Research & Reviews: Journal of Pharmaceutical Analysis
Menue-ISSN: 2320-0812
e-ISSN: 2320-0812
Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Mysore, India
Received date: 05/06/2018; Accepted date: 16/08/2018; Published date: 23/08/2018
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A simple, RP-UFLC method was established for determining Linagliptin in Active Pharmaceutical Ingredients. Linagliptin is a DPP-4 inhibitor developed by BoehringerIngelheim (German Pharmaceutical Company) for treatment of type II diabetes. Linagliptin was approved by the US FDA on 2 May 2011 for treatment of type II diabetes. Linagliptin and their degradation products were separated using C18 column with Acetonitrile: Methanol (50:50 (v/v) as the mobile phase. Detection was performed at 238 nm using a PDA detector and flow rate 1.5 ml/min and retention time was 4.4 min respectively. The method was validated using ICH guidelines and was linear in the range 2-10 μg /ml (r2=0.995) for Linagliptin. The method showed good linearity, accuracy, precision, ruggedness, robustness, and specificity. Good separation of the analytes and their degradation products was achieved using this method. The developed method can be applied successfully for the determination of Linagliptin. The present method was validated with respect to system suitability, accuracy (recovery) precision, linearity, limit of detection (LOD) and limit of quantification (LOQ) and robustness according to the ICH Guidelines.
Linagliptin is a DPP-4 inhibitor developed by BoehringerIngelheim (German Pharmaceutical Company) for the treatment of type II diabetes. Linagliptin was approved by the US FDA on 2 May 2011 for treatment of type II diabetes. It is being marketed by BoehringerIngelheim and Lilly. Linagliptin is an inhibitor of DPP-4 (dipeptidyl peptidase 4) an enzyme that degrades the incretion hormones, Glucagonlike peptide-1 (GLP-1) and Glucose dependent Insulinotropic polypeptide (GIP). Both GLP-1 and GIP increase insulin biosynthesis and secretion from pancreatic beta cells in the presence of normal and elevated blood glucose levels. GLP-1 also reduces glucagon secretion from pancreatic alpha cells, resulting in a reduction in hepatic glucose output. Thus, Linagliptin stimulates the release of insulin in a glucose-dependent manner and decreases the levels of glucagon in the circulation. Linagliptin showed that the drug can effectively reduce blood sugar. In summary, Linagliptin reduces blood glucose levels by inhibiting DPP-4 and increasing the levels of GLP-1 and GIP. Linagliptin was approved by the FDA in May 2011[1].
Drug Profile:
| Molecular structure: |  |
|---|---|
| Synonyms | Tradjenta, Trajenta |
| Melting point | 202ºC |
| Solubility | Soluble in methanol; sparingly soluble in ethanol; very slightly soluble in isopropanol, alcohol |
| Physical appearance | White to yellow solid |
| Category | Antidiabetic agent |
Dose and Administration
Oral administration of a single 5-mg dose to healthy subjects, peak plasma concentrations of Linagliptin occurred at approximately 1.5 hours post dose (Tmax); the mean plasma Area Under the Curve (AUC) was 139 nmol*h/L and maximum concentration (Cmax) was 8.9 nmol/L.
Mechanism of Action
Linagliptin belongs to a class of drugs called DPP-4 inhibitors. DPP-4 inhibitors prevent the hormone incretin from being degraded, allowing insulin to be released from the pancreatic beta cells. While incretin remains in the blood stream, the pancreas is stimulated to produce more insulin. Meanwhile, glucagon release from the pancreas is staggered, preventing glucose level increase. In other words, Linagliptin, along with diet and exercise, can help the body produce more insulin and lower blood glucose. Managing blood sugar can mean a lower HbA1c, an index for Glycaemia control that theoretically correlates with glucose level in the blood [2-4].
