ICH-Compliant HPTLC Method for the Quantitative Determination of Sacubitril and Valsartan in Pharmaceutical Tablets

Manisha P. Puranik¹*, Debarshi Kar Mahapatra², Shital D. Tiple¹, Pornima G. Zade¹

¹Department of Quality Assurance, Institute of Pharmaceutical Education and Research,

Borgaon (Meghe), Wardha 442001, Maharashtra, India.

²Chitkara College of Pharmacy, Chitkara University, Rajpura 140401, Punjab, India.

*Corresponding Author E-mail: manisha68_12@yahoo.com

ABSTRACT:

The multiple sample analysis of Sacubitril (SAC) and Valsartan (VAL) in raw materials of drugs and tablets as pharmaceutical products was developed and validated through advanced thin-layer chromatographic procedures. This method was characterized to be efficient, reliable, repeatable, as well as being user-friendly. The target compounds were separated using aluminum sheets that had previously been coated with silica gel 60 F254 and uses a toluene, ethyl acetate, formic acid and triethylamine solvent mixture in order to accomplish its separation, where toluene was used in a triethylamine ratio of 7:3: 0.1:0.3 v/v/v/v respectively. The CAMAG LINOMAT V applicator was used to apply sample bands, the chromatograms scanned by means of the CAMAG TLC Scanner III. Collection and analysis of the data was processed with WinCATS software 1.4.3. SAC and VAL produced well-differentiated retention factor (Rf) values of 0.66 and 0.72, respectively. Throughout the analysis, methanol was used as the diluting medium. The developed method was validated following the ICH Q2(R1) standards, evaluating key parameters such as calibration curve linearity, recovery, reproducibility, and method consistency. Excellent correlation coefficients were observed, with r² values of 0.9977 for SAC and 0.9947 for VAL, confirming linear response within the tested range. Accuracy assessments via recovery at 50%, 100%, and 150% levels showed average retrieval rates of 99.03% for SAC and 100.74% for VAL. The repeatability of the technique, reflected by %RSD values, was found to be 0.716% for SAC and 0.465% for VAL. Further robustness testing through intra-day and inter-day variability studies yielded %RSD figures of 0.582 and 0.149 for SAC, and 0.466 and 0.416 for VAL, verifying the method's stability and dependability across different testing conditions.

KEYWORDS: Sacubitril, Valsartan, High-performance thin-layer chromatography, Validation, Estimation, Tablet.

INTRODUCTION: 

The co-administration of Sacubitril and Valsartan, classified under angiotensin receptor–neprilysin inhibition therapy, has revolutionized therapeutic approaches for individuals suffering from heart failure with reduced pumping efficiency.¹

Sacubitril (depicted in Figure 1A) blocks the action of neprilysin and undergoes metabolic conversion to LBQ657, its bioactive derivative. This compound prevents the enzymatic degradation of beneficial peptides that support cardiovascular health, thereby promoting effects such as blood vessel dilation, increased urinary sodium elimination, and reduced tissue scarring.² In contrast, Valsartan (shown in Figure 1B), which belongs to the angiotensin II receptor antagonist group, interferes with the renin–angiotensin–aldosterone cascade, easing vascular tension and lowering the strain on the heart.³ When used in tandem, their synergistic pharmacology leads to superior clinical results, such as decreased death rates from cardiac causes and fewer admissions due to heart failure. These favorable outcomes were first confirmed in the PARADIGM-HF trial and later reinforced by numerous observational studies in routine medical settings.⁴

A

B

Figure 1: Structure of (A) Valsartan and (B) Sacubitril.

