Analytical Method Development and Validation of Tirzepatide in Bulk and Pharmaceutical Formulation by RP-HPLC

Dr. K. Patil; Y Patil; K  Patil; K Khadake; K Suryawanshi; V Patil; S Joshi; N Prowar; Dr. S. Barhate

doi:10.5281/zenodo.19810158

Research Paper | Open Access
Volume 04 | Issue 04 | Article Id IJPS/260404617

Analytical Method Development and Validation of Tirzepatide in Bulk and Pharmaceutical Formulation by RP-HPLC
Dr. K. Patil* Y Patil K Patil K Khadake K Suryawanshi V Patil S Joshi N Prowar Dr. S. Barhate
Department of Pharmaceutical Chemistry, Shellino Education Society’s, Arunamai College of Pharmacy, Mamurabad, Jalgaon, MH 425002.

Abstract

Tirzepatide is a novel dual agonist of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors, widely used in the management of type 2 diabetes mellitus and obesity. Reliable analytical methods are essential to ensure its quality, safety, and efficacy in bulk and pharmaceutical formulations. This study aimed to develop and validate a simple, accurate, precise, and stability-indicating RP-HPLC method for the estimation of tirzepatide in accordance with ICH Q2(R1) guidelines. Chromatographic analysis was performed using an Agilent 1100 HPLC system with a C18 column (4.6 mm × 250 mm, 1.8 µm). The mobile phase consisted of methanol and 0.1 N citric acid (50:50 v/v), delivered at a flow rate of 0.9 mL/min. Detection was carried out at 250 nm with an injection volume of 20 µL. The method was validated for linearity, accuracy, precision, robustness, LOD, and LOQ.The method showed excellent linearity over 5–25 µg/mL (R² = 0.9994). Recovery ranged from 99.10% to 101.40%, with %RSD < 0.2%. LOD and LOQ were 0.034 µg/mL and 0.104 µg/mL. The assay was 98.58%. The method is reliable and suitable for routine analysis

Keywords

Tirzepatide, RP-HPLC, Method Development, Method Validation

Introduction

Tirzepatide that acts as a binary agonist for the glucose-dependent insulinotropic polypeptide (GIP) and glucagon- suchlike peptide- 1 (GLP- 1) receptors. This two- way process produces bettered insulin perceptivity, increased insulin stashing, and dropped appetite, rotundity and type 2 diabetes are treated with tirzepatide [1-2]. The International Diabetes Federation (IDF) estimates that 89.8 million Indians between the periods of 20 and 79 would have diabetes in 2024. The International Diabetes Federation (IDF) estimates that 89.8 million Indians between the periods of 20 and 79 had diabetes as of 2024. also, the World Health Organization (WHO) reports that 77 million Indians over the age of 18 have type 2 diabetes, and the number of pre-diabetics is rising [3-6]. According to data, further than 43 of diabetics people who are 60 times of age or aged, whereas roughly 37.5 of people are in the 39 – 58 age range. The prevalence is slightly advanced in men (69) than in women. With notable, cure-dependent weight loss and advancements in rotundity- related diseases like sleep apnea and diabetes, tirzepatide is a prospective, veritably effective treatment for rotundity [6-8]. Encyclopedically, the frequence of rotundity has increased, with roughly 880 million grown-ups worldwide suffering from rotundity in 2022 (16 of grown-ups, or one in eight people). The frequence rates are 24.0 for women and 22.9 for males. While the FDA has approved tirzepatide, phrasings of tirzepatide are experimental, compounded, and have no scientific support [8-12]. Tirzepatide, a drug created by Eli Lilly and Company, has been approved to treat diabetes. It's vended in the US under the names Mounjaro for type 2 diabetes (T2D) and Zepbound for weight operation. It also provides advantages including better cardio metabolic pointers and dropped fat mass. Common side goods Not everyone should have gastrointestinal symptoms similar nausea, puking, and diarrhea. It can be fitted at any time of day, with or without refections, into the upper arm, ham, or tummy. Subcutaneous injections of tirzepatide are given once a week [12,13].

Fig No.1 Structure of Tirzepatide

2. MATERIALS AND METHOD

Tirzepatide Injection was purchased in market as Mounjaro prefilled injection 20mg/ml (Eli Lilly and Company) and mobile phase is Methanol and 0.1N Citric Acid were supplied by All other ingredients were used analytical grade.

Instrumentation:

HPLC Analysis was performed using AGILENT (1100) and CHEMSTATION software. AGILENT (1100) system equipped with G1310A ISO PUMP, with Auto Sampler (DAD) Detector, 0.001 to 5 ml discharge rate, 400 bar pressure limit range, 5% pressure display accuracy, 04 no. of mobile phase, 0 to 100% mixing ratio range, pump unit HP-1100 reciprocating pump and column (4.6 mm x 250 mm, 1.8μm), C-18(AGILENT- ECLIPSA X DB) used as stationary phase.

