Separation and Quantification of Sacubitril-Valsartan Combination in Tablets by a New Ion-pair HPLC

 

Saleh Trefi¹*, Yaser Bitar¹, Véronique Gilard²

 

ABSTRACT:

The objective of this study was to develop and validate a new ion-pair reversed phase high performance liquid chromatographic method on a standard C18-type stationary phase with UV detection for the analysis of the recent two-drugs combination sacubitril/valsartan in tablets. The mobile phase consisted of a mixture of 45% of 10-3 M of cetyltrimethylammonium bromide (cetrimide) as the ion-pairing agent and 55% acetonitrile. The method validation was based on linearity, accuracy, precision, robustness and specificity. This method exhibits good linearity and accuracy with mean recovery values between 95.0-105.0%, precision with relative standard deviations of the calculated concentrations less than 5.0% and specificity in the presence of degradation products. These results indicates that the proposed method is simple and applicable for the separation and determination of sacubitril, valsartan combination in tablets and could be a relevant method to implement in quality control laboratories.

 

 

 

INTRODUCTION:

The association of valsartan and sacubitril (Fig.1) was recently proposed as a first-in-class angiotensin receptor-neprilysin inhibitor for the treatment of heart failure^1,2. The formulation Entresto (Novartis) was approved in 2015 by U.S. Food and Drug Administration and European Medicines Agency. Nowadays, sacubitril/valsartan combination has been approved in more than fifty countries¹. Few chromatographic^3-7 or spectroscopic⁸ methods for analyzing sacubitril and valsartan in formulations were reported. The previous reported HPLC methods proposed reversed phase separation with a classical elution with phosphate^4,6 or ammonium³ buffers.

 

Many chromatographic methods for analyzing valsartan in other combined formulations were also reported^9-15.

 

Very few papers about the utility of ion pair chromatography in drug analysis, were reported¹⁶.

 

In this study, we propose a novel ion pair HPLC method. This proposed method, may be applied to the analysis of sacubitril/valsartan combination with some advantages. The use of cetrimide as a surfactant in the mobile phase, instead of buffers improves its flow ability through the HPLC chain and reduces problems of precipitation of salts in the pump, arising from the use of buffers. Moreover, this elution using a surfactant such as cetrimide decreases the pressure applied on the column.

 

Therefore, the objective of this work was to develop and validate a novel ion pair HPLC method, for the assay sacubitril, valsartan in tablets. This quick and easy method could be of interest for updating analytical method used for drug quality control in generic pharmaceutical industries. To the best of our knowledge, this is the first ion pair liquid chromatography method proposed to determine both sacubitril and valsartan in pharmaceutical formulations. This new method was utilized to determine another pharmaceutical combination recently¹⁷.

 

Figure 1 Chemical structures of Sacubitril and Valsartan.

 

MATERIAL AND METHODS:

Chemicals and reagents:

Standards of sacubitril and valsartan were gifted by Golden Med Pharma Industries (Tartous, Syria). The tablet samples whose brand names are Sacuval 24/26 and Sacuval 49/51 produced by Alzahabia Industries (Tartous, Syria), Arni 24/26 manufactured by Unipharma (Damascus, Syria) and Sacuplus 49/51 and Sacuplus 97/103 by UltraMedica (Damascus, Syria) were analyzed. As received, they were stored in the dark at ambient temperature and humidity. They were all analyzed within expiry dates. All the other used reagents were of HPLC grade: acetonitrile (PROLABO), cetrimide (TCI Chemicals), deionized water for HPLC. All solutions were filtered through a 0.45µm Millipore membrane.

 

Chromatography:

The HPLC instruments used were an Agilent 1260 infinity (Agilent Technologies, CA, USA), equipped with a UV detector and a Shimadzu LC 20-AT (Shimadzu, Kyoto, Japan) with diode array detector. The analysis conditions were: NUCLEODUR-C18 ec- Machrey-Nagel reversed phase column (250 mm × 4.6 mm i.d; 5µm particle size), detection wavelength 254 nm. The final mobile phase consisted of 45% of 10-3 M cetrimide and 55% acetonitrile, leading to a measured pH of 10.0. The flow rate was 1mL/min and the separation was performed at room temperature. The pH meter used was from Crison (Madrid, Spain).

 

For the surfactant concentration optimization, the same chromatographic conditions were used except that the mobile phase mobile phase consisted of 45% of 10-2 M cetrimide and 55% acetonitrile (pH=10).

