Analytical Method Development and Validation of Rimegepant by Using RP-HPLC

Recurrent headache attacks are the hallmark of migraine, the most prevalent interdisciplinary and complex neurologic condition. There are two types of migraine: episodic and chronic, with or without aura. A typical migraine attack includes four phases: premonitory, aura, headache, and postdrome.^[1,2] Symptoms commonly include unilateral throbbing pain, nausea, and sensitivity to light and sound. Diagnosis is typically based on medical history and clinical examination. Migraine affects about 15% of the general population, with a higher prevalence in women.^[3,4,5]

Management involves both non-pharmacological and pharmacological approaches. Lifestyle modifications such as regular sleep, hydration, stress management, and exercise play a key preventive role. Acute treatment includes NSAIDs and triptans, while antiemetics are used as adjuncts. Preventive therapy is considered in frequent or severe cases.  ^[6,7]

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuromodulating vasodilator that was first discovered in the trigeminovascular system in 1985. CGRP is produced in both peripheral and central neurons and is released during severe migraine episodes ^[8]. Calcitonin gene-related peptide (CGRP) has been shown to play a pivotal role in the pathophysiology of migraine and therefore offers a novel targeted approach for both the acute and preventive treatment of migraine ^[9].  Rimegepant is an oral CGRP receptor antagonist created by Biohaven Pharmaceuticals. It received FDA approval on February 27, 2020 for the acute treatment migraine headache, and was subsequently approved by the European Commission in April 2022 for both the treatment and prevention of migraines.^[10,11,12] Recently it received CDSCO approval on February 27, 2025 for Acute treatment of migraine with or without aura in adults with a previous insufficient response to triptans.^[13] Rimegepant is a small molecule calcitonin gene-related peptide (CGRP) receptor antagonist^.[14] IUPAC name of Rimegepant is :- (5S,6S,9R)-5-amino-6-(2,3-difluorophenyl)-5H,6H,7H,8H,9H-cyclohepta[b]pyridin-9-yl4-{2-oxo-1H,2H,3H-imidazo[4,5-b]pyridine-1yl}piperidine-1carboxylate.^[15]  

The Chemical Structure of Rimegepant is as shown in fig 1.

Figure 1:- Chemical Structure of Rimegepant

2. MATERIAL AND METHODS

2.1 Reagent and chemicals

Rimegepant API (purity>99.99%) was received as gift sample from Reliable’s Shree Industrial Training Centre and research labolatory, Jalgaon (MH). Other reagent such as Methanol[CH₃OH], Formic acid [CH₂O₂], Water [HPLC grade] were purchased from the Merck life science pvt ltd [INDIA]. All reagent used for analysis were analytical grade.

2.2 Instruments

Table 1 : Instruments Used In Method Development

2.3 preparation of 0.1% formic acid

A 0.1% formic acid solution was prepared by accurately measuring 1.0 mL of formic acid and transferring it into a 1000 mL volumetric flask. The volume was then made up to the mark with HPLC-grade water

2.4 preparation of standard solution

Accurately weighed 5 mg of Rimegepant was transferred to a 50 ml volumetric flask, dissolved in 10ml methanol to obtain a concentration of 500 µg/ml, and sonicated for 2 minutes to ensure complete dissolution.

2.5 sample preparation

Accurately weighed 5 mg of Rimegepant was transferred to a 50 ml volumetric flask, dissolved in 10ml methanol to obtain a concentration of 500 µg/ml, and sonicated for 2 minutes to ensure complete dissolution. From this solution 0.1, 0.2, 0.3, 0.4 & 0.5 ml was taken and transfer to 10 ml of volumetric flask and make up the volume by methanol (mobile phase) to get 5, 10, 15, 20 & 25 µg/ml concentration respectively. These solutions were used for calibration curve preparation.

2.6 marketed tablet preparation

for marketed tablet preparation we prepare lab solution. We get estimate weight of tablet (ie. 95 mg weight for 1 tablet). Twenty tablets were weighed and average weight was calculated. Powder equivalent to 6.33 mg of Rimegepant was transferred into a 50 ml volumetric flask, dissolved in 10 ml methanol, (ie. 600 µg/ ml Rimegepant stock II) and sonicated for 15 minutes to ensure complete extraction of drug.

2.7 assay preparation

An aliquot of 0.3 ml from stock solution II was diluted up to 10 ml with mobile phase for assay determination. The prepared solutions were filtered before injection to avoid particulate interference.