Chromatographic Condition:

The following chromatographic conditions were established by trial and error and were kept constant throughout the experimentation. Chromatographic separation was performed on, Column (4.6 mm x 250 mm, 1.8μm) with C-18(AGILENT- ECLIPSA X DB) as stationary phase. Mobile phase was used as METHANOL: 0.1 N CITRIC ACID) (3.202 gm citric acid was added to 500 ml HPLC water) in 90:10 ratio respectively at 0.9 ml/min flow rate with 250 nm wavelength and particle size was 20 μl at ambient temperature Ambient.

Preparation of Mobile Phase:

Combination of mobile phase is Methanol and 0.1 N Citric acid (3.202 gms citric acid was added to 500 ml HPLC water) 50:50 filtered through 0.45μ membrane filter and degassed by sonication.

Preparation of Standard Sample Solution:

The standard solution of tirzepatide was prepared by adding 5 mg in 10 ml Methanol. Stock solution was prepared as 500 μgm/ml of tirzepatide. From stock solution 0.1, 0.2, 0.3, 0.4 and 0.5 add in 10 ml of mobile phase to get 5, 10, 15, 20, 25 μg/ml of tirzepatide solution. 2.880 mg in 10 ml of methanol was used to get 500 μg/ml of tab solution. Take 0.4 from stock solution and make up volume 10 ml with mobile phase in volumetric flask to get 20 μg/ml for assay.

Injection assay:

For Injection assay take Tirzepatide Injection 20mg/ml(Mounjaro) calculated for 5mg and used in stock solution and 0.25 ml in 10 ml of methanol in volumetric flask and sonicate to dissolve it completely and make up the volume was used to get 500 μg/ml of injection solution. Mix well and filter through 0.45 μm filter. Further pipette 0.2ml of the above stock solution in 10 ml with mobile phase in volumetric flask to get 10μg/ml for assay. The simple chromatogram of test Tirzepatide. The amounts of Tirzepatide per injection were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated five times with injection formulations. Injection Assay for % Label claim for % RSD Calculated.

3. METHOD VALIDATION

In method validation for current method was carried as per International Conference on Harmonization (ICH) Q2R1 guidelines. Validation was done through linearity, accuracy, precision, repeatability and robustness

3.1 Preliminary studies on Tirzepatide

3.1.1 Melting point

The procured reference standard of Tirzepatide was found to melt in the range of 123-125oC. respectively.

3.1.2. Solubility

The drug was found to be

• freely soluble in methanol

• poorly soluble in acetic acid

3.1.3. UV Spectroscopy

UV absorption of 10 µg/mL solution of Tirzepatide in methanol was generated and absorbance was taken in the range of 200-400 nm λmax is 250 nm.

Fig No.2 UV Spectrum of Tirzepatide

3.2. Analytical of Method Validation

In method validation for current Method was carried as per International Conference on Harmonization (ICH) Q2R1 guidelines. Validation was done through linearity, accuracy, precision, repeatability and robustness.

1. Linearity: To establish linearity, the stock solutions were prepared (500 μg /ml) of Tirzepatide using mobile phase as the solvent, again from the stock solution further dilutions were made to yield solutions in the concentration range of 5-25 μg/ml. 20 μl of each solution was injected and records the chromatogram at 250 nm. The chromatogram optimized in given Figure, the calibration curve was plotted using concentration against peak area. The procedure was repeated for six times. The correlation coefficient was found to be above 0.9994 of tirzepatide.

Table No.1 Linearity of Tirzepatide

Sr. No.	Conc.	Mean	SD	%RSD
1.	5	453.2135	0.44	0.10
2.	10	943.5960	0.87	0.09
3.	15	1503.0895	0.71	0.05
4.	20	2023.6315	2.40	0.12
5.	25	2503.5380	0.97	0.04
		Avg	1.08

Fig No.3 Calibration Curve of Tirzepatide

2. Precision: The precision of an analytical method is the closeness of replicate results obtained from analysis of the same homogeneous sample. To study precision, five replicate standard solutions of TERZEPARIDE (500 μg/ml) were prepared and analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responses was calculated as,

Table No.2 Results of Intraday and Interday

Drug	Conc (ug/ml)	Intraday Precision			Interday Precision
Drug	Conc (ug/ml)	Mean±SD	%Amt Found	%RSD	Mean±SD	%Amt Found	%RSD
PER	10	970.21±1.42	100.29	0.15	971±2.37	100.45	0.24
	15	1505.48±1.18	101.30	0.08	1507.27±1.46	101.42	0.10
	20	200.11±0.49	99.90	0.02	2002.87±1.24	99.98	0.06

3. Repeatability: Repeatability is the closeness of agreement between mutually independent test results obtained with the same method on identical test material in the same laboratory by the same operator using the same equipment within short intervals of time. Repeatability was ascertained by getting the sample analyzed by different and the results are shown in table.

