INTRODUCTION:

Heart failure (HF), a public health concern, remains a leading cause of morbidity and mortality worldwide. Heart failure with reduced ejection fraction (HFrEF) presents in approximately 50% of all cases of heart failure.

Though advanced therapies have prolonged survival in patients in end-stage HF, pharmacotherapeutic optimization remains the mainstay of treatment in patients unsuitable for advanced strategies. It has been nearly ten years since the last mortality‐reducing medication has approved by Food and Drug Administration¹. After Several ground breaking studies, a new entity containing sacubitril and valsartan (previously called LCZ696) got manufactured and approved by the European Commission and USFDA². Sacubitril and valsartan combination significantly reduce the occurrence of the composite of cardiac death and hospitalizations for worsening HF events in a real-world HFrEF population³. Since approval, international HF treatment guidelines have given sacubitril and valsartan combination a class I recommendation to be used as first line therapy in HFrEF patients⁴.

Sacubitril and valsartan is a co-crystal complex of the sodium salts of two individual active components, in hydrated form. The chemical name of the sacubitril-valsartan complex is Octadecasodiumhexakis(4-{[(1S,3R)-1-([1,1´-biphenyl]-4-ylmethyl)-4-ethoxy-3-methyl-4-oxobutyl] amino}-4-oxobutanoate) hexakis (N-pentanoyl-N-{[2´-(1H-tetrazol-1-id-5-yl )[1,1´-biphenyl]-4-yl]methyl}-L-valinate) pentadecahydrate with molecular formula C₂₈₈H₃₃₀N₃₆O₄₈Na₁₈.15 H₂O and relative molecular mass 5747.96 g/mol. This complex comprised of anionic forms of sacubitril and valsartan, sodium cations, and water molecules in the molar ratio of 1:1:3:2.5 respectively as indicated in Figure 1⁵. Following oral administration, the sacubitril and valsartan complex dissociates where the neprilysin inhibitor component sacubitril gets metabolized to the active form sacubitrilat (LBQ657)⁶.

Figure 1: Structure of sacubitril and valsartan complex

A formulation that contains both sacubitril and valsartan was manufactured and approved by the FDA in July 2015 for the reduction of mortality and hospitalization in systolic heart failure patients. The new medication offers a potentially superior alternative to ACE inhibitor therapy in the management of systolic heart failure.

It is a first-in-class angiotensin receptor neprilysin inhibitor (ARNI) that simultaneously suppresses Renin-Angiotensin-Aldosterone System (RAAS) activation through blockade of angiotensin II type 1 receptor and enhances vasoactive peptides including NPs (natriuretic peptides) through inhibition of neprilysin, the enzyme responsible for their degradation⁷. It reduces the risk of cardiovascular death in HFrEF patients. The improved clinical advantages of sacubitril and valsartan increase the number of formulations in the market in the recent past.

Different analytical methods have been reported for simultaneous estimation of sacubitril and valsartan in their combined dosage form viz. HPLC-UV methods^8-11, ion pair HPLC method¹², LC-MS method¹³, spectrofluorometric method¹⁴and spectrophotometric methods^15-17. However, for routine analysis, a simple, rapid, and cost-effective analytical method is preferred in place of chromatographic techniques which are time-consuming, costly, and require expertise. The reported spectrophotometric methods were developed in methanol which is harmful to the environment. Moreover, methanol being a volatile organic solvent, it cannot be applied for in vitro dissolution study of pharmaceutical dosage forms. Hence, a new UV spectrophotometric method need to be developed and validated in 0.1N HCl and pH 6.8 phosphate buffer for routine analysis of drugs by dissolution study of pharmaceutical formulations.

The objective of the present study was to develop a simple, precise, accurate, economical, and validated analytical method for the estimation of sacubitril and valsartan in in vitro dissolution study of pharmaceutical formulations, performed in 0.1N HCl and pH 6.8 phosphate buffer. Sacubitril and valsartan complex showed absorption maxima at 253 nm in both 0.1N HCl and pH 6.8 phosphate buffer. The analytical method developed was validated as per ICH guidelines and USP requirements¹⁸.

MATERIALS AND METHODS:

Chemicals and reagents:

Sacubitril and valsartan is a gift sample from Dr. Reddy’s Labs, Hyderabad. The marketed pharmaceutical dosage form used in this study was Azmarda 50 mg tablets manufactured by Novartis Farma SpA (Italy) labelled to contain 24 mg of sacubitril and 26 mg of valsartan was purchased from Apollo Pharmacy, Visakhapatnam, Andhra Pradesh. All other chemicals and reagents used are of analytical grade.

