Method Development and Validation of RP-HPLC Method For Determination of Selexipag in Bulk and Formulation

The Project entitled Method development and validation of selexipag in its bulk and dosage forms has not been reported by using RP-HPLC method. Hence, there is a need of new analytical method development for the estimation of selexipag. Aim of work is to develop a new, simple, fast, rapid, accurate, efficient and reproducible RP-HPLC method by optimizing the chromatographic conditions for the analysis of selexipag and to perform stress degradation studies. The developed method will be validated according to ICH guidelinesQ2 (R1).¹ Mechanism of Selexipag is a selective prostacyclin (IP, also called PGI2) receptor agonist. The key features of pulmonary arterial hypertension include a decrease in prostacyclin and prostacyclin synthase (enzyme that helps produce prostacyclin) in the lung. Prostacyclin is a potent vasodilator with anti-proliferative, anti- inflammatory and anti-thrombotic effects; therefore, there is strong rationale for treatment with IP receptor agonists. Selexipag is administered orally; maximum concentration of drug and its active metabolites were observed with the bioavailability of 57% and 29% in  rats and monkeys. ² Drug material and products are assessed with precision, accuracy, and dependability thanks to the development and validation of analytical methods, which are essential parts of pharmaceutical analysis. Reverse Phase High- Performance Liquid Chromatography (RP-HPLC) has become the most popular method among the several that are available because of its high sensitivity, specificity, repeatability, and robustness³. It is especially useful for assays that indicate stability, Additionally, ICH guideline Q2(R1) lists the parameters linearity, accuracy, precision, specificity, limit of detection (LOD), limit of quantitation (LOQ), and robustness that are required for the validation of analytical methods. While some techniques for estimating selexipag using LC-MS and HPLC have been previously reported, many of them are either expensive or lack validation in accordance with ICH guidelines. Therefore, the current study's goal is to create a straightforward, accurate, precise, and validated RP-HPLC method for measuring selexipag in pharmaceutical dosage forms and bulk drugs. ^{4, 5}

2. MATERIAL AND METHOD:

2.1MATERIALS

API: Selesipag

• Solvents and Reagents: Methanol, Acetonitrile, 0.1% Orthophosphoric Acid, Water (HPLC grade) 0.1% Acetic Acid (HPLC grade)
• Marketed Sample: Selepeg 200 mg tablet
  2. Preliminary Characterization

Physical Properties

Selexipag: White crystalline powder.

Solubility

• Selexipag is freely soluble in Methanol and poorly soluble in water PH adjusted 0.1% Orthophosphoric Acid, Buffer pH 3.2.
  3. UV Spectroscopy

• Solvent: Methanol
• Wavelength scan: 200–400 nm
• λmax identified: 270 nm
  4. Method Development via RP-HPLC ^6,7

• Column: AgilentC₁­₈ (250mmX 4.6mm,5µm)
• Mobile phase trials: Methanol+0.1% (OPA)water,(75:25 % v/v)
• Optimized condition (Trial 6):

Chromatographic Conditions:

• Detector: U.V. Detector
• Column: AgilentC18
• Column Dimension: 250mmX 4.6mm,5µm
• Injection Volume: 20μl
• Wavelength: 270 nm
• Mobile phase: Methanol+0.1% (OPA)water,(75:25 % v/v)
• Flow Rate: 0.7 ml/min
• Run time: 15 Minutes
• Preparation of System suitability test (Selexipag standard solution):

Accurately weigh and transfer 10 mg Selexipag working standard into 10 ml volumetric flask as about dilute Methanol prepared in completely and make volume up to the mark with the same solvent to get 1000 µg/ml standard (stock solution) and 15 min sonicate to dissolve it and from the resulting solution 0.1 ml was transferred to 10 ml volumetric flask and the volume was made up to the mark with mobile phase Methanol:(0.1% OPA) Water solvent.

• The resulting 10 µg/ml of solution was subjected to chromatographic analyses using mobile phases of different strengths with chromatographic conditions.
  5. System Suitability
  6. Sample Analysis

• Formulation: Selepag 200 mg tablets
• Sample preparation involved methanol extraction, filtration, and dilution with mobile phase.
• % Assay calculated using validated formula.

3. Validation as per ICH Guidelines

3.1 Specificity

• No interference from placebo or excipients.
• Blank and placebo injections confirmed the method's specificity.
  1. Linearity

• Concentration range: 80–120 µg/mL
• Correlation coefficient: >0.998
• %RSD: <2%
  2. Accuracy (% Recovery)

• Recovery within 98–102% at all levels (80%, 100%, and 120%)
• %RSD: <2%

3.3. Precision

• Six replicates analyzed
• % Assay: 90–110%
• %RSD: <2%

Intermediate Precision

• Performed on different day
• %RSD: <2% across 12 samples

3.4. Reputability

• 3. Limit of Detection and Quantification
• LOD and LOQ calculated from calibration curve:
  • LOD = 3.3 σ / S
  • LOQ = 10 σ / S

1. 4. Robustness

• Parameters varied:
  • Flow rate ±0.1 mL/min
  • Temperature ±2°C
  • Wavelength ±3 nm
• System suitability maintained across variations
  5. Solution Stability
• Standard and test solutions stable for 24 hours under lab conditions.

