Short-Term Stability of Serum and Liver Extracts for Untargeted Metabolomics and Lipidomics

Abstract

Thermal reactions can significantly alter the metabolomic and lipidomic content of biofluids and tissues during storage. In this study, we investigated the stability of polar metabolites and complex lipids in dry human serum and mouse liver extracts over a three-day period under various temperature conditions. Specifically, we tested temperatures of −80 °C (freezer), −24 °C (freezer), −0.5 °C (polystyrene box with gel-based ice packs), +5 °C (refrigerator), +23 °C (laboratory, room temperature), and +30 °C (thermostat) to simulate the time between sample extraction and analysis, shipping dry extracts to different labs as an alternative to dry ice, and document the impact of higher temperatures on sample integrity. The extracts were analyzed using five fast liquid chromatography-mass spectrometry (LC-MS) methods to screen polar metabolites and complex lipids, and over 600 metabolites were annotated in serum and liver extracts. We found that storing dry extracts at −24 °C and partially at −0.5 °C provided comparable results to −80 °C (reference condition). However, increasing the storage temperatures led to significant changes in oxidized triacylglycerols, phospholipids, and fatty acids within three days. Polar metabolites were mainly affected at storage temperatures of +23 °C and +30 °C.

Exploring the Impact of Organic Solvent Quality and Unusual Adduct Formation during LC-MS-Based Lipidomic Profiling

“…The internal standards used for the system suitability test (SST) were obtained from Avanti Polar Lipids/Merck and contained the following analytes: CE 22:1, Cer d18:1/17:0, cholesterol- d 7 , DG 12:0/12:0/0:0, DG 18:1/2:0/0:0, Hex-Cer d18:1/17:0, LPC 17:1, LPE 17:1, MG 17:0/0:0/0:0, PC 15:0/18:1- d 7 , PE 17:0/17:0, PG 17:0/17:0, PI 15:0/18:1- d 7 , PS 17:0/17:0, SM d18:1/17:0, sphingosine d17:1, TG 17:0/17:1/17:0- d 5 , and TG 20:0/20:1/20:0- d 5 [ 24 ].…”

Hydrophilic Interaction Liquid Chromatography–Hydrogen/Deuterium Exchange–Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics

“…The metabolites were extracted using a biphasic solvent system of cold methanol, methyl tert -butyl ether, and water [ 52 , 61 ].…”

“…For the extraction of biofluids (plasma, serum), a 25 µL aliquot in a 1.5 mL tube was shaken (30 s) with 165 µL ice-cold methanol and 600 µL MTBE, both containing internal standards [ 61 ]. Subsequently, 165 µL of 10% methanol with internal standards [ 61 ] was added, vortexed (10 s), and then centrifuged (24,328× g , 5 min, 4 °C). A 70 µL aliquot of the bottom phase was collected and evaporated.…”

“…Subsequently, 1 mL of MTBE was added, and this mixture was shaken (30 s). Finally, 275 μL of 10% methanol was added, and after vortexing (10 s), the tubes were centrifuged (24,328× g , 5 min, 4 °C) [ 61 ]. A 70 µL aliquot of the bottom phase was collected and evaporated.…”

“…The LC-MS system comprised a Vanquish UHPLC system (Thermo Fisher Scientific, Bremen, Germany) and a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) equipped with a heated electrospray ionization (HESI-II) probe (Thermo Fisher Scientific), [ 52 , 61 ].…”