INTRODUCTION:

Heart failure is a complex clinical syndrome, which occurs due to the reduced ability of the heart to pump an adequate supply of the blood throughout the body. It has become a growing epidemiologic problem and a leading cause of morbidity and mortality.^1,2 Globally, around 26 million people suffer from heart failure. Blockade of renin angiotensin aldosterone system system using angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, mineralocorticoid receptor antagonists, etc. have been pivotal to treat heart failure and to reduce associated mortality.³ The study was to develop and validate rapid, specific, and sensitive stability indicating HPLC method for the estimation of SAC/ VAL in the presence of their degradation impurities from the drug substances. The HPLC method was developed and validated as per ICH Q2 (R1) guideline.^4,5

Sacubitril/valsartan (SAC/VAL) is a novel acting, first in a new class of drugs approved for the treatment of heart failure. It consists of neprilysin inhibitor prodrug “Sacubitril” (SAC) and angiotensin receptor blocker “Valsartan” (VAL) in their anionic forms along with sodium cations and water. This single molecule having two distinct moieties has been proven superior to conventional ACE inhibitors in the treatment of chronic heart failure.⁶ The combination is sold under the brand name “Entresto” by Novartis and has been approved in more than 57 countries, including India. It was approved by U.S. Food and Drug Administration in July 2015 in the United States (US) and in September 2015 in the European Union. It was approved by Therapeutic Goods Administration in September 2016 in Australia.⁶

VAL, (S)-2-(N-((2ʹ-(2H-tetrazol-5-yl)-[1,1ʹ-biphenyl]- 4-yl)methyl)pentanamido)-3-methylbutanoic acid, is official in United States Pharmacopeia (USP). The estimation of VAL and its related impurities using High-Performance Liquid Chromatography (HPLC) are described in the official monograph (“United States Pharmacopeia and National Formulary (USP 35- NF 30), Rockville, MD: United States Pharmacopeia Convention, 2012.,” n.d.). Different analytical methods have been reported in the literature for the estimation of VAL alone or in combination with another drug from the drug substance, dosage forms or human plasma.⁷ SAC, 4-(((2S,4R)-1-([1,1ʹ-biphenyl]-4-yl)-5-ethoxy-4- methyl-5-oxopentan-2-yl)amino)-4-oxobutanoic acid, is a prodrug that metabolize into sacubitrilat (LBQ657).^8,9 Structure of Valsartan and Sacubitril are shown in figure 1 and figure 2, respectively.^10,11 The simultaneous estimation of SAC/VAL complex from drug substance, formulation or human/rat plasma has been reported using various analytical methods, including UV spectroscopic method, High-Performance Thin-Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS).^12-33 The stability indicating High-Performance Liquid Chromatography (HPLC) method provides sensitive, selective output with better resolution in a shorter duration, which offers cost-effective outcomes. From the literature search, no such literature is reported till date for SAC/VAL.^12-33

Figure 1. Valsartan Structure and Sacubitril Structure

MATERIALS AND METHODS:

Samples, reagents, and chemicals:

Pure standard of SAC/VAL, as well as individual standard of VAL was obtained as a gift sample from Lincoln Pharmaceutical Ltd., Gujarat, India and SAC from Cipla Pharmaceutical Ltd., Mumbai, India.

HPLC grade methanol and acetonitrile were purchased from Merck and Ammonium acetate and glacial acetic acid were purchased from RENKEM (Thane, India). Analytical grade sodium hydroxide pellet, hydrogen peroxide, hydrochloric acid etc. were procured from Merck (Mumbai, India). Purified water used during the entire studies was obtained using Millipore Milli-Q plus (Model: Millipore Milli Q Plus ZD5211584) purification system.

Instrument and apparatus:

The HPLC analysis was performed on Seperation Modules 2695 (Waters Corporation, USA), which was equipped with a quaternary solvent manager, sample manager, an auto-injector, a photodiode array, and TUV detector. Empower 2.0 software was utilized for system monitoring, control, and data acquisition.