As the fixed-dose pairing of Sacubitril and Valsartan continues to demonstrate significant clinical benefit and widespread adoption in therapeutic settings, ensuring the integrity, performance, and safety of its oral dosage forms becomes critically essential.⁵ Robust, accurate analytical strategies are indispensable for tasks ranging from routine batch verification to regulatory documentation, formulation refinement, and long-term stability testing.⁶ Though sophisticated techniques like liquid chromatography and mass spectrometry are routinely employed for such analyses, there is a growing emphasis on developing faster, more economical, and environmentally responsible methodologies that can be feasibly adopted by both manufacturing sectors and research institutions.⁷

Among these alternatives, High-Performance Thin-Layer Chromatography (HPTLC) has emerged as a preferred platform due to its user-friendly nature, minimal solvent requirements, capacity for high-throughput screening, and simultaneous evaluation of multiple test samples. These attributes make it particularly well-suited for settings with limited financial or technological resources.⁸ However, for any HPTLC procedure to be considered trustworthy, it must undergo stringent assessment aligned with international standards, such as those prescribed by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines.⁹

This study outlines the creation and verification of a novel HPTLC protocol specifically designed for concurrent estimation of Sacubitril and Valsartan in combined oral dosage forms. The procedure was carefully refined for optimal solvent system selection, plate development parameters, and detection wavelength. Comprehensive validation was carried out in alignment with ICH Q2(R1) specifications, assessing metrics such as response consistency, measurement precision, and calibration reliability. The developed method is intended to serve as a tool dependable for QA and regulatory review of Sacubitril–Valsartan pharmaceutical products.

MATERIALS AND METHODS:

Instrumentation:

The analysis steps were performed on an HPTLC system (CAMAG, Muttenz, Switzerland) with an automated Linomat V sample applicator, an HPTLC Scanner 3 and the application and analysis of results was done by use of the visionCATS software. The silica gel 60 F₂₅₄ aluminum-backed plates purchased at E. Merck, Mumbai were used in the chromatographic separation in a twin-trough glass chamber; purchased at CAMAG, Switzerland. Supplementary instruments utilized for sample handling and visualization included an ultrasonic bath, a thermostatic drying oven, and a UV cabinet.

Materials:

Sacubitril was sourced from Pharmaffiliates Analytics & Synthetics Pvt. Ltd., located in Panchkula, Haryana, while Valsartan was acquired through Dhamtec Pharma and Consultants, based in Navi Mumbai, Maharashtra. A commercially available fixed-dose combination tablet—LASTAVIN-S 50, produced by Ajanta Pharma Ltd. (Mumbai, India), comprising 24mg SAC and 26mg VAL—was purchased from a retail drugstore situated in Wardha, Maharashtra. All chemicals and solvents utilized in this research, including ethyl acetate, toluene, methanol, formic acid, and triethylamine, were of analytical purity and obtained from Loba Chemie Pvt. Ltd., Mumbai.

Standard solution preparation:

An accurately weighed 10mg sample of either Sacubitril or Valsartan was introduced into a 10mL volumetric container. The compound was initially dissolved using methanol, and the solution was then made up to the mark with the same solvent, yielding a primary stock solution at a concentration of 1000µg/mL. From this stock, 1mL was carefully pipetted into another 10 mL volumetric flask, and the volume was again adjusted with methanol to obtain a working standard solution containing 100µg/mL.¹⁰

HPTLC Method development:

Multiple preliminary experiments utilizing thin-layer chromatography (TLC) were conducted to determine the most suitable solvent composition for concurrently analyzing Sacubitril and Valsartan. After evaluating the migration patterns and resolution of both compounds, a suitable mobile phase composed of toluene, ethyl acetate, formic acid, and triethylamine in a volumetric proportion of 7:3:0.1:0.3 v/v/v/v was finalized as the most effective for separation. This combination yielded sharp, distinctly separated bands for each analyte. The finalized mobile phase was then employed for establishing and validating the HPTLC procedure. A freshly made combined standard solution was prepared, comprising 100µg/mL of both analytes. From this solution, 10microliters were precisely applied as 6 mm-wide streaks onto silica gel 60 F₂₅₄ plates using the CAMAG Linomat V applicator under a nitrogen environment to ensure uniform application. The pre-coated plates were subjected to chromatographic development in a CAMAG twin-trough chamber, which had been pre-saturated with the selected mobile phase for 20minutes before initiating the process. The development process proceeded until the solvent front reached 80% of the plate’s total length under standard room conditions. After solvent evaporation, densitometric analysis was performed at a wavelength of 254nm utilizing the CAMAG HPTLC Scanner 3 instrument.¹¹