2.8 Chromatographic condition

The following chromatographic conditions were established by trial and error and were kept constant throughout the experimentation

Table 2: Chromatographic Condition

3. VALIDATION OF DEVELOPED METHOD

After developing the RP-HPLC method, validation was carried out to confirm the suitability of the method for its intended purpose. The validation of the developed method for Rimegepant was performed as per ICH guidelines.

The following parameters were evaluated:

A. Accuracy

Accuracy is defined as the closeness of agreement between the true value and the value found. It is also referred to as trueness. In this study, accuracy was determined by injecting known

concentrations of the drug at different levels and calculating the mean peak area. The standard deviation and %RSD were calculated.

Acceptance Criteria: The percentage relative standard deviation (%RSD) should be not more than 2%.

B. Precision

Precision of the method was evaluated by intraday and interlay studies. In intraday precision, the sample solution was injected multiple times within the same day, while interday precision was performed on different days. The peak areas were recorded and %RSD was calculated.

Acceptance Criteria: RSD of the mean concentration of replicate readings should be NMT 2%.

C. Recovery Studies:

Recovery studies were carried out by spiking known amounts of standard drug into the pre-analysed sample. The concentration obtained was compared with the added amount to calculate % recovery.

Acceptance Criteria: For assay method, mean recovery should be in the range of 98–102%.

D. Linearity

Linearity was established by preparing standard solutions of Rimegepant in the concentration range of 5–25 µg/ml. A calibration curve was plotted between concentration (x-axis) and peak area (y-axis).

Acceptance Criteria: The correlation coefficient (r²) should be not less than 0.995.

E. Limit of Quantification (LOQ)

LOQ is the lowest concentration of analyte that can be quantitatively determined with acceptable precision and accuracy.

LOQ = 10 × SD / Slope

Acceptance Criteria: Signal-to-noise ratio should be approximately 10:1.

F. Limit of Detection (LOD)

LOD is the lowest concentration of analyte that can be detected but not necessarily quantified.

LOD = 3.3 × SD / Slope

Acceptance Criteria: Signal-to-noise ratio should be approximately 3:1.

G. Specificity

Specificity of the method was evaluated by comparing chromatograms of standard and sample. No interference was observed at the retention time of Rimegepant, confirming specificity of the method

Acceptance Criteria: No interference at retention time and peak purity should be acceptable.

H. Robustness

Robustness was studied by making small deliberate changes in chromatographic conditions such as: Flow rate (±1%) ,Wavelength (±1 nm). The effect on peak area and retention time was observed.

Acceptance Criteria: Deviation in results should be less than 2%.

I. System Suitability Studies:

System suitability studies were performed to confirm the adequacy and reliability of the chromatographic system prior to analysis. Various parameters, including column efficiency (theoretical plates), resolution, capacity factor, tailing factor, and repeatability (%RSD), were evaluated by repeated injections of standard solutions. The results obtained were compared with established acceptance criteria to ensure consistent system performance. These studies were conducted in compliance with USP guidelines.

4. RESULTS AND DISCUSSION

4.1 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY:

A reverse-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the estimation of Rimegepant. Various chromatographic parameters were systematically optimized to obtain a sharp, well-resolved peak with good symmetry and reproducibility. The standard solution was prepared and analyse under optimized conditions, and the chromatograms were recorded. The developed method was found to be simple, precise, and suitable for the quantitative analysis of Rimegepant.

4.2 OPTIMIZATION OF CHROMATOGRAPHIC CONDITIONS

1. Selection of Wavelength:

The selection of detection wavelength plays a significant role in determining the sensitivity of the analytical method. The UV spectrum of Rimegepant was scanned over a suitable wavelength range to identify the wavelength of maximum absorbance. Based on the spectral analysis, 280 nm was selected as the detection wavelength, as the drug exhibited adequate absorbance at this wavelength. This ensured reliable detection and consistent analytical performance.

Figure 2: UV Spectrum of Rimegepant

2. Method development

Various combinations of methanol and 0.1% Formic Acid (ph 2.8)  were evaluated in different ratios. Such as (90:10), (85:15), (80:20), (75:25) (70:30) and (60:40). Among these, Methanol : 0.1% Formic Acid (ph 2.8) (75:25 v/v) was found to be most suitable at flow rate 1.0 ml/min as it produced a sharp, symmetrical peak with good resolution and acceptable retention time. The selected mobile phase also minimized peak tailing and improved system performance.