The chemical and thermal stress degradation were carried, respectively, using a water bath and a hot air oven equipped with a vacuum. SE60US (Enertech, India) ultrasonicator was used to dissolve the samples. The pH of the mobile phase and solutions were measured using AD102U pH meter (ADWA, Hungary).

HPLC conditions:

Various trials were conducted for the method development. Final chromatographic separation was achieved on Zorbax SB C8, (150×4.6)mm; 5 μm column. In the optimized method, the mobile phase consisted of a mixture of 0.02 M ammonium acetate buffer and acetonitrile (55:45, %v/v) and pH of the mobile phase was set at 3.0 using glacial acetic acid. The flow rate was set at 1.0ml/minute, the injection volume was kept 20μl. The samples were scanned in the range of 200–400nm using a PDA detector and the monitoring wavelength was set at 254nm.

Sample preparation:

Preparation of stock solutions for the standard drugs:

The mixture of water and acetonitrile (50:50, %v/v) was used as a diluent in the preparation for samples. Accurately weighed 20mg of SAC and 20mg of VAL standards were transferred to volumetric flask of 100ml. Around 50ml of the diluent was added and sonicated for 15 minutes to dissolve the drugs completely. The volume was made up to the mark with the diluent to obtain the stock solution of 0.20mg/ml of SAC and 0.20mg/ml of VAL.

Preparation of standard solution for assay method:

Precise and appropriate volume from the stock solution for SAC and VAL was transferred into the volumetric flask and diluted with the diluent to obtain a solution containing SAC (20μg/ml) and VAL (20μg/ml) for the assay method.

Preparation of system suitability solutions:

System suitability solution was prepared by diluting appropriate volumes from the stock solutions for SAC and VAL to obtain a solution with SAC (20μg/ml) and VAL (20μg/ml). Six replicate injections of the sample were injected in the HPLC and the peak area, asymmetry, resolution, theoretical plates were calculated.

Preparation of SAC/VAL sample solution:

A Quantity of the tablet powder equivalent to 24mg of the SAC and 26mg VAL complex sample was weighed accurately and transferred to 100ml of volumetric flask. About 50ml of the diluent was added and sonicated for 15 minutes. The volume was made up to the mark. The resulting sample stock solution was filtered using Whatman filter paper, discarding the first few milliliters. One ml of Aliquot from the prepared stock solution was transferred into 10ml volumetric flask and the volume was adjusted upto the mark with the diluents. This solution was used for the assay method.

Analytical method validation:

The developed HPLC method was validated as per ICH Q2(R1) guideline for the validation of analytical procedures to ensure the acceptable performance of the method for the proposed determination.

Linearity:

Linearity was carried out to demonstrate the detector response is proportional to the concentration of SAC/VAL in the specified range. Linearity test solutions were prepared in triplicate at five concentration levels ranging from 50% to 150% with respect to sample concentration for the assay method. The calibration curves for peak area versus concentration (μg/ml) were plotted and determination coefficients (r²) were calculated.

Accuracy:

The closeness of agreement between the true value and the value found was determined by accuracy study. For the assay method, 80%, 100%, and 120% of each drug were spiked in the SAC/VAL sample in the triplicate and percentage recovery was calculated at each level.

Precision:

The closeness of agreement between a series of measurements was exhibited by the precision study. Repeatability and intermediate precision of the assay method were carried out using six replicate injections of the SAC/VAL at 100% level. The %RSD was calculated for each drug.

Specificity/stress degradation study:

Stress degradation study was performed on SCA/VAL drug complex to establish the stability indicating property and to prove the specificity of the developed analytical method. The stress degradation study was carried as per ICH recommended conditions. The acid and base hydrolysis were carried out using 1 N HCl for 24hours and 1N NaOH for 24 hours at room temperature, respectively. The acid and base degraded samples were neutralised before dilution. Oxidative degradation was carried out using 3% H₂O₂ for 24 hours, while photolytic degradation was carried out by exposing the samples with 1.2million lux hours of visible radiation for 24 hours. Thermal stress was given using a hot air oven at 105°C for 24 hours.