Calibration Curve:

To construct the calibration plots, measured volumes of 2, 4, 6, 8, 10, and 12 microliters from the standard working solutions of Valsartan and Sacubitril—each having a concentration of 100µg/mL—were precisely applied onto TLC plates coated with silica gel 60 F₂₅₄ using a CAMAG Linomat V system. This process yielded analyte loads ranging from 0.2 to 1.2 micrograms per spot. The developed plates were introduced into a CAMAG twin-trough chamber that had been pre-equilibrated with the chosen mobile phase for 10 minutes. The solvent front was allowed to ascend until it reached approximately 80% of the plate's height, after which the plates were removed and left to dry in ambient air. Spot detection and quantification were performed using densitometric scanning at 254nm. Calibration curves were constructed by plotting the amount of drug applied per spot (µg) on the horizontal axis against the corresponding peak areas on the vertical axis. A linear regression model was applied to evaluate the method’s linearity.¹²

Standard Laboratory mixture:

Precisely measured quantities of Valsartan (26 mg) and Sacubitril (24mg) were introduced into a methanol pre-filled volumetric flask. The contents were subjected to ultrasonic agitation for 15 minutes to guarantee thorough solubilization. Afterward, the solution was made up to the mark with methanol and passed through Whatman No. 42 filter paper to achieve a clear solution. A portion of this filtrate was then appropriately diluted using methanol to prepare a working solution containing 104 µg/mL VAL and 96µg/mL SAC. From this prepared sample, 10 microliters were applied as narrow 6 mm bands on a pre-coated HPTLC plate for subsequent chromatographic evaluation.¹³

Tablet formulation:

Twenty dosage units were accurately weighed and finely ground to obtain a homogeneous powder, ensuring even dispersion of the active ingredients From this powdered blend, an amount corresponding to 26mg of Valsartan (approximately 24mg of Sacubitril) was carefully measured (in five replicates) and placed into a 25mL volumetric flask. Methanol (10mL) was introduced into the volumetric flask, and the contents were sonicated for 20minutes to facilitate effective extraction of the active components. Following ultrasonication, the solution was brought up to the mark with methanol to achieve the desired volume. The prepared sample was then passed through Whatman No. 41 filter paper to obtain a clear solution for further chromatographic evaluation.¹⁴

Validation of HPTLC Method:

The established HPTLC technique was thoroughly validated following the ICH Q2A and Q2B protocols, aligned with the standards of the United States Pharmacopoeia (USP) and in accordance with recommendations set by the US Food and Drug Administration (USFDA).

Linearity and range:

An accurately weighed portion corresponding to 10mg of either Sacubitril or Valsartan was introduced into a 10 mL volumetric flask. Methanol was added to dissolve the compound, and the solution was made up to volume with the same solvent. To ensure complete dissolution, the mixture was subjected to sonication for five minutes. The resulting solution was then filtered to yield a clear stock solution with a concentration of 1000µg/mL. Subsequently, 1mL of this stock was precisely transferred to another 10mL volumetric flask and diluted with methanol to the mark, producing a working standard of 100µg/mL. Subsequently, varying volumes (1mL to 6mL) from this working standard were dispensed into individual 10mL flasks and diluted with methanol to produce a range of solutions with final concentrations between 10 and 60µg/mL. From each of these dilutions, 10 microliters were applied onto pre-coated silica gel TLC plates as narrow bands, each measuring 6mm in width. After chromatographic development and densitometric scanning, the corresponding chromatograms were analyzed. A calibration graph was plotted using the mean peak area values against their respective concentrations (µg/mL), and the obtained results were analyzed using linear regression to evaluate the method’s ability to produce a response directly proportional to analyte concentration.¹⁵