 

Preparation of samples:

Preparation of standard solutions:

A precise amount of each standard was accurately weighed and dissolved in methanol then mixed to obtain the stock standard solution at 49 mg in 500 mL (0.0098 % w/v) for sacubitril and 51 mg in 500 mL (0.0102 % w/v) for valsartan. This starting standard solution, was used for the preparation of all diluted solutions.

 

Sample preparation for tablets:

Twenty tablets of Sacuval 24/26 or Sacuval 49/51, were crushed and powdered, the powder of two tablets of Sacuval 24/26 doses or 1 tablet of Sacuval 49/51 doses was transferred into a 100 mL volumetric flask. Methanol was added and the content was dispersed under magnetic stirring during 20 min and then sonicated for 10 min, until the two active pharmaceutical ingredients were fully dissolved.

 

The same protocol was applied, in order to prepare Arni 24/26 and Sacoplus 49/51 tablets for analysis. To prepare the tablets of Sacoplus 97/103, quantity of the powder containing one tablets of Sacoplus 97/103 was transferred into a 200 mL volumetric flask containing methanol. For the HPLC analysis, these stock solutions were diluted before analysis (45 mL in a 100mL volumetric flask). Final concentrations after dilution were around 20 mg/100mL for both valsartan (22.95 to 23.4 mg/100mL) and sacubitril (20.05 to 21.825 mg/100mL). For the degradation test, the stock tablets solution Sacuval 49/51, was left at room temperature under sunlight for 60 days.

 

Method validation:

Method validation was performed including consideration of characteristics included in the international conference on harmonization (ICH) guidelines Q2 (R1)¹⁸. The method was fully validated in terms of linearity, accuracy, precision, specificity and robustness.

 

Linearity:

The linearity of analytical procedure is its ability within a given range to obtain test results which are directly proportional to the concentration of analyte in the sample¹⁸.The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method. The linearity was studied for the five following concentrations 0.0146, 0.029, 0.0585, 0.117, 0.234 mg/mL for valsartan, and 0.0135, 0.027, 0.054, 0.108, 0.216 mg/mL for sacubitril. Measurements were done in triplicate each week during three weeks (n=9).

 

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. For the quantitative approaches, at least nine determinations across the specified range should be obtained¹⁸. Here, for accuracy study, three concentration levels have been used to study the accuracy of both valsartan (0.029, 0.0585, and 0.117 mg/mL) and sacubitril (0.027, 0.054, and 0.108 mg/mL).

 

Precision:

The precision of an analytical procedure: expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions¹⁸. Precision was assessed by means of intermediate precision. Solutions 0.029 mg/mL of valsartan and 0.054 mg/mL of sacubitril have been prepared at three different times, by three analysts during three weeks, each solution was injected three times (n = 9). The calculation of intermediate precision was performed by estimating the mean, standard deviation and relative standard deviation of the six content values obtained.

 

Robustness:

A The robustness was evaluated by deliberate variation in the chromatographic conditions¹⁸ and peak areas measurements. Variations of flow rate (0.9, 1.0, 1.1 mL/min) and composition of the mobile phase (53, 55 and 57 % acetonitrile) were considered. These variations were evaluated by measuring the peak areas of sacubitril and valsartan and the resolution factor between the two peaks after injection of the stock standard solution.

 

Specificity:

Specificity is the ability to assess unequivocally the analyte in the presence of components, which may be expected to be present. Typically, these might include impurities, degradants, matrix, etc¹⁸. In this study, specificity was demonstrated by analyzing chromatograms of tablets before and after degradation. To induce a degradation of the samples, tablets solution after extraction was exposed to sunlight for 60 days at room temperature in a stoppered flask to prevent any evaporation.

 

RESULTS AND DISCUSSION:

Optimization and validation of the chromatographic method:

A simple HPLC Method on a reversed phase column was developed to separate and quantify sacubitril and valsartan in combination tablets. The method was developed and optimized for obtaining peak separation with appropriate peak symmetry and rather short retention times. In the development of the method, the influence of the concentration of cetrimide was evaluated by injection of the starting standard solution. In a first step, a mobile phase of 45% of 10^-2 M cetrimide and 55% acetonitrile was used and provide a good separation of the two species, but with a quite long retention times not compatible with routine analyses i.e. 18.85 min for sacubitril and 37.48 min for valsartan (Fig.1A). Then, the concentration of cetrimide in the mobile phase was reduced and the mobile phase was 45% of 10^-3 M cetrimide and 55% acetonitrile. This composition of the mobile phase allowed a complete separation with a shorter retention times: 6.06 min for valsartan and 8.08 min for sacubitril (Fig.2B).