All the prepared sample solutions were filtered through 0.22μm nylon syringe filters prior to the injection in HPLC.

Limit of Detection (LOD) and Limit of Quantification (LOQ)

According to the ICH recommendation, the approach based on the standard deviation (SD) of the response and slope was use. LOD was calculated using equation, LOD = 3.3 σ/S and LOQ was calculated using equation, LOQ = 10 σ/S.

Robustness:

The robustness of the proposed developed method was checked by deliberately altering the instrumental conditions. The flow rate of the developed method was 1.0ml/minutes, so it was changed ± 0.1ml/minute, i.e., 0.9ml/minutes and 1.1ml/ minute. Similarly, the effect of mobile phase pH was studied by varying it by ±0.05 units, i.e., 2.95 and 3.05 pH instead of 3.00pH. The resolution and selectivity between the peaks of SAC/VAL were assessed in all the altered conditions.

RESULTS AND DISCUSSION:

The objective of the present study was to develop rapid, economic, sensitive, and specific HPLC method for the estimation of SAC/VAL in presence of their degradation products.

Method development and optimization:

The study was aimed to develop a sensitive, accurate, precise, stability-indicating RP-HPLC method for estimation of SAC/VAL combination. A Zorbax SB C8, (150×4.6mm, 5μm) column was selected as the stationary phase for the separation and determination of SAC/VAL and their degradation products.

The mobile phase containing 0.02 M ammonium acetate buffer and acetonitrile (55:45, %v/v) and pH of the mobile phase was set at 3.0 using glacial acetic acid was finalized. Method was optimized with flow rate of 1.0 mL/minutes, wavelength 254nm, 20μL volume of injection and 25.0°C column temperature as chromatographic conditions for the complete study where Valsartan, Sacubitril and their degradation products were eluted forming symmetrical peak shape and good resolution. The use of this optimized method, resulted in elution of VAL and SAC at about 2.83 and 4.91 minutes, respectively. The representative chromatogram is shown in Figure 2 and 3.

Figure 2. Chromatogram of binary mixture of Standard 20 µg/ml VAL and 20 µg/ml SAC

Figure 3. Chromatogram of Test formulation 26 µg/ml VAL and 24 µg/ml SAC

System suitability

In order to verify the system performance, system suitability was evaluated. Six replicated injections of the system suitability solution were injected in the HPLC system and the parameters, such as resolution, tailing factor, theoretical plates, and peak area were measured. The results of the system suitability study are given in Table 1, which suggested that all the parameters were within the specified acceptance criteria.

Table 1. System suitability parameters

PARAMETER	SAC	VAL
Retention time (min) + % RSD (n=6)	4.98 + 0.16	2.63 + 0.24
Tailing factor + % RSD (n=6)	1.23 + 0.56	1.09 + 0.68
Theoretical plates + % RSD (n=6)	2354 + 0.43	2216 + 0.72
Resolution	4.25

Analytical method validation:

Analytical method validation was performed to prove the reliability and consistency of the results within the scope of its intended use. The parameters including precision, accuracy, linearity, LOD, LOQ, specificity, and robustness were evaluated as per ICH Q2 guideline. The results of method validation are summarized in Table 2.

Table 2. Summary of validation parameters

PARAMETER	SAC	VAL
Linearity	12-36 μg/mL	13-39 μg/mL
Accuracy (% Recovery) (n=3)	99.10-99.84 %	100.81-100.96%
Precision (% RSD)
Repeatability (n=6)	0.10	0.21
Intraday (n=3)	0.10 - 0.14	0.10 - 0.23
Interday (n=3)	0.11 - 0.20	0.15 - 0.31
LOD (μg/mL)	0.0797	0.1615
LOQ (μg/mL)	0.2416	0.4893
Robustness (% RSD)
Change in Flow Rate	0.12	0.12
Change in pH	0.11	0.14

Linearity:

The linearity of the method was demonstrated using a series of five concentrations in the range of 50% to 150% for each drug. The calculated linear regression equations and determination coefficients (r²) are given in Figure 5. The determination coefficients (r2) were found not less than 0.990 for SAC and VAL.

