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Abstract

This study provides a comprehensive overview of Type 2 Diabetes Mellitus (T2D), focusing on its pathophysiology, symptoms, causes, genetic influences, and global epidemiology. T2D is characterized by insulin resistance and inadequate insulin production, leading to chronic hyperglycemia and associated complications. The increasing global prevalence, especially in low-to-middle-income countries, underscores the urgency of effective diagnosis and management strategies. Additionally, the role of analytical chemistry in pharmaceutical research is highlighted, with emphasis on High Performance Liquid Chromatography (HPLC) as a reliable and precise tool for drug analysis. The process of analytical method development and validation is detailed, including key parameters such as accuracy, precision, linearity, specificity, and robustness. This framework ensures that pharmaceutical products meet stringent quality standards and regulatory requirements, which is crucial for the effective monitoring and treatment of chronic diseases like T2D.

Keywords

Type 2 Diabetes Mellitus, antidiabetic drugs, high-performance liquid chromatography (HPLC), method development, method validation, ICH guidelines, simultaneous estimation, pharmaceutical analysis, quality control, and drug formulations

Introduction

Type 2 Diabetes (T2D) is a chronic metabolic disorder characterized by high blood sugar levels due to insulin resistance and impaired insulin production. It is influenced by genetic, lifestyle, and environmental factors and has become increasingly prevalent globally due to rising obesity rates and sedentary lifestyles. Early detection and proper management are critical to prevent complications. In pharmaceutical research and quality control, accurate and reliable analytical methods—particularly High-Performance Liquid Chromatography (HPLC)—are essential for drug analysis. Method development and validation ensure that these techniques produce consistent, precise, and accurate results, supporting the safety and efficacy of medications.

Liragutide: Liraglutide is a synthetic analog of the human glucagon-like peptide-1 (GLP-1), a hormone that plays a key role in regulating blood sugar levels and promoting insulin secretion. It is primarily used as a medication for the treatment of Type 2 diabetes mellitus (T2DM). As a GLP-1 receptor agonist, liraglutide works by mimicking the action of natural GLP-1, enhancing insulin release in response to meals, suppressing glucagon secretion (which reduces glucose production in the liver), and slowing gastric emptying, which leads to improved satiety and reduced food intake. In patients with T2DM, insulin resistance and beta-cell dysfunction lead to chronic hyperglycaemia, and liraglutide helps manage these issues by improving glucose homeostasis. Liraglutide is administered via subcutaneous injection and is typically used in conjunction with diet and exercise, and sometimes alongside other antidiabetic agents, to achieve optimal glycemic control. One of the key benefits of liraglutide is its ability to not only lower blood glucose levels but also assist in weight loss, making it especially beneficial for overweight or obese individuals with T2DM. Liraglutide has demonstrated significant efficacy in clinical trials, reducing HbA1c (a marker of long-term blood sugar control) and promoting weight loss in patients with T2DM. It is generally well-tolerated, though potential side effects include gastrointestinal disturbances and, in rare cases, pancreatitis or thyroid cancer. Its effectiveness, weight-reducing properties, and relatively low risk of hypoglycaemia make liraglutide a valuable addition to the pharmacological management of Type 2 diabetes.

Generic Name

Liraglutide

Structure

        <a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-8.png" target="_blank">
            <img alt="T-1.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-8.png" width="150"</a>

Brand Names

Victoza (for Type 2 Diabetes), Saxenda (for Obesity)

Drug Class

GLP-1 receptor agonist (glucagon-like peptide-1 receptor agonist)

Mechanism of Action

Mimics the action of GLP-1, enhancing insulin secretion, inhibiting glucagon release, and delaying gastric emptying to help control blood glucose and promote weight loss[13].