Preparation of sacubitril and valsartan reference solution using 0.1N HCl:

A stock solution of sacubitril and valsartan in 0.1N HCl was prepared by dissolving 50 mg of sacubitril and valsartan in 50 mL of methanol initially to obtain 1000 μg/mL solution. 10 mL of primary stock was transferred to a 100 mL volumetric flask and made to final volume with 0.1N HCl to obtain the working standard of 100 μg /mL. The working standard solution was further diluted with buffer to obtain concentrations in the range of 1- 20 μg/mL.

Preparation of sacubitril and valsartan reference solution using pH 6.8 phosphate buffer:

A stock solution of sacubitril and valsartan in pH 6.8 phosphate buffer was prepared by dissolving 50 mg of sacubitril and valsartan in 50 mL of pH 6.8 phosphate buffer to obtain primary stock (1000 μg/mL)¹⁹. 10 mL of this solution was transferred to a 100 mL volumetric flask and made to final volume with the medium to obtain the working standard of 100 μg /mL. The working standard solution was further diluted to obtain concentrations in the range of 1- 20 μg/mL.

Preparation of sample solution:

To prepare the sample solutions, ten tablets (Azmarda 50mg) were weighed accurately and crushed to a ﬁne powder and extracted using the respective media. Amount of powdered tablets equivalent to 100 mg of sacubitril and valsartan transferred to two 100 mL volumetric ﬂasks. In case of 0.1N HCl, the sample was initially solubilised in 10-15ml of methanol and further diluted with 0.1N HCl. After the addition of 70 mL medium, the ﬂasks were shaken ultrasonically (5 min) and made up to volume with respective medium. Then contents of the flask were filtered using the Whatman filter of pore size 0.45 μm to get clear solution. 10 mL aliquots of the clear supernatant liquid were transferred to 100 mL volumetric ﬂasks and made up to volume with respective medium to give a ﬁnal concentration of 100 μg/mL.

Instrument details:

A double beam LAB INDIA 3000+ UV–VIS spectrophotometer and LAB INDIA T60 UV–VIS spectrophotometer connected to the computer using UV-Probe software was used in the study. Both the instruments have an automatic wavelength accuracy of 0.1 nm and matched quartz cells of 10 mm cell path length.

Determination of λ_max:

A clear solution of 10 μg/mL concentration of sacubitril and valsartan prepared using both the media and scanned using double beam LAB INDIA 3000+ UV–VIS spectrophotometer in the range of 200-400 nm against the respective medium (0.1N HCl/pH 6.8 phosphate buffer) as blank. Maximum absorbance was recorded²⁰.

Validation of the developed UV spectrophotometric method:

Validation of the developed analytical method is very important for its application in the analysis of the drug in the dosage forms and during dissolution studies. The UV spectrophotometric method was validated. Method validation includes determination of the following parameters: linearity, accuracy, precision, the limit of detection (LoD), the limit of quantitation (LoQ), ruggedness, robustness, molar absorptivity and Sandell’s sensitivity as per the ICH guidelines²¹

Linearity:

To establish linearity of the proposed method, six different concentrations of sacubitril and valsartan in the range of 1–20 μg/mL were prepared using the respective media (0.1N HCl/ pH 6.8 phosphate buffer) and analysed at 253 nm. The least square regression analysis was done for the obtained data (Table 1).

Accuracy:

Accuracy of the proposed method was determined using recovery studies. The recovery studies were carried out by adding different amounts (50%, 10% and 150%) of the pure drug to the pre-analysed formulation. The solutions were prepared in triplicates and the % recovery was calculated as

Measured concentration

% Accuracy= ------------------------------ x 100

Actual added concentration

Precision:

Precision studies were carried out to ascertain the reproducibility of the proposed method. It was determined by using different levels of drug concentrations (4%, 8%, 12%) prepared from independent stock solutions, and analysed. Different levels of drug concentrations in triplicates were prepared and studied three different times in a day for intra-day variation. The same procedure was followed for three different days to study inter-day variation. The results were reported as % RSD (% Relative Standard Deviation) (Table 3).

LOD and LOQ:

LOD and LOQ parameters are not a requirement for drug assay; however, it is always useful to demonstrate that the analysis is being conducted in a region that is above the LOQ value. The LOD and LOQ were calculated based on the standard deviation of the response (y-intercepts of regression lines) and the slope estimated from the calibration curve of the analyte, as defined by ICH (Table 1). LOD and LOQ were calculated by using following formula: LOD = 3.3×σ/S and LOQ = 10×σ/S, where, σ is the standard deviation of y-intercepts of regression line, S is slope of the calibration curve. ^22,23

Ruggedness:

Ruggedness of the proposed method was checked by applying developed method to assay 10 μg/ml of sacubitril and valsartan solution using two different instruments LAB INDIA 3000+ UV–VIS spectrophotometer (instrument 1) and LAB INDIA T60 UV–VIS spectrophotometer (instrument 2) on different days under same conditions. The obtained results indicate reproducibility of the method (Table 4).