Accuracy:

To assess the reliability of the proposed HPTLC procedure, a recovery experiment was conducted through the standard spiking technique. Known quantities of the reference compound were added to the sample matrix at three graded levels—half, equal to, and one-and-a-half times the intended concentration. Each concentration point was analyzed three times to verify consistency in results. The average recovery percentage was determined and reported as mean ± confidence limits. Additionally, the percentage relative error (%RE) was computed for each level, reflecting the deviation from the actual spiked values.¹⁶

Precision:

To evaluate the reproducibility of the optimized HPTLC technique, precision studies were carried out using a spike-recovery approach at three concentration tiers: 80%, 100%, and 120% of the nominal dose. For intra-day variation, each level was examined three times over the course of a single working day. To determine inter-day consistency, the same procedure was repeated in triplicate across three successive days. The consistency was quantified through %RSD for the observed responses at each concentration bracket.¹⁷

RESULTS AND DISCUSSION:

Chromatographic conditions:

Initial experiments confirmed that both Sacubitril and Valsartan exhibited good solubility in methanol, establishing it as the appropriate solvent for sample preparation throughout the investigation. During the optimization phase, multiple solvent systems with varying polarity profiles and volume proportions were assessed to attain distinct separation between the two compounds. Following extensive evaluation, the optimal separation was obtained using a mixture of toluene, ethyl acetate, formic acid, and triethylamine in a volumetric ratio of 7:3:0.1:0.3 v/v/v/v. Chromatographic development was performed after a 20-minute saturation period in the chamber, and detection was executed at a wavelength of 254nm. Under these refined experimental parameters, Sacubitril and Valsartan yielded retention factor values of 0.66 and 0.72, respectively, reflecting efficient and reliable peak separation (refer to Figure 2).

Figure 2: Chromatogram depicting (A) Effective separation of SAC and VAL (B) Track scanning at 254 nm.

Method validation:

Linearity and range:

Standard plots were generated for Valsartan and Sacubitril across a concentration span ranging from 0.2 to 12 micrograms per application. The analysis demonstrated excellent proportionality within this interval. For Valsartan, the determination coefficient (R²) was calculated as 0.994, with the linear regression model expressed as Y = 3944.5x + 644.83. In the case of Sacubitril, the R² value reached 0.992, corresponding to the equation Y = 4283.4x + 935.55. These elevated R² scores confirm a strong and consistent linear association between analyte concentration and detector response for both compounds. The respective graphical representations of these calibration models are presented in Figures 3A and 3B.

A                                                                                                       B

Figure 3: Linearity profile of (A) Valsartan and (B) Sacubitril.

Accuracy:

The reliability of the newly established HPTLC technique was assessed through recovery-based evaluation, employing the calibration line wherein both the slope and intercept significantly influenced the calculation of percentage recovery. The findings revealed that Valsartan and Sacubitril exhibited recovery rates of 99.27% and 99.43% by weight, respectively. Relative standard deviation values remained under the 2% threshold across three concentration levels—half, equal, and one and a half times the nominal dose—confirming that the results conformed to official pharmacopeial standards. This consistency demonstrates that the method possesses high accuracy and meets the required criteria for analytical validation.

Precision:

The consistency of the developed HPTLC method was evaluated by calculating the %RSD, which serves as a crucial metric for assessing repeatability. For Valsartan and Sacubitril, %RSD values were calculated as 0.0071 and 0.00465, respectively—both substantially beneath the 2% benchmark outlined by ICH standards, as detailed in Table 1. These minimal deviations emphasize the high level of precision achieved through this approach. Additionally, the method’s ruggedness was verified by intentionally modifying variables such as time intervals (same-day vs. multi-day testing) and analyst involvement. Even with these changes, %RSD values for both compounds consistently remained below the 2% limit, confirming the analytical procedure’s stability and dependability. These outcomes fulfill the validation expectations described in the ICH Q2(R1) framework and support the method’s applicability for routine assessment of fixed-dose pharmaceutical formulations containing Sacubitril and Valsartan. 

