D.China Babu1*, M. Alagusundaram1, G. Sai Sri Harsha1, Nem Kumar Jain1, G. Venkateshwarlu1, S. Angala Parameswari2 and SK. Aleesha3
Article Publishing History
Article Received on : 25 Jul 2023
Article Accepted on :
Article Published : 17 Nov 2023
Background: Lasmiditan is an agonist of 5HT1F receptor, acute migraine is treated and oral dose is safe and effective. No analytical methods were reported on Lasmiditan Drug. Objectives: The goal of the current project is to create a quick, effective, and reproducible LC/MS/MS method for the estimation of Lasmiditan. The mobile phase was used Acetonitrile: 0.1% formic acid (70:30) in the developed method, and it was running down a C18 column (SP) with dimensions of 150 mm, 4.6 mm i.d., and 3.5 µm at a rate of 1 mL/min. The [M+H]+ ion m/z ratio for the substance was observed in the mass spectrum was 378.24. The drug had a retention time of 2.3 minutes. The linearity range was found to be in between 12.50 ng/mL and 75 ng/mL. The regression coefficient value was found to be 0.999, which is good satisfactory value for linearity. The LOD and LOQ for baseline measurement and detection were 0.66 ng/mL and 2.22 ng/mL, respectively; the method was shown good accuracy and precision levels, and it was proved through the assay values. The method was proved as robust after deliberated changes in parameters of flow rate (±0.2mL) and organic phase (±3 mL) in mobile phase composition and values were found to be 99.50%-100.73% & 99.10%- 101.83% respectively. Conclusion: The LC/MS/MS method is more advanced and sensitive to determine the drugs at nanograms level. Hence developed method used for the regular assay. This is more sensitive, selective, and rapid for identification and quantification of LMT in the bulk and Pharmaceutical formulation.
KEYWORDS:Acetonitrile; Formic acid; Lasmiditan; LC/MS/MS
Download this article as: Copy the following to cite this article:Babu D. C, Alagusundaram M, Harsha G. S. S, Jain N. K, Venkateshwarlu G, Parameswari S. A, Aleesha S. K. LC/MS/MS Method Development and Validation for the Estimation of Lasmiditan in Bulk and Pharmaceutical Formulation. Orient J Chem 2023;39(6).
Babu D. C, Alagusundaram M, Harsha G. S. S, Jain N. K, Venkateshwarlu G, Parameswari S. A, Aleesha S. K. LC/MS/MS Method Development and Validation for the Estimation of Lasmiditan in Bulk and Pharmaceutical Formulation. Orient J Chem 2023;39(6). Available from: https://bit.ly/46j0yGr
Introduction
The Lasmiditan (Fig.1) drug is used for the treatment of acute migraine and available in tablet dosage form. It lacks vasoconstrictive properties, safe and effective for the treatment of acute migraine in the patients with cardiovascular risk factors in which triptans are contraindicated.1-3 Lasmiditan is a selective 5 HT1F receptor agonist and less affinity on 5 HT1B receptor does not cause vasoconstriction.4 For the treatment of an acute therapy for migraine Lasmiditan being developed to address significant unmet needs with risk factors with the cardiovascular system, poor response to the current treatment and stable cardiovascular disease.5-12 The oral dose of Lasmiditan is safe and more effective in the treatment of acute migraine.13&14 The RP-HPLC15-17, impurities in Lasmiditan18 and Bio-analytical19 methods were on reported LMT. The developed method was more sensitive and determined at lowest level compared with other reported methods.
Materials and Method
Materials
The Lasmiditan drug was gifted by Supriya Lifescience Ltd. Acetonitrile, formic acid and water for HPLC grade brought from the Merck chemicals. The supplier of these chemicals is Bros Scientifics, Tirupathi, Andhra Pradesh 517507. The HPLC system was coupled to SCIEX QTRAP 5500 (Manufactured by AgilentLC) and ZORBAXLC column was used, it was symmetry C18 150 mm × 4.6 mm i.d and 3.5 μ. The filters were used in the pore size of 0.44 µm, manufactured by Millipore.