Indications

Type 2 Diabetes, Obesity, Cardiovascular risk reduction in diabetic patients

Administration

Subcutaneous injection (daily)

Dosage Forms

Injectable solution

Usual Dose (for T2DM)

nitial: 0.6 mg once daily, increase to 1.2 mg after 1 week, may increase to 1.8 mg daily depending on response

Common Side Effects

Nausea, vomiting, diarrhea, constipation, decreased appetite, headache, dizziness

Drug Interactions

May interact with insulin or sulfonylureas, increasing the risk of hypoglycemia. Caution when used with drugs affecting gastric emptying[14]

Duration of Effect

24 hours per dose

MATERIAL AND METHOD

Table: 1 Instrument specification for High Performance LC

Make

Shimadzu

Model

LC 2010

Type

Binary Gradient

Detector

HPLC Detector

Software

LC Solution

Column

HYPERSIL ODS C18, 250 mm*4.6 mm

Pump

High Pressure Gradient (Reciprocating pump)

Table: 2 Instrument specifications for weighing balance

Make

Mettler Toledo

Sensitivity

0.1 milligram

Minimum weighing capacity

1 milligram

Table: 3 Instrument specification for melting point apparatus

Make

Gallenkamp

Design no.

889339

Table: 4 Instrument Specification for UV double beam Spectrophotometer

Make

Shimadzu

Model

HPLC 1800

Type

Double beam spectrophotometer

Detector

Photodiode

Scanning Range

190-1100

Output

%T & Absorbance

Software

U.V. Probe 2.42

Table: 5 Procurement of pure APIs

Sr. No.

Name of APIs

Source

1

Liraglutide

Sun Pharma

2. Identification of Drugs

1. Identification by Melting Point Determination

Melting point of Liraglutide has been determined. The melting points of the compounds were taken by open capillary method.

Table 6 Melting Point of Drugs

Sr. No.

APIs

Melting Point

Reported

Measured

1

Liraglutide

182.2 °C

>182 °C

3. Identification by FTIR Spectroscopy

Liraglutide

        <a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-7.png" target="_blank">
            <img alt="Structure of Liraglutide.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-7.png" width="150">
        </a>
Fig.1 Structure of Liraglutide

3. Solution Stability

The solubility of Liraglutide practically determined separately by taking 100 mg of the drugs in 100 ml volumetric flasks, adding required quantity of solvent at room temperature and shaken for few minutes. Solubility data for each study was observed and recorded in Table 9.

Table 9 Solubility Table

Description Terms

Relative Quantities of solvent for 1 Parts of solute

Very soluble

Less than 1 part

Freely soluble

From 1 to 10 parts

Soluble

From 10 to 30 parts

Sparingly soluble

From 30 to 100 parts

Slightly soluble

From 300 to 1000 parts

Very slightly soluble

From 1000 to 10000 parts

Practically Insoluble

More than 10000 parts

3. Development and Optimization of RP-HPLC Method [15-18]

1. Selection of Wavelength

To determine wavelength for measurement, standard spectra of Liraglutide was scanned between 200-400 nm against diluents. Absorbance maxima Liraglutide detected at 240 nm. Chromatogram was taken at 215 nm, drug give good peak height and shape. So, 215 nm was selected for estimation of Liraglutide formulation. The UV overlain spectrum for Liraglutide shown in fig No.7.7

2. Selection of Chromatographic Conditions

Proper selection of the HPLC method depends upon the nature of the sample (ionic or ionisable or neutral molecule), its molecular weight, pKa and solubility. RP-HPLC was selected for the initial separation based on literature survey and its simplicity and suitability. To optimize the chromatographic conditions the effect of chromatographic variables such as mobile phase, flow rate and solvent ratio were studied. Finally, the chromatographic condition was chosen that give the best resolution, symmetry and capacity factor for estimation of both drugs.            

3. Selection of Column

For RP-HPLC Method, various columns are available but based on literature survey C-18 (id 4.6 x 150 mm, 5 µm) was selected over the other columns.

