INTRODUCTION:

Selexipag (SLP) is a chemical compound with the full name 2-{4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide. It is a pharmacological agent that can be taken orally and selectively targets the prostacyclin receptor, acting as an agonist. The term "orphan prodrug" refers to a drug designed to undergo a specific metabolic process in the body to release its active form. To slow disease progression and reduce the risk of hospitalization, the U.S. Food and Drug Administration approved SLP in 2015 for the treatment of pulmonary arterial hypertension (PAH) in patients with functional class II or III. The active metabolite of SLP, known as ACT-333679, is a prodrug that shows significantly higher selectivity for the IP receptor, with a 130-fold increase in selectivity over other receptors¹.

SLP has the molecular formula C26H32N4O4S and a molecular weight of 496.63 g/mol. It is a pyrazine derivative with phenyl groups at the fifth and sixth positions. It also belongs to the classes of monocarboxylic acid amides, ethers, pyrazines, aromatic amines, tertiary amines, and N-(methylsulfonyl) acetamides. Functionally, it is related to ACT-333679. SLP appears as a light yellow crystalline powder that is nearly insoluble in water. It is highly stable in its solid form and does not exhibit hygroscopic or photosensitivity properties².

There is limited research on SLP analysis in the literature, which includes HPLC methods for analyzing both SLP formulations and bulk materials³, stability-indicating HPLC analysis⁴, and LC-MS/MS techniques⁵.

Additionally, spectrophotometric methods have been employed to determine SLP levels in bulk and tablet formulations^6-7. Further improvements in method optimization and system suitability parameters should be made in line with ICH (Q2) R1 guidelines³. This study proposes a rapid, accurate, and precise HPLC method for analyzing SLP in bulk and pharmaceutical formulations.

Fig. 1: Chemical Structure of Selexipag

Mechanism of Action:

Selexipag is a selective agonist of the prostacyclin (IP, also known as PGI2) receptor. A reduction in prostacyclin and prostacyclin synthase, an enzyme that aids in the making of prostacyclin, in the lung is one of the main characteristics of pulmonary arterial hypertension. Treatment with IP receptor agonists makes a lot of sense because prostacyclin is a strong vasodilator that has anti-proliferative, anti-inflammatory, and anti-thrombotic actions. Selexipag has a high selectivity for the IP receptor and is chemically unique since it is neither PGI2 nor a PGI2 analogue. Carboxylesterase 1 breaks it down to produce an active metabolite (ACT-333679) that is about 37 times more effective than selexipag. The IP receptor is the only prostanoid receptor that selexipag and its metabolite selectively bind to.

Pharmacodynamics of Selexipag:

It was discovered that selexipag did not significantly lengthen the QT interval at the highest tolerable dosage of 1600 mcg twice day. With IC50 values of 5.5µM and 0.21µM, respectively, selexipag and its metabolite both inhibited platelet aggregation in vitro in a concentration-dependent manner. However, after receiving numerous doses of selexipag in healthy people, there was no impact on platelet aggregation test parameters at clinically relevant amounts.

Metabolism:

The hepatic carboxylesterase 1 enzyme hydrolyzes the acylsulfonamide in selexipag to produce its active metabolite. CYP3A4 and CYP2C8 catalyze oxidative metabolism, which produces dealkylated and hydroxylated metabolites. The active metabolite's glucuronidation is facilitated by UGT1A3 and UGT2B7. Other metabolites in circulation, aside from the active metabolite, make up no more than 3% of the entire drug-related material.^8,9

Pharmacokinetics of Selexipag:

Selexipag is rapidly absorbed from the gut and hydrolyzed by carboxylesterases in the liver and intestines to ACT-333679. The high first-pass impact is probably the reason for the about 49% absolute bioavailability. After one to three hours for selexipag and three to four hours for the active metabolite, the blood plasma reaches its highest concentrations. Approximately 99 percent of both chemicals are bound to plasma proteins in the bloodstream, namely to albumin and alpha-1-acid glycoprotein in identical proportions.