Table No.3 Repeatability of Tirzepatide

Conc.	Mean	Amt found	% Amt found	SD	%RSD
10	952.66	9.86	98.60	0.77	0.08

Fig No.7 Chromatogram of Repeatability

4. Accuracy: Accuracy can be defined as the closeness of agreement between a test result and the accepted reference value. Accuracy of the method was determined by recovery study. Analytical method may be considered validated in terms of accuracy if the mean value is within ± 20 % of actual value. Recovery of specified impurities was found in the range of 80.0% to 120.0%, which was well within the acceptance criteria as shown below.

Table No.4 Accuracy of 80%

Sr. No

μg/ml

Amt.

added

Area

Amt found

Amt received

recvd

% Received

865.117

9.01454633

4.04

101.10

866.321

9.02616795

4.06

101.40

Mean

9.02

4.02

100.51

0.008

0.21

% RSD

0.091

0.204

0.20

Table No.5 Accuracy of 100%

Sr. No	μg/ml	Amt. added	Area	Amt found	Amt received recvd	% Received
1	5	5	962.565	9.95516409	4.9551641	99.10
2	5	5	965.265	9.98117761	4.9811776	99.62
			Mean	9.97	4.97	99.36
			SD	0.018	0.018	0.37
			% RSD	0.185	0.370	0.37

Table No.6 Accuracy of 120%

Sr. No	μg/ml	Amt. added	Area	Amt found	Amt received recvd	% Received
1	5	6	1073.358	11.0245946	6.0245946	100.41
2	5	6	1075.11	11.0415058	6.0415058	100.69
			Mean	11.03	6.03	100.55
			SD	0.012	0.012	0.20.
			% RSD	0.108	0.198	0.20

5. Robustness: The robustness is evaluated by the analysis of Tirzepatide under different experimental conditions such as making small changes in flow rate, wavelength and mobile phase concentration. In robustness overall %RSD should not be more than 2.0% for the results obtained at the control and variable conditions. The results are discussed in following table.

Table No.7. Mobile Phase Composition Change: 49ml Methanol+ 51ml 0.1 N Citric acid

Sr. No.	ug/ml	Area
1.	10	930.816
2.	10	9352.59
	Mean	931.70
	SD	1.25
	%RSD	0.13

Fig No.11 Chromatogram Mobile Phase Composition Change:49 Methanol+51%0.1N Citric acid

Table No.8 Mobile Phase Composition Change: 51ml Methanol+ 49ml 0.1 N Citric acid

Sr. No.	ug/ml	Area
1.	10	946.209
2.	10	948.68
	Mean	947.44
	SD	1.75
	% RSD	0.18

Fig No.12 Chromatogram Mobile Phase Composition Change:51ml Methanol+49ml 0.1N Citric acid

Wavelength Change:

Table No.9 Wavelength Composition Change:249nm

Sr. No	ugm/ml	Area
1.	10	961.779
2.	10	960.21
	Mean	961.0
	SD	1.11
	%RSD	0.12

Table No.10 Wavelength Composition Change: 251nm

Sr. No.	ugm/ml	Area
1.	10	900.892
2.	10	903.32
	Mean	902.11
	SD	1.72
	%RSD	0.19

Fig No. 13 Wavelength Composition (249 and 251nm)

LOD and LOQ:

LOD and LOQ were calculated from the linearity curve by using the formula

LOD = 3.3 X Avd. SD / Slope and

LOQ = 10 X Avd. SD / Slope

Table No. 11 LOD and LOQ Data of Tirzepatide

Parameters	Value
Slope	103.6
Intercept	68.79
Correlation coefficient R²	R² = 0.999
LOD	0.0344015
LOQ	0.1042471

Analysis of formulation: The % assay of TIRZEPTIDE was found to be 101.24% and 100.79% respectively, which was well within the limits i.e., 90-110%. Results for the assay are discussed in following table

Table No.12 Assay data of Tirzepatide

Conc.	Area	Amt found	% label claim
10.00	951.665	9.8499517	98.50
10.00	953.254	9.8652896	98.65
Mean	952.46	9.86	98.58
SD	1.124	0.011	0.108
% RSD	0.118	0.110	0.110

