Analytical methods development plays important role in drug discovery, generic product development and manufacture of pharmaceuticals. Method development is the process of identifying set of parameters which provides desired analytical performance. Below are the important parameters which needs to be considered during method development.
SOLUBILITY PROFILE:
Solubility of interested compound/API in different solvents such as water, acetonitrile, methanol, isopropyl alcohol etc. is useful while selecting diluents for standard solutions and extraction solvents for test solutions. The pH solubility data of API also helps in selecting diluent during sample and standard preparation. BCS and saturation solubility data is used for selecting media for dissolution method.
ANALYTICAL PROFILE:
The spectral profile is useful in understanding the absorption characteristics, which helps in selection of detector and the wavelength for analysis. Understanding the degradation profile will help in developing the method for separation and estimation of all possible impurities and degradants. Information regarding possible process related impurities and degradants shall be obtained.
SELECTION AND OPTIMIZATION OF MOBILE PHASE:
The primary objective in selection and optimization of mobile phase is to achieve optimum separation of all the individual impurities and degradants from analyte (API) peak. The selection of mobile phase is always done in combination with selection of column (stationary phase). Following are the parameters which shall be taken into consideration while selecting and optimizing the mobile phase.
(a) Buffer, if any and its strength and pKa.
(b) pH of the buffer or pH of the mobile phase.
(c) Mobile phase composition.
SELECTION OF COLUMN:
Following are the parameters of a chromatographic column which are to be considered while choosing a column for separation of interested compounds such as impurities and degradants.
(i) Length and diameter of column.
(ii) Packaging material
(iii) Shape of the particles
(iv) Size of the particles
SELECTION OF SOLVENT DELIVERY SYSTEM:
A chromatographic separation with isocratic elution is always preferable. However, gradient elution is a powerful tool in achieving separation between closely eluting compounds. The important feature of the gradient elution which makes it a powerful tool is the polarity and ionic strength of the mobile phase can be changed (can be increased or decreased) during the run. Conduct experiments using different mobile phase combinations and different gradient program to achieve separation of all the impurities and degradants from API peak.
In general, while running a gradient, two mobile phases having different composition is kept in different channels. Two mobile phases are then introduced into the column by two different ways.
(i) By low-pressure gradient – where, the mobile phases are mixed at the predetermined ratios and then pumped using a single pump.
(ii) By high-pressure gradient – where, the mobile phases are pumped at different flow rates so as to achieve the required composition and then mixed in a chamber and then introduced into the column.
While optimizing the separation of impurities, it is to be decided whether to opt for low-pressure gradient or high-pressure gradient. Low-pressure gradients can be adopted when not more than 80% organic phase is to be pumped. High-pressure gradient is preferred when more than 80% of the organic phase is to be pumped.
While optimizing gradient program, especially using low viscous solvents like acetonitrile, it is recommended to mix about 10% aqueous portion in the organic port to avoid pumping problems. So instead of 100% acetonitrile- it is preferred to have 90% acetonitrile and 10% buffer solution.
While optimizing gradient program, it is important to monitor following:
(i) Pressure graph is to be monitored so as to ensure that overall system pressure will not cross 300 bar at any point during the run for HPLC.
(ii) Flow rate is to be physically cross checked by collecting output from detector during run at different time intervals, especially when gradient is running with higher organic phase composition so as to ensure that there were no pumping problems during the run when mobile phases of different compositions are pumped.
Also it is important to optimize the program for initialization after each run and before going for next injection. The program for initialization is required to avoid carry over to the next run and to stabilize the system with initial composition.
SELECTION OF FLOW RATE:
Flow rate can be selected based on the following data:
(i) Retention times.
(ii) Column backpressures.
(iii) Separation of impurities
(iv) Peak symmetries.
Preferably the flow rate shall be not more than 2.5 ml/min. Select the flow rate which gives least retention times, good peak symmetries, least backpressures and better separation of impurities from API peak.
SELECTION OF DETECTOR WAVELENGTH:
Selection of detector wavelength is a critical step in finalization of the analytical method for impurities and degradants. Inject API standard solutions into the chromatographic system with photodiode array detector and collect the spectra. Select a wavelength, which gives higher response for all compounds.
Note: If the compound is non chromaphoric, go for alternative detectors like RID, ELSD or mass and also if the compound is volatile and thermally stable, GC with FID or TCD detector can also be used for method development. Verify the linearity of response before finalizing method on ELSD.
METHODS OF EXTRACTION:
In general methods followed for extraction are sonication, rotary shaking or both. In some cases where the API is not extracted by the above procedures heating can be adapted if the substance is stable and do not precipitate upon cooling to room temperature.
In case of finished products such as tablets, capsules, ointments etc., conduct experiments to optimize the extraction of API in presence of excipients at different test concentrations using diluent chosen on solubility and select the test concentration at which the extraction is most efficient.
SELECTION OF TEST CONCENTRATION AND INJECTION VOLUME:
Test concentration is generally chosen based upon response of API peak and impurities at the selected detector wavelength. However, the test concentration shall be finalized after it is proved that API is completely extractable at the selected test concentration.
Generally an injection volume of 10 to 20 µL is recommended for HPLC. However, if the extraction is found to be difficult, then the test concentration can be kept low and the injection volume can be increased up to 50 µL or more. But it is to be ensured that at the selected injection volume the column is not overloaded, resolution between individual impurities from API peak and peak symmetry are not compromised.
After test concentration and diluent is finalized, prepare a test solution and keep the filtered solution in closed condition in a stopper flask on bench top and observe for any precipitation or turbidity up to suitable time (12hr/24hr). The solution should not show any turbidity/precipitation.
Note: Preferably use lower injection volume, when only organic phase is used as diluent.
ESTABLISHMENT OF STABILITY OF TEST PREPARATION:
Prepare test & standard solution and conduct stability of solution for at least 12 hours. As far as possible select a diluent in which test & standard solution is most stable. If the solution is found to be unstable at room temperature by its nature, then conduct solution stability at 2-8°C
ESTABLISHMENT OF SYSTEM SUITABILITY:
System suitability parameters can be selected based on criticality of the method. In general, standard reproducibility, tailing factor, plate count, resolution etc. can be adopted as a system suitability requirement. The acceptance criteria can be defined as per the requirement of individual test.
ESTABLISHMENT OF SPECIFICITY OF THE TEST METHOD:
Conduct forced degradation study by stressing API using acid, base, H2O2, water, light and heat. Subject the drug substance to stress with different strengths of stressing agents to obtain a degradation of about 5% to 20%. Inject samples into HPLC system equipped with PDA and check for separation of degradants formed under stressed conditions and the peak purity of the API peak.
If the purity of the peak is found to be satisfactory as per the individual software requirements, then the method can be considered as stability indicating.
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