The active ingredient is rendered inactive by the hydroxylation and dealkylation processes of the liver enzymes CYP2C8 and, to a lesser degree, CYP3A4. Additionally, UGT1A3 and UGT2B7 enzymes glucuronidate ACT 333679. The active metabolite has a terminal half-life of 6.2 to 13.5hours, while selexipag has a terminal half-life of 0.8 to 2.5hours.

Selexipag and Act-333679:

A wider search for substances with IP receptor activation resulted from the oral prostanoids' limited half-life and bioavailability. Selexipag is the first nonprostanoid IP receptor agonist authorized for therapeutic usage; its production was initially documented in 2007. Selectipag is quickly absorbed when given orally, and liver carboxylesterase then hydrolyzes it to produce its more active metabolite, ACT-333679¹⁰. Although both ACT-333679 and selexipag are IP receptor agonists with a high degree of selectivity against prostaglandin family members, ACT-333679 is thought to be the primary factor in selexipag's effectiveness because it is around 37 times more potent at activating the IP receptor¹¹. Selexipag was demonstrated to decrease right ventricular systolic pressures, pulmonary artery wall thickness, and right ventricular hypertrophy in an animal model of PAH produced by monocrotaline, while also increasing survival¹².

The first nonprostanoid IP receptor agonist authorized for therapeutic use is selexipag. It is quickly absorbed and converted into the more powerful ACT-333679 by liver carboxylesterase. A major factor in Selexipag's effectiveness is ACT-333679, which is 37 times more potent at activating the IP receptor. According to research on animals, Selexipag increases survival, decreases right ventricular hypertrophy, and lowers pulmonary artery pressure.^12,13

Drug Interactions & Safety:

Metabolites of selexipag are eliminated by the liver. Drug levels are greatly impacted by hepatic impairment, which lengthens half-life. It has a modest risk of medication interactions but inhibits CYP2C8/2C9 and stimulates CYP3A4. No impact on the metabolism of midazolam or warfarin. Half-life is shortened by co-administration of Rifampicin (a CYP2C8 inducer) and prolonged by Gemfibrozil (a CYP2C8 inhibitor)^14,15

Analytical Method Development:

The process of developing an accurate assay to ascertain the composition of formulations is known as analytical technique development. Developing an analytical approach entail determining whether it is appropriate for use in a laboratory. Analytical methods must adhere to GMP and GLP procedures and fulfill ICH guidelines' approval requirements Q2 (R1)¹⁶.

Method Validation:

In the United States, validation was first used in 1978. It has since broadened to include other endeavors in domains such as medical research and device production. It involves ensuring the proper operation of computer systems, control protocols, medical item labels, and testing. Usability, or ease of use, is important, especially when adhering to good manufacturing practices (GMP) regulations. Verification is the process of determining whether something is accurate or legitimate. In a manufacturing, several departments work together to follow this protocol. Verification is the process of obtaining proof that an equipment or product satisfies its specifications.

The Validation Parameters are as Follows:

1. Accuracy

2. Precision

3. Linearity

4. Limit of detection

5. Limit of quantitation

6. Specificity

7. Range

8. Robustness

9. Ruggedness

1. Accuracy: The degree of accuracy of an analytical technique is indicated by the distance between the measured value and the actual or expected value of the quantity under evaluation. It is comparable to confirming whether our measurement is consistent with our prior knowledge. The accuracy of the results is evaluated by comparing samples with and without the extra addition (spiking).

2. Precision: The consistency of an analytical procedure while measuring numerous samples of the same object under various conditions is known as precision. Repeatability, intermediate precision, and reproducibility are the three categories of precision. They aid in our comprehension of the method's dependability in producing comparable outcomes under diverse circumstances.

· Repeatability: This refers to the degree of consistency in measurements made rapidly and repeatedly throughout the same procedure.