4. Preparation of Solution

1. Preparation of Mobile Phase

HPLC method was followed by isocratic elution technique. Mobile phase comprised of 1% v/v orthophosphoric acid and acetonitrile in a ratio of (55:45% v/v) ratio because it elutes both drugs peak efficiently in short time with satisfactory resolution, tailing factor and theoretical plates.

2. Preparation of Standard Stock Solution:

Accurately weighed quantity of Liraglutide 10 mg was transferred into 10 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 100 μg/mL. Withdraw 0.4 ml from Stock Solution and make up to 10 ml with to get 4 μg/mL.

3. Chromatographic condition

The chromatographic separation of Liraglutide was achieved on C-18 (id 4.6 x 150 mm, 5 µm) by using mobile phase composed of Acetonitrile: Water (25:75 v/v/v %), at flow rate 1.0 ml/min with

run time of 10 minutes. Detection of drug was carried out at 225 nm by using diluent as mobile phase.

4. Method of validation[19,20]

As per ICH guideline (Q2R1), the method validation parameters studied were specificity, linearity, accuracy, precision, limit of detection, limit of quantitation and robustness.

1. Specificity

The analytical method for specificity was evaluated by injecting the following solutions. Diluent was prepared and inject into the HPLC system in triplicate. Sample solution was prepared with appropriate levels of excipients as a placebo sample and inject into the HPLC system in triplicate for all the dosage strengths. Placebo was prepared by mixing all excipients without active ingredients. Standard and sample solutions were prepared for assay (100% Conc.) and inject into the HPLC system in triplicate.

2. Linearity and Range

Preparation of Solution for linearity studies: For the purpose of linearity, accurately weighed amount of Liraglutide (10 mg) was taken into the volumetric flask (10 ml) and volume of the flask was raised to 10 ml with methyl alcohol to give stock solution containing 100 µg/ml of Liraglutide. Various aliquots from this stock solution were transferred to another 10 ml volumetric flask and volume was raised to the mark with mobile phase to give final solutions containing 2, 4, 6, 8 and 10 µg/ml of Liraglutide respectively.

3. Precision

1. Repeatability

Prepared standard working solution of mixtures having concentration of Liraglutide (4 μg/ml) was injected at volume of 20 μL into column by employing optimized chromatographic conditions. Each standard mixture was injected 5 time and peak area was monitored. Each concentration was monitored for repeatability by RSD. Intra-day and Inter-day Precision.

4. System Suitability Parameters

Solution of Liraglutide (4 μg.ml-1) was injected 3 times for determination of System suitability parameters which includes Retention time (Rt), Tailing factor (Tf), Resolution (Rs) and number of theoretical plates. System suitability parameters for selected concentration were determined by C.V.

5. Accuracy

Accuracy of the analytical method has been performed by spiking of sample with the standard. Spiking of the placebo was performed at 50,100 and 150 % of the target concentration

6. Limit of detection and Limit of Quantification

The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the standard deviation of y-intercept of calibration curve.

7. Robustness

Following parameters were altered one by one for determination of robustness of the method and their effect was observed by comparing with the standard preparation. Mobile phase flowrate (± 0.1 mL/min), optimized flowrate was 1.0 mL/min. Mobile phase composition (± 2 mL), in optimized ratio 2 determinations of Liraglutide = 2 µg/mL for each alteration were carried out and RSD was measured.

8. Assay

Sample preparation

Label claim: (Total contents 30grams-Equivalent to 2%w/w of Liraglutide). Squeeze all the contents in beaker and extract all the quantity with 100 ml methanol. Filter the phase, if necessary, that gives the solution containing 6000 µg/ml of Liraglutide. Dilute 50 µl of previous solution to 10 ml with mobile phase to give solution containing 300 µg/ml of Liraglutide.