 

 

 

Figure 2 Chromatogram of the stock standard solution containing a mixture of Sacubitril and Valsartan. Elution was done using a mobile phase consisting of (A) 45% of 10-2 M cetrimide and 55% acetonitrile (B) 45% of 10-3 M cetrimide and 55% acetonitrile. Chromatograms A and B were recorded using the same column but different HPLC equipment.

 

In these chromatographic conditions, the tailing factors of valsartan and sacubitril were 0.95 and 1.12 respectively whereas the resolution (Rs) between the two peaks was 3.2. We noticed that the retention time of valsartan is shorter than the sacubitril's one when using10^-3 M cetrimide and it became longer than sacubitril when using10^-2 M. This is explained by the fact that valsartan has two acidic functions, the tetrazole ring (pKa=4.7) and the carboxylic acid group (pKa=3.9), while sacubitril has only one acidic function in his structure, the carboxylic acid group (pKa=4.2). So when the concentration of the surfactant is of 10^-2 M, the two acidic functions of valsartan will form two ion pairs with two molecules of cetrimide, this double ion pairs with valsartan delay its retention time, due to increase in its affinity to the stationary phase.

 

Linearity:

Linearity between peak area and concentration was analyzed using the calibration curves obtained with standard solutions of valsartan and sacubitril at five concentrations. The linear equation obtained by the least squares method was y = 43315x + 422.78 (n=9) for valsartan and y = 23113x –169.48 (n=9) for sacubitril as shown in (Fig. 3). The resulting correlation coefficients allowing estimating the quality of the curves were 0.9989 and 0.9963 respectively, these values of R2 greater than 0.99 indicates a satisfactory linearity. The linearity was demonstrated in the concentration range (0.0146-0.234 mg/mL) for valsartan, (0.0135-0.216 mg/mL) for sacubitril.

 

 

 

 

Figure 3 Calibration curves obtained from the results of the linearity tests for sacubitril and valsartan.

 

Accuracy:

Accuracy was determined from three concentrations for valsartan and sacubitril, with three replicates. Results are reported in (Table 1). For valsartan, the results indicated that the individual recovery ranged from 92.60% to 102.04%. The recovery of Valsartan by the proposed method was acceptable, as the mean recovery value was 97.56% in the range 95.0-105.0% and RSD was 4.85%, less than 5%. For sacubitril accuracy, the same approach was used and the individual recovery ranged from 102.29% to 105.21 %. Considering the same criteria, the recovery of sacubitril by the proposed method was also satisfactory, with a mean recovery value of 103.59% and a RSD of 1.43%.

 

Table 1: Mean recoveries of three concentration levels solutions of sacubitril and valsartan.

	Valsartan		Sacubitril
Mean concentration level1 (0.117mg/ml) %a	92.60	Mean concentration level1 (0.108mg/ml) %a	102.29
Mean concentration level2 (0.058mg/ml) %a	98.03	Mean concentration level2 (0.054mg/ml) %a	103.28
Mean concentration level3 (0.029mg/ml) %a	102.04	Mean concentration level3 (0.027mg/ml) %a	105.21
Mean recovery % (±) SD	97.56 ± 4.73	Mean recovery % (±) SD	103.59 ± 1.48
%RSD	4.85	%RSD	1.43

^amean, n = 3.

 

Precision:

The precision data are shown in (Table 2). The results show coefficient of variation (RSD) under the limit of 5%. RSD of 4.80% and 1.35 % were measured for valsartan and sacubitril respectively. These results indicated that the intermediate precision of this method was acceptable for sacubitril and valsartan.

 

Table 2: Relative standard deviation of the nine determinations of solutions of Sacubitril and Valsartan.

