RP-HPLC Method Development and Validation for the Estimation of Sacubitril and Valsartan in Pharmaceutical Dosage Form

 

G.M. Kadam¹, A.L. Puyad^1*, T.M. Kalyankar²

¹School of Chemical Sciences, S. R. T. M. University, Nanded – 431606, Maharashtra – India.

²School of Pharmacy, S. R. T. M. University, Nanded – 431606, Maharashtra – India.

 

ABSTRACT:

A new, economical, simple, accurate, and precise RP-HPLC method was developed for simultaneous assay and content uniformity determination of Sacubitril and Valsartan in bulk and pharmaceutical dosage form. The separation of Sacubitril and Valsartan was achieved within 6 minutes on Phenomenex Luna C18 250 mm x 4.6mm and 5µm Particle Size, column using Acetonitrile: Methanol: Water (30:55:15% v/v/v) as the mobile phase. Detection was carried out at 250 nm wavelength. The retention time of Sacubitril and Valsartan was found to be 2.361 and 3.304 min, respectively. The validation of the developed method was performed in terms of specificity, accuracy, precision, linearity, the limit of detection, the limit of quantification as mentioned in International Conference on Harmonization (ICH) guidelines. The method showed adequate sensitivity concerning linearity, accuracy, and precision over the range 12-36 μg/ml and 13-39 μg/ml for Sacubitril and Valsartan, respectively. The percentage recoveries obtained for Sacubitril and Valsartan were found to be in the range of 98.00 – 102.00 %. The proposed method is suitable for use in quality-control laboratories for quantitative analysis.

 

 

 

INTRODUCTION:

Sacubitril is used in combination with Valsartan. The combination drug Sacubitril and Valsartan, known during trials as LCZ696 and is marketed under the brand name Entresto, is used for the treatment of heart failure. Sacubitril is a neprilysn (NEP) inhibitor prodrug with natriuretic activity¹.

 

The drug Valsartan, is an orally active compound of nonpeptide triazole-derived antagonist of Angiotensin (AT) II with antihypertensive properties². The Chemical Structures of Sacubitril and Valsartan were shown in Figure 1.

 

Figure 1: Structure of Sacubitril (a) and Valsartan (b)

 

From the literature survey^3-18 compared the method performed by us with earlier reported methods, it is observed that there are few assay methods available, however to the best of our knowledge, it was observed that a single method for simultaneous assay & content uniformity (content of single dosage unit) determination of Sacubitril and Valsartan in bulk and pharmaceutical dosage forms by RP-HPLC (PDA) is not available. Hence the present work was undertaken. The proposed method is economical and can be used for routine as well as stability batches analysis in quality control and research laboratory.

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Standard and API were obtained as a gift sample from Sura Pharma Labs, Hyderabad, India. The marketed formulation in the brand name Entresto (Sacubitril - 24 mg and Valsartan - 26 mg) procured from the local pharmacy. All the chemicals and reagents used in this work were HPLC grade water, Acetonitrile, methanol, and were obtained manufactured by Rankem.

 

Instrumentation:

A HPLC system of waters 2695 separation module equipped with a photodiode array detector, autosampler injection with Empower-2 software. Electronic balance, Ultra Sonicator, Hot air oven were used.

 

Chromatographic Conditions:

The chromatographic separations achieved on a Phenomenex Luna C18 250mm x 4.6mm and 5µm Particle Size, Make Phenomenex as a stationary phase. The mobile phase was composed of Acetonitrile: Methanol: Water (30:55:15% v/v/v) at a flow rate of 1.0 mL/minute and injection volume is 20 μL. The column oven temperature was maintained at ambient, and the drugs were detected at 250 nm.

 

Preparation of Mobile Phase:

Mixed 300 mL of Acetonitrile, 550 mL of Methanol and 150 mL of HPLC Grade water (30:55:15 v/v/v) respectively. The mobile phase was sonicated for 15 minutes and filtered through a 0.45μm membrane filter.

 

Preparation of Diluent:

Mixed Acetonitrile, Methanol and HPLC Grade water in the ratio of 30:55:15 v/v/v and used as a diluent.

 

Preparation of Standard Solution:

Weighed and transferred 24 mg of Sacubitril and 26 mg of Valsartan working standard into 100 mL of clean and dry volumetric flask, added about 70 mL of diluent, and sonicated to dissolve the contents and made volume up to the mark with diluent carefully (Standard stock solution). Further pipetted 1mL of the Sacubitril and Valsartan standard stock solution into a 10 mL volumetric flask and diluted up to the mark with diluent carefully (24 ppm of Sacubitril and 26 ppm of Valsartan).

