**Abstract**

PH46A, a new chemical entity developed by our group, has shown potent antiinflammatory activities through various pre-preclinical studies. The aim of this work was to develop and validate a sensitive and robust LC-MS/MS analytical method to determine the levels of PH46 in human plasma, urine and faeces. The linearity (0.5–500 ng/mL for plasma/urine, and 10–2000 ng/g for human faeces), accuracy (within 100 15% for plasma/urine or 100 20% for faeces), precision (≤ 15% CV for plasma/urine or ≤ 20% CV for faeces) and the method's specificity were demonstrated to be acceptable. No significant matrix effects or carry-over was observed for PH46 and IStd, and the recovery was consistent. About 10- and 100-fold dilutions in control matrix were found not to affect the assays' performance. PH46 was proven to be stable: at room temperature for >24 hrs in plasma through 3 freeze-thaw cycles, at –20°C for 83 days in plasma/32 days in urine/33 days in faeces, and at –80°C for 154 days in plasma/ 33 days in faeces. The re-injection reproducibility of PH46 in matrix extracts was at least 239 hrs at 4°C in plasma/25 days in urine/6.5 days in faeces. This method was successfully applied to the pharmacokinetic evaluation of the Phase I clinical studies.

**Keywords:** bioanalytical, development, validation, LC-MS/MS, PH46A, human plasma, urine, faeces

## **1. Introduction**

The therapeutic effect of indane derived molecules has been clinically evident for treatment of many disease conditions, ranging from inflammation [1], cancer [2],

neurological conditions [3] to HIV [4]. Several classes of indane dimers have been developed, characterised and investigated by our research group [5–7] for various biological activities, including smooth muscle relaxation, mediator release inhibition and inflammatory conditions [8–14]. In particular, A lead, first-in-class molecule, [6-(Methylamino)hexane-1,2,3,4,5-pentanol 4-(((1S,2S)-1-hydroxy-2,3-dihydro-1H,10 H-[2,2-biinden]-2-yl)methyl)benzoate (PH46A) (**Figure 1**) with S, S configuration [15] has been considered as a potential new treatment for inflammatory bowel disease (IBD) based on the observation of its biological effect in two different wellestablished preclinical models of murine colitis: the acute dextran sodium sulphate model and the chronic and spontaneous Interleukin-10 (IL-10�/�) knock-out mouse model. During the course of our work, PH46A was subject to a range of preclinical studies [16–19] prior to entering a Phase I clinical trial which has recently been completed [20].

This manuscript describes the development and validation of a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical method for determining PH46 (the free acid form of PH46A salt) (**Figure 1**) in human plasma, urine and faeces samples according to the US Food and Drug Administration (FDA) Guidance Document on Bioanalytical Method Validation [21] and the European Medicines Agency (EMA) Guidelines on Bioanalytical Method validation [22]. The method was subsequently used to analyse the clinical samples from healthy volunteers in the Phase 1 trial [20]. The lower limit of quantification (LLOQ) in plasma and urine was 0.5 ng/mL and the LLOQ in faeces was 10 ng/g faecal equivalent.

### **2. Results and discussion**

#### **2.1 Stability of PH46 and IStd in stored stock solutions**

The mean accuracy of the stored solutions compared to freshly prepared solutions met the acceptance criteria. Both PH46 and Compound 1 (Internal Standard, IStd) (**Figure 1**) stock solutions were found to be stable at 4°C for at least 200 and 250 days, respectively. The stability of IStd working solutions for at least 192 days at 4°C, and PH46 stock solutions for at least 24 h at room temperature (RT) was demonstrated.

**Figure 1.** *Chemical structures of PH46A, PH46 and compound 1 (internal standard, IStd).*

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

#### **2.2 Linearity and specificity**

The calibration data from reported human plasma (12 batches), urine (3 batches) and faeces (3 batches) standards over a range of 0.5–500 ng/mL (plasma or urine) or 10–2000 ng/g (faecal equivalent), duplicate run at each concertation, were analysed. The data are presented in **Table 1** for human plasma and **Table 2** for human urine and faeces batches.