Chromatographic Conditions
| Column | Kinetex 5 μ C18 (250 x 4.6 mm. 5 μ) |
| Flow rate | 1.5 mL/min |
| Run time | 10 min |
| Wavelength | 238 nm |
| Injection Volume | 10 μL |
| Detector | PDA Detector |
| Elution | Binary Gradient |
| Mobile Phase | Methanol and Acetonitrile (50:50) (v/v) |
| Column oven temperature | 25 ± 5ºC |
Preparation of Diluent
The diluent is a mixture of 50 parts of methanol and 50 parts of acetonitrile.
Preparation of Mobile Phase
Mobile phase is Methanol and Acetonitrile was used in the ratio of (50:50) (v/v).
Preparation of Standard Stock Solution for Linagliptin
100 mg of Linagliptin was taken into 100 mL volumetric flask. To this add 50 mL of diluent and sonicate to dissolve and the volume was made up to the mark with diluent (1000 μg/ml). Pipette 1ml of the above solution into 10 ml volumetric flask and make up the volume using diluent (100 μg/ml).
Preparation of Solutions for Linearity
The solutions for linearity were prepared from the stock solution by diluting with diluent. The concentration ranging from 2, 4, 6, 8, 10 μg/mL were prepared for Linagliptin. Pipette 0.2, 0.4, 0.6, 0.8, 1.0 mL in 10 ml volumetric flasks and make up the volume using diluent to get the above concentrations.
Preparation of Calibration Curve
From the stock solution (100 μg/mL) aliquots of Linagliptin were pipetted into a series of 10 mL volumetric flask. The volume was made up to the mark by using Acetonitrile and Methanol as diluent so as to obtain concentration range of 2-10 μg/mL and filtered through membrane filter of 0.45 μ pore size. 10 μL solutions were injected and peak areas were recorded. The calibration curve was established. The Beer’s law is obeyed in the concentration range of 2-10 μg/mL
Selection of Wavelength for Analysis (λ max)
The standard solutions of Linagliptin were scanned in the range of 200-400 nm against mobile phase as blank. Linagliptin showed maximum absorbance at 238 nm. Thus the wave length selected for the determination of Linagliptin is 238 nm.
Method Development
The RP-UFLC strategy created in this examination was gone for finding the chromatographic framework fit for eluting and resolving Linagliptin and its degradation product with fulfilling framework appropriateness conditions. To build up the conditions different parameters, for example, versatile stage, pH, stream rate and dissolvable proportion were changed and reasonable chromatographic condition has been created for routine investigation of medication tests. Beginning trails were done by utilizing same segment taking Methanol and Acetonitrile in different extents with stream rate of 1.5 ml/min. The column was kept up with gradient phase. The chromatograms got subsequent to injection drug tests and kept up with run time of 10 min detailed in separation and peaks were watched wide with thick peak heads and high retention time.
Method Validation
Method validation is the process used to confirm that the analytical method used for a particular test is reasonable for its expected utilize. Results from method validation can be utilized to reliability, judge the quality and consistency of analytical results; it is a necessary piece of any great diagnostic practice. It is the way toward characterizing a scientific necessity, and affirms that the technique under thought has execution abilities steady with what the application requires.
The different approval parameters incorporate linearity, precision, accuracy, Selectivity and Specificity, range, robustness and LOD, LOQ [5-10].
In developing the method, systematic study of the effects of various parameters is carried out. Initially the solubility of the Teneligliptin drug is determined. In UFLC method, chromatographic conditions stand advanced to obtain great peak. Initially, various mobile phase compositions were tried to elute the drug. Mobile phase and flow rate selection was based on peak parameters (height, capacity, theoretical plates, tailing or asymmetry factor), run time and resolution. The system with mobile phase containing Mobile Phase A (Methanol): Mobile Phase B (Acetonitrile): Mobile Phase C (Potassium dihydrogen ortho phosphate of pH 4.6) is used in the ratio of 40:20:40 (v/v) with 1 mL/min flow rate is very strong. The ideal wavelength for identification is 246 nm at which better detector response for the drug is acquired. The chromatogram for blank and Teneligliptin with retention time at 3.308 min were shown in Figures 1 and 2 respectively.