4. RESULTS AND DISCUSSION
   1. Preliminary Characterization and Identification of Drug
      1. Color, Odour, and Appearance

The physical examination of Selexipag revealed that it is a White to off-white powder (pure API) powder. These results are in line with the reported physical characteristics, confirming the identity and physical form of the drug.

4.1.2. Melting point

The procured reference standard of Selexipag was found to melt in the range of 134⁰C respectively

4.1.3. Solubility Study

This study was carried out to find an ideal solvent in which drugs are completely soluble. Various solvents were tried for checking solubility of Selexipag. From solubility studies it was concluded that of Selexipag is freely soluble in Methanol and poorly soluble in water PH adjusted 0.1% Orthophosphoric Acid, Buffer pH 3.2.

4.2. UV Spectral Analysis

4.2.1. Selection of Solvent

UV absorption of 10 µg/mL solution of Selexipag in methanol was generated and absorbance was taken in the range of 200-400 nm.270 nm λmax

4.2.2. Selection of Analytical Wavelength

1) Blank Methanol

Figure. No.1: UV spectrum of Methanol as a blank

2) selexipag standard solution

Figure. No. 2: UV spectrum of Selexipag

Standard solutions were scanned in the range of 200-400nm,against 10 ml methanol and volume make with methanol solvent system as reference Selexipag in methanol was found to be 270 nm, selected wavelength is 270 nm.

4.3. Method Development by RP-HPLC

4.3.1. Optimization of Chromatographic Conditions

The final chromatographic conditions selected were as follow:

• Analytical column:  Agilent C₁₈ Column (250mm x 4.6 mm, 5µm    particle size).
• Injection volume:   20µl
• Flow Rate:   0.7 ml/min
• Mobile Phase: Methanol: water (75: 25 % V/V)
• Detection:   270 nm
• Run Time: 15 min

Figure. No. 3: Typical chromatogram of Trial 6

4.4. System Suitability Test

All five replicate injections of the standard solution passed the system suitability criteria:

• % RSD of area: 0.05% (Limit: NMT 2.0%)
• Theoretical plates: >15000 (Limit: NLT 2000)
• Asymmetry: 1.13–1.14 (Limit: <2.0)

These results confirmed the system’s suitability for routine analysis.

Figure No.4: Chromatogram of System suitability No- 2

4.4.1. Analysis of Marketed Test samples (Assay)

1. Selepag 200 mg Tablet
2. Total weight of    20 tab Powder wt. = 63.12 gm
3. Avg. Powder Weight = 315.6 mg
4. Eq.Wt for 10 mg=   10 x 315.6 / 200 =   15.78    MG
5. Take 15.78 mgs in 100 ml Methanol i.e. =   1000 µg/ml tab solution -TAB STOCK –II

Figure No.5: Chromatogram of standard Selexipag

4.5.1. Solution Stability

Standard and test solutions were stable at room conditions for up to 24 hours, with % absolute differences well below 2.0%.

4.5.2. Specificity

No interference was observed at Selexipag retention time from blank or placebo solutions, confirming specificity.

4.5.3. Linearity and range

Table No 3. Linearity of Selexipag (HPLC)

Table No 4. Regression equation data for Selexipag

Linearity of of Selexipag was observed in the range of 10-50 μg/ml. Detection wavelength used was 270 nm. The calibration curve yielded correlation coefficient (r²) 0.999 & 0.999 for Selexipag respectively

4.5.4. Accuracy (Recovery Study)

1) Accuracy (Recovery)

Recovery studies were performed to validate the accuracy of developed method. To pre analyzed Tablet solution, a definite concentration of standard drug (80%, 100%, and 120%) was added and then its recovery was analyzed .Statistical validation of recovery studies shown in

*mean of each 2 reading

*Denotes average of three determinations.

Figure. No. 6 Chromatogram of Accuracy 80%

Figure. No.7 Chromatogram of Accuracy 120%-02

2) Precision

The method was established by analyzing various replicates standards of Selexipag. All the solution was analyzed thrice in order to record any intra-day & inter-day variation in the result that concluded. The result obtained for intraday is shown in table respectively.

*Mean of each 3 reading

Chromatpgram:

Figure No. 8: Chromatogram of Precision

To evaluate the robustness of the proposed method, small but deliberate variations in the optimized method parameters were done. The effect of changes in mobile phase composition and flow rate, wavelength on retention time and tailing factor of drug peak was studied.  The mobile phase composition was changed in (±1 ml/min^-1) proportion and the flow rate was varied by of optimized chromatographic condition. The results of robustness studies are shown in table. Robustness parameters were also found satisfactory; hence the analytical method would be concluded.

1) Flow Rate Change 0.9 ml

3) Wavelength Change 269 nm