Method Development, Validation and Forced Degradation Study of Lasmiditan by HPLC Method

Pharmaceutical Analysis is that core branch of pharmacy education and research, which is advancing very fast. It can be categorized as synthesis of new drugs molecules and pharmaceutical analysis. Method development for Lasmiditan” refers to the scientific process of creating a standardized analytical technique, typically using high-performance liquid chromatography (HPLC), to accurately measure the concentration of Lasmiditan (a medication used to treat migraines) in a pharmaceutical sample, like a tablet or solution, by optimizing parameters like mobile phase composition, column selection, and detection wavelength to ensure reliable and precise quantification of the drug within a given formulation. Validation of lasmiditan refers to the process of developing and testing methods to measure and quantify lasmiditan. This includes developing a method that is sensitive, precise, and accurate. A “forced degradation study of lasmiditan” refers to a laboratory experiment where the drug lasmiditan is intentionally exposed to extreme conditions like high heat, light, acidity, or alkalinity to deliberately break down the molecule and identify its potential degradation pathways and products, thereby assessing its stability and providing valuable information for formulation development and storage conditions; essentially, it’s a stress test to see how lasmiditan reacts under harsh conditions that might be encountered during its shelf life.

MATERIALS AMD METHODS

Drug Profile

Lasmiditan, approved in the U.S. in October 2019 and by the European Commission in 2022, is an oral 5HT1F agonist used to treat migraines with or without aura. Unlike triptans, which can cause vasoconstriction and cardiovascular side effects, lasmiditan selectively targets the 5-HT1F receptor, avoiding these issues. This unique selectivity allows it to effectively terminate migraines without vasoconstriction, marking the creation of a new class of anti-migraine drugs: neurally-acting medications.

CAS NO: 439239-90-4

Chemical formula: C19H18F3N3O2

Molecular weight: 377.37 g/mol.

Water Solubility: 0.0175 mg/mL

Melting point: 143-145°C.

Boiling point: 433.3°C

Structure:

Mechanism of action:

The acute treatment of migraines was once based on constricting cerebral blood vessels, as vasodilation was believed to cause the pain. However, the neurogenic hypothesis suggests that migraine pain stems primarily from increased firing of trigeminal nerve pathways, with cerebral vasodilation being secondary. Lasmiditan, a medication that supports this hypothesis, selectively agonizes the 5-HT1F receptor on trigeminal neurons, likely inhibiting their activity to reduce pain. It is rapidly absorbed orally, with a bioavailability of 40%, and penetrates the blood-brain barrier. Lasmiditan undergoes metabolism primarily via non-CYP enzymes, and is mostly eliminated through metabolism, with a small amount excreted in urine. Its half-life is 5.7 hours.

Diluent:

Based up on the solubility of the drugs, diluent was selected, Acetonitrile and Water taken in the ratio of 50:50

Preparation of Standard stock solutions: Accurately weighed 10mg of Lasmiditan transferred 50ml and volumetric flasks, ¾ Th of diluents was added and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution (200µg/ml of Lasmiditan).

Preparation of Standard working solutions (100% solution): 1ml of Lasmiditan from each stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (20µg/ml of Lasmiditan).

Preparation of Sample stock solutions: 5 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 100 ml volumetric flask, 50ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (500µg/ml of Lasmiditan).

Preparation of Sample working solutions (100% solution): 0.4ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (20µg/ml of Lasmiditan)

Preparation of buffer:

0.1%OPA Buffer: 1ml of Perchloric acid was diluted to 1000ml with HPLC grade water.

Buffer:0.1N Potassium dihyrogen Ortho phosphate

Accurately weighed 1.36gm of Potassium dihyrogen Ortho phosphate in a 1000ml of Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make up the volume with water then added 1ml of Triethylamine then PH adjusted to 4.8 with dil. Orthophosphoric acid solution

Validation:

System suitability parameters:

The system suitability parameters were determined by preparing standard solutions of Lasmiditan (20ppm) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined.

