Thursday, December 16, 2010

Effective HPLC method development

Effective HPLC method development

In order to have an efficient method development process, the following three questions must be answered:
1.1.  What are the critical components for a HPLC method?
The 3 critical components for a HPLC method are: sample preparation, HPLC analysis and standardization (calculations).  During the preliminary method development stage, all individual components should be investigated before the final method optimization. This gives the scientist a chance to critically evaluate the method performance in each component and streamline the final method optimization.

1.2. What should be the percentage of time spent on different steps of the method development?
The rest of the article will discuss the recommended sequence of events and the percentage of time that should be spent on each step in order to meet the method development timeline.  One common mistake is that most scientists focus too much on the HPLC chromatographic conditions and neglect the other 2 components of the method (i.e., sample preparation, standardization).  The recommended timeline would help scientists investigate different aspects of the method development and allocate appropriate time in all steps.

1.3. How should a method development experiment be designed?
A properly designed method development experiment should consider the following
important questions:
What sample should be used at each stage?
What should the scientists look for in these experiments?
What are the acceptance criteria?

2.        Method Development Timeline

The following is a suggested method development timeline for a typical HPLC-UV related substance method.  The percentage of time spent on each stage is proposed to ensure the scientist will allocate sufficient time to different steps.  In this approach, the three critical components for a HPLC method (sample preparation, HPLC analysis and standardization) will first be investigated individually.  Each of these steps will be
Discussed in more detail in the following paragraphs.

Step 1: Define method objectives and understand the chemistry (10%) Determine the goals for method development (e.g., what is the intended use of the method?), and to understand the chemistry of the analytes and the drug product.

Step 2: Initial HPLC conditions (20%)
Develop preliminary HPLC conditions to achieve minimally acceptable separations.  These HPLC conditions will be used for all subsequent method development experiments.

Step 3: Sample preparation procedure (10%)
Develop a suitable sample preparation scheme for the drug product

Step 4: Standardization (10%)
Determine the appropriate standardization method and the use of relative response factors in calculations.

Step 5: Final method optimization/robustness (20%)
Identify the “weaknesses” of the method and optimize the method through experimental design. Understand the method performance with different conditions, different instrument set ups and different samples.

Step 6: Method validation (30%)
Complete method validation according to ICH guidelines 3. Define Method Objectives There is no absolute end to the method development process. 
The question is what is the “acceptable method performance”?  The acceptable method performance is determined by the objectives set in this step.  This is one of the most important considerations often overlooked by scientists.  In this section, the different end points (i.e., expectations) will be discussed in descending order of significance.

All these Steps are further discussed in my next post.

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