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Validating Analytical Procedures

The following parameters must be considered to have a validated HPLC method for use with SOP, GMP, GLP and other pharmaceutical, environmental and forensic applications:
  • Accuracy: How close the observed value is the to true value. This is analogous to an archer hitting the “intended” point each time he shoots his arrow. If the center is hit, it is accurate. It is unrelated to Precision.

  • Detection Limit: The lowest reliable, detectable amount of your analyte is considered the detection limit of your method.

  • Linearity: If the raw data (actual results without any correction) of your run is proportional to the concentration of your analyte of interest (within the established range of testing), your method will be considered linear.

  • Octanol-Water Partition Coefficient: The Octanol-Water Partition Coefficient is a physical property used to describe a chemical's lipophilic or hydrophobic properties. It is the ratio of the concentration of your compound in the octanol phase to its concentration in the aqueous phase at equilibrium. Commonly measured and labeled as Log P. Compounds with large non polar structures are usually high values and for compounds with highly polar groups, it is usually very low. The Log P value combined with the pI of the molecule can help predict a compounds retention time on a stationary phase.

  • Precision: How “tight” the observed values are relative to each other that is independent of personnel, time and instrumentation; and without any intervention in the method. Precision is analogous to an archer hitting the same place each time on a target. It can be very far off the center, but it is still precise if he is hitting the same point each time.

  • Quantitation Limit: This is closely related to Detection limit. The lowest amount of your analyte that can be reliably quantified is considered the quantitation limit of your method.

  • Recovery: This is the amount of your analyte that is detected during your method.

  • Repeatability: This is very closely related to Precision. This takes into account your sample. A method can be considered repeatable when, from a homogeneous sample, Precision is achieved.

  • Reproducibility: This is closely related to Precision. A method will be considered Reproducible when Precision can be achieved in separate methods and independent of location and time and operator of the method.

  • Robustness: When variations of the analytical conditions do not change the reliability of your data no matter how small the variations are then your method is considered Robust.

  • Specificity: How well a species is separated from or resolved from interferences in the actual run.