· Intermediate precision: This degree of precision verifies accuracy in a variety of scenarios, such as various analysts, items, or days. It illustrates how several conditions cause the lab to alter.

· Reproducibility: This indicates how well data from several labs match up.

3. Linearity: When you measure something in a sample, such as the amount of a chemical present, linearity means that the test findings shift in a straight line as the item's concentration varies.

4. Limit of Detection: The least amount of a chemical that can be found in a sample using the analytical method, even if the measurement isn't exact, is known as the detection limit.

5. Limit of Quantitation: The smallest quantity of a material that can be accurately determined in a sample is known as the limit of quantitation in analytical procedures.

6. Specificity: Specificity now refers to how clearly, we can tell if a test has the right properties

7. Range: The range of the analytical method is the difference between the highest and lowest concentrations of a material in a sample where the method works well.

8. Robustness: When a method is robust, it maintains its dependability even when minor adjustments are made to the procedure.

9. Ruggedness: The consistency of test results while assessing the same samples in various settings, such as different labs or with different analysts, is known as ruggedness. It demonstrates whether or not results are reliable in various contexts.

Importance of validation

1. Quality guaranteed at a high level.

2. Time limits recognized and process improved.

3. Fewest product failures per batch.

4. Better productivity and manufacturing.

5. Reduced costs for maintaining quality.

6. Less rejection of products.

7. More products produced successfully.

8. Fewer complaints about process problems.

9. Equipment starts quickly and reliably.

10. Employees become more aware of the process¹⁷.

Chromatographic Technique:

1. UV spectrophotometry:

Ultraviolet visible, or UV-Vis, spectroscopy is widely utilized to furnish characterisation information for a range of materials. UV-visible spectroscopy can be used to view inorganic or organic, solid or liquid groups, such as organic molecules and functional groups. It can also be used to assess reflectance for coatings, paints, textiles, biochemical studies, dissolution kinetics, band gap measurements, etc. The UV-Vis delivers these details based on the varying responses of samples and the degree of transmittance or absorption of a given wavelength of beam light.¹⁸

2. Gas Chromatography:

A group of analytical separation techniques known as "gas chromatography" are employed to assess volatile materials in the gas phase. By dissolving a sample's component parts in a solvent and vaporizing them to separate the analytes, gas chromatography separates a sample into two phases: a stationary phase and a mobile phase. The mobile phase, a chemically inert gas, transports the analyte molecules through the heated column. One of the few chromatography techniques that does not involve the analyte interacting with the mobile phase is gas chromatography. While gas-liquid chromatography (GLC) uses a liquid on an inert support, gas-solid chromatography (GSC) uses a solid adsorbent as the stationary phase¹⁹.

3. Ultra-performance liquid chromatography:

Another important turning point was the introduction of Ultra-performance Liquid Chromatography (UPLC) in the early 2000s. This technique offers remarkable improvements in resolution, speed, and sensitivity by using sub-2-μm particles and operating at pressures higher than 6000 psi²⁰. UPLC was developed to meet the increasing demand for faster analytical times without sacrificing separation quality. Technological breakthroughoughs have led to substantial improvements in column chemistry, such as hybrid organic particles, monolithic columns, and superficially porous particles. Modern software, automated sample handling, and detection technologies²¹.

4. High Performance Liquid Chromatography:

High Performance Liquid Chromatography (HPLC) is an essential separation method for the pharmaceutical industry. At or close to the head of a stainless-steel tube (column) filled with microscopic particles, a minute amount of sample is injected into the flowing liquid (mobile phase). After that, the mobile phase—which is supplied by a pump and forced through the column by high pressure at a constant pace-transports the sample's component parts along the column²².

5. High-performance thin layer chromatographyl:

An improved instrumental technique that capitalizes on thin layer chromatography's potential is high-performance thin layer chromatography. Its benefits in automation, scanning, full optimization, the selective detection principle, minimum sample preparation, hyphenation, and other areas make it a potent analytical tool for chromatographic information of complex mixtures of pharmaceuticals, natural products, clinical samples, food items, and so on²³.