Recovery:

The recovery analysis revealed that Sacubitril and Valsartan achieved average retrieval rates of 99.03% ± 1.626 and 100.74% ±1.120, respectively, accompanied by relative standard deviation values of 0.0164 for Sacubitril and 0.0110 for Valsartan. These outcomes strongly affirm that the proposed HPTLC procedure delivers both high trueness and reliability. The remarkably low RSD percentages highlight outstanding reproducibility and negligible variation during repeated testing (n = 5) conducted under uniform experimental setups. Additionally, all observed figures comfortably comply with the acceptance thresholds specified by the United States Pharmacopeia (USP), which stipulates that validated analytical procedures should maintain RSDs below 2%. The summarized data in Table 2 further corroborates the method’s robustness and underscores its practicality for routine assessment of Sacubitril and Valsartan in combined dosage products.

Table 1: Precision study.

S. No	Drug	Wt. taken (mg)		Peak area		% Estimated
		VAL	SAC	VAL	SAC	VAL	SAC
1.	Standard	26.1	24	3892.3	5783.6	-	-
2.	Tablet	223		3951.8	5831.2	99.17	99.01
				3931.6	5821.3	99.57	98.84
				3950.6	5849.3	100.06	99.32
				3929.1	5858.9	99.51	99.48
				3988.5	5891.3	101.02	100.03
					Mean	99.87	99.34
					±S.D.	0.7153	0.4620
					R.S.D.	0.0071	0.00465
					C.V.	0.716	0.465

Table 2: Recovery study.

S. No.	Level of accuracy	Weight of tablet powder taken (mg)	% of drug Found on pre Analysed basis		Amount of Pure drug Added (mg)		Peak area		% Recovery
			VAL	SAC	VAL	SAC	VAL	SAC	VAL	SAC
1.	80%	223	98.74	100.05	20.8	19.2	4698.8	7021.6	97.45	99.84
2.	100%	220	100.88	100.36	26	24	4931.6	7231.3	98.96	100.4
3.	120%	219	101.14	101.37	31.2	28.8	5150.6	7562.3	100.7	102
								Mean	99.03	100.74
								±S.D.	1.626	1.120
								R.S.D.	0.0164	0.0111
								C.V.	1.64	1.111

CONCLUSION:

This study effectively developed and validated an HPTLC method for the simultaneous quantification of Sacubitril and Valsartan, both as individual compounds and within fixed-dose tablet formulations, following the criteria outlined in the ICH Q2(R1) framework. A carefully selected solvent mixture of toluene, ethyl acetate, formic acid, and triethylamine in a ratio of 7:3:0.1:0.3 v/v/v/v delivered distinct and consistent separation of both components, yielding reproducible Rf values of 0.66 for Sacubitril and 0.72 for Valsartan. Validation through recovery trials at varied concentrations confirmed the method’s reliability, while precision assessments conducted within the same day and across different days verified its repeatability and consistency under diverse analytical scenarios. The method also demonstrated robustness, maintaining stable performance despite intentional modifications in experimental settings. Due to its ease of execution, low operational cost, reduced solvent consumption, and swift processing capability, this HPTLC protocol presents an excellent tool for ongoing quality surveillance and regulatory scrutiny of fixed-dose Sacubitril–Valsartan pharmaceutical combinations. It serves as a viable, resource-efficient substitute for more intricate analytical systems and holds promise for broad adoption across quality control environments in the pharmaceutical sector.

CONFLICT OF INTEREST:

No conflict of interest is declared.

ACKNOWLEDGEMENT:

MPP acknowledges IPER, Wardha and DKM acknowledges Chitkara University, Rajpura for the technical support provided by them.

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Received on 06.07.2025      Revised on 16.09.2025

Accepted on 22.10.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5395-5400.

DOI: 10.52711/0974-360X.2025.00778

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