INTRODUCTION:

Valsartan (VAL) is a nonpeptide, orally active and specific angiotensin II receptor blocker acting on the AT1 receptor subtype. Valsartan is chemically (2S)-3-methyl-2-[N-({4-[2-(2H-1,2,3,4-tetrazol-5-yl) phenyl] phenylmethyl) pentanamido] butanoic acid.

Sacubitril (SAC) is chemically 4- [[(2S,4R)-5-ethoxy-4-methyl-5-oxo-1-(4-phenylphenyl) pentan-2 yl] amino]-4-oxobutanoic acid. Sacubitril is a prodrug that is activated to sacubitrilat 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. Sacubitril is an antihypertensive drug used in combination with valsartan for the treatment of heart failure. On literature survey, it was found that RP-HPLC methods and few spectrophotometric methods reported for the simultaneous estimation of Sacubitril and Valsartan in combined dosage forms and no method is available in the pharmacopoeias.

Few analytical methods have been developed for the determination of Sacubitril and Valsartan individually, and in combination with other drugs. So we have developed a novel, simple and highly sensitive UV spectrophotometric methods for assay of Sacubitril and Valsartan in bulk and pharmaceutical formulations and validated according to ICH guidelines.

Fig 1. Structure of Valsartan

Fig 2. Structure of Sacubitril

MATERIALS AND METHODS:

Reagents and Chemicals:

Active pharmaceutical ingredient (API) working standards of Sacubitril and Valsartan were received as gift samples. The pharmaceutical dosage form used in this study was Vymada tablets manufactured by Novartis pharmaceutical Co. Ltd. which were purchased from local market. Methanol and water were procured from Fisher chemicals Ltd., Mumbai.

Instrumentation:

The analysis was carried on a double beam UV–Visible spectrophtometer (Lab India, T60) attached to a microwave software with a spectral width of 2nm, wavelength accuracy of 0.5nm and pair of 1cm matched quartz cells.

Preparation of standard stock solution:

100mg each of Sacubitril and Valsartan were weighed separately and transferred into two different 100ml volumetric flasks. Both the drugs were dissolved in 100 ml of Methanol to obtain a final concentration of 1000 μg/ml of each component. From the above stock solution, 10ml was pipette out into a 100ml volumetric flask and the volume was made up to the mark with methanol to obtain the final concentration of 100μg/ml for both drugs.

Preparation of Sample Solution:

Twenty tablets of the combined dosage form of sacubitril and valsartan were weighed and powdered. The quantity of the powder equivalent to 10mg of Sacubitril was transferred to 100ml volumetric flasks. The content was mixed with methanol (70ml) and sonicated for 20 min to dissolve the drug as completely as possible. The solutions were then filtered through a Whatman filter paper no. 41 and volumes were adjusted up to the mark with methanol. An aliquot of each solution (1ml) was transferred to a 10ml volumetric flask and the volume was adjusted up to the mark with methanol to obtain the required concentration of Sacubitril (10µg/ml) and Valsartan (12µg/ml).

Selection of Analytical Wavelength:

The scanning for solution of SAC (100μg/ml) and VAL (100μg/ml) were carried out in the range of 200-400nm against using methanol as a blank. The maximum absorption (λmax) of SAC and VAL was found at 230 nm and 290nm (Fig. 2 and Fig. 3). Absorption and absorptivity for a series of standard solutions were recorded at selected wavelengths. The two wavelengths selected should be such that at each wavelength the difference in the absorptivity between the two components should be as large as possible.

Figure 3: Overlain absorption spectra of Sacubitril and Valsartan

Simultaneous equation method:

If a sample contains two absorbing drug each of which absorbs at the λmax of the other, it may be possible to determine both drugs by the technique of simultaneous equation. Two wavelengths selected for the development of the simultaneous equations are 220nm and 240nm. The absorptivity values determined for SAC are 0.0643 (ax₁), 0.0242 (ax₂) and for VAL are 0.0055 (ay₁), 0.0351 (ay₂) at 220nm and 240nm respectively. These values are means of six estimations. The absorbance’s and absorptivity at these wavelengths were substituted in equation 1 and 2 to obtain the concentration of both drugs.

The amount Sacubitril and Valsartan present in the sample solutions were determined by solving the following simultaneous equations

c _x= (A₁ a_Y2 - A₂ a_Y1) / (a_X1 a_Y2 - a_X2 a_Y1) equation 1

c _y = (a_X1 A₂ - a_X2 A₁) / (a_X1 a_Y2 - a_X2 a_Y1) equation 2

Where,

A₁ = Absorbance of sample solution at 230nm

A₂ = Absorbance of sample solution at 290nm

a_X1 = Absorbitivity of Sacubitril at 230nm,

a_X2 = Absorbitivity of Sacubitril at 290nm,

a_Y1= Absorbitivity of Valsartan at 230nm,

a_Y2= Absorbitivity of Valsartan at 290nm.

Method validation:

The method was developed and validated as per the ICH guidelines for linearity, precision, accuracy, LOD and LOQ.

Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solution of SAC and VAL. The beer-Lambert’s concentration range was found to be 2-14μg/ml for SAC and 2-50μg/ ml VAL. The linearity of the relationship between absorbance and the concentration was determined by plotting the calibration curve for SAC and VAL respectively shown in Fig. 4 and Fig. 5. The linearity data was shown in Table 1.

