In bioanalysis studies and tests, HPLC method validation establishes that a method’s performance characteristics meet the intended purpose.
Validation of an analytical procedure is as important as testing the drug compound during drug development and research. HPLC method validation forms the basis of accurate and reproducible data, which is then used for quality control of drug formulations and dosage. Ergo, analytical procedures such as HPLC method development require the high-quality instruments and methodologies to provide specificity, precision, robustness, and range.
Modern liquid chromatography instruments for high-performance testing were used successfully for the first time in the 1960s. These instruments have since become much better and more precise with time and are, today, a necessity in the lab of every contract research organization to conduct high-performance liquid chromatography or HPLC. In the biotech and pharma industry, the role of an analytical technique in identifying, separating, and quantifying formulations and compound is widely understood and appreciated.
Understanding How HPLC Analysis Works
The chromatography technique uses a mobile and stationary phase. During the HPLC Method Validation process, an analyte or drug compound for the pharma industry, usually in liquid form (mobile), is pumped through a bed of particles (stationary). The interaction between the analyte and the finely-packed particles determines the partition coefficient, which is collected by integration software to form the basis for the suitability of the HPLC method.
Different Types of HPLC Method
During the procedure, the modern HPLC Analysis has two common variants-
Normal Phase HPLC– In this methodology, tiny silica particles and a non-polar solvent are used.
Reversed-Phase HPLC- The internal diameter of the column is less than 4.6 mm while the length can go up to 150-250mm. The phase tests the interaction between polar silica particle and the non-polar solvent.
In this methodology, the silica particles are modified to make them non-polar with the use of long hydrocarbon chains. The phase test s interaction with a polar solvent.
Guidelines For Analytical Method Validation
The Food and Drug Administration of the United States has laid down process the HPLC Method Validation parameters:
- Selectivity and specificity– Quantifying the analyte in the presence of inerferents is selectivity. Specificity uses a single analyte to create a measurable pr detectable interaction.
- Precision– Multiple results from the same sample express the closeness of agreement at three levels: repeatability, precision, and reproducibility
- Range– Linearity studies determine the range between the lower and upper concentration of analyte
- Accuracy– HPLC analysis does not have an idea measurement process. Hence, accuracy is tested on a minimum of nine determinations over three concentration levels.
- Limit of Detection– It is the level of analyte’s presence, which can be detected in a compound or process, but can or cannot be quantified.
- Limit of Quantitation– It tests the lowest quantifiable presence of a compound with suitable accuracy and precision.
- Robustness– It measures the capacity of an analyte to remain unaffected and stable due to variations in parameters, which further helps to establish the reliability
- System Suitability Testing– Equipment, samples, and analytical procedures are tested to assure scientists that the results produced by the method are precise and accurate.
All the above parameters are identified, tested for impurities (quantitatively), and assays are run during HPLC method development. CRO services are required to choose the HPLC analysis parameters depending upon their requirements.