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