Development and Validation of an Analytical Method for the Determination of Active Ingredient or Formulated Test Item in Matrix

Besides classical physical-chemical studies, like a water solubility test, more and more (ecotoxicological) studies performed at ibacon require the analytical determination of the concentration of active ingredient or formulated product in all kinds of different matrices due to new guideline demands.

This can be a dose verification in aqueous samples taken from an acute fish test, the analysis of a spraying solution from a plant study to confirm the targeted nominal application rate or the determination of residues in bees and bee products sampled during a field trial.

Additionally, for manufacturers seeking pesticide authorisation it is necessary to provide analytical methods for post-registration monitoring and control in matrices like plants and plant products, foodstuff of plant and animal origin, drinking-, ground- and surface water and soil.

For all these purposes it is essential to have a reliable and robust method of analysis to produce meaningful data. This will be ensured by performing a method validation:

Validation is the confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled. [ISO/IEC 17025 definition]

The extent of a validation should reflect its intended purpose and should include but is not limited to: establishment of a Limit of Quantification (LOQ) and Limit of Detection (LOD), linearity, specificity, accuracy and precision.

Primary methods for post-registration monitoring and control purposes are validated according to requirements given by SANCO guideline 825/00. For some matrices like plants, plant products, foodstuff of plant origin and in feeding stuff, etc. an additional validation of the primary method must be performed by an independent laboratory. This independent laboratory validation (ILV) shall confirm the LOQ of the primary method.

Study Design

Course of the test

The challenge for our analytical team is to develop, adapt, implement and/or validate analytical methods which are suitable for the determination of concentrations of the active ingredient from e.g. several g/L down to sub-ppb levels in all kinds of matrices (e.g. water, plants, foodstuff of plant or animal origin, soil, etc.). De

We use a wide range of analytical instruments at our disposal that allows us to analyse almost every kind of substance e.g.

  • UPLC and HPLC with UV, DAD, or ELSD detection
  • LC MS-MS
  • GC-MS
  • GF-AAS (graphite furnace atom absorption spectroscopy)
  • IC (ion chromatography)

Method Development

Our analytical team is used to develop an appropriate method with the required sensitivity for the forced concentration range. Method development is carried out under non-GLP conditions. During this stage of method development an appropriate instrument and detection method is chosen (e.g. LC-MS/MS detection using a specific column), along with any necessary sample preparation (e.g. liquid-liquid extraction, SPE, pre-concentration, dilution, filtration, centrifugation, etc.).

Main Study and Method Verification

The final method will be validated under GLP conditions alongside the analytical measurements of the main biological study samples. The validity of the method will be determined according to the validity criteria set out in the SANCO 825/00 guidelines. Fortified samples are prepared to check for the precision and accuracy of the method, and various blank samples are used to confirm the absence of analytical contaminants or interferences which could have entered the samples during sampling, sample preparation, or analysis. A calibration curve across the required concentration range is also prepared to confirm the linearity of the instrument response, and to be able to quantify the test substance in the biological samples. These data will be included in the final study report.

Guidelines and Literature

  • SANCO/825/00 rev.8.1