Medical sciences

Raw data from the manuscript entitled "Recent solid-phase microextraction-based analytical approaches for the profiling of biliary bile acids in pre-transplant assessments of liver grafts subjected to normothermic ex vivo liver perfusion".

Kamil Łuczykowski^a, Natalia Warmuzińska^a, Karol Jaroch^a, Dagmar Kollmann^b,c, Markus Selzner^b, Barbara Bojko^a

^a Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Bydgoszcz, Poland

^b Department of Surgery, Ajmera Transplant Centre, Toronto General Hospital, University Health Network, Toronto, ON M5G 2C4, Canada

^c Department of General Surgery, Medical University of Vienna, 1090 Vienna, Austria

Liver transplantation is the definitive treatment for end-stage liver failure, but the scarcity of donor organs remains a significant challenge. Leveraging organs from extended criteria donors (ECD) offers a potential avenue to address worldwide shortages, though these organs are more susceptible to post-reperfusion injury. This study explores the use of normothermic ex vivo liver perfusion (NEVLP) as a method for organ preservation – an approach that sustains liver metabolism and facilitates pre-transplant assessments of organ viability via bile analysis. The focal point of this study revolves on the development of analytical methods for determining the bile acid profile throughout the peritransplantation period as a potential indicator of liver function and viability.

The study optimized and validated a high-throughput analytical method to quantify selected bile acids in bile samples using a thin-film microextraction-liquid chromatography-mass spectrometry (TFME-LC-MS) platform. Furthermore, it introduced a solid-phase microextraction-microfluidic open interface-mass spectrometry (SPME-MOI-MS) method for rapid direct analysis of bile acid isobar groups. In the animal study, discernible variations in the concentrations of specific bile acids were observed between donors after circulatory death (DCD) and heart-beating donors (HBD), particularly following normothermic perfusion and reperfusion. Noteworthy fluctuations in individual bile acid concentrations were observed throughout the entire organ transplantation process, with taurocholic acid (TCA), glycocholic acid (GCA), and glycochenodeoxycholic acid (GCDCA) emerging as promising indicators of organ quality. The efficacy of the SPME-MOI-MS platform in corroborating these trends highlights its potential for real-time bile acid analysis during liver transplantation procedures.

Our findings underscore the efficacy of NEVLP in tandem with advanced bile acid analysis methods as a reliable strategy for pre-transplant assessments of organ viability, potentially increasing the use of ECD organs and reducing organ shortages. The ability to monitor bile acid profiles in real-time provides crucial insights into liver function and ischemic injury, making significant strides in improving transplant outcomes and patient survival rates.

Animal study

The study utilized bile samples from two types of porcine model donors (Yorkshire pigs weighing 29–35 kgs): HBD, and DCD. The experiments were approved by the Animal Resource Centre at the University Health Network, and all animals were treated humanely in accordance with the National Institute of Health's “Guide for the Care of Laboratory Animals”. The research material was provided by the Department of Surgery at Toronto General Hospital (University Health Network, Toronto). Bile samples were collected at specific time points during NEVLP and after liver transplantation: before organ procurement, during perfusion, 3 and 5 h after reperfusion, and on days 1 and 3 post-transplantation (Fig. 1). For the DCD group, a model with 90 min warm ischemia time was used (n = 5 in each group). 

Sample preparation 

Preparation of samples was carried out using SPME, with each step of the process conducted on a high-throughput 96-manual SPME system (Professional Analytical System (PAS) Technology, Magdala, Germany), enabling simultaneous analysis of all samples. Steel blades coated with a 10 mm octadecyl silica (C18) sorbent were used. Steel blades were purchased from PAS Technology (Magdala, Germany) and polypropylene Nunc 96 DeepWell plates were purchased from Merck (Poznań, Poland). 

