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Controlled iontophoretic delivery in vitro and in vivo of ARN14140, a multi target compound for Alzheimer’s disease

5. Supplementary information

5.1. Validation of HPLC-UV method for the quantification of ARN and ACE

A P680A LPG-4 pump equipped with an ASI-100 autosampler and a UV170U detector (Dionex; Voisins LeBretonneux, France) were used to quantify ARN14140 (ARN) in the samples from the in vitro experiments. Isocratic separation was performed using a 125 mm x 4 mm LiChrospher column (BGB Analytik AG; Boeckten, Switzerland) packed with 5 μm C18 end-capped silica reversed-phase particles. The flow rate and injection volumes were 1.0 mL/min and 25 μL, respectively; the column temperature was maintained at 25°C. The mobile phase consisted of 45:55 (% v/v) of acetonitrile and water (both with 0.1% trifluoroacetic acid), and ARN was assayed using its UV absorbance at 215 nm. Acetaminophen (ACE) was analyzed separately using the same column at a flow rate of 1.0 mL/min, with a column temperature of 30 °C and using 243 nm as the detection wavelength. The mobile phase consisted of 20%

methanol and 80% citrate buffer (30 mM citrate buffer, pH 3.0).

The specificity of the method for ARN and ACE was assessed in an Acetonitrile: water mixture (60:40) spiked with skin extract by using a six point calibration curve over a concentration range from 2 to 200 µM for ARN and 6.6 to 330.8 µM for ACE. No interference or overlapping with the skin extract was detected at the retention time of 4.5 min for ARN. The method was linear in the concentration range of 0.9 to 200 µM with a R2 of 0.99. The limit of detection (LOD) and limit of quantification (LOQ) were determined using the linear regression method and found to be 0.7 and 2 µM, respectively for ARN and 2.2 and 6.6 µM, respectively for ACE.

Intra- and inter-day precision and accuracy were assessed using 5, 50 and 200 µM ARN solutions in extraction media. Table 3 shows a summary of intra- and inter-day accuracy and precision for the method to quantify ARN. The HPLC-UV method to quantify ACE was also

96 precise and accurate.

Table 3. Intra- and inter-day accuracy and precision for ARN in HPLC-UV quantification method.

Intra-day Inter-day 1 Inter-day 2

[ARN]theo

The results obtained were highly comparable and within the acceptance limits of analytical method validation guidelines (ICH Q2).

5.2. Validation of UHPLC-MS/MS method for the in vivo samples

An isocratic UHPLC−MS/MS method was developed and validated to determine the plasma concentration of ARN. A Waters Acquity UPLC core system (Baden-Dättwil, Switzerland) comprising a binary solvent pump and sample manager with a Waters XEVO TQ-MS tandem quadrupole detector (Baden-Dättwil, Switzerland) equipped with electron spray ion source was used for the UHPLC−MS/MS analysis together with a Waters XBridge BEH C8 column (2.1 × 50 mm and 2.5 μm particle size). The mobile phase comprised Milli-Q water:acetonitrile (80:20, v/v) with 0.1% formic acid maintained at 40 °C with a flow rate of 0.5 mL/min. The injection volume was 5 μL. The Waters XEVO TQ-MS detector was operated in positive ion mode using multiple reaction monitoring. The mass transition ion pair of ARN was m/z 508.5→329.5 and IS (chloroquine) m/z 320→247.5. MS source parameters were as follows:

ion spray capillary voltage, 1 kV; cone voltage, 30 V; desolvation gas temperature, 500 °C;

cone gas flow rate, 1000 L h−1; and collision energy, 25 V. Data acquisition was carried out using MassLynx V4.1 software.

A simple protein precipitation extraction method was developed and validated for quantification of ARN in blood plasma. Each plasma sample (100 µL) was thawed at room temperature and plasma protein was precipitated using 400 µL of acetonitrile containing 0.1%

formic acid and chloroquine (0.313 nmol) as internal standard (IS). Sample was vortexed for 10 s and centrifuged at 10,621g for 10 min to collect the supernatant for UPLC-MS/MS

97 analysis.

The method was linear in the concentration range of 8-1000 nM for plasma samples and 10-2000 nM for brain and skin samples with no interference or overlapping due to matrix was observed at the retention time of 1.8 min for ARN. The LOD/LOQ for plasma, skin and brain samples were 3/8, 3/10 and 3/10 nM, respectively. Intraday precision and accuracy in plasma, skin and brain samples were assessed using 10, 200 and 1000 nM ARN solutions in their relevant extraction solvents. Table 4 shows a summary of intraday accuracy and precision for the method to quantify ARN in plasma, skin and brain samples.

Table 4. Intra- and inter-day accuracy and precision for ARN in in plasma, skin and brain

5.3. Extraction validation of porcine skin

Table 5. Validation of the ARN extraction method used to quantify the amount of drug retained within the porcine, human and rat skin.

Skin source Control

98 Acknowledgements

We would like to thank the University of Geneva for financial support and for providing teaching assistantships for MS. We would also like to thank Prof. Brigitte Pittet-Cuénod and her colleagues from the Department of Plastic, Aesthetic and Reconstructive Surgery, Geneva University Hospital (Geneva, Switzerland) for providing human skin samples. We would like to thank the University of Geneva, the Fondation Ernst and Lucie Schmidheiny and the Société Académique de Genève for providing financial support to enable the acquisition of the Waters Xevo® TQ-MS detector.

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PART 2: UNDERSTANDING THE IONTOPHORETIC