The % RSD for the area of six standard injections results should not be more than 2%.

Specificity: Checking of the interference in the optimized method. We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

Precision:

Preparation of Standard stock solutions: Accurately weighed 10mg of Lasmiditan transferred 50ml and volumetric flasks, ¾ Th of diluents was added and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution (200µg/ml of Lasmiditan).

Preparation of Standard working solutions (100% solution): 1ml of Lasmiditan  and 1ml of saxagliptin from each stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (75 µg/ml of Lasmiditan) 

Preparation of Sample stock solutions: 5 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 100 ml volumetric flask, 5ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters.(500µg/ml of Lasmiditan)

Preparation of Sample working solutions (100% solution): 0.4ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (20µg/ml of Lasmiditan) 

Linearity:

To demonstrate the linearity of assay method, inject 6 standard solutions with concentrations of about 18.75 ppm to 112.5ppm of Lasmiditan. Plot a graph to concentration versus peak area. Slope obtained was 34826Y-Intercept was10389and Correlation Co-efficient was found to be 0.999 and Linearity plot

Preparation of Standard stock solutions: Accurately weighed 10mg of Lasmiditan transferred 50ml and volumetric flasks, ¾ Th of diluents was added and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution (200µg/ml of Lasmiditan).

25% Standard solution: 0.25ml each from two standard stock solutions was pipetted out and made up to 10ml. (5µg/ml of Lasmiditan)

50% Standard solution: 0.5ml each from two standard stock solutions was pipetted out and made up to 10ml. (10µg/ml of Lasmiditan)

75% Standard solution: 0.75ml each from two standard stock solutions was pipetted out and made up to 10ml. (15µg/ml of Lasmiditan)

100% Standard solution: 1.0ml each from two standard stock solutions was pipetted out and made up to 10ml. (20µg/ml of Lasmiditan)

125% Standard solution: 1.25ml each from two standard stock solutions was pipetted out and made up to 10ml. (25µg/ml of Lasmiditan)

150% Standard solution: 1.5ml each from two standard stock solutions was pipettede out and made up to 10ml. (30µg/ml of Lasmiditan)

Accuracy:

Preparation of Standard stock solutions: Accurately weighed 10mg of Lasmiditan transferred 50ml and volumetric flasks, ¾ Th of diluents was added and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution (200µg/ml of Lasmiditan).

Preparation of 50% Spiked Solution: 0.2ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Preparation of 100% Spiked Solution: 0.4ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Preparation of 150% Spiked Solution: 0.6ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Acceptance Criteria:

The % Recovery for each level should be between 98.0 to 102

Robustness:

Small deliberate changes in method like Flow rate, mobile phase ratio, and temperature are made but there were no recognized change in the result and are within range as per ICH Guide lines.

Robustness conditions like Flow minus (0.9ml/min), Flow plus (1.1ml/min), mobile phase minus, mobile phase plus, temperature minus (25°C) and temperature plus (35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much effected and all the parameters were passed. %RSD was within the limit.

LOD sample Preparation: 0.25ml of Standard stock solution was pipetted out and transferred to 10ml volumetric flasks and made up with diluents. From the above solution 0.1ml Lasmiditan, were transferred to 10ml volumetric flasks and made up with the same diluents

LOQ sample Preparation0.25ml of Standard stock solution was pipetted out and transferred to 10ml volumetric flasks and made up with diluents. From the above solution 0.3ml Lasmiditan, were transferred to 10ml volumetric flasks and made up with the same diluents

LOD: Detection limit of the Lasmiditan in this method was found to be 0.13µg/ml.

Based on drug solubility and Pka Value following conditions has been used to develop the method Estimation of Lasmiditan.

Trial 1

Chromatographic Conditions

Column: ODS 150mm x 4.6 mm, 5?.