Chromatogram:

Figure 3: Chromatogram for Rimegepant

Figure 4 : Chromatogram Of Rimegepant Accuracy At 80%

Table 3: Drug Accuracy Studies of Rimegepant (80%)

Sr. No.	Concentration (µg/ml)	Amount added	Area	Amount found	Amount recovered	% Recovery
1	5	4	853.49	9.00	4.00	99.90
2	5	4	854.50	9.01	4.01	100.16
			Mean	9.00	4.00	100.03
			SD	0.007	0.007	0.19
			%RSD	0.083	0.186	0.19

Figure 5: Chromatogram Of Rimegepant Accuracy At 100%

Table 4: Drug Accuracy Studies of Rimegepant (100%)

Sr. No.	Concentration (µg/ml)	Amount added	Area	Amount found	Amount recovered	% Recovery
1	5	5	946.86	9.960	4.960	99.21
2	5	5	950.63	9.999	4.999	99.99
			Mean	9.98	4.98	99.60
			SD	0.028	0.028	0.55
			%RSD	0.277	0.554	0.55

Chromatogram:

Figure 6: Chromatogram of Rimegepant Accuracy At 120%

Table 5 : Drug Accuracy Studies of Rimegepant (120%)

Sr. No.	Concentration (µg/ml)	Amount added	Area	Amount found	Amount recovered	% Recovery
1	5	6	1086.074	11.038	6.038	100.64
2	5	6	1084.54	11.022	6.022	100.38
			Mean	11.03	6.03	100.51
			SD	0.011	0.011	0.19
			%RSD	0.102	0.186	0.19

Figure 7 : chromatogram of Rimegepant intra-day precision

Table 7 : Intra-day Precision Data of Rimegepant

Drug name: Rimegepant
Sr. No.	Concentration (µg)	Area	Average	SD	%RSD
1	10	963.2721	966.32	4.31	0.45
	10	969.3697
2	15	1437.757	1440.84	4.36	0.30
	15	1443.742
3	20	1950.536	1947.14	4.80	0.25
	20	1943.742
	Range of %RSD				0.25 - 0.45

Chromatogram:

Figure 8: chromatogram of Rimegepant inter-day precision

Table 8 : Inter-day Precision Data of Rimegepant

Drug name: Rimegepant
Sr. No.	Concentration	Area	Average	SD	%RSD
DAY 1	10	969.271	966.01	4.61	0.48
	10	962.756
DAY 2	15	1439.307	1454.681	21.75	1.49
	15	1470.055
DAY 3	20	1953.917	1954.01	0.14	0.01
	20	1954.11
	Range of %RSD				0.01 – 1.49

Figure 9 : Linearity Curve Of Rimegepant

Figure 10: chromatogram of system suitability of Rimegepant

Table 13: System Suitability of Rimegepant

Sr no	Parameter	Injection 1	Injection 2	Mean	SD	%RSD	Acceptance Criteria
1	Retention time(time)	4.254	4.254	4.254	-	-	consistent
2	Peak area (mAUs)	950.25	958.24	954.24	5.65	0.59	NMT 2%
3	Peak height (mAU)	117.90	117.90	-	-	-	-
4	Theoretical plates (N)	6662	5972	-	-	-	NLT 2000
5	Tailling factors (T)	0.99	0.99	0.99	-	-	NMT 2.0

Figure 11:-  Test Solution 1 (Marketed Prep.) Chromatogram

Figure 12:-  Test Solution 2 (Marketed Prep.) Chromatogram

Table 14: Marketed Test Sample Result

Name	Area	RT(min)	Rimegepant in mg	% Lable claim
Test solution 1	1924.622	4.321	20mg	100.30
Test solution 2	1924.825	4.308	20mg	100.75
Mean	1924.72	-	-	100.53
SD	0.144	-	-	0.319
%RSD	0.007	-	-	0.317

5. CONCLUSION:

A rapid, simple, and user-friendly RP-HPLC method was developed and validated for the determination of Rimegepant in pharmaceutical dosage form. All the validation parameters such as accuracy, precision (intra-day and inter-day), linearity, robustness, LOD, and LOQ were found to be within acceptable limits as per ICH guidelines. The method showed good linearity over the selected concentration range with a high correlation coefficient (r² = 0.9999), indicating reliable performance for quantitative analysis. The developed method also demonstrated good reproducibility and precision. Hence, the method is suitable for routine estimation of Rimegepant in pharmaceutical dosage forms.

