1St Mary’s College of Pharmacy, Secunderabad, Andhra Pradesh, India, 2IPT, Sri Padmawathi Mahila Vishwa Vidyalayam, Tirupathi, Andhra Pradesh, India
Email: naazneenshaikphd@gmail.com
Received: 29 Sep 2016, Revised and Accepted: 02 Dec 2016
ABSTRACT
Objective: A stability-indicating high performance liquid chromatographic (HPLC) method was developed and validated for the estimation of combined tablet formulation of valsartan and sacubitril.
Methods: Chromatographic separation was optimized by gradient HPLC on a C18 column [Xterra, 250 x 4.6 mm, 5µ] utilizing a mobile phase consisting acetonitrile, methanol and potassium dihydrogen phosphate, pH 3.8 in the ratio of 30: 50:20 v/v at a flow rate of 1 ml/min with UV detection at 263 nm.
Results: The retention time of sacubitril and valsartan was 3.01 min and 4.22 min respectively. Good linearity obtained over the range of 20μg/ml to 160μg/ml for valsartan and sacubitril. The correlation coefficient was found to be 0.999and0.998 for sacubitril and valsartan respectively. The % RSD of precision for sacubitril and valsartan was found to be 0.31 and 0.27 respectively. The % mean recovery was found to be 99.20-99.54% for valsartan and 99.85-100.90% for sacubitril. The results obtained for accuracy, precision, LOD, LOQ and ruggedness were within limits.
Conclusion: The proposed HPLC method was found to be simple, specific, precise, accurate, rapid and economical for simultaneous estimation of valsartan and sacubitril in bulk and tablet dosage form. Thus the validated economical method was applied for forced degradation study of valsartan and sacubitril tablet.
Keywords: Degradation study, HPLC method, Sacubitril, Valsartan
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijap.2017v9i1.15448
INTRODUCTION
Valsartan is a nonpeptide, orally active and specific angiotensin II receptor blocker acting on the AT1 receptor subtype. Valsartan is chemically N-(1-oxopentyl)-N-[[2'-(1Htetrazol-5-yl)[1,1'-biphenyl] -4-yl]methyl]-Lvaline [1-5]. Methods such as HPLC [6-8], LC-MS [9-11], Protein precipitation [12], Capillary electrophoresis [13] and Simultaneous UV-spectrophotometric methods [14-15] are reported for estimation of valsartan alone or in combination with other agents. sacubitril is chemically 4-[[(2S,4R)-5-ethoxy-4-methyl-5-oxo-1-(4-phenylphenyl)pentan-2-yl]amino]-4-oxobutanoic acid [16]. Sacubitril is an antihypertensive drug used in combination with valsartan for the treatment of heart failure. [17-19] Literature search reveals that only two analytical methods were reported for simultaneous estimation of valsartan and sacubitril from rat plasma using LC-MS/MS [20] and from a synthetic mixture using HPLC [21]. There is no stability indicating analytical methods were reported for simultaneous estimation of atovaquone and proguanil. Hence a simple, rapid, sensitive and accurate stability indicating HPLC method was developed for the simultaneous estimation of valsartan and sacubitril from bulk and pharmaceutical dosage form.
Sacubitril structure from pubchem | Valsartan structure from pubchem |
MATERIALS AND METHODS [21]
Chemicals and reagents
HPLC grade methanol, acetonitrile and analytical grade trifluoro acetic acid were purchased from Merck (Mumbai, India). Sacubitril and valsartan standards were received as gift samples from Manus Akketeva and Lupin Ltd, India, respectively.
Instrumentation
The HPLC system consisted of Alliance waters 2695 with dual λ Absorbance UV detector. HPLC column BDS 250 mm x 4.6 mm, 5µ. Mobile phase filtration unit (Pall Life sciences, Mumbai, India), LAB-INDIA U. V with UV Win software, Sonicator, PH meter (LAB-INDIA), digital balance (Denver).
Preparation of sample solution
Twenty tablets each containing 24 mg of sacubitril and 26 mg of valsartan were weighed and powdered equivalent to dose, transferred to a 100 ml volumetric flask, and extracted with methanol. The mixture was sonicated for 20 min in an ultrasonic bath. The volume was adjusted to 100 ml with the same solvent and then filtered. Transfer 1 ml of solution into a 10 ml volumetric flask and diluted up to the mark with diluents. to obtain a Final concentration of sacubitril and valsartan was found to be 24 and 26μg/ml respectively.