Figure 1: Chromatogram of Blank (Diluent).
Figure 2: Chromatogram for linagliptin.
From the standard chromatograms various system suitable parameters were recorded.
System Suitability
System suitability tests are used to verify the reproducibility of the chromatographic system. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stock solutions.
Data Interpretation
From the above tabulated data Table 1, it was observed that the system suitability parameters were within the acceptance criteria (Figures 3-10, Table 2).
| Parameters | Acceptance criteria | Results |
|---|---|---|
| Tailing factor | NMT 2.0 | 1.215 |
| Theoretical plates | NLT 2000 | 4078.495 |
Table 1: Results for System Suitability
Linearity
| Level | Concentration ( µg/mL) | Peak Area of Linagliptin |
|---|---|---|
| 1 | 2 | 88571 |
| 2 | 4 | 125745 |
| 3 | 6 | 156769 |
| 4 | 8 | 190716 |
| 5 | 10 | 214721 |
| Regression Equation | y = 15864x + 60123 | |
| Correlation Coefficient (R2) | R²=0.995 | |
| Slope | 15864 | |
| Intercept | 60123 | |
Table 2: Results for linearity.
Figure 3: Calibration curve of linagliptin.
Figure 4: Chromatogram for standard linagliptin (2 μG/ML).
Figure 5: Chromatogram for standard linagliptin (4 μG/ML).
Figure 6: Chromatogram for standard linagliptin (6 μG/ML).
Figure 7: Chromatogram for standard linagliptin (8 μG/ML).
Figure 8: Chromatogram for standard linagliptin (10 μG/ML).
Figure 9: Graph for blank linagliptin.
Figure 10: Graph for standard linagliptin (10 ΜG/ML).
Acceptance Criteria
Correlation coefficient should be NLT 0.95.
Data Interpretation
From the statistical treatment of linearity data from Table 2 of Linagliptin it is clear that the response of Linagliptin is linear between 15% to 50% level of working concentration. The correlation was not less than 0.95, so linearity parameter was within the acceptance criteria.
Precision
The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple sampling of homogeneous sample.
System Precision
The system precision is to ensure that the analytical system is working properly (Table 3).
| Sl.No | 2µg | 6 µg | 10 µg |
|---|---|---|---|
| 1 | 88571 | 156769 | 214721 |
| 2 | 88575 | 158999 | 215780 |
| 3 | 88756 | 158950 | 216330 |
| 4 | 88780 | 159795 | 218690 |
| 5 | 88570 | 158989 | 219800 |
| 6 | 85811 | 159790 | 218547 |
| Average | 88177.16667 | 158882 | 217311.3 |
| Standard Deviation | 1061.829776 | 1012.633859 | 1809.184 |
| % RSD | 1.204200379 | 0.637349643 | 0.832531 |
Table 3: Results for system precision studies, SD: Standard Deviation RSD: Relative Standard Deviation.
Acceptance Criteria
The %RSD of the area response for Linagliptin peak obtained from 6 injections of Standard preparation should be NMT 2.0%.
Data Interpretation
From the above Table 3, it can be concluded that area response are consistent as evidenced by relative standard deviation.
Method Precision
Method precision indicates whether a method is giving consistent results for a single material or not (Table 4).
| Sl.No | 2µg | 6 µg | 10 µg |
|---|---|---|---|
| 1 | 88571 | 156769 | 214721 |
| 2 | 89678 | 159658 | 215858 |
| 3 | 89751 | 153658 | 215455 |
| 4 | 89652 | 158954 | 215885 |
| 5 | 90581 | 158655 | 215888 |
| 6 | 91874 | 158666 | 225858 |
| Average | 90017.83333 | 157726.6667 | 217277.5 |
| Standard Deviation | 1014.590133 | 2018.733899 | 3859.338 |
| % RSD | 1.127099038 | 1.279893845 | 1.776225 |
Table 4: Results for method precision studies, SD: Standard Deviation RSD: Relative Standard Deviation.
