril are prepared from stock solution.

These dilutions are injected separately and correlation coefficient (r2) was calculated.

 

2.    ACCURACY:

50%, 100%, 150% concentration of samples were prepared and injected. Then percent recovery was calculated as follows

 

                        Sample peak area x weight of standard

% Recover = --------------------------------------------X 100

                             Standard peak area x weight of sample

 

3.    PRECISION:

Six replicates of sample solutions were prepared from the tablet powder, injected and the chromatograms were developed as per the test procedure.

 

4.    Intermediate Precision (Ruggedness):

Six replicates of sample solutions were prepared from the tablet powder, injected and the chromatograms were developed as per the test procedure.

 

5. ROBUSTNESS:

(A). The flow rate was varied at 0.8ml/min to 1.2ml/min. Standard solution 10ppm of Valsartan and 15ppm of Sacubitril was prepared and analysed. The chromatograms were developed.

 

(B). The organic composition in the mobile phase was varied from 65% to75 % standard solution 10µg/ml of Valsartan and 15µg/ml of Sacubitril were prepared and analysed using the varied mobile phase composition along with the actual mobile phase composition in the method. The chromatograms were developed.

 

6.    LIMIT OF DETECTION (LOD):

LOD’s can be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve (S) as follows:

 

                          σ

LOD=  3.3  X---------

                           S

Where

σ - Standard deviation (SD)

S  - Slope

 

7.    LIMIT OF QUANTIFICATION (LOQ):

LOQ’s can be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve (S):

 

                           σ

LOQ=  10  X---------

                           S                                                          

8.    SYSTEM SUITABILITY:

System is suitable for analysis if the relative standard deviation (RSD) of area counts in the six replicate injections for each peak should not be more than 2.0%. The USP plate count of peak should not be less than 2000 theoretical plates for HPLC. The tailing factor for each peak should not be more than 2.0 and the resolution for two peaks should not be less than 2.0.


 

RESULTS:

UV SPECTRAL RESULTS:

 

1.     METHOD DEVELOPMENT TRAILS:

Chromatogram of Valsartan and Sacubitril

S.No

Peak Name

Rt

Area

Height

USP Plate Count

USP Tailing

USP Resolution

1

Valsartan

2.605

2233704

365596

4456

1.4

 

2

Sacubitril

3.781

1328106

174637

5823

1.3

6.5

 

3. METHOD VALIDATION:

3.1 Specificity:

 

S. No

Peak Name

Rt

Area

Height

USP Plate Count

USP Tailing

USP Resolution

1

Valsartan

2.589

2004682

342227

5167

1.3

6.5

2

Sacubitril

3.711

1184227

162666

6389

1.2

 

3.2 Linearity:

Table 3.1 Linearity of Valsartan and Sacubitril

Sample ID

valsartan

Sacubitril

Conc. (mcg/ml)

Area

Conc. (mcg/ml)

Area

20%

20

1224140

10

740046

40%

30

1595681

15

990204

60%

40*

1992966

20*

1183023

80%

50

2356546

25

1439886

100%

60

2797214

30

1682302

Correlation Coefficient

0.999

 

4.    ACCURACY:

 

 

Table: 4.1 Accuracy of Valsartan:

Recovery  level

                                         Accuracy of  Valsartan

Average  %

Recovery

Amount taken (mcg/ml)

Area

Average area

Amount recovered

(mcg/ml)

Percentage Recovery

50%

5.05

1011326

1017498.5

101.3927

101.3927

 

 

 

 

100.599%

5.05

1015029

5.05

1026141

100%

10

1986534

1987384.8

100.0106

100.0106

10

1987425

10

1988195

150%

15

2989367

2992493.4

100.3936

100.3936

15

2991556

15

2996557

 

Table: 4.1 Accuracy of Sacubitril:

Recovery level

Accuracy of Sacubitril

Average % Recovery

Amount taken (mcg/ml)

Area

Average area

Amount recovered (mcg/ml)

% Recovery

 

50%

8.1

646754

 

648293.3

 

101.91

 

101.91

 

 

 

 

101.22%

8.1

648998

8.1

649128

 

100%

15

1172743

 

1174011.1

 

99.66

 

99.66

15

1174031

15

1175259

 

150%

23.3

1866742

 

1868236.3

 

102.09

 

102.09

23.3

1867956

23.3

1870011

 


5. PRECISION:

Valsartan: Table 5.1: Method precision: Valsartan

S. No

Injection

Rt

Area

Height

1

Injection-1

2.586

2010800

346322

2

Injection-2

2.588

2002956

340800

3

Injection-3

2.590

2012800

346911

4

Injection-4

2.590

2005243

344089

5

Injection-5

2.591

2011092

345720

Average

2008578.1

Standard Deviation

4237

%RSD

0.2

 

Sacubitril: Table 5.2: Method precision: Sacubitril:

S. No

Injection

Rt

Area

Height

1

Injection-1

3.713

1184689

162348

2

Injection-2

3.714

1188199

163120

3

Injection-3

3.734

1195842

163500

4

Injection-4

3.737

1184210

160362

5

Injection-5

3.741

1198327

162484

Average

1190253.2

Standard Deviation

6483.1

%RSD

0.5

 

DISCUSSION:

A) Method development:

Trial 6 was optimized for the method development of deliberately changing the chromatographic conditions. Column used was Thermosil C18 (100 mm x 4.6 mm) 5µg.mobile phase composition of ortho phosphoric acid and Acetonitrile in the ratio (25:75% V/V). The Flow rate set to 0.8ml min-1 with UV detection was carried out at 254 nm.