Test solution:

Withdraw 100µl from above filtrate in 10 mL volumetric flask; make up the volume with mobile phase, which contain Liraglutide = 3 µg/ml. Inject the above solution for 3 times under optimized

1. Selection of Wavelength

To determine wavelength for measurement, standard spectra of Liraglutide was scanned between 200-400 nm against diluents. Absorbance maxima of Liraglutide have detected at 263 nm & 240 nm. Chromatogram was taken at 215 nm, both drugs give good peak height and shape. So, 215 nm was selected for Simultaneous estimation of Liraglutide in their formulation.

Selection of Mobile phase

Trail 1

Column

C-18 (id 4.6 x 150 mm, 5 µm)

Mobile Phase

1% v/v orthophosphoric acid and acetonitrile in a ratio of 80:20% v/v.

Detection

225 nm

Flow rate

1 ml/min

Run Time

10 min

Observations

Peaks detected and separated, but broad peaks observe.

        <a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-6.png" target="_blank">
            <img alt="Chromatogram of Liraglutide.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-6.png" width="150">
        </a>
Fig 2 Trial 1: Chromatogram of Liraglutide (4 µg, ml-1)

Trail 2

Column

C-18 (id 4.6 x 150 mm, 5 µm)

Mobile Phase

1% v/v orthophosphoric acid and acetonitrile in a ratio of 50-50% v/v.

Detection

215 nm

Flow rate

1 ml/min

Run Time

10 min

Observations

Peaks detected and separated, but broad peaks observe.

        <a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-5.png" target="_blank">
            <img alt="Fig 3.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-5.png" width="150">
        </a>
Fig 3 Trial 2: Chromatogram of Liraglutide (4 µg,ml-1)

Trail 3

Column

C-18 (id 4.6 x 150 mm, 5 µm)

Mobile Phase

1% v/v orthophosphoric acid and acetonitrile in a ratio of 50-45% v/v.

Detection

225 nm

Flow rate

1 ml/min

Run Time

10 min

Observations

Good peak with Adequate solution was observed.

        <a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-4.png" target="_blank"> <img alt="Fig 4.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-4.png" width="150">
        </a>
Fig 4 Trial 3: Chromatogram of Liraglutide (4 µg,ml-1)

Chromatographic conditions for optimized mobile phase trial

Stationary Phase

C-18 (id 4.6 x 150 mm, 5 µm)

Mobile Phase

1% v/v orthophosphoric acid and acetonitrile in a ratio of 55-45% v/v.

Detection

215 nm

Flow rate

1 ml/min

Run Time

10 min

Detector

UV detector

Injection Volume

20 μl

Column Temperature

40ºC

Mode

Isocratic

Observations

Good peak with Adequate solution was observed.

        <a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-3.png" target="_blank">
            <img alt="Fig 5.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-3.png" width="150">
        </a>
Fig 5 Optimized mobile phase trial for optimized chromatogram of Std. Liraglutide:2.225 min

            <a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-2.png" target="_blank">
            <img alt="Fig 6.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-2.png" width="150">
        </a>
Fig 6 Chromatogram of blank 1% v/v orthophosphoric acid and acetonitrile in a ratio of (55:45% v/v)

3. Method Validation

1. Linearity

For the purpose of linearity, accurately weighed amount of Liraglutide(10 mg) was taken into the volumetric flask (10 ml) and volume of the flask was raised to 10 ml with methyl alcohol to give stock solution containing 100 µg/ml of Liraglutide. Various aliquots from this stock solution were transferred to another 10 ml volumetric flask and volume was raised to the mark with mobile phase to give final solutions containing 2, 4, 6, 8 and 10µg/ml of Liraglutide.

Table 10 Linearity data for Liraglutide

Liraglutide

Conc.