Preparation of standard solution
“Lasmiditan was weighed 10 mg, dissolved with diluent and volume was made up to mark (10 mL) with diluent (1000 µg/mL). This is used as a primary standard stock solution. The further dilution was made by transferred 4.5 mL of Apalutamide into 100 mL volumetric flask and made the volume up to mark with diluent. The final concentration of the solution was obtained 45µg/mL and used as a standard working solution.”
Preparation of marketed formulation (Assay Procedure)
Accurately weighed 20 tablets and calculated average weight of the tablets. The tablets were crushed into powder with the help of mortar and pestle, the powder equivalent to 114.14 mg of Apalutamide was transferred into 10 mL volumetric flask added few quantities of diluent and dissolved by sonicated for 5 min, volume was made up to mark with diluent (Primary stock sample solution). The solution was filtered through 0.22 µL. Further dilution was made with 4.5 mL of solution to 100mL with diluent and concentration of the solution was obtained 45µg/mL and it was used as a working sample solution.
Results and Discussion
Mass Spectrometry condition
The mass spectrometer was operated with mode of positive ion with MRM. Multiple reaction monitoring (MRM) was applied to quantify the Lasmiditan. The HPLC system was coupled to SCIEX QTRAP 5500 and set up the different optimized conditions for the analysis of the selected molecule: collision energy 15V, Ion spray voltage 5500℃, source of temperature 550℃, 120℃ – 250℃ temperature of drying gas, nitrogen was used as collision gas, flow stream of drying gas maintained at 5L/min, entrance potential was 10V and declustering potential was 40V. The monitored [M+H]+ ion m/z ratio was 378.24 (Fig.2). The exit potential was 7V and dwell time was set to 1 second.
Method Development
The method was optimized with different conditions and shown in the table 1.
Table 1: Optimized conditions for estimation of Lasmiditan by LC/MS/MS method.
S. No
Parameter
Description
1.
Stationary Phase
Symmetry C18 (150X4.6X5)
2
Mobile Phase
ACN:0.1% Formic acid (70:30)
3
Rate of flow
1 mL/min
4
m/z ratio
378.24
5
Detector
Tandem Quadrupole (Triple Quadrupole) MS
6
Injection
Auto sampler
7
Volume of injection
10 μL
8
Temperature of column
Ambient
9
Run time
4mins
10
Diluent
Mobile Phase
11
Rt
Lasmiditan: 2.33
Analytical Method Validation
The optimized method was validated according to ICH Q2R1 guidelines and discussed different validation parameters. (20)
System Suitability
To evaluated tailing factor, resolution and theoretical plates in the system suitability test. The % RSD was calculated for the 100% of standard solution. The chromatograms were recorded for six replicated injections.
Specificity
The standard and sample chromatograms (Fig 3a &3b) were shown specificity of the method and record the chromatograms at100% concentration of sample & standard solution. The optimized conditions were shown in table no 2.
Table 2: System suitability parameters of LMT
S.NO
Parameters
LMT
Results
Limits
1
Plate Count
15890
˃2000
2
USP Tailing factor
1.02
T˂2
3
USP Resolution
–
R˃2
4
Retention time
2.33 min
–
Linearity
The calibration curve of the LMT was linear over concentration range, a graph was plotted response of the area on X-axis and concentration on Y-axis and (R2) regression coefficient value, was found 0.999. The concentration of standard solution shown linearity range from 12.50 ng/mL to 75 ng/mL and graph shown in figure 4.
Precision
The precision was studied at intraday and intermediate precision and every level of precision was evaluated (Table.3) with six replicated injections of Lasmiditan.
Intraday Precision
The intraday precision was performed with 100% sample solution at three time intervals like 9:00 am, 1:00 pm and at 5:00 pm. Record the chromatograms at these three intervals at each level six replicated injections were injected and calculate %RSD.
Interday Precision
The interday precision was performed at day‑1, day‑2 and day‑3 with 100% concentration of solution. Record the chromatograms of six replicated injections at each level and calculated average of %RSD for three days.
