Analytical Dose Verification

Every test substance sent to ibacon for testing has unique physical and chemical properties which can influence the way in which the aquatic biology studies are carried out. Substances may be poorly water soluble, prone to hydrolysis, photolysis, biological degradation, or may be extremely volatile. This can therefore falsify the results if degradation of the test substances occurs over the course of the study.  Ensuring that the test plant or animal was continuously exposed to the correct concentration is therefore an essential part of any of the wide variety of biological studies conducted here at ibacon.

A dedicated team of analytical and environmental chemists works together in close collaboration with our biology team to monitor the behavior and verify the concentrations of every test substance. This allows us to decide on the most suitable test design for each test substance (e.g. semi static or flow-through test design for unstable test substances, closed vessel design for volatile test substances, etc).

The main challenge for our analytical team is to establish and adapt analytical methods which are suitable for the verification of even the lowest test concentrations down to the low µg L-1 range and below. We have a wide range of analytical instruments at our disposal that allows us to analyse almost every kind of substance. Our available instruments are:

  • UPLC and HPLC with UV, DAD, or ELSD detection
  • LC MS-MS
  • GC-MS
  • IC (Ion Chromatography)
  • AAS
  • TOC (Total Organic Carbon)

Method Development

If a suitable analytical method cannot be provided by the sponsor, our analytical team can develop an appropriate method with the required sensitivity for the concentration range used in the biological study. 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. HPLC with UV detection at a specific wavelength using a specific column), along with any necessary sample preparation (e.g. dilution, filtration, or centrifugation).

After initial method development the biological pre-test will be analysed to produce information concerning the dosing of the test substance, its stability during the test and during storage of the samples, and the suitability of the analytical method. This information is vital for choosing the appropriate test design and appropriate analytical method for the main study. At this stage the development of complex sample preparation steps such as enrichment via liquid-liquid extraction or solid phase extraction, or derivatisation, may be necessary if the limit of quantification of the initial analytical method is not suitable.

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 3029/99 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

  • Chemicals Act (ChemG) of the Federal Republic of Germany, Annex 1, in the version published on 28 August 2013 (Federal Law Gazette I, p. 3498) last amended by Article 1 of the Ordinance of 20 June 2014 (Federal Law Gazette I, p. 824)
  • OECD Principles of Good Laboratory Practice, adopted by Council on 26th November 1997 [C(97)186/Final], Environment Directorate, Organisation for Economic Co-operation and Development, ENV/MC/CHEM(98)17, Paris 1998
  • SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414