6. Reversed Phase Chromatography:

It is the most used technique for analytical and preparative separations of chemicals of interest in the fields of chemistry, biology, pharmacology, food, and biomedicine. This technique uses a polar solvent as the mobile phase and a non-polar hydrophobic packing containing an octal or octadecyl functional group bonded to silica gel as the stationary phase. Polar chemicals elute first in this phase, while non-polar molecules are retained for a longer amount of time.

Since they are not stored for long periods of time, most drugs and pharmaceuticals elute more quickly due to their polar nature. The several columns that are used include OctaDecylSilane (ODS), also known as C18, C8, and C4, in order of increasing stationary phase polarity. An aqueous mobile phase enables the use of secondary solute chemical equilibrium (ionization control, ion suppression, ion pairing, and complexation) to control retention and selectivity²⁴.

Guidelines for Validation:

1. Clarifications and language used in the ICH Q2A guide for validating analytical methods (March 1995).

2. Steps and procedures detailed in the ICH Q2B guide for validating analytical techniques (June 1997).

3. FDA's (Draft) Industry Guidance for Analytical Procedures and Method Validation.

4. Standards and guidelines established by the United States Pharmacopeia (USP) and European Pharmacopeia (EP) for pharmaceutical products¹⁷.

Reported Method for Selexipag:

1. Saloni Borade et al (2023) Was developed Validation and forced degradation by RP-HPLC of selexipag drug in bulk and dosage form A simple, accurate, and stability-indicating HPLC method was developed for determining Selexipag using a Grace C18 column (250mm x 4.6mm, 5µm) with a mobile phase of 60% buffer (pH 7.4) and 40% methanol at a flow rate of 1.0 mL/min. Detection was at 294nm, with Selexipag’s retention time at 4.296 min. The method showed linearity in the range of 20-100 μg/mL (r > 0.999), and recovery ranged from 100.99% to 101.06%. Stress testing demonstrated the method’s specificity, separating degradation products. This validated method is suitable for Selexipag analysis in pharmaceutical formulations, following ICH guidelines²⁵.

Parameters	Values
Column	Grace C18
Mobile Phase	Methanol: Phosphate Buffer (40:60)
Wavelength	294 nm
Flow rate	1.0 ml/min
Injection Volume	20µl
Temperature	Ambient
Run time	7.78min
Retention time	4.296 min SLG

2. Saniye Ozcan et al (2023) Was developed A new HPLC method for selexipag analysis in pharmaceutical formulation and bulk formA new HPLC method was developed for analyzing Selexipag in bulk and pharmaceutical formulations using a SupelcoAscentis® Express column (100×4.6 mm, 2.7 µm) with an isocratic elution of acetonitrile: water (60:40 v/v) containing 0.1% formic acid. The method showed linearity in the range of 15.7-117.6 µg/mL, with LOD and LOQ of 2.4 and 3.1 µg/mL, respectively. Validation following ICH Q2(R1) guidelines demonstrated high accuracy and precision. The method is suitable for Selexipag analysis in both bulk and pharmaceutical formulations²⁶.

Parameters	Values
Column	SupelcoAscentis® Express column (100×4.6 mm, 2.7 µm)
Mobile phase	acetonitrile: water (60:40 v/v)
Linearity range	15.7-117.6 µg/mL
Limit of detection LOD	2.4 µg/mL
Limit of Quantification LOQ	3.1 µg/mL

3. Burhan Ceylan et al (2023) Was study A novel HPLC method for selexipag in human plasma and application to a prototype pharmacokinetic study A simple, cost-effective HPLC method was developed for quantifying Selexipag (SLP) in human plasma, suitable for pharmacokinetic studies. Separation was achieved using a C18 column (150 mm × 4.6 mm, 5 μm) with isocratic elution (acetonitrile:0.5% formic acid, 65:35 v/v) at a flow rate of 1.2 mL/min and detection at 300 nm. The linearity range was 10-150 ng/mL, with a retention time of 8.20 ± 0.02 min. The method's LOD was 3.3 ng/mL, and recovery was 97.83%. Validation followed EMA guidelines. The method was applied to a pharmacokinetic study in a healthy volunteer, assessing AUC, Cmax, Tmax, and half-life after administering 800 μg of SLP.²⁷