Chromatographic conditions
Chromatographic Conditions the HPLC system consisted of Shimadzu gradient HPLC (JAPAN) with dual λ Absorbance UV detector. The wavelength of detection as set at 263 nm. Separation was carried out in isocratic mode on Xterra C18 column (4.6x250 mmx5 µm) and the retention time of sacubitril and valsartan was found to be 3.01 min and 4.22 min respectively. (fig. 1), using mobile phase consisting acetonitrile, methanol and potassium dihydrogen phosphate, pH 3.8 in the ratio of 30: 50:20 v/v at a flow rate of 1 ml/min with UV detection at 263 nm. The mobile phase filtered through nylon millipore (0.2 µm) membrane filter, purchased from pall life sciences, Mumbai and degassed with Ultrasonicator prior to use. Chromatography was carried out at room temperature 25 °C and maintains the column temperature at 32 °C.
Fig. 1: Chromatogram of sacubitril and valsartan
The developed Method was validated for linearity, precision, accuracy, ruggedness and is applied for forced degradation studies as per the ICH guidelines [22-26].
RESULTS AND DISCUSSION
Method validation
Linearity
Linear concentrations of both drugs were prepared, and the best fit line was calculated. Wide range calibration was determined by solutions containing 20μg/ml to 160μg/ml (table 1) for valsartan and sacubitril. The correlation coefficient was found to be 0.999and0.998 for sacubitril and valsartan respectively (shown in fig 2 and 3).
Limit of detection (LOD) and limit of quantification (LOQ)
The LOD is calculated using the formula 3.3 times σ/s where “σ” is the standard deviation of the intercept obtained for calibration curve and “s” is the slope of the calibration curve. Similarly, LOQ is calculated using the formula 10 times σ/s. The calculated LOD and LOQ are shown in table 2 and 3.
Table 1: Linearity data for valsartan and sacubitril
S. No. |
Sacubitril |
Valsartan |
||
Concentration(µg/ml) |
Peak area |
Concentration(µg/ml) |
Peak area |
|
1 |
20 |
5284 |
20 |
6041 |
2 |
40 |
9728 |
40 |
10734 |
3 |
60 |
14260 |
60 |
15849 |
4 |
80 |
19689 |
80 |
21347 |
5 |
100 |
24135 |
100 |
25761 |
6 |
120 |
29541 |
120 |
31474 |
7 |
140 |
34581 |
140 |
35250 |
8 |
160 |
39517 |
160 |
41502 |
Fig. 2: Calibration curve of sacubitril
Fig. 3: Calibration curve of valsartan
Table 2: LOD and LOQ results of sacubitril
Conc (µg/ml) | Area 1 | Area 2 | Area 3 | Avg area |
20 | 5373 | 5198 | 5281 | 5284 |
40 | 9826 | 9613 | 9746 | 9728 |
60 | 14119 | 14290 | 14372 | 14260 |
80 | 19492 | 19124 | 20452 | 19689 |
100 | 23865 | 23677 | 24865 | 24135 |
120 | 29238 | 29148 | 30238 | 29541 |
140 | 34611 | 34521 | 34611 | 34581 |
160 | 39184 | 39284 | 40084 | 39517 |
Intercept | -18.57 | -228.9 | -41.57 | -96.6 |
slope | 244.2 | 245.4 | 249.9 | 246.5 |
Intercept standard deviation | 115.36 | |||
LOD (µg/ml) | 1.54 | |||
LOQ(µg/ml) | 4.68 |
Table 3: LOD and LOQ results of valsartan
Conc (µg/ml) | Area 1 | Area 2 | Area 3 | Avg Area |
20 | 6039 | 5997 | 6087 | 6041 |
40 | 10178 | 11012 | 11013 | 10734 |
60 | 15817 | 15675 | 16057 | 15849 |
80 | 21156 | 21346 | 21541 | 21347 |
100 | 25195 | 26101 | 25987 | 25761 |
120 | 31034 | 31274 | 32114 | 31474 |
140 | 35273 | 35192 | 35287 | 35250 |
160 | 41312 | 41178 | 42018 | 41502 |
Intercept | 639.2 | 1040 | 968.1 | 882.4 |
Slope | 251.2 | 249.2 | 253.2 | 251.2 |
Intercept standard deviation | 213.69 | |||
LOD (µg/ml) | 2.80 | |||
LOQ(µg/ml) | 8.50 |
Precision
The intraday precision was demonstrated by injecting standard solutions of valsartan and sacubitril with 40µg/ml and 140µg/ml respectively as per the test procedure (table 4) and recording the chromatograms of six standard solutions. The % RSD of Sacubitril and Valsartan was found to be 0.31 and 0.27 respectively.
Intermediate precision
Intermediate precision of the analytical method was determined by performing method precision on in three successive days by different analysts under same experimental condition by injecting six replicate standards preparations was determined and the mean % RSD of sacubitril (40µg/ml) and valsartan (140µg/ml) was found to be 0.31 and 0.27 respectively (table 5).