Prior to extraction, SPME blades were conditioned for 30 min in 1.0 mL of a methanol: water (50:50; v/v) solution in 96-well-plates, with agitation at 1000 rpm at room temperature. This was followed by a 10-s wash step. Subsequently, extractions were carried out using 1 mL of diluted bile (100-fold and 10000-fold in PBS) with an internal standard (IS) added to achieve a final concentration of 50 ng/mL, under the same conditions, for 60 min. The concentration of organic solvents in the studied samples did not exceed 1 % (v/v), thereby ensuring optimal extraction efficiency [12]. Post-extraction, the blades were washed in 1 mL of nanopure water for 10 s. Desorption was subsequently performed in 1 mL of methanol with agitation at 1000 rpm for 45 min at room temperature.

LC-MS analysis

Chromatographic separation was performed on the Nexera UHPLC system. Bile extracts were injected in a volume of 5 μL on an ACQUITY UPLC CSH C18 Column (1.7 μm, 2.1 mm ×100 mm, Waters). The autosampler and column temperatures were maintained at 4 ^◦C and 55 ^◦C, respectively, throughout the analysis. The flow rate was set to 0.4 mL/min. Mobile phase A consisted of water with formic acid (99.9:0.1; v/v), while mobile phase B was composed of acetonitrile with formic acid (99.9:0.1; v/v). The total analysis time per sample was 13 min. The initial mobile phase conditions were set at 40 % B from 0 to 4.0 min, followed by a linear gradient to 70 % B from 4.0 to 10.0 min, an isocratic hold at 100 % B from 10.1 to 11.0 min, and a 2-min column re- equilibration time with 40 % B. The analyses were performed on a LC–MS 8060 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan), equipped with an electrospray ionization source operating in negative-ion mode and utilizing multiple reaction monitoring (MRM). The MRM transitions, along with the collision energies for individual bile acids, are detailed in Table S1. The ESI source parameters were set as following: interface voltage at 3.0 kV, interface temperature at 300 ^◦C, DL temperature at 250 ^◦C, heat block temperature at 450 ^◦C, nebulizing gas flow at 3.0 L/min, drying gas flow at 10.0 L/min, and heating gas flow at 10.0 L/min.

Solid phase microextraction - microfluidic open interface – mass spectrometry (SPME-MOI-MS) analysis

A rapid analysis of bile acids without chromatographic separation was performed using a microfluidic open interface (MOI) coupled directly to a mass spectrometer. Analytes were extracted from bile samples using 8 mm C18 SPME fibers. Prior to sampling, fibers were preconditioned for 60 min in a methanol:water (50:50 v/v) solution and then rinsed with purified water. Extractions were carried out from 1 mL of diluted bile samples (1:99 in PBS) spiked with CA-d₄ to a final concentration of 50 ng/mL, for 15 min under agitation at 1000 rpm. Following extraction, SPME fibers were quickly rinsed with ultrapure water for 5 s to remove residual salts and non-specific bile components loosely adhered to the sorbent surface. Individual fibers were then placed in the desorption chamber of the MOI for 10 s for desorption, and subsequently removed from the chamber. The resulting solution containing the desorbed bile acids was transferred from the MOI to the MS, and ionized by electrospray, with a dwell time of 10 msec. The construction and functional principles of the MOI device are described in detail elsewhere. The desorption solution, consisting of MeOH with formic acid (99.9:0.1; v/v), was delivered to the MOI device by a Harvard Apparatus Pump 11 Elite pump (Holliston, Massachusetts, USA). The desorption step was performed by equilibrating the pump flow rate, set at 165 μL/min, with the electrospray ionization (ESI) aspiration. Similar to the LC-MS analysis described above, a triple quadrupole mass spectrometer operating in negative ionization mode was utilized, employing previously optimized MRM settings for bile acids. The MS parameters were configured as follows: interface voltage at 3.0 kV, interface temperature at 300 ^◦C, DL temperature at 250 ^◦C, heat block temperature off, nebulizing gas flow at 3.0 L/min, drying gas flow at 10.0 L/min, and heating gas flow at 10.0 L/min. 

This study was funded by National Science Centre (Poland), grant Preludium No. 2022/45/N/ST4/01177