**3. Materials and methods**

The research work reported in this article was carried out following the Principles of the Organisation for Economic Co-operation and Development (OECD) of Good Laboratory Practice (GLP) as accepted by international regulatory authorities, including EMA (EU), FDA & EPA (USA) and MHLW, MAFF & METI (Japan).


*\* Nominal concentration of PH46 (ng/ml for urine and ng/g eq. for faeces). # samples diluted 10-fold prior to extraction and analysis. A. freezing stability (urine); B. freezing stability (faeces); C. reinjection reproducibility (urine); D. reinjection reproducibility (faeces).*

#### **Table 10.**

*Stability tests (one month storage and reinjection reproducibility) of PH46 in human urine and faeces.*

Ethical approval was obtained through Trinity College Dublin which complies with the Council for International Organisations of Medical Sciences' (CIOMS), International Guiding Principles for Biomedical Research.

#### **3.1 Chemical and reagents**

PH46A [purity 97.1% with moisture and solvent free; correction factor (purity and salt content): 0.6429] and Compound 1 as IStd (**Figure 1**) were obtained from Trino Therapeutics Ltd. (Ireland). Control human plasma (using lithium heparin as anticoagulant), urine and faeces were obtained from Charles River Laboratories (UK) and stored at 20°C when not in use. All control plasma was mixed and centrifuged prior to use. Control human faeces was homogenised prior to use with addition of deionised H2O (1:3, faeces:H2O, *w/v*). HPLC grade solvents and additives, including methanol (MeOH), ACN, H2O, ammonia solution (28.0–30.0%), acetic acid (AA), acetone, dimethylsulphoxide (DMSO), chloroform (CHCl3), TFA and FA, were purchased commercially between VWR (Ireland), Fisher Scientific (Ireland) and Sigma-Aldrich (Ireland) and were of analytical/HPLC grade or equivalent.


*Bioactive Indanes: Development and Validation of a Bioanalytical Method of LC-MS/MS… DOI: http://dx.doi.org/10.5772/intechopen.112275*

*M: month. – : not applicable. A. stability test at 20°C; B. stability test at 80°C. \* Nominal concentration of PH46. <sup>+</sup> Repeat timepoint experiment. # Samples diluted 10-fold prior to extraction and analysis.^Does not meet acceptance criteria (100 15%).*

#### **Table 11.**

*Storage stability tests of PH46 in human plasma at 20°C and 80°C.*
