Analytical Method Development and Validation of a Cardiovascular Drug By HPLC

Analytical chemistry concerns itself with the analysis of material samples to gain a thorough understanding of their chemical composition and structural architecture. It is, at its core, a measurement science — a collection of powerful ideas and methods that find use across every field of science and medicine. The discipline seeks ever-improved means of measuring the chemical composition of both natural and artificial materials [1,2].

In recent decades, the centre of gravity in analytical chemistry has shifted decisively toward instrumental methods. The speed and sensitivity of these physical and physicochemical approaches have far surpassed what classical gravimetric and volumetric analysis can deliver. Currently, most instrumental analysis rests on comparing a signal from the sample against that from a standard of known composition [3,4].

Analytical chemistry divides into qualitative analysis, which identifies the elements, ions or compounds present in a sample, and quantitative analysis, which determines how much of one or more constituents is present [29]. Classical methods rely on precipitation, extraction or distillation to separate components. Instrumental methods exploit the interaction of matter with energy to extract analytical information. The principal instrumental families include spectroscopy, electrochemistry, chromatography, thermal analysis and microscopy [5–8].

Chromatography is unique in the history of analytical methodology and is probably the most powerful and versatile separation technique available [9]. In a single procedure it can separate a mixture into its individual components and simultaneously provide quantitative data on the amount of each component present [10]. HPLC stands for high-resolution, high-pressure, high-speed liquid chromatography. It has several times the resolving power of open-column liquid chromatography and is used for speedy resolution of complex mixtures [11-12]. It provides a specific, sensitive and precise method for analysis, with ease of sample preparation and rapid turnaround [13–15]. Validation is defined as documented evidence that provides a high degree of confidence that a process will consistently produce a product meeting its predetermined specifications. The ICH Q2(R1) guideline identifies the following parameters: accuracy, linearity, precision (repeatability and intermediate precision), detection limit, quantitation limit, specificity, range and robustness [16–18].

Fig. 1: Classical Analytical Methods

2. DRUG PROFILE

Vericiguat was approved by the United States FDA on December 22, 2015 for the treatment of pulmonary arterial hypertension (PAH) to delay disease progression and reduce risk of hospitalization. PAH is a relatively rare disease with usually a poor prognosis requiring more treatment options to prolong long-term outcomes. Marketed by Actelion Pharmaceuticals under brand name Uptravi, Vericiguat and its active metabolite, ACT-333679 (MRE-269), act as agonists of the prostacyclin receptor to increase vasodilation in the pulmonary circulation and decrease elevated pressure in the blood vessels supplying blood to the lungs [19,20].

Fig. 2: Structure of Vericiguat

Table 1: Profile of drug

Heart failure involves the impaired synthesis of nitric oxide (NO) and decreased activity of soluble guanylate cyclase (sGC). Vericiguat directly stimulates sGC by binding to a target site on its beta-subunit, bypassing the need for NO-mediated activation, and causes an increase in the production of intracellular cGMP, resulting in vascular smooth muscle relaxation and vasodilation.

3. LITERATURE SURVEY

Mandhare et al. (2024) developed a QbD-based RP-HPLC method using a C18 column with methanol and 0.1% OPA (76:24) at 331 nm. LOQ was 0.7209 µg/mL, LOD was 0.2379 µg/mL [21].

Mustafa et al. (2023) used an Inertsil ODS-C18 column with water:acetonitrile (70:30) at 332 nm. Retention time was 4.500 min, linearity 2–20 µg/mL, r² = 0.9996 [22].

Madhavi et al. (2024) used a Kromacil C18 column with KH?PO?:acetonitrile (50:50) at 252 nm. RT was 4.42 min, linearity 30–70 µg/mL, recovery 99.92–100.61% [23].

Bodke et al. (2023) used a Hemochrom C18 column with 0.1% TFA:acetonitrile (65:35) at 327 nm. RT was 8.3 min, linearity 5–150 µg/mL [24].

Patel et al. (2023) used a Zorbax Eclipse Plus C18 column with KH?PO?:methanol (60:40) at 256 nm. RT was 6.9 min, linearity 50–150 µg/mL, r² = 0.9995 [25].

Gadapa N. LC-Based Analytical And Assay Methodologies For Multiclass Drug Quantification. Authors Click Publishing; 2026 Jan 23 [27].

Sahu PK, Ramisetti NR, Cecchi T, Swain S, Patro CS, Panda J. An overview of experimental designs in HPLC method development and validation. Journal of pharmaceutical and biomedical analysis. 2018 Jan 5;147:590-611 [28].