Robustness:

The robustness of the method was determined by studying small changes in the assay wavelength (±1 nm). The results are expressed as % RSD (Table 1).

Molar absorptivity:

Molar absorptivity is absorbance of light per unit path length (cm) and per unit of concentration (in moles /L). The term explains how strongly a chemical species absorbs light at a particular wavelength.²⁴

Sandell’s Sensitivity:

Sandell’s sensitivity, the concentration of the analyte (in μg/mL or μg/cm²) which will give an absorbance of 0.001 in a cell of path length 1 cm, was calculated. It represents the sensitivity of the method.

Analysis of sacubitril and valsartan in pharmaceutical formulations

The sample solution of 10 μg/mL strength of sacubitril and valsartan was prepared from 100 μg/mL stock using both media²⁵. The concentration of sacubitril and valsartan present in the sample solution was determined by measuring the absorbance at 253 nm. The procedure was replicated for 5 times and the amount of drug present in the tablets was calculated using average values (Table 5).

RESULTS AND DISCUSSION:

0.1N HCl and pH 6.8 phosphate buffer were selected as vehicles in the proposed method as they were suggested as dissolution media for the tablet dosage forms²⁶containing sacubitril and valsartan. The drug combination is fairly soluble in pH 6.8 phosphate buffer and slightly soluble in 0.1N HCl. Hence, in order to make the drug combination completely soluble in 0.1N HCl, methanol was used initially to prepare stock solution.

Selection of λ_maxby UV Spectroscopy

(a)

(b)

Figure 2: Wavelength scan of 10 μg/mL sacubitril and valsartan in (a) 0.1 N HCl (b) 6.8 Phosphate buffer

The absorption spectra of sacubitril and valsartan in 0.1N HCl and pH 6.8 phosphate buffer indicate λ_max was 253 nm (Figure 2). The observed λ_max was close to the expected λ_max calculated using Woodward Fieser rule to predict wavelength of absorption maximum in an UV-VIS spectrum²⁷. As the drug used in this study possess a biphenyl structure in the molecule the expected λ_max was found to be near to 252 nm.

Validation of the developed UV spectrophotometric method

Linearity:

(b)

The linearity of sacubitril and valsartan was found to be in the range of 1-20 μg/ml with linear correlation coefficient 0.999 in both the media as shown in calibration curve (Figure 3a and 3b) indicating that the proposed method could be successfully used in quantitative analysis of pharmaceutical tablet dosage form. The results of analytical parameters indicated in Table 1.

(a)

(b)

Figure 3: Calibration curve of sacubitril and valsartan (a) 0.1 N HCl (b) pH 6.8 Phosphate buffer

Accuracy:

The results of recovery studies at three different levels, i.e., 50, 100 and 150% were excellent and mean % recovery values (Table 2) close to 100%, with low standard deviation (s.d.< 1.0) represents high accuracy of the analytical method in both the media.

Precision:

The % RSD values for intra-day precision was found to be in the range of 0.494-0.945 and 0.579-0.897 in 0.1N HCl and pH 6.8 phosphate buffer respectively and % RSD values for inter-day precision was found to be in the range of 0.986-1.083 and 0.831-1.287 in 0.1N HCl and pH 6.8 phosphate buffer respectively (Table 3). The % RSD value < 2 indicates that the method is precise.

LOD and LOQ:

The LOD and LOQ for pure drug were found to be 0.5353 and 1.6222 μg/mL in 0.1N HCl and 0.395 μg/mL, 1.198 μg/mL in pH 6.8 phosphate buffer (Table-1).

Ruggedness:

Ruggedness of the proposed method was studied with the help of two instruments and the results listed in Table 4 indicate good reproducibility of the developed method. Thus, the proposed method was considered as rugged (Table-4).

Table 1: Analytical and validation data of the proposed method for estimation of sacubitril and valsartan

Regression analysis
Medium	0.1N HCl	pH 6.8 phosphate buffer
λ_max (nm)	253nm	253nm
Beer's law limits (μg/mL) *	1–20	1–20
Regression equation	y=0.0376x+0.0021	y=0.0384x+0.0049
Slope	0.0376	0.0384
Intercept	0.0021	0.0049
Correlation coefficient (r)	0.9991	0.9995
Molar Absorptivity (L mole^-1cm^-1)	3.84 x 10⁴	3.68 x 10⁴
Sandell’s sensitivity (μg cm^-1/0.001 absorbance unit)	0.024	0.025
Limit of detection (LOD) (μg/mL)	0.5353	0.395
Limit of quantification (LOQ) (μg/mL)	1.6222	1.198
Robustness (mean %recovery ± s.d.)^*	100.09 ± 1.29	100.54 ± 0.87