The following formula used for the calculation of percentage assay
% Assay = Area in sample / Area in STD× Weight of STD / Dilution of STD× Dilution of sample / Weight of sample× Average weight / Labelled claim× Purity of ST
Table 3: Precision results of Intraday and Interday of LMT
S.No
Lasmiditan
Intraday
Interday
Peak Area
% Assay
Peak Area
% Assay
1
2.014
98.8
2.012
98.9
2
2.019
99.5
2.020
99.1
3
2.021
99.5
2.011
98.6
4
2.013
99
2.009
99.0
5
2.035
100.3
2.023
99.5
6
2.045
101
2.035
100.4
Mean
2.024
99.7
2.018
99.3
SD
0.01
0.828
0.01
0.635
%RSD
0.49
0.83
0.49
0.64
*Average of three interval
Accuracy
The accuracy at three distinct concentration levels of 50%, 100%, and 150% was evaluated using the recovery studies (Table.4). Six repeated injections at 50% and 150% concentrations and three replicated injections at 100% concentration were used to obtain the accuracy-based recovery percentage.
Table 4: Accuracy results of Lasmiditan
Parameters
Peak Area
Amount added(ng)
Amount recovered (ng)
% of Recovery
% Mean recovery
Lasmiditan
**50%
1.027
2.523
2.536
100.53
100.53
*100%
2.045
5.049
5.054
100.10
100.10
**150%
3.057
7.513
7.550
100.52
99.52
*Average of three replicates *Average of six replicates
Detection limit and quantification limit
The detection limit and quantification limit were determined through the linearity curve of slope and the response of the standard deviation (precision). The LOD and LOQ for LMT were determined to be 0.66 ng/mL and 2.22 ng/mL, respectively.
Robustness
The robustness was performed (Table.5) with only minor adjustments to the method’s flow rate and composition of mobile phase, both of which were assessed at 100% sample concentration. The rate of flow was changed ± 0.2 mL/min and recorded the chromatograms for six replicate injections. The mobile phase composition of the method was changed to ± 7mL of organic phase and recorded the chromatograms for six replicated injections.
Table 5: Robustness results of Lasmiditan
Parameter
Condition
LMT
Rt (min)
Peak Area
% Assay
Flow rate
0.8 mL/min
2.91
2.323
99.82
1 mL/min
2.33
2.022
99.50
1.2 mL/min
1.95
1.865
100.73
Mobile Phase
M:B 63:37 v/v
2.52
2.155
100.22
M:B 70:30 v/v
2.33
2.021
99.10
M:B 77:23 v/v
2.20
1.938
101.83
Conclusion
The Lasmiditan was estimated using LC/MS/MS in both its bulk and commercial formulation. The technique was successfully validated by following the ICH Q2R1 recommendations. Multiple reaction monitoring was applied to determine the Lasmiditan. The HPLC system was coupled to SCIEX QTRAP 5500 and set up the different optimized conditions for the analysis of the selected molecule. The monitored [M+H]+ ion m/z ratio was 378.24. The method was shown a good linearity range and values found to be 12.50 ng/mL to 75 ng/mL. The regression coefficient value was found to be within the limits, 0.999. The % RSD for accuracy and precision was found to be <2%, it indicated that the parameters are within the limits as per guidelines. The method was proved as robust after deliberated changes in parameters of rate of flow (±0.2mL) and organic phase (±0.7mL) in the composition of mobile phase and values were found to be 99.50%-100.73% & 99.10%- 101.83% respectively.. The LOD and LOQ of the study proved the sensitivity of the method and determine the drug at nanogram level and values were found to be 0.66 ng/mL and 2.22 ng/mL, respectively. The developed method was more sensitive and selective, hence the recommended method allows industrialists and researchers to identify and estimate Lasmiditan in bulk and in commercial formulations.
Acknowledgement
The authors express gratitude to the dean and administration of ITM University for providing the tools required to complete the task.
Conflict of Interest
The authors have declared that they have no competing interests.
References
Jessica C Oswald.; Nathaniel M Schuster. Lasmiditan used for the treatment of acute migraine: a review and potential role in clinical practice. J Pain Res, 2018, 11, 2221-2227. doi: 10.2147/JPR.S152216.
This work is licensed under a Creative Commons Attribution 4.0 International License.
Related Posts
留言 (0)