Table 4: Method precision data of valsartan and sacubitril
S. No |
Sacubitril (40µg/ml) |
Valsartan (140µg/ml) |
Area |
Area |
|
1 |
9826 |
34987 |
2 |
9741 |
35016 |
3 |
9798 |
35126 |
4 |
9814 |
35195 |
5 |
9804 |
35203 |
6 |
9818 |
35183 |
Mean |
9800 |
35118 |
SD |
30.64 |
94.87 |
%RSD |
0.31 |
0.27 |
Table 5: Precision data for sacubitril and valsartan
Sacubitril area for 40µg/ml |
Valsartan area for 140µg/ml |
|||||||
S. No. |
day-1 |
day-2 |
day-3 |
avg |
day-1 |
day-2 |
day-3 |
avg |
1 |
9806 |
9797 |
9787 |
9797 |
34917 |
34882 |
34847 |
34882 |
2 |
9722 |
9712 |
9702 |
9712 |
34946 |
34911 |
34876 |
34911 |
3 |
9778 |
9769 |
9759 |
9769 |
35056 |
35021 |
34985 |
35021 |
4 |
9794 |
9785 |
9775 |
9785 |
35125 |
35089 |
35054 |
35089 |
5 |
9784 |
9775 |
9765 |
9775 |
35133 |
35097 |
35062 |
35097 |
6 |
9798 |
9789 |
9779 |
9789 |
35113 |
35077 |
35042 |
35077 |
Mean |
9781 |
9771 |
9761 |
9771 |
35048 |
35013 |
34978 |
35013 |
SD |
30.6 |
30.6 |
30.5 |
30.6 |
94.69 |
94.59 |
94.5 |
94.59 |
% RSD |
0.31 |
0.31 |
0.31 |
0.31 |
0.27 |
0.27 |
0.27 |
0.27 |
Accuracy
Accuracy of the method was established by performing recovery studies according to the ICH guidelines. Spiked samples were prepared by spiking pre-analyzed sample solutions with the pure drug at three different concentration levels each in triplicate. Mean percentage recovery values at three different concentrations of the two drugs were calculated. The % mean recovery of sacubitril (99.20-99.54%) and valsartan (99.85-100.90. %) at each level was within the limits of 98% and 102% (table 6).
Table 6: Accuracy of valsartan and sacubitril
Accuracy of sacubitril |
|||||||
S. N0. |
Conc. |
Calculated concn. |
% recovery |
Mean recovery |
SD |
%RSD |
|
1 |
80 |
79.54 |
99.43 |
||||
2 |
80 |
79.43 |
99.29 |
99.43 |
0.14 |
0.15 |
|
3 |
80 |
79.66 |
99.58 |
||||
1 |
160 |
159.38 |
99.61 |
||||
2 |
160 |
158.96 |
99.35 |
99.54 |
0.16 |
0.17 |
|
3 |
160 |
159.44 |
99.65 |
||||
1 |
240 |
240.05 |
100.02 |
||||
2 |
240 |
236.88 |
98.70 |
99.20 |
0.71 |
0.72 |
|
3 |
240 |
237.35 |
98.89 |
||||
Accuracy of valsartan |
|||||||
S. No. |
Conc. |
Calculated concn. |
% recovery |
Mean recovery |
SD |
%RSD |
|
1 |
80 |
80.45 |
100.56 |
|
|
|
|
2 |
80 |
80.55 |
100.69 |
100.90 |
0.48 |
0.48 |
|
3 |
80 |
81.16 |
101.45 |
|
|
|
|
1 |
160 |
160.86 |
100.53 |
|
|
|
|
2 |
160 |
160.98 |
100.61 |
100.57 |
0.038 |
0.04 |
|
3 |
160 |
160.92 |
100.57 |
|
|
|
|
1 |
240 |
239.83 |
99.92 |
|
|
|
|
2 |
240 |
239.51 |
99.79 |
99.85 |
0.069 |
0.07 |
|
3 |
240 |
239.59 |
99.83 |
|
|
|
Ruggedness
The ruggedness of method for Valsartan and Sacubitril was calculated with six injections of 68μg/ml in two batches using two different columns. The % CV of ruggedness for sacubitril was 0.14 with column-1 and 0.04 with column-2, and the % CV of ruggedness for valsartan was 0.04 with column-1 and 0.03 with column-2 (table 7), which is within acceptance limits.
