Targeted quantification of lipid mediators in skeletal muscles using restricted access media-based trap-and-elute liquid chromatography-mass spectrometry

Abstract

Lipid mediators (LMs) are a class of bioactive metabolites of the essential polyunsaturated fatty acids (PUFA), which are involved in many physiological processes. Their quantification in biological samples is critical for understanding their functions in lifestyle and chronic diseases, such as diabetes, as well allergies, cancers, and in aging processes. We developed a rapid, and sensitive LC-MS/MS method to quantify the concentrations of 14 lipid mediators of interest in mouse skeletal muscle tissue without time-consuming liquid-liquid or solid-phase extractions. A restricted-access media (RAM) based trap was used prior to LC-MS as cleanup process to prevent the analytical column from clogging and deterioration. The system enabled automatic removal of residual proteins and other biological interferences presented in the tissue extracts; the target analytes were retained in the trap and then eluted to an analytical column for separation. Matrix evaluation tests demonstrated that the use of the combined RAM trap and chromatographic separation efficiently eliminated the biological or chemical matrix interferences typically encountered in bioanalytical analysis. Using 14 LM standards and 12 corresponding deuterated compounds as internal standards, the five-point calibration curves, established over the concentration range of 0.031 to 320 ng mL−1, demonstrated good linearity of r2 > 0.9903 (0.9903 to 0.9983). The lower detection limits obtained were 0.016, 0.031, 0.062, and 0.31 ng mL−1 (0.5, 1, 2, and 10 pg on column), respectively, depending on the specific compounds. Good accuracy (87.1–114.5%) and precision (<13.4%) of the method were observed for low, medium, and high concentration quality control samples. The method was applied to measure the amount of 14 target LMs in mouse skeletal muscle tissues. All 14 analytes in this study were successfully detected and quantified in the gastrocnemius muscle samples, which provided crucial information for both age and gender-related aspects of LMs signaling in skeletal muscles previously unknown. This method could be applied to advance the understanding of skeletal muscle pathophysiology to study the role of LMs in health and disease. Furthermore, we will expand the application of this methodology to humans and other tissues/matrices in the near future.

Quantification of aminobutyric acids and their clinical applications as biomarkers for osteoporosis

“…Linear regression lines were constructed for each biological matrix versus 5% bovine serum albumin (BSA) in phosphatebuffered saline (PBS, pH 7.4) at 11 concentrations, and linear coefficient values were calculated as presented in Supplementary Table 2. A slope of K = 1 from the linear equation implies correlation and thus demonstrates similar matrix effects exerted by these two matrices [23][24][25][26] . As shown in Supplementary Table 2, the slopes of the linear equations from four sets of solutions are close to unity (0.87-1.12) for these analytes, indicating parallelism of matrix effects presented between the surrogate matrix selected and biological fluids.…”

Free fatty acid profiling in marine algae extract by LC‐MS/MS and isolation as well as quantification of the ω‐3 fatty acid hexadeca‐4,7,10,13‐tetraenoic acid

“…To date, profiling and quantification of Wakame GC-MS is a widely used method for the analysis of fatty acids [14]. However, LC-MS has been rarely used but its importance for fatty acid elucidation is increasing [15][16][17][18][19][20][21]. Typically, these methods use a targeted multiple reaction monitoring (MRM) approach [22].…”

Mouse skeletal muscles infected with Plasmodium berghei and Plasmodium chabaudi reveal a crosstalk between lipid mediators and gene expression

“…Then we further determined the absolute concentrations of 16 key LMs, related to six metabolic pathways, from mouse skeletal muscles in this study. This quanti cation method, developed in the Brotto Muscle Research Lab in collaboration with the Shimadzu Research Institute, was recently applied and showed substantial differences during aging as well sex related differences in mice [27]. In the present study, both quanti cation and pro ling results indicated signi cant differences (p < 0.05) in LM levels in skeletal muscles of mice infected with P. berghei and P. chabaudi compared to that of the muscles from the control mice (Table 2, Table S1, and Fig.…”

“…The MS/MS analysis was performed on Shimadzu LCMS-8050 triple quadrupole mass spectrometer. The instrument following previously published quanti cation method[27]. All analyses and data processing were completed on Shimadzu LabSolutions V5.91 software (Shimadzu Scienti c Instruments, Inc., Columbia, MD, USA).Tissue preparation for lipidomics analysesStriated muscles were isolated after mice sacri ce, then snap frozen in liquid nitrogen immediately and stored at -80°C.…”