S. No	Valsartan (0.029mg/ml)	Sacubitril (0.054mg/ml)
1	0.0314	0.0569
2	0.0317	0.0565
3	0.0315	0.0565
4	0.0294	0.0551
5	0.0281	0.0547
6	0.0293	0.0558
7	0.0277	0.0553
8	0.0300	0.0560
9	0.0297	0.0552
Mean (±) SD	0.0298±0.0014	0.0558±0.0008
RSD%	4.80	1.35

 

Robustness:

The evaluation of the robustness considering variation of the flow rate resulted in almost no variation of the resolution Rs that remains in all conditions lower than 3 between the two peaks but relatively small variations of the peak area of both peak (-3.71 % and -1.11 % ≤ 5 %) indicating an acceptable robustness with respect to the flow rate (Table 3). Similarly, variation of the percentage of acetonitrile in the mobile phase has no significant influence on the peak area and resolution as reported in (Table 3).

 

 

 

 

 

 

 

Table 3: Relative standard deviation of peak area for valsartan and sacubitril under small variations of chromatographic method

Parameter	range	RSD % (Peak area of valsartan)	RSD % (Peak area of sacubitril)
Flow rate	0.9 mL/min	2.46	-0.81
	1.1 mL/min	-3.71	-1.11
Composition of the mobile phase	Acetonitrile 53 %	1.93	1.02
	Acetonitrile 57 %	-3.58	1.41

 

Specificity:

At last, for specificity evaluation, we have compared chromatograms of the initial tablets solution Sacuval 49/51, before and after a forced degradation process over sunlight exposure. The chromatogram of the tablets solution before degradation, indicated no additional peaks other than those of sacubitril and valsartan (Fig.4A) Then, a new chromatogram was recorded after forced degradation of the sample. The chromatogram of the solution of sacubitril and valsartan showed additional peaks with retention times of 3.68, 10.10 and 14.48 min, but they were well resolved from the two main peaks of valsartan and sacubitril. The resolution between the first impurity and valsartan being 3.85. We must moreover notice a slight variation of the retention times of valsartan and sacubitril (relative variation ≈ 10%) that do not affect their separation (Fig.4B) with a Rs of 3.4.

 

 

 

Figure 4 Chromatograms of the commercial tablets solution (Sacuval 49/51) of sacubitril and valsartan (A) initial solution (B) after forced degradation.

 

Application to tablet assays for quality control:

The data of commercial tablets contents are reported in (Table 4). All the commercial formulations contain the active ingredient in the range 96.61-103.73% of the stated concentration of valsartan with CV not more than 5% and 95.68-103.51% of the stated concentration of sacubitril with CV not more than 2%. These results showed that the proposed method is suitable for the analysis of commercial formulations and allow a quick and easy quality control of the valsartan and sacubitril content.

 

Table 4: Results of tablets assay by the proposed HPLC method

Formulation name	Active Ingredient and dosage	Batch number	Manufacturer name	% of nominal concentration sacubitril /valsartan	SD sacubitril /valsartan	% RSD sacubitril /valsartan
Sacuval 24/26	sacubitril24mg /valsartan26mg	001	Golden Med Pharma- Syria	103.15 / 103.73	0.47 /0.08	0.45 /0.07
Sacuval 49/51	sacubitril49mg /valsartan51mg	001	Golden Med Pharma- Syria	103.51 / 100.85	0.43/0.84	0.41 /0.84
Arni 24/26	sacubitril24mg /valsartan26mg	18 ARLT 1	UNIPHARMA- Syria	101.52 / 100.10	1.26 /2.25	1.24 /2.25
Sacoplus 49/51	sacubitril49mg /valsartan51mg	H0662	Ultra Medica- Syria	95.68 / 97.62	0.19 /2.37	0.20 /2.42
Sacoplus 97/103	sacubitril97mg /valsartan103mg	H0662	Ultra Medica- Syria	96.14 / 96.61	0.97/1.01	1.00 /1.04

 

CONCLUSION:

A simple, accurate and novel ion pair HPLC method has been developed and validated for the determination of sacubitril and valsartan. This method was used to separate a mixture of sacubitril and valsartan and for the determination of their concentrations in tablets manufactured in Syria. This simple and easy method to implement could be particularly useful in the quality control of raw materials, active pharmaceutical ingredients and especially for the assay of pharmaceutical formulations.

 

ACKNOWLEDGEMENT:

This research work was supported by University of Aleppo. ST acknowledge Golden Med Pharma Industries, Tartous-Syria, Unipharma Damascus-Syria and Ultra Medica Damascus- Syria for providing analytical standard and/or tablet samples as reported in the experimental part.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 04.12.2018         Modified on 10.01.2019

Accepted on 16.01.2019      © RJPT All right reserved