*3.5.6.1 Determination of short-term frozen storage stability and freeze/thaw stability of PH46 in matrix*

Six aliquots (200 μL) at each concentration were extracted and analysed immediately to confirm the suitability of the stability samples. The remainder of each bulk was aliquoted into tubes (n = 18 for plasma, n = 48 for urine and n = 46 for faeces) containing a greater volume than required for analysis. Half of the replicates in each matrix (urine and faeces) were stored at 20°C and the other half of the replicates in each matrix were stored at 80°C. The effects of the corresponding stability samples were investigated after at least 1 month storage. The freeze/thaw stability of PH46 in plasma matrix assay was performed by using QCs samples (n = 6) at each concentration subjecting to freeze/thaw cycles (3) in a freezer set to maintain 20°C (initial freeze cycle 24 h as minimum, freeze cycles 2 and 3 at least 12 h and thaw cycle for one h 6 min at RT unprotected from light). At the time point, replicate samples (n = 6) at each concentration and each condition were then extracted and analysed with fresh CSs and QCs. The samples were deemed to be stable if the accuracy was within 100 15% (urine and plasma) or 100 20% (faeces) of the nominal concentration and the CV was ≤15% (urine and plasma) or ≤ 20% (faeces) at each concentration level.

#### *3.5.6.2 Determination of re-injection reproducibility*

The accuracy and precision batch for each assay was re-injected, having stored the extracts in an autosampler at 4°C for at least 239 h for plasma extracts, at least 25 days for urine extracts and at least 6.5 days for faeces extracts.

#### *3.5.6.3 Determination of ambient RT stability of PH46 in matrix*

Ambient RT stability of PH46 in plasma was assessed on further QCs (n = 6) at each level after being left at ambient temperature (unprotected from light) for 24 h 30 min before extraction with fresh CSs and QCs.

#### *3.5.6.4 Determination of extended frozen storage stability of PH46 in matrix*

Six aliquots (200 μL) at each concentration were extracted and analysed immediately to show that the stability samples had been suitably prepared. The remainder of each bulk was aliquoted into glass tubes (n ≥ 48) containing a greater volume than required for analysis. The extended frozen storage stability of PH46 in human plasma was investigated after at least one, two and four months at 20°C and the effects of storing samples at 80°C were also assessed after at least one, three and five months. At each time point, replicates (n = 6) at each concentration were extracted and analysed with fresh CSs and QC samples. The samples were deemed to be stable if the *Bioactive Indanes: Development and Validation of a Bioanalytical Method of LC-MS/MS… DOI: http://dx.doi.org/10.5772/intechopen.112275*

accuracy was within 100 15% of the nominal concentration and the CV was ≤15% at each concentration level.

#### **3.6 Data handling, processing and calculations**

Analyst® 1.4.2 software was used for data collection during the study. Thermo Fisher Scientific Watson™ 7.0 software was employed to determine the calibration parameters/values, including slope, intercept and coefficient of determination, the mean, standard deviation and accuracy data. Three significant digits were presented for the nominal matrix concentration (CS and QC samples) and the determined concentration results. The calculations of the accuracy and precision data were made from the rounded mean values and from the unrounded determined concentration data and peak area data, respectively, and were displayed to one decimal place. Four significant digits were reported for slopes and intercepts, four decimal places were for coefficients of determination, and six decimal places for instrument responses.

### **4. Conclusions**

In summary, we developed and validated a sensitive and specific bioanalytical method for the determination of PH46, a potential anti-inflammatory bowel disease agent, in human plasma, urine and faeces. A range of testing, including solution stability, specificity, assay linearity, accuracy, precision, recovery, matrix effect, carry-over, dilution effect and stabilities, were established for the matrices. The method meets EMA validation criteria. This method was successfully applied to the clinical pharmacokinetic study of PH46A in human.

### **Acknowledgements**

This work was supported by The Wellcome Trust (grant reference no. 067033/Z/ 02/A), Enterprise Ireland. The authors would like to acknowledge the contributions of Charles River Laboratories for providing the human plasma (proposal no. 313701) and the technical input throughout this study. The authors also acknowledge NatPro for the funding for APC of this publication.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author contributions**

Conceptualisation, N.F. and H.S.; investigation, H.S.; funding acquisition, N.F. and H.S.; data curation, G.A.S. and T.Z.; data analysis, T.Z. and G.A.S.; methodology, G.A. S. and T.Z.; data supervision, G.A.S.; writing—original draft, T.Z.; writing—review and editing: T.Z., G.A.S., N.F., H.S. All authors have read and agreed to the published version of the manuscript.