 

Preparation of sample solution (Assay):

10 tablets weighed and crushed to a fine powder. Crushed fine tablet powder equivalent to 24 mg of Sacubitril and 26 mg Valsartan was weighed and transferred into a 100 mL volumetric flask. Added about 70 mL of diluent and sonicated for 30 minutes to extract the drug contents and made up to volume with diluent carefully. The sample solution was filtered through 0.45μm PVDF filter (Sample stock solution). Further, pipetted 1 mL of the Sacubitril and Valsartan stock solutions into a 10 mL volumetric flask and diluted up to the mark with diluent carefully (24 ppm of Sacubitril and 26 ppm of Valsartan).

 

Preparation of sample solution (Content uniformity):

weighed one tablet and transferred into a 100 mL volumetric flask. Added about 70 mL of diluent and sonicated for 30 minutes to extract the drug contents and made up to volume with diluent. The sample solution was filtered through 0.45μm PVDF filter (Sample stock solution). Further, pipetted 1 mL of the Sacubitril and Valsartan stock solutions into a 10 mL volumetric flask and diluted up to the mark with diluent carefully (24 ppm of Sacubitril and 26 ppm of Valsartan).

 

Validation of the RP-HPLC Method:

The developed RP-HPLC method was subjected to validation as per ICH guidelines.

 

System Suitability Parameters:

The system suitability parameters were determined by preparing standard solutions of Sacubitril (24 μg/mL) and Valsartan (26 μg/mL), injected six times and the parameters like retention time, peak tailing, resolution and USP plate count were determined.

 

Linearity:

The stock solution of Sacubitril and Valsartan was prepared using diluent. From it, various working standard solutions were prepared in the range of 12-36 μg/mL, 13-39 μg/mL, and injected into the HPLC system. The calibration plot (peak area vs. concentration) was generated by replicate analysis of prepared solutions (n=3) at all concentration levels. The linearity of the method evaluated using the least square method.

 

Accuracy:

The accuracy method was carried out by standard addition method at different concentration levels 50%, 100%, and 150% and then comparing the theoretical value and found value.

 

Precision:

The precision of the method performed by actual determination of six replicates of sample of the drug (24μg/mL Sacubitril, 26μg/mL Valsartan). The precision was also determined in terms of intraday and interday. The peak area for a set of sample solutions was calculated in terms of relative standard deviation (% RSD).

 

Specificity:

ICH guideline defines "Specificity" as the ability of the method to specifically separate the particular API or analyte in the presence of other components. The specificity of the developed method was proved by studying stress conditions using acid, alkaline, peroxide, thermal, UV, water degradations. The sample was exposed to these stress conditions and the main peak of the drug was studied for peak purity that indicating the method effectively separated the degradation products from the pure active ingredient.

 

Preparation of sample stock solution:

20 tablets were weighed and crushed to a fine powder. Resultant tablet powder equivalent to 24 mg of Sacubitril and 26 mg Valsartan were weighed and transferred into a 100 mL volumetric flask. Added about 70 mL of diluent and sonicated for 30 minutes to extract the drug contents and made up to volume with diluent. The sample solution was filtered through 0.45μm PVDF filter (Sample stock solution).

 

Degradation by acidic condition:

Further pipetted 1 mL of the Sacubitril and Valsartan stock solution into a 10 mL volumetric flask. Added 1 mL of 2N HCl solution and refluxed for 30 minutes at 60 ºC. The resultant degradation solution was neutralized with 1 mL of 2N NaOH and diluted up to the mark with diluent. Finally, degraded sample solution was filtered through 0.45-micron PVDF syringe filter. (24 ppm of Sacubitril and 26 ppm of Valsartan).

 

Degradation by alkaline condition:

Further pipetted 1 mL of the Sacubitril and Valsartan stock solution into a 10 mL volumetric flask. Added 1 mL of 2N NaOH solution and refluxed for 30 minutes at 60 ºC. The resultant degradation solution was neutralized with 1 mL of 2N HCl and diluted up to the mark with diluent. Finally, degraded sample solution was filtered through 0.45-micron PVDF syringe filter. (24 ppm of Sacubitril and 26 ppm of Valsartan).

 

Water Degradation (Hydrolysis):

Further pipetted 1 mL of the Sacubitril and Valsartan stock solution into a 10 mL volumetric flask. Added 1 mL of water and refluxed for 30 minutes at 60 ºC and diluted up to the mark with diluent. Finally, degraded sample solution was filtered through 0.45-micron PVDF syringe filter. (24 ppm of Sacubitril and 26 ppm of Valsartan).