*Average of 6 determinations

Table 2: Standard addition method for accuracy

Medium	Drug in formulation (μg/mL)	Pure drug added (μg/mL)	Percent of recovery level	Amount recovered^* (μg/mL) (Mean±S.D.)	% Recovery (Mean±S.D.)
0.1N HCl	8	4	50%	11.99±0.051	100.05±0.324
	8	8	100%	15.96±0.126	99.98±0.401
	8	12	150%	20.02±0.089	100.11±0.44
pH 6.8 phosphate buffer	8	4	50%	12.01±0.068	100.13±0.565
	8	8	100%	16.02±0.065	100.16±0.405
	8	12	150%	19.99±0.083	99.96±0.419

*Average of 3 determinations

Table 3: Precision study

Medium	Concentration (μg/mL)	Intra-day precision			Inter-day precision
Medium	Concentration (μg/mL)	Absorbance**	S.D.	%RSD	Absorbance**	S.D.	%RSD
0.1N HCI	4	0.156	0.873	0.494	0.147	0.783	1.083
	8	0.326	0.358	0.932	0.317	0.864	0.986
	12	0.418	0.576	0.945	0.435	0.684	1.049
pH 6.8 phosphate buffer	4	0.129	0.663	0.897	0.132	0.982	0.831
	8	0.308	0.985	0.926	0.311	0.753	0.948
	12	0.439	0.846	0.579	0.442	0.592	1.287

**Average of 3 determinations

Table 4: Ruggedness of the method by two instruments

Medium	Test parameters	Instrument 1	Instrument 2
0.1N HCI	Concentration ( 8 μg/mL)	7.87	7.93
	Absorbance**	0.315	0.321
	s.d.	0.069	0.187
	%RSD	0.462	0.586
pH 6.8 phosphate buffer	Concentration ( 8 μg/mL)	7.72	7.89
	Absorbance**	0.311	0.318
	Standard deviation	0.132	0.148
	%RSD	0.593	0.694

**average of 3 determinations

Robustness:

The proposed method remained unaffected by small changes in wavelength indicate robustness of the method (Table 1).

Molar absorptivity and Sandell’s Sensitivity:

The molar absorptivity and Sandell’s sensitivity values of drug in 0.1 N HCI and pH 6.8 phosphate buffer were found to be 3.84x10⁴, 3.68x10⁴L mole^-1cm^-1 and 0.0249 and 0.025 μg cm^-1/0.001 absorbance unit respectively (Table 1) indicate high sensitivity of the method as the analysis can be performed at lowest concentrations with good correlation.

Analysis of sacubitril and valsartan in pharmaceutical formulations:

The proposed method was applied to pharmaceutical tablet formulation and % amount of drug estimated was 99.86% and 99.12% in 0.1N HCl and pH 6.8 phosphate buffer (Table 5) respectively and was found to be in good agreement with the label claim. Hence, indicating that the method could be effectively applied to analyse marketed tablet dosage forms containing sacubitril and valsartan.

Table 5: Assay of marketed formulation

Buffer	Formulation	Label claimed (mg)	*Amount recovered (mg)**	% Drug recovered	% RSD
0.1N HCl	AZMARDA	50	49.93± 0.393	99.86	0.787102
pH 6.8 phosphate buffer	AZMARDA	50	49.563± 0.284	99.12	0.573043

*Average of 5 determinations

CONCLUSION:

The UV spectroscopic method developed and validated for estimation of sacubitril and valsartan combination in 0.1 N HCI and pH 6.8 phosphate buffer was simple, sensitive, accurate, precise and reproducible. The estimated λ_max of sacubitril and valsartan combination in 0.1N HCl and pH 6.8 phosphate buffer is in compliance with Woodward Fieser rule and reported for the first time in this investigation. It is possible to construct the calibration curves in the concentration range 1-20 μg/mL in both 0.1N HCl and pH 6.8 phosphate buffer and hence, this method is more sensitive and appropriate for simultaneous estimation of sacubitril and valsartan combination in pure form, pharmaceutical tablet dosage forms and dissolution testing of lowest dilutions also. The method is completely developed using stock solution containing sacubitril and valsartan combination (LCZ 696) instead of individual stock solution of drugs as reported in earlier methods. The proposed method is economical and useful for routine analysis of drug, as it was developed in eco-friendly medium suitable for in vitro dissolution studies using UV spectrophotometer instead of expensive equipment and organic solvents which are volatile, analytical grade, and harmful to environment.

ACKNOWLEDGEMENT:

This research received no specific grant from any funding agency.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 10.11.2021 Modified on 29.03.2022

Research J. Pharm. and Tech 2022; 15(11):5232-5238.

DOI: 10.52711/0974-360X.2022.00881