Table 7: Results of ruggedness
Sacubitril 160μg/ml |
Valsartan 160μg/ml |
|||
S. No. |
Column 1 |
Column 2 |
Column 1 |
Column 2 |
1 |
159.06 |
159.12 |
159.18 |
159.14 |
2 |
159.06 |
159.2 |
159.21 |
159.01 |
3 |
159.34 |
159.09 |
159.14 |
159.04 |
4 |
159.54 |
159.22 |
159.02 |
159.02 |
5 |
159.15 |
159.11 |
159.15 |
159.09 |
6 |
159.55 |
159.24 |
159.04 |
159.11 |
Mean |
159.28 |
159.16 |
159.12 |
159.06 |
±SD |
0.22 |
0.06 |
0.07 |
0.052 |
% CV |
0.14 |
0.040 |
0.048 |
0.033 |
% accuracy |
99.55 |
99.47 |
99.45 |
99.41 |
Results of stress degradation studies
Stress degradation studies were performed as per the ICH guidelinesQ1A (R2) Stability Testing of New Drug Substances and Products, using the proposed validated analytical method (table 10 and 11)
Acid degradation studies
To 1 ml of stock solution valsartan and sacubitril, 1 ml of 2N HCl was added and refluxed for 30 min at 60 °c. From the above solution10 µl was injected into the system and the chromatograms were recorded to detect the stability of the sample. (fig. 2)
Fig. 4: Chromatogram of acid degradation
Alkali degradation studies
To 1 ml of stock solution of standard drug and sample valsartan and sacubitril, 1 ml of 2N NaOH was added and refluxed for 30 min at 60 °c. From the above solution10 µl was injected into the system and the chromatograms were recorded to detect the stability of the sample. (fig. 5).
Oxidative degradation
To 1 ml of stock solution of standard drug and sample of valsartan and sacubitril, 1 ml of 20% H2O2was added and refluxed for 30 min at 60 °c. From the above solution10 µl was injected into the system and the chromatograms were recorded to detect the stability of sample (fig. 6).
Fig. 5: Chromatogram of base degradation
Fig. 6: Chromatogram of oxidative degradation
Photostability studies
The photochemical stability of the drug was also studied by exposing the 36 µg/ml solution to UV Light by keeping the beaker in UV Chamber for 7days or 200 Watt-hours/m2in photostability chamber. For HPLC study, from the above solution10 µl was injected into the system and the chromatograms were recorded to detect the stability of the sample. (fig. 7).
Fig. 7: Chromatogram of UV degradation
Thermal degradation studies
The 1 ml of stock solution of standard drug and sample of valsartan and sacubitril was exposed to temperature 105 °C for 24 h for HPLC study, from the above solution10 µl was injected into the system and the chromatograms were recorded to detect the stability of sample (fig. 8).
Fig. 8: Chromatogram of thermal degradation study
Table 8: Results of stress degradation studies of sacubitril
S. No. | Stress conditions | Time | % assay | % degradation | Purity angle | Purity threshold |
1 | Acid Degradation | 30 min | 91.2 | 8.8 | 0.17 | 0.21 |
2 | Base Degradation | 30 min | 92.6 | 7.4 | 0.16 | 0.22 |
3 | Peroxide Degradation | 30 min | 97.5 | 2.5 | 0.22 | 0.25 |
4 | UV Degradation | 7 d | 96.6 | 3.4 | 0.21 | 0.28 |
5 | Thermal Degradation | 24 h | 97.8 | 2.2 | 0.14 | 0.18 |
Table 9: Results of stress degradation studies of valsartan
S. No. | Stress conditions | Time | % assay | % degradation | Purity angle | Purity threshold |
1 | Acid Degradation | 30 min | 97.3 | 2.7 | 0.12 | 0.16 |
2 | Base Degradation | 30 min | 97.1 | 2.9 | 0.19 | 0.25 |
3 | Peroxide Degradation | 30 min | 91.7 | 8.3 | 0.16 | 0.20 |
4 | UV Degradation | 7 d | 95.8 | 4.2 | 0.17 | 0.23 |
5 | Thermal degradation | 24 h | 98.4 | 1.6 | 0.19 | 0.26 |
Valsartan and sacubitril undergoes significant degradation in acidic, oxidation, alkaline, and UV. Comparatively, More degradation was found with acid and base for sacubitril and with peroxide for valsartan. As per ICH guidelines peak, purity angle should be less than peak purity threshold. Hence, a method of the analysis of valsartan and sacubitril in tablet dosage form shows that the degradation product doesn’t interfere with the analytical determination. Hence the proposed analytical method is also useful for the determination of valsartan and sacubitril stability in a sample of the pharmaceutical dosage form.
CONCLUSION
In present study valsartan and sacubitril simultaneously estimated by HPLC, good linearity obtained for both drugs (20μg/ml-160μg/ml) with Correlation coefficient of 0.999and0.998 for sacubitril and valsartan respectively. The results for precision, recovery and ruggedness were within limits. Hence the method was successfully applied for degradation studies, the developed stability indicating HPLC-UV method for simultaneous estimation of valsartan and sacubitril was novel, simple, precise, accurate, robust and cost-effective method. There is no HPLC method reported till now on selected combination drugs. Hence the developed method suitable for the routine analysis and quality control and percentage degradation of pharmaceutical preparations containing these drugs either individually or in combination.
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