 

 

Oxidative degradation:

Further pipetted 1 mL of the Sacubitril and Valsartan stock solution into a 10 mL volumetric flask. Added 1 mL of 3% H₂O₂solution and refluxed for 15 minutes at 60 ºC and diluted up to the mark with diluent. Finally, degraded sample solution was filtered through 0.45-micron PVDF syringe filter. (24 ppm of Sacubitril and 26 ppm of Valsartan).

 

Thermal degradation:

The Sacubitril and Valsartan sample was transferred in petridish and exposed in Hot air oven at 110⁰ C for 24 hours. For sample preparation, 10 tablets were weighed and crushed to a fine powder. Resultant tablet powder equivalent to 24 mg of Sacubitril and 26 mg Valsartan were weighed and transferred into a 100 mL volumetric flask. Added about 70 mL of diluent and sonicated for 30 minutes to extract the drug contents and made up to volume with diluent. The degraded sample solution was filtered through 0.45μm PVDF filter (Sample stock solution). Further pipetted 1 mL of the Sacubitril and Valsartan sample stock solution into a 10 mL volumetric flask and diluted up to the mark with diluent carefully (24 ppm of Sacubitril and 26 ppm of Valsartan).

 

Photolytic degradation:

The photostability of the drug was studied by exposing the stock solution to UV light for 200 Watt-hours/m² in photostability chamber. For sample preparation, 10 tablets were weighed and crushed to a fine powder. Resultant tablet powder equivalent to 24 mg of Sacubitril and 26 mg Valsartan were weighed and transferred into a 100 mL volumetric flask. Added about 70 mL of diluent and sonicated for 30 minutes to extract the drug contents and made up to volume with diluent. The degraded solution was filtered through 0.45μm PVDF filter (Sample stock solution). Further pipetted 1 mL of the Sacubitril and Valsartan sample stock solution into a 10 mL volumetric flask and diluted up to the mark with diluent carefully (24 ppm of Sacubitril and 26 ppm of Valsartan).

 

Robustness:

The Robustness of the method performed by small and deliberate changes in method parameters such as flow rate (±0.1), Mobile Phase organic phase ratio (±5%). System suitability parameters were evaluated.

 

RESULTS AND DISCUSSION:

Results of the validated method proved, performance & characteristics to meet the requirement of the standard analytical application.

 

System Suitability:

System suitability was performed to evaluate the parameters like tailing factor, theoretical plates, resolution, and % RSD for replicate injections. The representative chromatogram is shown in Figure 3. The results were within limits and given in Table 1.

 

Linearity:

The linearity of the measurement was evaluated by analyzing different concentrations (50% to 150%) of the standard solutions of Sacubitril and Valsartan. The calibration curve was plotted, concentration against mean peak area, and the regression equation was computed. The coefficient of correlation (R²) for Sacubitril and Valsartan were found to be 0.999 and 0.999 respectively. The summary of the parameters is given in Table 1 and linearity plots are shown in Figure 2.

 

Accuracy:

Recovery studies were conducted at three different levels 50 %, 100 % and 150% of working concentration. Accuracy was calculated as the percentage of recovery, and the results were shown in Table 1.

 

Figure 2: Calibration graph of Sacubitril (a) and Valsartan (b)

 

Table 1: a) System suitability b) Linearity and c) Accuracy results for Sacubitril and Valsartan

Injection	Retention Time		Peak Area		USP plate Count		USP Tailing		Resolution
	Sacubitril	Valsartan	Sacubitril	Valsartan	Sacubitril	Valsartan	Sacubitril	Valsartan
1	2.361	3.308	870728	102317	3107	6269	1.3	1.2	5.0
2	2.363	3.312	871910	103125	3171	6171	1.3	1.2	5.0
3	2.365	3.304	873040	103169	3200	6073	1.3	1.2	5.0
4	2.365	3.312	875684	103178	3159	6083	1.3	1.2	5.0
5	2.361	3.304	872657	102697	3158	6077	1.3	1.2	5.0
6	2.363	3.308	874158	103245	3185	6082	1.3	1.2	5.0
Mean			873030	102955			1.3	1.2	5.0
SD			1732.57	369.36
% RSD			0.19	0.35

 

Drug(s)	% Linearity level	Peak area	Concentration (µg/ mL)	Correlation Coefficient
Sacubitril	50	431494.7	12.0	0.999
	75	642216.7	18.0
	100	858051.3	24.0
	125	1059996	30.0
	150	1268304	36.0
Valsartan	50	49597	13.0	0.999
	75	73701	19.50
	100	96836	26.0
	125	119802	32.5
	150	142938	39.0