Acceptance Criteria
The % RSD calculated on 6 determinations should be NMT 2.0%.
Data Interpretation
From the above Table 4, it can be concluded that the method is precise.
Limit of Detection & Limit of Quantitation
Limit of Detection (LOD) is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions.
Limit of Quantitation (LOQ) is the lowest amount of analyte in a sample that can be quantitated with acceptable accuracy and precision, under the stated experimental conditions.
The LOD & LOQ is calculated according to slope, intercept and correlation coefficient and the relative standard deviation from the linearity curve.
Data Interpretation
From the above Table 5, it can be concluded that distinct visible peaks were observed at LOD level concentration. The LOD and LOQ for Linagliptin were found to be 0.097 and 1.023 μg/mL respectively.
| LOD | 0.097 (µg/mL) |
| LOQ | 1.023 (µg/mL) |
Table 5: Results of limit of detection & limit of quantitation.
Accuracy
The accuracy of an analytical method is the closeness of test results obtained by that method to the true value (Standard value).
Acceptance Criteria
Individual and Mean % recovery at each level should be between 98.0% and 102.0%.
Data Interpretation
From Table 6, it can be concluded that the recovery is well within the limit. Hence the method is accurate.
| Sl.No | Level of % Recovery |
Amount of std formulation (µg/mL) |
Amount of drug added (µg/mL) | Total amount of drug (µg/mL) |
Total amount of drug found | % Recovery |
|---|---|---|---|---|---|---|
| 1. | 50 | 4 | 2 | 6 | 5.9 | 98.3 |
| 5.8 | 96.6 | |||||
| 6.2 | 103.3 | |||||
| Mean | 99.4 | |||||
| 2. | 100 | 4 | 4 | 8 | 8.3 | 103.7 |
| 7.8 | 97.5 | |||||
| 8.1 | 101.2 | |||||
| Mean | 100.8 | |||||
| 3. | 150 | 4 | 6 | 10 | 9.8 | 98 |
| 9.7 | 97 | |||||
| 10.4 | 104 | |||||
| Mean | 99.6 |
Table 6: Results for method accuracy studies.
Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.
Acceptance Criteria
► The Tailing factor should be NMT 2.0.
► The Theoretical plates should be NLT 2000
► The relative standard deviation should be NMT 2.0%
Data Interpretation
From Table 7, it can be concluded no significant changes were observed due to change in above said chromatographic conditions, hence the method is robust.
| Condition | Tailing | %RSD | Theoretical plates | %RSD | |
|---|---|---|---|---|---|
| As such condition (optimized method) | 1.215 | -- | 4478.4 | -- | |
| Mobile phase ratio as such (50:50) |
25:75 | 1.341 | 0.9789 | 4417.8 | 0.6811 |
| 45:55 | 1.358 | 1.6086 | 4415.6 | 0.7060 | |
| Change in pH | Decreased (-0.2 units) | 1.328 | 0.4918 | 4419.3 | 0.6642 |
| Increased (+0.2 units) | 1.322 | 0.2654 | 4421.3 | 0.6415 | |
| Flow rate | Decreased (-0.2 mL/min) | 1.358 | 1.6086 | 4425.9 | 0.5896 |
| Increased (+0.2 mL/min) | 1.366 | 1.9022 | 4430.5 | 0.5376 | |
| Column temperature | Decreased (-5°C) | 1.354 | 1.4612 | 4435.6 | 0.4801 |
| Increased (+5°C) | 1.333 | 0.6797 | 4440.9 | 0.4204 | |
| Wave length | Decreased (1 nm) | 1.348 | 1.2392 | 4445.3 | 0.3709 |
| Decreased (2 nm) | 1.352 | 1.3873 | 4447.9 | 0.3416 | |
| Increased (1 nm) | 1.356 | 1.5350 | 4449.9 | 0.3192 | |
| Increased (2 nm) | 1.358 | 1.6086 | 4321.9 | 1.7783 | |
Table 7: Results of robustness.