Mobile phase.: Water: Methanol (50:50)

Flow rate.: 1.0 ml/min

Detector: PDA 260nm

Temperature :300C

Injection Volume. : 10µL

Trial 2

Chromatographic Conditions

Column:    ODS 150mm x 4.6 mm, 5(.

Mobile phase:     0.1% OPA: Methanol (50:50)

Flow rate:     1.0 ml/min

Detector:     PDA 260

Temperature:     300C

Injection Volume:      10µL

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Fig 4 trial chromatogram -2

Trial 3

Chromatographic Conditions

Column:      ODS 150mm x 4.6 mm, 5(.

Mobile phase:     Acetonitrile:0.1% OPA (50:50)

Flow Rate:     1.0 ml/min

Detector:     PDA 260nm

Temperature:     300C

Injection Volume:     10µL

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Fig 5 trial chromatogram -3

Trial 4

Chromatographic Conditions

Column: Agilent 150mm x 4.6 mm, 5(.

Mobile phase: 0.1% OPA: Methanol (60:40)

Flow rate: 1.0 ml/min

Detector: PDA 260nm

Temperature:  300C

Injection Volume: 10µL

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Fig 6 trial chromatogram -4

Trial 5:

Optimized Chromatographic Conditions:

Column:      Agilent 150mm x 4.6 mm, 5m.

Mobile phase:     Acetonitrile; water [50;50]

Flow rate:     1.0 ml/min

Detector:     PDA 260nm

Temperature:     300C

Injection Volume:     10µL

Degradation studies were performed with the formulation and the degraded samples were injected. Assay of the injected samples were calculated and all the samples passed the limit of degradation

Degradation Procedure:

Oxidation:

To 1 ml of stock solution ofLasmiditan1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 min at 1c. For HPLC study, the resultant solution was diluted to obtain (20ppm) solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Acid Degradation Studies:

To 1 ml of stock solution Lasmiditan1ml of 2N Hydrochloric acid was added and refluxed for 30mins at 60 ⁰c. The resultant solution was diluted to obtain (20ppm) solution and 10µl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.

Alkali Degradation Studies:

To 1 ml of stock solutionLasmiditan1 ml of 2 N sodium hydroxide was added and refluxed for 30mins at 1c. The resultant solution was diluted to obtain (20ppm) solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Dry Heat Degradation Studies:

The standard drug solution was placedinovenat105⁰c for6h to study dry heat degradation.For HPLC study, the resultant solution was diluted to (20ppm) solutionand10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Photo Stability Studies:

The photochemical stability of the drug was also studied by exposing the (750ppm) solution to UV Light by keeping the beaker in UV Chamber for 7days or 200Watt hours/m² in photo stability chamber^. For HPLC study, the resultant solution was diluted to obtain (20ppm) solutions and 10µl were injected into the system and the chromatograms were recorded to asssess the stability of sample.

Neutral Degradation Studies:

Stress testing under neutral conditions was studied by refluxing the drug in water for 6hrs at a temperature of 60º. For HPLC study, the resultant solution was diluted to (20ppm) solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Degradation Data of Lasmiditan

RESULT AND DISCUSSION:

Selection of Solvent:

Lasmiditan shows maximum solubility in Acetonitrile and water (50:50 v/v), hence it was selected as the solvent (diluents for further studies)

Selection of wavelength:

5µ of Lasmiditan exhibited maximum absorbance at 260nm

Linearity and Range

To demonstrate the linearity of assay method, inject 6 standard solutions with concentrations of about 18.75 ppm to 112.5ppm of Lasmiditan. Plot a graph to concentration versus peak area. Slope obtained was 34826Y-Intercept was10389and Correlation Co-efficient was found to be 0.999 and Linearity plot.

Table 1: Linearity Concentration and Response

Table :2 Accuracy Data

Table :4 Intermediate precision data

Table :5 Assay of Formulation

Table :6 Degradation Data of Lasmiditan

Table: 7 System suitability Chromatogram

Table :8 Peak Name

Table: 10 Linearity plot