 

Drug(s)	% Accuracy level	Average peak area	Amount Added (µg/ mL)	Amount Found (µg/ mL)	% Recovery	% Mean Recovery
Sacubitril	50	43127	12	12.085	100.71	100.55
	100	85625	24	24.151	100.63
	150	127750	36	36.110	100.31
Valsartan	50	48914	13	13.037	100.28	100.56
	100	97268	26	26.259	101.00
	150	144425	39	39.154	100.39

 

Precision:

Precision of the method was carried out in terms of system precision, repeatability, and intermediate accuracy. These are assessed by using six replicates at a concentration of 24μg/mL of Sacubitril and 26μg/mL of Valsartan The % RSD was found to be < 2, indicating the repeatability of the method. The data was given in Table 2.

 

Specificity:

In the blank chromatogram, there were no peaks observed at the retention times of Sacubitril and Valsartan. The retention times of Sacubitril and Valsartan were 2.361 min and 3.304 min for standard and 2.381 min and 3.347 min for sample respectively.

 

Since no interference of blank and degradants, peak purity of Sacubitril and Valsartan passed, hence the proposed method was specific and is stability-indicating. Degradation is observed when exposed to acidic, basic, oxidative, thermal conditions compared to hydrolysis and photolytic condition. The results of the forced degradation study were summarized in Table 2.

 

Robustness:

The robustness of the method was evaluated for flow rate (± 0.1) and solvent composition (± 5%), and the influence of these changes on specific peak characteristics like peak area, peak tailing and number of theoretical plates were evaluated. The results were shown in Table 3.

 

Figure 3: Chromatogram of Blank (Diluent), standard solution and sample solution

 

Table 2: Results of Method Precision, Intermediate precision and Forced Degradation Studies

S. No.	Method repeatability		Intermediate precision		Stress condition(s)	% Assay			% Degradation
	Sacubitril	Valsartan	Sacubitril	Valsartan		Sacubitril	Valsartan		Sacubitril	Valsartan
1	885966	103737	898658	104526	Acid degradation	63.28	61.55		36.72	38.45
2	886678	103946	895874	104238	Alkali degradation	80.14	79.52		19.86	20.48
3	888046	104197	895746	104526	Water degradation	97.03	96.14		2.97	3.86
4	888565	104210	896525	104254	Oxidative degradation	81.35	82.31		18.65	17.69
5	890142	104406	897854	103989	Thermal degradation	64.51	62.72		35.49	37.28
6	889654	103895	890253	104787	Photolytic degradation	95.24	94.37		4.76	5.63
Mean	888175	104065	895818	104387
SD	1633.76	247.13	2955.95	281.76
% RSD	0.18	0.23	0.32	0.27

 

Table 3: Results for Robustness for Sacubitril and Valsartan

Parameters	Changed value	Sacubitril			Valsartan
		Retention time (minutes)	USP Plate Count	USP Tailing	Retention time (minutes)	USP Plate Count	USP Tailing
Flow rate (mL/min.)	0.9	2.636	3278	1.34	3.712	6685	1.22
	1.0	2.361	3107	1.30	3.312	6269	1.20
	1.1	2.171	3034	1.30	3.048	5846	1.20
Change in organic composition in the mobile phase (Acetonitrile: Methanol: Water (30:55:15 v/v)	Low organic	2.640	3299	1.33	3.716	6704	1.25
	As per method	2.361	3107	1.30	3.312	6269	1.20
	High organic	2.169	3653	1.31	3.048	5779	1.21

 

Advantages:

Advantages of the newly developed and validated method compared to other reported methods, determination of assay as well as content uniformity in single analysis which save time and resources used in experimentation, economical and useful in decision for the selection or rejection of manufactured batches.

 

CONCLUSION:

A new, simple and accurate, and precise RP-HPLC method was developed for simultaneous assay and content uniformity determination of Sacubitril and Valsartan in the bulk and pharmaceutical dosage form. The method was validated as per ICH guidelines. The method was applied to the analysis of marketed combination product. The results obtained lead to the conclusion that the target of developing a common chromatographic method for both assay and content uniformity has been achieved. This simple, economical, and efficient method can be used to estimate Sacubitril and Valsartan as independent components as well as in combination.

 

ACKNOWLEDGEMENT:

The authors are thankful to Sura Pharma Labs., Hyderabad, India for providing gift samples.

 

CONFLICT OF INTEREST:

Hereby author(s) declares that there is no conflict of interest.

 

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18.   Validation of Analytical Procedure: Text and Methodology, Q2(R1)

 

 

Accepted on 08.03.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(11):5797-5802.