Fig No.14 Chromatogram of Pharmaceutical formulation

CONCLUSION

A simple, precise, accurate, and robust Reverse Phase High-Performance Liquid Chromatographic (RP-HPLC) method was successfully developed and validated for the estimation of Tirzepatide in its pharmaceutical formulation (Mounjaro injection). The optimized chromatographic conditions using a C18 column with a mobile phase of methanol and 0.1 N citric acid (50:50 v/v), a flow rate of 0.9 ml/min, and detection at 250 nm produced sharp, well-resolved peaks with excellent linearity (R² = 0.9994) over the concentration range of 5–25 µg/mL.The method demonstrated high precision and reproducibility with % RSD values below 0.2%, while accuracy results (recoveries of 99.36–101.55 %) confirmed the reliability of the procedure. Robustness studies indicated that small variations in analytical parameters did not significantly affect the results, proving the method’s stability. The low LOD (0.034 µg/mL) and LOQ (0.104 µg/mL) values further established its high sensitivity. The assay of the marketed formulation showed 98.58% of the labeled claim, which lies within acceptable pharmacopoeial limits. Overall, the developed RP-HPLC method is rapid, cost-effective and efficient, making it suitable for routine quality control, quantitative estimation, and purity assessment of Tirzepatide in bulk and pharmaceutical dosage forms.

REFERENCES

Munirah S. O. Alhar, Walaa I. El-Sofany, Aljazi Abdullah Al Rashidi, Khaled Hamden, Protective Effects of Isolated Curcumin From Curcuma longa on Key Enzymes Involvedin the Insulins signaling Pathway and Digestive and Metabolic Enzymes Associated With Obesity, Type 2 Diabetes, and Hypertension, Journal of Diabetes Research.
Chavda, V.P.; Ajabiya, J.; Teli, D.; Bojarska, J.; Apostolopoulos, V. Tirzepatide, a New Era of Dual-Targeted Treatment for Diabetes and Obesity: A Mini-Review. Molecules 2022; 27:4315.
Ashraf AR, Mackey TK, Vida RG, Kulcsár G, Schmidt J, Balázs O, Domián BM, Li J, Csákó I, Fittler AMultifactor Quality and Safety Analysis of Semaglutide Products Sold by Online Sellers without a Prescription: Market Surveillance, Content Analysis, and Product Purchase Evaluation StudyJ Med Internet Res 2024;26:e65440.
Hamza, M., Papamargaritis, D., & Davies, M. J. (2024). Tirzepatide for overweight and obesity management. Expert Opinion on Pharmacotherapy, 26(1), 31–49.
Rangraze, I., Patoulias, D., Karakasis, P., El-Tanani, M., & Rizzo, M. (2024). Tirzepatide, a novel, dual glucose-dependent insulinotropic polypeptide/glucagon-like peptide-1 receptor agonist for the ongoing diabesity epidemic: the dawn of a new era? Expert Review of Clinical Pharmacology, 17(1), 853–856.
Willard, F. S., Douros, J. D., Gabe, M. B. N., Showalter, A. D., Wainscott, D. B., Suter, T. M., et al. (2020). Tirzepatide is an imbalanced and biased dual GIP and GLP-1 receptor agonist. JCI Insight, 5(17).
Yang, J., Gu, Y., Chen, H., Wang, H., Hong, L., Li, B., & Yang, L. (2024). Tirzepatide’s innovative applications in the management of type 2 diabetes and its future prospects in cardiovascular health. Frontiers in Pharmacology, 15.
Janapati YK, Junapudi S, Dachani SR. Optimization of diabetes by herbal medicine. Technol Innov Manag Rev. 2021;6:1-8.
ICH. Q2(R1) Validation of Analytical Procedures: Text and Methodology Guidance for Industry. International Conference on Harmonisation, Geneva; 2005.
Kazakevich Y, Lobrutto R, editors. HPLC for Pharmaceutical Scientists. Hoboken: Wiley-Interscience; 2006.
Nauck MA, D‘Alessio DA. Tirzepatide, a dual GIP/GLP-1 receptor co-agonist for the treatment of type 2 diabetes with unmatched effectiveness regrading glycaemic control and body weight reduction. Cardiovasc Diabetol. 2022;21:169.
Schneck K, Urva S. Population pharmacokinetics of the GIP/GLPreceptor agonist tirzepatide. CPT Pharmacometrics Syst Pharmacol.2024;13(3):494–503.
Frías JP, Davies MJ, Rosenstock J, Pérez Manghi FC, Landó LF, Bergman BK, et al. Tirzepatide versus Semaglutide Once Weekly in Patients with Type 2 Diabetes. N Engl J Med. 2021;385(6):503-515.