The results of the linearity study for method exhibited an excellent correlation between proportional concentration (μg/ml) of analytes and their peak areas and method was linear in the given range.

Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD was calculated using following equation,

LOD = 3.3 σ/S

Where σ = Standard deviation of response and S = Slope of calibration curve.

LOD = 0.0797 μg/ml for Sacubitril

LOD = 0.1615 μg/ml for Valsartan

LOQ was calculated using following equation,

LOQ = 10 σ/S

Where σ = Standard deviation of response and S = Slope of calibration curve.

LOQ = 0.2416μg/ml for Sacubitril

LOQ = 0.4893μg/ml for Valsartan

Robustness:

The robustness study of HPLC method was established by making deliberate minor variations in the chromatographic parameters as mentioned in the experimental section. The effect of the change in the chromatographic parameters on the resolution, theoretical plates, and asymmetry was observed. The results showed that the theoretical plates and asymmetry for SAC and VAL were within acceptance criteria. The %RSD calculated for the obtained results between nominal and deliberately changed parameters were within acceptance criteria, which demonstrated that the developed RP-HPLC method was robust and reliable within the given range.

Estimation of Sacubitril and Valsartan in the marketed formulation by the proposed Stability indicating RP-HPLC method:

Applicability of the proposed method was tested by analyzing the commercially available formulation. The assay data are shown in the Table 4.

The assay results were comparable to labelled value of each drug in tablet formulation. These results indicate that the developed method is accurate, precise, simple and rapid. It can be used in the routine quality control of formulation in industries.

Table 4. Analysis of marketed formulation

Sacubitril		Valsartan
Label claim (mg)	Amt found in assay (μg)	Label claim (mg)	Amt found in assay (μg)
24	24.32	26	26.56
	24.53		25.81
	24.89		26.24
	24.72		25.62
	24.87		25.97
Mean Amt Found (mg) ± SD	24.67 ± 0.24	Mean Amt Found (mg) ± SD	26.04 ± 0.37
% RSD	0.98	% RSD	1.42
% labelled claim	102.79	% labelled claim	100.15
Standard Limit	90 - 110	Standard Limit	90 - 110

CONCLUSION:

A rapid and economical RP-HPLC method was developed for the estimation of SAC and VAL. The method was validated as per ICH Q2 R2 guideline and the results showed the ability of the method to produce precise, accurate, linear, specific, sensitive, and robust results within the validated range. The stability indicating potential of the method was confirmed using a forced degradation study. The SAC/VAL complex was labile to acidic, alkaline, and oxidative stress conditions, whereas it was stable to thermal and photolytic stress conditions. The developed method can be useful to ensure the quality and stability of active pharmaceutical ingredients and finished products.

ACKNOWLEDGMENTS:

The authors would like to thank God and Family.

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Received on 12.07.2021 Modified on 15.12.2021

Research J. Pharm. and Tech. 2022; 15(8):3627-3633.

DOI: 10.52711/0974-360X.2022.00607

Stress condition	Valsartan			Sacubitril
Stress condition	Area	R.T	% Degradation	Area	R.T.	% Degradation
As such	289762	2.63	-	305744	4.91	-
Acid	259076	2.62	10.59	267867	4.96	12.38
Base	248850	2.62	14.12	214324	4.98	29.90
Oxidative degradation	235647	2.60	18.67	294949	4.99	3.53
Photo degradation	268950	2.67	7.18	295613	4.95	3.31
Thermal	259549	2.70	10.42	292431	4.94	4.35