 

B) Validation Parameters:

The calibration was linear with correlation coefficient 0.999 for Valsartan and 0.999 for Sacubitril.

 

In precision it was found that % RSD is less than 2% which indicates that the proposed method has good reproducibility.

 

The system suitability parameter indicates good resolution of both the peaks   > 2.

 

From the Accuracy was found that % Recovery of the drug was found to be in the range of 99.77%  and 101.22% for Valsartan and Sacubitril respectively.

 

Robustness, When pH was altered RT has no changed significantly, when mobile phase was altered there was no change in the RT significantly.

 

ACKNOWLEDGEMENT:

The Authors acknowledge the management of the Jyothishmathi College of Pharmacy, Turkapally to provide facilities to perform this research work and KP Labs Pvt Ltd. to promote the method evaluation of the same.

 

CONFLICT OF INTEREST:

No Conflict of Interest by the authors.

 

REFERENCES:

1.     S. U. Kokil and M. S. Bhatia, Simultaneous Estimation of Nebivolol Hydrochloride and using RP HPLC. Indian J Pharm Sci. 2009 Mar-Apr; 71(2): 111–114.

2.     Ravishankar et al, “A review on analytical method development.” Indian J. Res. in Pharmacy and Biotechnology, 2014, 2(3), 1183-1195.

3.     Indian Pharmacopoeia, Govt. of India Ministry of Health and Family Welfare, The Indian Pharmacopoeia Commission, Ghaziabad, 2010, 3, 228.

4.     The United States Pharmacopoeia (USP 30), The National Formulary (NF 25),United State Pharmacopeial Convention Inc. Rockville, U. S. A, 2007, 32(1), 3445.

5.     The Merck Index, An Encyclopedia of Chemicals, Drugs And Biological; 14th Edition; Published by Merck Research Laboratories, 2006, 9916.

6.     Srinath Nissankararao, Anil Kumar. A, et al- Method development and validation for the estimation of valsartan in bulk and tablet dosage forms by RP-HPLC. Der Pharma Chemica, 2013, 5(2):206-211.

7.     ICH Harmonized Tripartite Guideline, Q2 (R1) Validation of Analytical Procedures: Text and Methodology, International Conference on Harmonization, Geneva, Switzerland, 2005.

8.     Black, H. R.; et al- Valsartan, a new angiotensin II antagonist for the treatment of essential hypertension: efficacy, tolerability and safety compared to an angiotensin-converting enzyme inhibitor, lisinopril. Journal of Human Hypertension 1997, 11 (8), 483-9.

9.     S. U. Kokil and M. S. Bhatia, Simultaneous Estimation of Nebivolol Hydrochloride and using RP HPLC. Indian J Pharm Sci. 2009 Mar-Apr; 71(2): 111–114.

10.   Mustafa Çelebier, Mustafa Sinan Kaynak, et al- Validated HPLC Method Development: The Simultaneous Analysis of Amlodipine and Valsartan in Samples for Liver Perfusion Studies. Hacettepe University Journal of the Faculty of Pharmacy Volume 28 / Number 1 / January 2008 / pp. 15-30.

11.   Gandla. Kumara Swami, N. Ravindra, et al- A New RP-HPLC Method Development and Validation for the Simultaneous Estimation of Amlodipine and Valsartan in Tablet Dosage Forms. Asian J. Pharm. Ana. 2014; Vol. 4: Issue 3, Pg 103-107.

12.   SS Chitlange , Kiran Bagri et al- Stability Indicating RP- HPLC Method for Simultaneous Estimation of Valsartan and Amlodipine in Capsule Formulation. Asian J. Research Chem. 1(1): July-Sept. 2008.

13.   Weber, M. A., Angiotensin II receptor blockers and cardiovascular outcomes: what does the future hold? Journal of the Renin-Angiotensin- Aldosterone System 2003, 4 (2), 62- 73.

14.    Holwerda, N. J.; Fogari, R. et.al, Valsartan, a new angiotensin II antagonist for the treatment of essential hypertension: efficacy and safety compared with placebo and enalapril Journal of Hypertension 1996, 14 (9), 1147-51.

15.   Tamboli, A. M; Chavan, Cretan, et al, Development and Validation of a RP- HPLC method for simultaneous determination of Amlodipine Besylate and Enalapril Maleate. Journal of Pharmacy Research; Nov2010, Vol. 3 Issue 11, p25 – 64.

 

 

 

 

 

 

 

 

 

 

 

 

 

Received on 21.12.2017                Modified on 19.01.2018

Accepted on 11.02.2018            © A&V Publications All right reserved

Asian J. Res. Pharm. Sci. 2018; 8(1):09-16.

DOI: 10.5958/2231-5659.2018.00003.6