Development And Validation Method for Estimation for Liraglutide By RP-HPLC From Its Injectable Dosage Form

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.

MATERIAL AND METHOD

Table: 1 Instrument specification for High Performance LC

Table: 2 Instrument specifications for weighing balance

Table: 3 Instrument specification for melting point apparatus

Table: 4 Instrument Specification for UV double beam Spectrophotometer

Table: 5 Procurement of pure APIs

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

3. Identification by FTIR Spectroscopy

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

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

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

        <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

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.

        <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

Table 11 Linearity results for Liraglutide

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.

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.

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.

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.

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

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.

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

Summary of Method Validation

Table 21 Summary of validation parameter of RP-HPLC method