Amaar NM, Awad FS, Mortada WI, Abdallah AB. Innovative approaches in electrochemical sensing for the sensitive and selective label-free detection of vericiguat in real blood samples. Microchemical Journal. 2025 Jan 1;208:112488 [49].

Manasa K, Shiva S, Gowtham C, Akanksha S, Sreeja K, Sandhya N. A Review On Method Development And Validation Ofselected Anticancer And Cardiovascular Drugs Using Different Analytical Techniques. Journal For Innovative Development in Pharmaceutical and Technical Science (JIDPTS). 2026 Jan;9(1) [50].

Prakash L, Himaja M, Subbaiah BV, Vasudev R, Srinivasulu C, Haribabu R. Isolation, identification and characterization of degradant impurities in Tolterodine tartrate formulation. Journal of Pharmaceutical and Biomedical Analysis. 2014 Mar 5;90:215-21 [51].

4. MATERIALS AND METHODS

4.1 Drug, Chemicals and Reagents

Table 2: Drug and Drug Supplier

Table 3: List of Reagents and Chemicals used

4.2 Instrumentation

Table 4: Instrument (HPLC) Details used during Method Development

4.3 UV Spectrum

A 10 µg/mL solution of Vericiguat in methanol was scanned over 200–400 nm. The drug showed maximum absorbance at 258 nm

Fig. 2: UV spectrum of Vericiguat (λmax 258 nm)

4.4 Method Validation

The developed method was validated as per ICH Q2(R1) guidelines for linearity, accuracy, precision, robustness, specificity, LOD and LOQ [31,34,35].

5. RESULTS AND DISCUSSION

5.1 Method Development

Several mobile phase combinations were screened [25-26,30]. The trials and outcomes are summarised below.

Table 5: Different Trials of Chromatographic Condition

The selected mobile phase (50:50 v/v), pH 3.2, flow rate 0.8 mL/min gave retention at 5.268 min with TP = 12,255 and TF = 0.85.

Blank chromatogram:

Chromatogram of blank

Standard chromatogram of Vericiguat:

Fig. 3: Chromatogram of standard Vericiguat

Table 6: Result for standard Chromatogram of Vericiguat

5.2 Linearity

Working standard solutions at five concentration levels (10–50 µg/mL) were injected in duplicate and the peak areas recorded.

Fig. 4: Chromatogram of Linearity

Table 7: Result for standard Chromatogram of Vericiguat

Fig. 5: Chromatogram of Linearity 10mcg-01

Table 8: Result for Chromatogram of linearity 10mcg-01

Fig. 6: Chromatogram of Linearity 10mcg-02

Table 9: Result for Chromatogram of linearity 10mcg-02

Fig. 7: Chromatogram of Linearity 20mcg-01

Table 10: Result for Chromatogram of Linearity 20mcg-01

Fig. 8: Chromatogram of Linearity 20mcg-02

Table 11: Result for Chromatogram of linearity 20mcg-02

Fig. 9: Chromatogram of Linearity 30mcg-01

Table 12: Result for Chromatogram of linearity 30mcg-01

Fig. 10: Chromatogram of Linearity 30mcg-02

Table 13: Result for Chromatogram of linearity 30mcg-02

Fig. 11: Chromatogram of Linearity 40mcg-01

Table 14: Result for Chromatogram of Linearity 40mcg-01

Fig. 12: Chromatogram of Linearity 40mcg-02

Table 15: Result for Chromatogram of Linearity 40mcg-02

Fig. 13: Chromatogram of Linearity 50mcg-01

Table 16: Result for Chromatogram of linearity 50mcg-01

Fig. 14: Chromatogram of Linearity 50mcg-02

Table 17: Result for Chromatogram of Linearity 50mcg-02

Table 18: Linearity of Vericiguat (HPLC)

Fig. 15: Calibration curve of Vericiguat (HPLC)

Table 19: Regression equation data for Vericiguat

5.3 Accuracy (Recovery)

Recovery studies were performed at 80%, 100% and 120% levels to validate accuracy.

Fig. 16: Chromatogram of Accuracy 80%

Table 20: Result for Chromatogram of Accuracy 80%

Fig. 17: Chromatogram of Accuracy 80%-02

Table 21: Result for Chromatogram of Accuracy 80%-02

Fig. 18: Chromatogram of Accuracy 100%

Table 22: Result for Chromatogram of Accuracy 100%

Fig. 19: Chromatogram of Accuracy 100%-02

Table 23: Result for Chromatogram of Accuracy 100%-02

Fig. 20: Chromatogram of Accuracy 120%

Table 24: Result for Chromatogram of Accuracy 120%

Fig. 21: Chromatogram of Accuracy 120%-02

Table 25: Result for Chromatogram of Accuracy 120%-02

Table 26: Result of Recovery data for Vericiguat

Table 27: Statistical Validation of Recovery Studies Vericiguat

5.4 System Suitability (Repeatability)

Fig. 22: Chromatogram of System suitability No. 1

Table 28: Result for Chromatogram of System suitability No. 1

Fig. 23: Chromatogram of System suitability No. 2

Table 29: Result for Chromatogram of System suitability No. 2

Table 30: Repeatability studies on Vericiguat (HPLC)

5.5 Precision

The method was established by analyzing various replicates of Vericiguat. Solutions were analyzed to record intra-day and inter-day variation.