Parameters	Values
Column	C18 column (150 mm × 4.6 mm, 5 μm)
Mobile phase	acetonitrile:0.5% formic acid, 65:35 v/v
flow rate	1.2 mL/min
Detection rate	300 nm
Linearity range	10-150 ng/mL
retention time	8.20 ± 0.02 min
Limit of detection LOD	3.3 ng/mL
Recovery rate	97.83%

4. Santosh SS et. al. (2021) was developed Stability Indicating Method Development and Validation of Selexipag in Bulk and Pharmaceutical Dosage form by using RP-HPLC A simple and efficient HPLC method was developed and validated for determining Selexipag in tablet dosage form. Using a Symmetry C18 column (150×4.6 mm, 5µ) with a mobile phase of methanol: water (50:50 v/v), the method achieved a retention time of 2.682 minutes at 281 nm, with a flow rate of 0.6 ml/min and 25°C column temperature. The method showed high accuracy (99.9% purity), precision (%RSD: 0.2), and recovery (100%), with a correlation coefficient (r²) of 0.999. Validated per ICH guidelines, this economical and reproducible method is suitable for routine analysis and related substances determination in Selexipag tablets²⁸

Parameters	Values
Column	C18 column (150×4.6 mm, 5µ)
mobile phase	methanol: water (50:50 v/v)
retention time	2.682 minutes at 281 nm
flow rate	0.6 ml/min
Temperature	25°C column
Accuracy	100%
Linearity and Range	10 μg/ml-50 μg/ml
Regression coefficient (r2)	0.999

5. Koya Prabhakara Rao et al (2021) This study presents a validated LC–MS/MS bioanalytical method for pharmacokinetic analysis of Selexipag and its related impurities in rat plasma. Utilizing a gradient elution with a flow rate of 1 ml/min on an X-Bridge phenyl column (150×4.6 mm, 3.5 µ), the mobile phase comprised formic acid buffer and acetonitrile. Separation of Selexipag, its impurities, and the active metabolite (impurity-D) was achieved within 30 minutes, with Ambrisentan as the internal standard. The method demonstrated linearity (10–200% plasma concentration, R² = 0.999), precision, accuracy, recovery, and stability, complying with USFDA guidelines. It is suitable for pharmacokinetic studies in rat plasma²⁹.

Parameters	Values
Column	X-Bridge phenyl column (150×4.6 mm, 3.5 µ)
mobile phase	formic acid buffer and acetonitrile
Run rate	30 min
flow rate	1 ml/min

Banothu Bhadrua et al. (2019) validated LC-MS/MS method using liquid-liquid extraction was developed to estimate Selexipag in human plasma with Selexipag-D6 as the internal standard. Chromatographic separation was performed on a CORTECS C18 column (100×4.6 mm, 2.7 µ) with a mobile phase of acetonitrile and 10 mM ammonium formate (pH 4.0) in an 80:20 ratio at a flow rate of 0.5 ml/min. Detection employed turboionspray (API) in positive mode with MRM transitions at 498.20 → 344.20 m/z for Selexipag and 503.70 → 344.20 m/z for Selexipag-D6. The method demonstrated excellent linearity (r² > 0.999) over a range of 10–25,600 pg/ml with a total run time of 10 minutes ³⁰.