In all cases, all concentrations determined for these standards met the acceptance criteria being at least 75% of standards [including at least one replicate at the LLOQ and the upper limit of quantification (ULOQ) levels] used to construct each calibration line met the acceptance criterion of the determined concentrations being within 100 15% (plasma and urine batches) or 100 20% (faeces batches) of the nominal concentrations (100 20% for plasma and urine batches or 100 25% for faeces batches of the nominal concentration at the LLOQ).

Regression analysis of the peak area ratios of PH46:IStd against the concentration showed good linearity for human plasma, urine and faeces over the range of concentration tested (0.5–500 ng/mL for plasma/urine samples and 10–2000 ng/g faecal equivalent for faeces samples) using a linear regression with a weighting factor of 1/x<sup>2</sup> (**Tables 3** and **4**). All calculated concentrations for these standards met the acceptance criteria.

The quality control (QC) sample data for the supporting QC samples are presented in **Table 5**, which met the acceptance criteria. The selectivity of the assay for PH46 and IStd was determined by extraction and analysis of the following samples: one double blank (DB) sample (control matrix only without PH46 or IStd) from six independent sources of control matrix; three single blank (SB) samples (control matrix with IStd only) in a single source of control matrix; three ULOQ samples (no IStd) in the same source of control matrix as for the SB samples. The assay specificity demonstrated there were no significant interfering substances (IStd, PH46 or any impurity) at the retention times of PH46 and IStd, respectively. Samples containing either PH46 or IStd showed no significant interfering substances at the retention time of the other compound. Therefore, the assays were deemed to be specific to PH46. Representative chromatograms of human plasma extracts containing PH46 and IStd (LLOQ and ULOQ) are shown in **Figure 2**.

#### **2.3 Assay recovery and matrix effects**

For PH46 and IStd in plasma, the recovery was consistent. When the concentration of PH46 at 1.25 ng/mL, the IStd-normalised Matric Factors (MFs) (six sources) were calculated: 1.40, 1.35, 1.46, 1.51, 1.57, 1.25, 1.37 and the associated precision was determined to be 7.8%. When the PH46 concentration at 400 ng/mL, the IStdnormalised MFs were 1.35, 1.26, 1.45, 1.38, 1.44, 1.21, 1.25 and the corresponding precision was found to be 7.2%. These results confirmed that the matrix effects met the acceptance criteria, and no significant matrix effect was present for PH46 and IStd in human plasma by the current method.

#### **2.4 Assay carry-over and matrix dilution**

For human plasma, urine and faeces samples, no significant carry-over was observed in the matrix blank and solvent samples injected after a ULOQ sample for PH46 or IStd using needle-wash solution: [H2O:acetonitrile (ACN):trifluoroacetic acid


*–: not applicable.*

*\* Outside acceptance criteria (100 15%), value not used in statistical calculations or regression analysis. NR: no result as no internal standard peak detected in sample, value not used in statistical calculations.*

#### **Table 1.**

*Calibration curve data and parameters for PH46 in human plasma.*

(TFA) (25:75:0.1, *v/v/v*); H2O:ACN:formic acid (FA):TFA (90:10:0.1:0.005, *v/v/v/v*)]. The accuracy and precision for plasma and urine samples prepared at 4000 ng/mL or faeces samples at 16000 ng/g eq., and diluted at 10- and 100-fold, also met the acceptance criteria (**Table 6**). The results showed that dilutions of samples in control plasma, urine and homogenised faeces had no effect on the accuracy and precision of the method.

#### **2.5 Intra- and inter-batch assay accuracy and precision**

The intra-batch accuracy and precision of the assay for PH46 in plasma, urine and faeces met the acceptance criteria at each of the four concentrations assessed. The inter-batch accuracy and precision for PH46 in plasma also meet the acceptance criteria on each occasion (**Table 7**).


