INTRODUCTION:

Sacubitril [Figure 1], chemically designated as 4-{[(2S,4R)-1-(4-Biphenylyl)-5-ethoxy-4-methyl-5-oxo-2-pentanyl]amino}-4-oxobutanoic acid. Sacubitril is a prodrug that is activated to LBQ657 by de-ethylation via esterases. LBQ657 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 volume¹. It is used as antihypertensive agent. Literature survey reveal few analytical methods are reported for estimation of sacubitril with valsartan in combined dosage form includes spectrophotometric^2-4 and RP-HPLC^5-12 methods but no UV spectrophotometric method reported yet for estimation of sacubitril alone in pharmaceutical dosage form, synthetic mixture or biological fluids. Hence an attempt was made to develop a rapid, accurate, sensitive and economical UV spectrophotometric method for estimation of Sacubitril from synthetic mixture.

Figure 1: Structure of Sacubitril

MATERIALS AND METHOD:

Instrumentation:

An ELICO UV/Visible spectrophotometer model SL 210 with 10 mm matched quartz cells was used for all spectral measurements.

Chemicals and reagents:

Mixture of Methanol and Doubled distilled Water (25:75 v/v) was used as a solvent in the present study. Both Methanol and Double distilled Water were of Merck made. Pure Sacubitril obtained as gifted sample.

Determination of λ_max:

Sacubitril (100 mg) was accurately weighed and dissolved in 100 ml of mixture of methanol and doubled distilled water (25:75 v/v) to form a stock solution (1000 μg/ml). The stock solution was further diluted suitably with the same solvent mixture to get a working standard solution of concentration 100 μg/ml. This working standard solution was suitably diluted to give a concentration of 10 μg/ml and this was then scanned in UV range. This showed an absorption maximum at 242 nm.

Figure 2: UV spectrum of Sacubitril

Procedure for calibration curve:

Aliquots (0.2,0.4……1.2 ml) of working standard solution corresponding to 2-12 μg/ml were taken in a series of 10 ml volumetric flasks and volume was made up with solvent mixture of methanol and doubled distilled water. The absorbance measurements of these solutions were carried out against solvent mixture as blank at 242 nm. A calibration curve of sacubitril was plotted. The concentration of the unknown was read from the calibration graph.

Preparation of test solution:

Synthetic mixture was prepared by mixing 50 mg of sacubitril, 2 mg povidone, 2 mg magnesium stearate and 46 mg avicel. 100 mg of this mixture was transferred to 50 ml volumetric flask and the solution was made up to required volume using methanol and doubled distilled water (25:75 v/v) . The solution was filtered and the first 10 mL of the filtrate is discarded. 1 ml of this filtrate was transferred to a 100-mlvolumetric flask and made up to final volume using methanol and doubled distilled water (25:75 v/v) to obtain a concentration of 10 μg.mL^-1. The drug concentration was calculated from the calibration curves.

RESULTS AND DISCUSSION:

Validation of method parameters¹³:

Linearity:

Six points calibration curve were obtained in a concentration range from 2-12 µg/ml for Sacubitril. The response of the drug was found to be linear in the investigation concentration range and the linear regression equation was y = 0.024x + 0.001 with correlation coefficient 0.999 (Table 2, Figure 3).

Table 1: Optical characteristics and analytical parameters

Analytical Parameters

λ _max(nm)

Beer’s law limits (µg/ml)

Molar absorptivity (lit / mol^-1 cm^-1)

Limit of Detection (LOD/ µg/ml)

Limit of Quantification (LOQ/ µg/ml)

Sandell’s sensitivity (µg/cm²/0.001 A.U.)

Regression equation (Y*)

242

2 – 12

2.5348× 10⁴

0.466

1.412

0.0395

Slope (b)

Intercept (a)

0.024

0.001

Correlation coefficient (r)

% RSD**

Confidence limits with 0.01 level

0.999

0.778

0.9624

* Y= bX+a, where X is the concentration of Sacubitril in μg/ml and Y is the absorbance at respective λ_max

** For six replicate samples.

Table 2: Calibration curve data

S. No	Conc (µg/ml)	Absorbance
1	2	0.0559
2	4	0.0976
3	6	0.1490
4	8	0.1971
5	10	0.2502
6	12	0.3010

Figure 3: Calibration curve of Sacubitril

Precision:

The precision of analytical procedure expresses the closeness of agreement between a series of measurement obtained from multiple sampling of the same homogenous sample under the prescribed condition. System precision, intraday precision and inter day precision were done. The system precision was analysed by 6 different solutions of same concentration and absorbances were noted. The result was indicated by % RSD. The results are shown in Table 2.