Chromatogram of Intra-day Precision:

Fig. 24: Chromatogram Intra-day precision (10 mcg)-01

Table 31: Result for Chromatogram of Precision (10 mcg)

Fig. 25: Chromatogram Intra-day precision (10 mcg)-02

Table 32: Result for Chromatogram of Precision (10 mcg)-02

Fig. 26: Chromatogram Intra-day precision (30 mcg)

Table 33: Result for Chromatogram of Precision (30 mcg)

Fig. 27: Chromatogram Intra-day precision (30 mcg)-02

Table 34: Result for Chromatogram of Precision (30 mcg)-02

Fig. 28: Chromatogram Intra-day precision (50 mcg)

Table 35: Result for Chromatogram of Precision (50 mcg)

Fig. 29: Chromatogram Intra-day precision (50 mcg)-02

Table 36: Result for Chromatogram of Precision (50 mcg)-02

Inter-day Precision:

Fig. 30: Chromatogram Inter-day precision (10mcg)

Table 37: Result for Chromatogram of Precision (10mcg)

Fig. 31: Chromatogram Inter-day precision (10mcg)-02

Table 38: Result for Chromatogram of Precision (10mcg)-02

Fig. 32: Chromatogram Inter-day precision (30mcg)-01

Table 39: Result for Chromatogram of Precision (30mcg)-01

Fig. 33: Chromatogram Inter-day precision (30mcg)-02

Table 40: Result for Chromatogram of Precision (30mcg)-02

Fig. 34: Chromatogram Inter-day precision (50mcg)

Table 41: Result for Chromatogram of Precision (50mcg)

Table 42: Result for Chromatogram of Precision (50mcg)-02

Table 43: Result of Intraday and Inter day Precision for Vericiguat HPLC

5.6 Robustness

Small deliberate variations in flow rate, mobile phase composition and wavelength were made to evaluate robustness.

1) Flow Rate Change 0.7 mL:

Fig. 36: Chromatogram of Flow rate change 0.7ml-01

Table 44: Result for Chromatogram of Flow rate change 0.7 ml-01

Fig. 37: Chromatogram of Flow rate change 0.7 ml-02

Table 45: Result for Chromatogram of Flow rate change 0.7 ml-02

2) Flow Rate Change 0.9 mL:

Fig. 38: Chromatogram of Flow rate change 0.9 ml

Table 46: Result for Chromatogram of Flow rate change 0.9 ml

Fig. 39: Chromatogram of Flow rate change 0.9 ml-02

Table 47: Result for Chromatogram of Flow rate change 0.9 ml-02

3) Mobile phase composition: 49 mL MeOH + 0.1% acetic acid 51 mL Water:

Fig. 40: Chromatogram of Mobile phase composition change (49:51)

Table 48: Result for Chromatogram of Mobile phase composition change

Fig. 41: Chromatogram of Mobile phase composition change (49:51)-02

Table 49: Result for Chromatogram of Mobile phase composition change-02

4) Mobile phase composition: 51 mL MeOH + 0.1% acetic acid 49 mL Water:

Fig. 42: Chromatogram of Mobile phase composition change (51:49)

Table 50: Result for Chromatogram of Mobile phase composition change

Fig. 43: Chromatogram of Mobile phase composition change (51:49)-02

Table 51: Result for Chromatogram of Mobile phase composition change-02

5) Wavelength Change 257 nm:

Fig. 44: Chromatogram of wavelength change 257nm

Table 52: Result for Chromatogram of wavelength change 257 nm

Fig. 45: Chromatogram of wavelength change 257nm-02

Table 53: Result for Chromatogram of wavelength change 257 nm-02

6) Wavelength Change 259 nm:

Fig. 46: Chromatogram of wavelength change 259nm

Table 54: Result for Chromatogram of wavelength change 259 nm

Fig. 47: Chromatogram of wavelength change 259nm-02

Table 55: Result for Chromatogram of wavelength change 259 nm-02

Table 56: Result of Robustness Study of Vericiguat