Fig 4: Calibration curve of Valsartan at 290 nm

Fig 5: Calibration curve of Sacubitril at 230 nm

Table 1: Linear regression analysis of calibration curves

Particulars	SAC	VAL
Linearity Range(µg/ml)	2-14	2-50
λmax (nm)	230	290
Correlation Coefficient(r²)	0.998	0.9951
Regression Equation	y= 0.041x + 0.3229	y = 0.0184 x + 0.0959
LOD(µg/ml)	0.232	0.154
LOQ(µg/ml)	0.751	0.521

Limit of Detection and limit of quantitation:

Limit of detection (LOD) is the lowest amount of an analyte that can be detected but not necessarily as an exact value. Limit of quantification (LOQ) is the lowest amount of an analyte in a sample that can be quantitatively determined with suitable precision and accuracy. The LOD and LOQ were determined based on calibration curve method. Results are shown in Table 1.

LOD = 3.3 x σ/s, LOQ= 10 x σ/s

Where,

σ = Standard deviation of y intercept of regression lines,

S = Slope of calibration Curve

Precision:

Repeatability of method was established by analyzing various replicates samples of SAC and VAL. Precision was carried out by performing an inter day variation and Intraday variation. In Inter day variation the sample was analyzed on three consecutive days and intra-day variation the absorbances were measured three times in a day. The percentage relative standard deviation (%RSD) was calculated. The (%RSD) less than 2 indicates the precision of the method.

Table 2: Intraday and Interday Precision Data of Sacubitril and Valsartan

Drug	Concentration (µg/ml)	Intraday precision % RSD (n=3)	Interday precision %RSD (n=3)
SAC	6	0.611	0.939
	8	0.409	0.899
	10	1.877	1.997
VAL	10	0.562	0.961
	20	0.701	1.202
	30	0.928	1.585

Accuracy:

The accuracy study was performed using the standard addition method. The pre-quantified sample solutions of SAC and VAL were spiked with an extra 50, 100, and 150% of the standard SAC and VAL. The percentage recovery of the samples were calculated at each concentration level.The percentage recovery for SAC and VAL were found to be 98.33-99.75% and 99.03-99.93% indicates the accuracy of the method.

Table 3: Accuracy data of Sacubitril & Valsartan.

DRUG	% Level	Amount present in Formulation (µg/ml)	Amount of standard added (µg/ml)	Amount of standard recovered^* (µg/ml)	*%Recovery^ ± SD**
Sacubitril	50%	10	5	4.83	98.33 ± 1.791
	100%	10	10	9.84	99.20 ± 0.613
	150%	10	15	14.92	99.75 ± 0.519
Valsartan	50%	12	6	5.98	99.93 ± 0.210
	100%	12	12	11.965	99.58 ± 0.295
	150%	12	18	17.971	99.03 ± 0.154

*mean of three determinations

RESULTS AND DISCUSSION:

Overlain spectra of the SAC and VAL depicted occurrence of two peaks at 230nm and 290nm, shown in Fig. 4. To optimize the UV parameters, several conditions were tried to achieve a good absorpation and peak shape for SAC and VAL. Several solvents of different compositions were tried to provide sufficient selectivity towards the drugs. Methanol as organic components resulted in better sensitivity.

The methods discussed in the present work provide a convenient and accurate way for simultaneous analysis of SAC and VAL by simultaneous equation method. In this method wavelengths selected for analysis were 230 nm for SAC and 290nm for VAL (Fig. 4). The linearity was observed in the concentration range of 2-20µg/ml with correlation coefficient were 0.988 for SAC and 2-50µg/ml correlation coefficient 0.999 for VAL. In this method, concentration of the individual drug was determined by solving the simultaneous equation at 230 nm and 290nm using the respective absorptivity value. Percent recovery for SAC and VAL, was found in the range of 98.33% to 101.92 %. Intra-day precision of the method ranged from 0.899 to 1.997 % RSD for SAC and 0.961 to 1.585 for VAL (Table 2). Inter-days precision of the method was found to be 0.408 to 1.877% RSD for SAC and 0.961 to 1.585 for VAL (Table 2), which indicate that the developed method is precise. The LODs for SAC and VAL were found to be 0.232 and 0.154 μg/ml, while the LOQs for SAC and VAL were 0.751 and 0.521μg/ml, respectively (Table 1). The accuracy experiments were carried out by the standard addition method. The percentage recoveries for SAC and VAL were found in the range of 98.33-99.75% and 98.03-99.93% respectively. The values of the recovery (%) are shown in table III, which indicated the accuracy of the proposed method.

CONCLUSION:

All the above factors lead to the conclusion that proposed method is simple, accurate, precise, sensitive and cost effective and can be applied successfully for the estimation of sacubitril and valsartan in combined dosage form.

ACKNOWLEDGMENTS:

The authors are grateful to Dr. L. Rathaiah, Chairman of Lavu educational society for providing necessary facilities to carry out the above research work.

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Received on 24.05.2019 Modified on 17.08.2019

Asian J. Research Chem. 2021; 14(2):111-114.

DOI: 10.5958/0974-4150.2021.00020.1