**Table 2.** *Calibration*

 *curve data and parameters*

 *for PH46 in human urine and faeces.*

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


*acceptance regressionanalysis.*

> **Table 3.**

 *plasma.*


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

*Bias: difference between determined concentration and nominal concentration. Conc.: concentration. \* Value outside acceptance criteria 100 15% (urine) and not used in regression analysis.*

#### **Table 4.**

*Assay linearity for PH46 in human urine and faeces.*

#### **2.6 Stability experiments of PH46 in matrix**

The results of different stability experiments are summarised in **Table 8** with the detailed data presented in **Tables 9**–**11**.

The mean stability results of PH46 in human plasma, at low, high and diluted concentrations, after at least one-month (33 days) storage at 20°C did not meet the acceptance criteria. This experiment was repeated after 35 days storage and similar results were obtained (**Table 11**). The data were unexpected as no stability issues had been observed in the previous studies of PH46 in dog and rat plasma [16]. Stability studies of PH46 in human plasma at 80°C, was subsequently assessed after at least one month (30 days) storage alongside the assessment of the 20°C stability after at least two months (83 days) storage (**Table 11**). The mean stability results at all concentration levels met the acceptance criteria for both storage temperatures and durations. The data generated for the two-month stability timepoint at 20°C contradict that of the one-month timepoint, but provide confidence that there should not be stability issue at 20°C. At one-month timepoint at 20°C, fresh calibration


*-: no applicable. Conc.: concentration.*

*\* Outside acceptance criteria value included in statistical analysis.*

#### **Table 5.**

*Quality control data relating to PH46 in human plasma, urine and faeces.*

standard (CS) and QC stock solutions were prepared and tested to ensure the stocks were suitable for use. The stock solutions were then used to prepare fresh bulk CSs and QC samples in control matrix., and the stability samples were extracted alongside these fresh CS and QC samples. The acceptance criteria for both CSs and supporting QC samples were met for each of the stability batches, therefore giving no reason to suspect the failing stability data after one-month storage at 20°C.

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

#### **Figure 2.**

*Chromatograms of a human plasma extract (left) containing PH46 (LLOQ, 0.5 ng/mL) & internal standard and a human plasma extract (right) containing PH46 (ULOQ, 500 ng/mL) & internal standard.*


#### **Table 6.**

*Effects of dilution of PH46 with human plasma, urine and faeces homogenate.*

At the one-month timepoint for 80°C and two-month timepoint for 20°C, a further fresh set of CS and QC stock solutions were made and used to prepare the fresh bulk CS and QC samples extracted alongside the stability samples. The acceptance criteria for these CSs and supporting QC samples were met. As the stability samples stored at 20°C and 80°C extracted in this batch also met the acceptance criteria, this demonstrates that both sets of samples prepared at different times were appropriate for use. Although there is still no explanation for the anomalous results at


#### **Table 7.**

*Intra- and inter-assay accuracy and precision for PH46 in human plasma, urine, and faeces.*

one-month timepoint for 20°C storage, it does provide confidence that there is not a stability issue for PH46 in human plasma at 20°C for the timeframe tested (**Table 11**). After at least four months (153 days) storage at 20°C, the mean stability results met the acceptance criteria at high and diluted concentrations but did not meet at the low concentration. The low concentration samples were repeated and the failure to meet the acceptance criteria was confirmed. The mean stability results, after storing PH46 in human plasma 80°C for at least three months (100 days) and five months (154 days) met the acceptance criteria at all levels of low, high and diluted concentration (**Table 11**).

Following the successful method establishment, this validated bioanalytical method was applied to determine PH46 in human samples obtained from the healthy volunteering during the phase I clinical study (ISRCTN90725219) [20].

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


**Table 8.**

*Summary of stability results of PH46 in human plasma, urine and faeces.*


*A. Freeze/thaw stability; B. Ambient temperature stability; C. Autosampler stability (plasmas extract stored at 4°C, reinjection reproducibility). B.L. Baseline.*

*\* Nominal concentration of PH46. # Samples diluted 10-fold prior to extraction and analysis.*

#### **Table 9.**

*Stability tests (freeze/thaw, ambient temperature & reinjection reproducibility) of PH46 in human plasma.*