Forced Degradation Studies
The stress studies were performed for Linagliptin drug at 50 μg/ml concentration. Here the bulk drug is exposed to acidic stress by addition of 1.0 ml of 0.1M HCl to drug solution and counteracted with 1.0 ml of 0.1M NaOH, at 0 min, 30 min, 1 hrs, 2 hrs, 4 hrs, 8 hrs, 6 hrs and 32 hrs respectively. Similarly, the basic stress studies were performed by adding 1.0 ml of 0.1 M NaOH and neutralized with 1 ml of 0.1M HCl. Oxidation studies were achieved on bulk drug by addition of 1.0ml of 3% H2O2, Thermal studies were performed by heating the sample at 600C and UV studies were also carried out by sample at UV- Lamp 450ºC respectively.Entire samples were placed in different volumetric flask (10 ml) and dissolved in HPLC grade methanol. Final drug concentration for assay was made up with methanol and injected in the chromatographic system. For all these stability study, the development of degradable item was affirmed by contrasting and the chromatogram of the arrangement kept under ordinary unstressed conditions. Every stressed sample was analyzed by improved UFLC method. The degradation data for Linagliptin was shown [11-16].
Acid Stress
For 2 ml sample add 2 ml 0.1N HCl keep aside for 5 min and then add 2.ml of 0.1N NaOH, then inject this sample for 36 hours at intervals 30 min, 1 hr., 1.30 min respectively (Figure 11).
Figure 11: Chromatogram for acid stress.
Basic Stress
For 2 ml sample add 2 ml of 0.1N NaOH keep aside for 5 min and then add 2 ml of 0.1N HCL, and inject the sample (Figure 12).
Figure 12: Chromatogram for basic stress.
Peroxide Stress
For 2 ml sample add 1 ml of 3% peroxide solution and inject this sample (Figure 13).
Figure 13: Chromatogram for peroxide stress.
Heat Stress
Take 2 ml sample and heat for 1 hr. at 80 c and inject the sample (Figure 14).
Figure 14: Chromatogram for heat stress.
Photolytic Stress
Take 2 ml sample and place in a UV chamber for 1 hr. UV- Lamp 450oCrespectively and then inject the sample (Figure 15, Table 8).
Figure 15: Chromatogram for photolytic stress.
| Time | Drug | UV | THERMAL | 0.1N HCL | 0.1N NaOH | 3%H2O2 |
|---|---|---|---|---|---|---|
| 0 Min | Linagliptin | |||||
| 89.23% | 80.76% | 87.79% | 89.35% | 81.34% | ||
| 30 Min | Linagliptin | |||||
| 85.34% | 77.31% | 84.14% | 87.34% | 74.34% | ||
| 1 hr | Linagliptin | |||||
| 82.43% | 60.16% | 78.86% | 80.34% | 68.23% | ||
| 2 hr | Linagliptin | |||||
| 77.34% | 57.14% | 74.78% | 78.38% | 60.87% | ||
| 4hr | Linagliptin | |||||
| 69.34% | 31.69% | 67.27% | 70.34% | 44.34% | ||
| 8hr | Linagliptin | |||||
| 52.23% | 20.15% | 59.65% | 57.23% | 32.62% | ||
| 16hr | Linagliptin | |||||
| 43.87% | 19.6% | 44.64 | 43.24% | 22.23% | ||
| 32hr | Linagliptin | |||||
| 34.24% | --- | -- | --- | -- |
Table 8: Results for recovery studies linagliptin after the stress conditions (% recovery of drug).
The above RP-UFLC analytical method satisfies all validation parameters like accuracy, precision, system suitability, specificity, linearity of detector response, ruggedness and robustness. At the same time the method satisfies the forced degradation study. Hence, the validated method can be used for routine determination of stability studies in quality control laboratories in the pharmaceutical industry.
The authors express their sincere thanks to the Principal, JSS College of Pharmacy, Mysuru, for providing the necessary facilities to carry out the research work.