Repeatability or Intra-day precision was investigated on six replicate sample solutions on the same day. Inter-day precision was assessed by analyzing newly prepared sample solutions in triplicate over three consecutive days. Both inter day and intraday precision was expressed as % RSD. The % RSD values for intraday precision was found as 0.98. The % RSD for inter day precision was 0.79. The results were summarized in Table 3 and 4. The low value of % RSD for both methods indicates the high precision of the both methods.

Table 3: Precision (repeatability) data

S. No	Conc (µg/ml)	Absorbance
1	6	0.1421
2	6	0.1435
3	6	0.1442
4	6	0.1420
5	6	0.1421
6	6	0.1412
	SD= 0.001109	% RSD = 0.778

Table 4: Interday and Intraday precision

Sample No.	% Assay
Set	Intraday	Interday
1	101.8	99.3
2	101.2	98.2
3	101.3	99.0
4	101.5	99.0
5	100.6	98.6
6	99.1	100.5
Mean	101.1	99.1
SD	0.99	0.78
%RSD	0.98	0.79

Accuracy:

Accuracy of the method was ascertained by recovery studies at three levels. Standard quantity equivalent to 80%, 100% and 120% was to be added in sample. The result shown that best recoveries (99.35 - 99.71%) of the spiked drug were obtained at each added concentration, indicating that the method was accurate (Table 5).

Table 5: Accuracy results by recovery method

% Level

% Recovery

Mean % Recovery ± SD

99.63

99.13

99.45

99.40 ±0.0054

100

99.54

99.23

99.61

99.46±0.0063

120

99.56

99.67

99.84

99.69±0.0047

Robustness:

The evaluation of robustness should be considered during the development phase and depends on the type of procedure under study. It should show the reliability of an analysis with respect to deliberate variations in method parameters.

If measurements are susceptible to variation in analytical conditions, the analytical condition should be suitably controlled or a precautionary statement should be included in the procedure. The result of robustness study of the developed assay method was established in Table 6. The result shown that during all variance conditions, assay value of the test preparation solution was not affected and it was in accordance with that of actual. Hence the analytical method would be concluded as robust.

Table 6: Robustness studies

Sl no.	240 nm	242 nm	244 nm
1	0.142	0.143	0.141
2	0.143	0.142	0.141
3	0.142	0.142	0.142
4	0.141	0.141	0.143
5	0.142	0.143	0.141
6	0.143	0.142	0.141
Mean	0.142	0.142	0.141
SD	0.000753	0.0005164	0.0005164
%RSD	0.53	0.36	0.36

Solution stability study:

The solution stability was studied at different time intervals for test preparation. It was concluded that the test preparation solution was found stable up to 10 hr at room temperature, as during this time the result was not decrease below the minimum percentage. The result was summarized in Table 7.

Table 7: Stability studies

Time (Hrs.)	Standard	Sample
0	0.142	0.141
2	0.141	0.141
4	0.141	0.140
6	0.140	0.140
8	0.140	0.140
10	0.139	0.139

Table No. 8: Assay of synthetic mixture

Sr. No.	Amount taken (μg/ml)	Absorbance	Amount of drug found (μg/ml)	% Amount found
1	10	0.252	10.11	101.1
2	10	0.249	9.99	99.90
3	10	0.247	9.91	99.10
4	10	0.251	10.07	100.70
5	10	0.255	10.23	102.30
6	10	0.253	10.15	101.50

CONCLUSION:

The results and the statistical parameters demonstrate that the proposed UV spectrophotometric method is simple, specific, accurate and precise. Therefore, this method can be used for the determination of sacubitril either in bulk or in synthetic mixture without interference with commonly used excipients.

ACKNOWLEDGEMENT:

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 19.12.2018 Modified on 20.01.2019

Asian J. Research Chem. 2019; 12(1): 07-10.

DOI: 10.5958/0974-4150.2019.00002.6