Maharashtra college of pharmacy, Dept of Quality Assurance, Nilanga-413521 Dist. Latur Maharashtra, India.
Another methodology was set up for synchronous estimation of a Terlipressin by RP-HPLC system. The chromatographic conditions were viably created for the unit of Terlipressin by using Inertsil – ODS C18 (250 x 4.6 mm 5µ), stream is 1.0 ml/min, convenient stage extent was Methanol:Acetonitrile (30:70), recognizable proof wave length was 225 nm.
Terlipressin is a synthetic analogue of vasopressin, which is an endogenous neurohormone that accts as a vasoconstrictor. It is a prodrug of lypressin, or lysine vasopressin. Compared to endogenous vasopressin, Terlipressin has a longer half-life and increased selectivity for the V1 receptor. Molecular formula is C52H74N16O15S2.
Figure no. - 1
The Literature survey indicates that there are no methods for the Estimation of Terlipressin. Therefore, an attempt was made to develop and validate a simple and economical RP-HPLC method as per ICH guidelines for the estimation of Terlipressin pharmaceutical dosage forms.
MATERIALS AND METHODS:
Instrument:
Experimental conditions:
Quantitative HPLC was performed on isocratic HPLC of Waters model no. 2690/5 with software Empower-2 infinity isocratic LC manual injector with variable wavelength detector. For method development several trials were carried out. After many trials, the chromatographic conditions were decided. The separation was conducted by using column of Inertsil- ODS C18 (5µ, 4.6 mm×250) with mobile phase consisting of methanol and Acetonitrile in the ratio of (30:70). The mobile phase delivered at the flow rate of 1.0ml/min. The eluent was monitored at wavelength 225 nm and found a sharp and symmetrical peak with retention time of 3.68 min. The run time observed was 6 min.
Preparation of standard solution:
Take 100mg Terlipressin working standard in 100ml V.F add methanol sonicate it 30min, (That is 1000ppm solution).
Preparation of sample solution:
Take 10ml of above solution in 100ml V.F add Methanol up to mark sonicate it 10min (That 100ppm solution)
Diluent:
The methanol was used as diluent.
Method validation:
Validation establish a documented evidence which provides which a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes.
A Standard solution was prepared by using Terlipressin working standard as per test method and was injected Five times into the HPLC system. The system suitability parameters were evaluated from standard chromatograms by calculating the % RSD from five replicate injections for Terlipressin, retention times and peak areas.
2. Precision:
3.Accuracy:
A study of Accuracy was conducted. Drug Assay was performed in triplicate as per test method with equivalent amount of Terlipressin into each volumetric flask for each spike level to get the concentration of Terlipressin equivalent to 50%, 100%, and 150% of the labelled amount as per the test method. The average % recovery of Terlipressin was calculated.
4.Linearity:
A Series of solutions are prepared using Terlipressin working standard at concentration levels from 20ppm to 70 ppm of target concentration.
5. Ruggedness:
System to system variability study was conducted on different HPLC systems, under similar conditions at different times. Six samples were prepared and each was analysed as per test method. Comparison of both the results obtained on two different HPLC systems, shows that the assay test method is rugged for System to system variability
6.Robustness:
A study was conducted to determine the effect of variation in flow rate. Standard solution prepared as per the test method was injected into the HPLC system using flow rates, 1.0ml/min and 1.2ml/min. The system suitability parameters were evaluated and found to be within the limits for 1.0ml/min and 1.2ml/min flow. Terlipressin was resolved from all other peaks and the retention times were comparable with those obtained for mobile phase having flow rates 1.0ml/min.
7.LODAnd LOQ (Limit Of Detection And Limit Of Quantitation):
From the linearity plot the LOD and LOQ are calculated:
Figure no. 2 - chromatogram for diluent
Figure no. 3 - Blank Chromatograph
Figure no. 4 - Chromatogram Standard
RESULTS AND DISCUSSION:
Mobile Phase:
Methanol: Acetonitrile (30:70)V/V. Sonicate it 30min, Filter this mobile phase through 0.45micron filter paper.
Optimized Method Stock Solution Preparation : Take 100mg Terlipressin working standard in 100ml V.F add methanol sonicate it 30min, (That is 1000ppm solution).
Further Dilution (or) Optimized Method Solutions Preparation:
Take 4ml of above solution in 100ml V.F add Methanol up to mark sonicate it 10min (That 40ppm solution).
Chromatographic conditions :
Table 1 : chromatographic condition
Table 2 : Data of System Suitability
Table 3 : Data System precision
Table 4 : Data Method precision
Table 5 : Data of Accuracy
Table 6 : Data of Linearity
Fig no. 5 - Linearity Plot (Concentration Vs Response)
Table 7 : Data on System Variability System(Ruggedness)
Table 8 : Data for Effect of variation in flow rate(Robustness)
Table 9 : Assay of formulation
CONCLUSION :
Different parameters were studied to create the analytical approach. For starters, the maximum absorbance of Terlipressin was discovered to be 225nm. The injection volume was set at 20µl, which resulted in a nice peak area. The Inertsil C18 column was employed in this work, and ODS picked a nice peak shape. The temperature of the ambient environment was determined to be adequate for the type of the medication solution. Because of the good peak area, adequate retention duration, and good resolution, the flow rate was set at 1.0ml/min. Different mobile phase ratios were investigated, however the mobile phase with a Methanol: Acetonitrile (30:70) ratio was chosen because to its symmetrical peaks and high resolution. As a result, the planned research made use of this mobile phase. The accuracy of both the system and the procedure was determined to be precise and well within range. The correlation coefficient and curve fitting were discovered during the linearity investigation. For both medicines, the analytical approach was shown to be linear throughout a range of 20-70ppm of the target concentration. Both robustness and ruggedness tests were passed by the analytical. The relative standard deviation in both circumstances was excellent.
ACKNOWLEDGEMENTS :
Authors are thankful to for providing working standard & express their sincere gratitude towards Dr M. A. Shetkar (Research guide) Maharashtra college of pharmacy Nilanga), And Dr S. S. Patil Principal, Maharashtra College of Pharmacy, Nilanga for providing necessary facilities to carry out research work.
REFERENCES :
Bhagyashri S. Dhange, Madhav A. Shetkar, Sidheshwar S. Patil, Analytical Method Development And Validation For The Terlipressin In Pharmaceutical Doasage Form By RP-HPLC, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 5, 947-953. https://doi.org/10.5281/zenodo.11214748
10.5281/zenodo.11214748