(µg/ml)

Mean

Area

± SD (n=5)

% RSD

2

206575

206575 ± 2935.65

1.42

4

315873

315873 ± 2441.81

0.77

6

398027

398027 ± 1245.003

0.31

8

486460

486460 ± 1882.09

0.37

10

573993

573993 ± 608.92

0.11

        <a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-1.png" target="_blank">
            <img alt="Overlain Linearity Spectra of Liraglutide.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-1.png" width="150">
        </a>
Fig 7 Overlain Linearity Spectra of Liraglutide

        <a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-0.png" target="_blank">
            <img alt="Calibration curve of Liraglutide.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250606145106-0.png" width="150">
        </a>
Fig.8 Calibration curve of Liraglutide

Table 11 Linearity results for Liraglutide

Regression Analysis

Liraglutide

Concentration Range

2-10 μg/mL

Regression equation

y = 7422.2x + 20622

Correlation co-efficient

0.9956

2. Precision

1. Repeatability

The data for repeatability for Liraglutide is shown in table 13. The % R.S.D For Repeatability data was found to be 1.10 % for Liraglutide.

Table 13 Repeatability data for Ranitidine and Ondansetron

Drugs

Conc. (µg/ml)

Mean Peak Area ± SD

%RSD

Liraglutide

4

724860 ± 1041.54

1.10

2. Inter-day precision

The data for interlay precision for Ranitidine and Ondansetron is shown in table 7.14. The % R.S.D for intraday precision was found to be 0.30-1.57 % for Liraglutide.

Table 14 Inter-day precision data for estimation of Liraglutide

Liraglutide

Mcg/ml

2

4

6

 

207689

381580

574389

205567

384567

573321

201345

381456

570934

MEAN

204867

382534.3333

572881.3333

± SD

3229.409234

1761.432466

1768.964763

RSD

1.576344279

0.460463889

0.308783802

3. Intra -day precision

The data for intra-day precision for Liraglutide is shown in table 14. The % R.S.D for intraday precision was found to be 0.12-0.56 % for Liraglutide.

Table 15 Intra-day precision data for estimation of Liraglutide

Liraglutide

Mcg/ml

2

4

6

 

206589

395476

574489

204378

395807

573209

204879

392657

573278

MEAN

205282

394646.6667

573658.6667

± SD

1159.282968

1731.031581

719.9168934

RSD

0.564727043

0.438628203

0.125495688

4. Accuracy

Accuracy of the method was confirmed by recovery study from synthetic mixture at three level standard additions. Percentage recovery for Liraglutide was found to be 99.48- 99.78%. The results are shown in table 16.

Table 16 Recovery data for Liraglutide

Liraglutide

 

50%

100%

150%

Amount of drug recovered (mg)

%Recovery

Amount of drug recovered (mg)

%Recovery

Amount of drug recovered (mg)

%Recovery

1.46

99.76

2.97

99.20

4.54

100.20

1.40

97.70

2.89

99.01

4.56

100.22

1.56

100.50

3.09

100.01

4.68

100.30

Mean

1.49

96.65

2.98

99.43

4.69

100.24

%RSD

0.02

1.30

0.04

1.75

0.05

0.68

5. LOD and LOQ

The limit of detection (LOD) and Limit of Quantification (LOQ) was found to be as per below:

Table 18 LOD and LOQ Limit for Liraglutide

Liraglutide

LOD(μg/ml)

LOD(μg/ml)

2.30

2.30

6. Selectivity

There is no interference in the mixture.

7. Robustness

The method is found to be robust as the results were not significantly affected by slight variation in Mobile Phase Composition and flow rate of mobile phase. The results are shown in table 7.19. Variation seen was within the acceptable range respect to peak asymmetry and theoretical plates, so the method was found to be robust.

Table 19 Robustness data for Liraglutide

Parameter

Level of Change

Effect on assay volume

Liraglutide

Assay ± SD

RSD

Flow rate

0.9 mL/min

97.70 ±0.50

0.49

1.1 mL/min

101.09 ±0.72

0.72

Mobile phase composition

50:50

97.47 ±0.53

0.53

60:40

97.39 ±0.99

0.98

30:70

99.51 ±0.67

0.67

8. Analysis of marketed product

The proposed method was successfully applied to analysis of the commercially available tablet formulation. The % drugs were found satisfactory, which is comparable with the corresponding label claim.

