Method development is a step of process that determines test method capabilities through the selection and optimization of analytical test parameters.
It provides a high degree of assurance that the test method will meet (or) deviate the established requirements.
Method Development starts with Literature search of the sample (i.e. Physical and chemical properties)
Physical properties:
· Chemical Structure
· Melting/Boiling point
· Solubility
· pKa
· Absorbance/ Spectral data
· Matrix
Chemical Properties:
· Thermal Stability
· Decomposition
· Reactivity
Factors influencing Method Development:
1. Selection of column
2. Selection of mobile phase & mobile phase composition
3. Role of pH
4. Role of Buffer
5. Role of Flow rate
6. Role of Column oven Temperature
1. Selection of column:
Column is considered as the heart of the method and plays a crucial role in the separation of components.
As most of the pharmaceutical compounds are polar in nature reverse phase columns (stationary phase is Non-polar) are preferable. The most commonly used reverse-phase columns are C18 (octadecyl silane), C8 (octyl silane), phenyl and cyano. These are chemically different bonded phases.
While performing method development, the selection of columns can be streamlined by starting with shorter columns as it reduces run time and helps to quickly optimize the mobile phase.
2. Selection of mobile phase & mobile phase composition:
A successful chromatographic separation depends upon differences in the interaction of the solutes with the mobile phase and the stationary phase.
In liquid chromatography choosing and varying the mobile phase is of critical importance in achieving optimum efficiency.
For LC-MS/MS ammonium acetate and ammonium formate are suitable buffer mobile phase as they are volatile in nature.
3. Role of pH:
pH is another factor in the resolution that will affect the selectivity of the separation in reverse-phase HPLC. Under unfavorable circumstances change in 0.1pH units can have a significant effect on the separation.
The selection of proper buffer pH is necessary for reproducible separation of ionizable compounds if we select an improper pH that leads to asymmetric, broad and split peaks.
When pH=pKa for the analyte, it is half ionized, i.e the concentration of ionized and unionized species are equal. As mostly all of the pH caused changes in the retention occurs within ±1.5 pH units of the pKa value, it is advisable to adjust the mobile phase to pH value ±1.5 pH units of above or below the pKa to ensure 100% unionization of analyte for retention purpose.
4. Role of Buffer:
A correctly chosen buffer will ensure that the analyte -ionizable functional group is in a single form.
If the sample solution is at pH damaging to the column, then the buffer will quickly bring the pH of the injected solution to a less harmful pH.
5. Role of Flow rate:
Change in flow rate shows a more significant effect in Isocratic than gradient separation and readily utilized to increase the resolution.
The slower flow rate will decrease the column backpressure and increase the resolution but the disadvantage is an increase in the run time.
6. Role of Column oven Temperature:
Temperature variation has a quite significant effect on separations, retention times generally decrease with an increase in temperature for neutral compounds.
Temperature variation plays a significant role in the separation of isomers. An increase in 1°C will decrease the retention time by 1 to 2%.