Parameters	Values
Column	CORTECS C18 column (100×4.6 mm, 2.7 µ)
mobile phase	Acetonitrile ammonium formate (80: 20, v/v)
Run rate	10 min
flow rate	0.5 ml/min

6. Kotwal T.S et al (2017) WasDevelopment and Validation of Simple Uv Spectrophotometric Method for the Determination of Selexipag in API and its Bulk Dosage Form A simple, accurate, precise, and cost-effective UV-Visible spectrophotometric method was developed and validated for the estimation of Selexipag using dimethyl sulfoxide (DMSO) as the solvent. The drug’s absorption maximum (λmax) was determined at 306 nm, and linearity was observed in the 5–25 μg/mL range, with a correlation coefficient (R²) of 0.9963. Recovery rates at 80%, 100%, and 120% were 99.16%, 99.7%, and 99.00%, respectively. Interday and intraday precision showed %RSD of 0.490% and 0.28%, with LOD and LOQ values of 0.477 μg/mL and 1.44 μg/mL, respectively. This method is precise, robust, and suitable for routine analysis of Selexipag in bulk and dosage forms³¹.

Parameters	Values
Detection wavelength(λmax)	306 nm
Solvent used	dimethyl sulfoxide (DMSO)
Linearity range	5–25 μg/mL
correlation coefficient (R²)	0.9963
Recovery rates	80%, 100%, and 120% were 99.16%, 99.7%, and 99.00%
Precision %RSD	0.490% and 0.28%
LOD	0.477 μg/mL
LOQ	1.44 μg/mL

7. Snigdha Damireddy et.al. (2017) A simple and precise RP-HPLC method was developed and validated for Selexipag estimation. Separation was achieved on an Inertsil ODS column (4.6×250 mm) with a mobile phase of acetonitrile and 0.1% OPA buffer (70:30 v/v, pH 3 adjusted with NaOH) at a 1 ml/min flow rate and UV detection at 270 nm. The retention time was 2.16 min, and Selexipag’s % purity was 100.43%. Linearity was observed in the 100–500 µg/mL range (r² = 0.999) with % recovery of 100.19%. System suitability met standards with theoretical plates (2832.72) and tailing factor (1.17). Validated as per ICH guidelines, the method is effective for Selexipag analysis ³².

Parameters	Values
Column	InertsilOdscolumn (4.6×250 mm)
Mobile phase ratio	ACN: OPA (70: 30 % v/v)
Detection wavelength	270 nm
Flow rate	1.0ml/min
Injection volume	10µl
Column temperature	Ambient
Run time	8 min

8. Priska Kaufmann et. al (2015) Selexipag, an oral selective prostacyclin receptor (IP receptor) agonist, targets the prostacyclin pathway to treat pulmonary arterial hypertension (PAH). In a phase I double-blind, placebo-controlled study involving 64 healthy males, the pharmacokinetics (PK) and tolerability of single and multiple ascending doses of Selexipag were assessed, with an additional group receiving a 400µg dose under fasted and fed conditions. Maximum plasma concentrations of Selexipag and its active metabolite, ACT-333679, occurred at 2.5 and 4 hours, respectively, with half-lives of 0.7–2.3 and 9.4–14.2 hours. Food reduced ACT-333679 exposure by 27%. Selexipag was well tolerated up to a single dose of 400 µg and multiple doses of 600 µg, with headache as the most common adverse event and no significant effects on vital signs or ECG. These findings support further investigation of Selexipag ³³.

CONCLUSION:

Selexipag is a novel, selective IP receptor agonist that effectively manages pulmonary arterial hypertension by targeting the prostacyclin pathway. Its active metabolite, ACT-333679, plays a crucial role in its therapeutic efficacy. The proposed HPLC method provides a reliable, accurate, and precise analytical technique for quantifying Selexipag in bulk and pharmaceutical formulations. This method, validated as per ICH guidelines, ensures robust quality control and stability assessment, paving the way for enhanced therapeutic monitoring and further pharmacokinetic studies.

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Received on 20.03.2025 Revised on 04.04.2025

Accepted on 17.04.2025 Published on 06.05.2025

Available online from May 10, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(2):116-122.

DOI: 10.52711/2231-5675.2025.00019

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