Table 20 Analysis of marketed formulations

Drug

Amount taken (µg/mL)

Amount found (µg/mL)

% Assy

Ranitidine

3

2.93±0.04

    1. 1.20

Summary of Method Validation

Table 21 Summary of validation parameter of RP-HPLC method

Optimized chromatographic Condition

Stationary Phase

C-18 (id 4.6 x 150 mm, 5 µm)

Mobile Phase

1% v/v Orthophosphoric acid and acetonitrile in a ratio of (55:45% v/v)

Detection wave Length

215 nm

Flow rate

1 ml/minute

Run time

10 minutes

Retention Time

2.225 min

 

Validation Parameters

Parameter

Limit

Result

Conclusion

Liraglutide

Linearity and Range

R2> 0.995

0.9956

(2-10µg/mL)

Method was linear

Repeatability

RSD<2

1.10

Method was repeatable

LOD

-

-

-

LOQ

-

-

-

Intra-day Precision

RSD<2

0.30.-1.57

Method was precise

Inter-Day Precision

RSD<2

0.12-0.56

Method was precise

%Recovery

98-102%

99.35 ±0.83– 100.01±0.03 %

Method was accurate

Robustness

RSD<2

0.41– 0.63

Method was robust

Assay%

-

99.80 ±1.20

-

CONCLUSION

A method for the development and validation of a Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) technique for the simultaneous determination of Liraglutide has been successfully established. Mobile phase for Liraglutide 1% v/v orthophosphoric acid and acetonitrile in a ratio of 55:45% v/v. 215 nm wavelength taken for Liraglutide. In conclusion, the developed and validated HPLC method is a promising, efficient, and reliable approach for the determination of Liraglutide in pharmaceutical products, contributing to better quality control practices and patient safety.

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Reference

  1. American Diabetes Association. What Are My Options for Type 2 Diabetes Medications? (https://diabetes.org/health-wellness/medication/oral-other-injectable- diabetes-medications) Accessed 11/8/2024.
  2. Centers for Disease Control and Prevention (U.S.). Type 2 Diabetes (https://www.cdc.gov/diabetes/basics/type2.html). Last reviewed 4/18/2023. Accessed 11/8/2024.
  3. Goyal R, Singhal M, Jialal I. Type 2 Diabetes (https://www.ncbi.nlm.nih.gov/books/NBK513253/). 2023 Jun 23. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan. Accessed 12/8/2024.
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Akhil Bhakhar
Corresponding author

Noble Pharmacy College, Faculty of Pharmacy, "Parth-Vatika", Junagadh- Bhesan Road, Via. Vadal, Nr. Bamangam, Junagadh - 362310, Gujarat, INDIA.

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Dhirendra Kumar Tarai
Co-author

Noble Pharmacy College, Faculty of Pharmacy, "Parth-Vatika", Junagadh- Bhesan Road, Via. Vadal, Nr. Bamangam, Junagadh - 362310, Gujarat, INDIA.

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Khyati Bhupta
Co-author

Noble Pharmacy College, Faculty of Pharmacy, "Parth-Vatika", Junagadh- Bhesan Road, Via. Vadal, Nr. Bamangam, Junagadh - 362310, Gujarat, INDIA.

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Dr. Santosh Kirtane
Co-author

Noble Pharmacy College, Faculty of Pharmacy, "Parth-Vatika", Junagadh- Bhesan Road, Via. Vadal, Nr. Bamangam, Junagadh - 362310, Gujarat, INDIA.

Akhil Bhakhar*, Khyati Bhupta, Dhirendra Kumar Tarai, Dr. Santosh Kirtane, Development and Validation Method for Estimation for Liraglutide By RP-HPLC From Its Injectable Dosage Form, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 6, 1134-1147. https://doi.org/10.5281/zenodo.15607231

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