6. ACKNOWLEDGEMENT

We are thankful to Reliable’s Shree Industrial Training Centre and research laboratory , Jalgaon (MH) for providing gift sample of  Rimegepant API. We are also thankful for Vidyabharati College of Pharmacy, Amravati, for providing infrastructure and facility to carry out present work.

REFERENCES

1. Khan J, Al Asoom LI, Al Sunni A, Rafique N, Latif R, Al Saif S, et al. Genetics, pathophysiology, diagnosis, treatment, management, and prevention of migraine. Biomed Pharmacother. 2021;139:111557. doi:10.1016/j.biopha.2021.111557.
2. Charles A. The pathophysiology of migraine: Implications for clinical management. Lancet Neurol. 2018;17(2):174–82. doi:10.1016/S1474-4422(17)30435-0.
3. Manorenj S. Acute migraine: An overview. Int J Community Med Public Health. 2025;12(4):1952–64.
4. Ashina M. Migraine. N Engl J Med. 2020;383(19):1866–76. doi:10.1056/NEJMra1915327.
5. Steiner TJ, Stovner LJ, Jensen R, Uluduz D, Katsarava Z. Migraine remains second among the world’s causes of disability, and first among young women: Findings from GBD2019. J Headache Pain. 2020;21:137. doi:10.1186/s10194-020-01208-0.
6. Aguilar-Shea AL, Membrilla MDJA, Diaz-de-Teran J. Migraine review for general practice. Aten Primaria. 2022;54(2):102208. doi:10.1016/j.aprim.2021.102208.
7. Lew C, Punnapuzha S. Migraine medications. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2026.
8. Berger AA, Winnick A, Carroll AH, Welschmeyer A, Li N, Colon M, et al. Rimegepant for the treatment of migraine. Health Psychol Res. 2022;10(5):38534. doi:10.52965/001c.38534.
9. Blair HA. Rimegepant: A review in the acute treatment and preventive treatment of migraine. CNS Drugs. 2023;37(3):255–65. doi:10.1007/s40263-023-00982-9.
10. Pfizer, Biohaven. VYDURA® (Rimegepant) granted first ever marketing authorization by European Commission for both acute treatment of migraine and prophylaxis of episodic migraine [Internet]. Pfizer website; [cited 2026 May 20]. Available from: https://www.pfizer.com
11. U.S. Food and Drug Administration. Nurtec ODT (Rimegepant) orally disintegrating tablets [Internet]. FDA Approved Drug Products database; [cited 2026 May 20]. Available from: https://www.accessdata.fda.gov
12. Biohaven Pharmaceuticals. Nurtec ODT FDA approval [press release]. [Internet]. [cited 2026 May 20]. Available from: https://www.biohaven.com
13. List of New Drugs approved 258april.pdf [Internet]. Available from: https://share.google/V0pHOhyWlgPue3O2r
14. Diener HC, Charles A, Goadsby PJ, Holle D. New therapeutic approaches for the prevention and treatment of migraine. Lancet Neurol. 2015;14(10):1010–22. doi:10.1016/S1474-4422(15)00198-2.
15. Luo G, Chen L, Conway CM, Kostich W, Macor JE, Dubowchik GM. Asymmetric synthesis of heterocyclic analogues of a CGRP receptor antagonist for treating migraine. Org Lett. 2015;17(24):5982–5. doi:10.1021/acs.orglett.5b02985.
16. Kabra, J. P., & Jadhao, M. (2024). Bioanalytical estimation of antihypertensive drug by using HPLC in human plasma. International Journal of Creative Research Thoughts, 12(5), 2320–2882. Link: https://ijcrt.org/papers/IJCRT24A5600.pdf
17. Dandge, V. D., Malve, V., Waghulkar, V. M., Baitule, A. W., & Jawarkar, S. G. (2024). Development and validation of a bioanalytical method for determination of Teneligliptin in human plasma by RP-HPLC. International Journal of PharmTech Research, 15(2), [page range]. ISSN: 0974-4304. Link: https://www.sphinxsai.com/2022/ph_vol15_no2/1/(48-57)V15N2PT.pdf