NEVERAS DE MANTENIMIENTO ( CÁMARAS DE REFRIGERACIÓN)

Propranolol standard was prepared at 0.5 mgmL-1 in distilled water for LESA and MALDI optimisation experiments. A serial dilution of propranolol was prepared in distilled water to give a concentration range between 0.5 µgµL-1 and 500 fgµL-1. Fenclozic acid standard was diluted to 0.5 mgmL-1 in 50 % ACN for both LESA and MALDI optimisation experiments. A serial dilution of fenclozic acid was prepared in distilled water to give a final concentration range between 0.5 µgµL-1 and 500 fgµL-1. Standard solutions of each drug compound were prepared at 25 ngµL-1 in 50 % ACN with 0.1 % HCOOH and analysed by ESI-MS with and without mobility separation. Data were acquired using a Synapt G2 HDMS mass spectrometer operated in ESI positive ion mode with and without mobility separation. A capillary voltage of 1.2

73 kV was applied. The TOF mass analyser was calibrated using sodium formate solution in 90% propan-2-ol. In mobility-TOF mode instrument acquisition parameters were varied to provide an optimal ion mobility separation. Optimal separation was achieved with a wave velocity of 800 m/s and a wave height of 40.0 V for both propranolol and fenclozic acid and was used in all subsequent LESA experiments.

Fragmentation of fenclozic acid was carried out using a collision energy of 20.0 eV, whilst fragmentation of propranolol required a collision energy of 28.0 eV. For MS/MS mobility experiments the CID was carried out in the trap cell of the mass spectrometer (prior to mobility separation) and these conditions were used for all LESA profiling experiments.

2.3.6 LESA profiling

For limit of detection experiments, 0.5 µL of each drug compound concentration was spiked onto glass slides and control rat kidney tissue sections to give a final amount of drug ranging from 0.25 µg to 250 fg.

The LESA enabled TriVersa NanoMate device was operated using Chipsoft (v8.3.1) and sampling positions were selected using LESA Points (v1.1) software. A nano- ESI voltage of 1.55-1.65 kV and a gas pressure of 0.45-0.50 psi were applied in all experiments. 50 % ACN with 0.1 % HCOOH (v/v) was used as the extraction solvent in all experiments. 1 µL droplet of extraction solvent was used on-tissue and the liquid microjunction was sustained on the tissue surface for 5 second before aspiration back into the tip.

Infusion was performed through a nanofabricated sample introduction chip with 400 spray-nozzles (each 5.5 µm ID x 28 µm OD x 75 µm long). On tissue spot size was around 2 mm in diameter, and sample positions were manually selected at appropriate distances to avoid spot overlap.

Data were acquired for a minimum of 2 minutes per sample, in MS/MS mobility mode from 100-600 m/z, with mobility separation after MS/MS fragmentation for all

74 experiments. After acquisition, data evaluation was performed using Driftscope and MassLynx (v4.1).

2.3.7 MALDI-MS analysis of standards

Propranolol at 0.5 mgmL-1 was diluted ten-fold and prepared 1:1 (v/v) with matrix solution. 1 µL of propranolol and matrix mix was spotted onto stainless steel target plates for MALDI analysis. CHCA (3.6 mgmL-1 in 50 % ACN + 0.05 % TFA (v/v)) and DHB (10 mgmL-1 in 50 % ACN + 0.05 % TFA (v/v)) were tested as matrix compounds across the propranolol concentration range. Matrix solution and propranolol standard were mixed 1:1 (v/v) to give a final concentration range between 0.25 µgµL-1 and 250 fgµL-1.

Fenclozic acid was diluted to 0.5 mgmL-1 in 50 % ACN; diluted ten-fold in distilled water and prepared 1:1 (v/v) with matrix solution. 1µL of fenclozic acid and matrix mix was spotted onto stainless steel target plates for MALDI analysis. CHCA (3.6 mgmL-1 in 50 % ACN + 0.05 % TFA (v/v)) was used as the matrix compound for fenclozic acid. Matrix solution and fenclozic acid standard were mixed 1:1 (v/v) to give a final concentration range between 0.25 µgµL-1 and 250 fgµL-1.

1 µL of each drug standard concentration were spotted onto control tissue sections (rat kidney) as 2 x 0.5 µL droplets. 1 µL of matrix solution was applied as 2 x 0.5 µL droplets to the same positions to determine limits of detection on tissue and establish the most selective method for successful detection of each drug.

Data were acquired using a Synapt G2 HDMS mass spectrometer operated in MALDI positive ion mode from 100-600 m/z with and without mobility separation. The TOF mass analyser was calibrated using phosphorus red (10 mgmL-1) prepared in acetone. In mobility-TOF mode instrument acquisition parameters were adjusted to provide an optimal ion mobility separation. Optimal separation was achieved using a wave velocity of 800 m/s and a wave height of 40.0 V for propranolol and fenclozic acid. These conditions were used for all subsequent MALDI experiments. MS/MS fragmentation was optimised using drug standards, with and without mobility separation. A collision energy of 30.0 V was applied in the transfer region

75 of the mass spectrometer after mobility separation, with 27.0 V applied in the trap region for MS/MS experiments without mobility separation for propranolol. Fragmentation of fenclozic acid was achieved using a collision energy of 20.0 V applied in the trap region of the mass spectrometer for MS/MS experiments with and without mobility separation.

Ion mobility separation following MS/MS fragmentation was determined to be the most selective approach for the identification and localisation of fenclozic acid and propranolol over a range of concentrations on tissue. Fenclozic acid was successfully detected at 250 fg spiked on tissue, whilst propranolol was successfully detected at 2.5 ng spiked on tissue. This method was used for all subsequent profiling and imaging experiments on dosed tissue.