The goal of untargeted lipidomics is to have high throughput, yet comprehensive and unambiguous identification and quantification of lipids. Novel stationary phases in LC separation and new mass spectrometric instruments capable of high mass resolving power and faster scanning rate are essential to achieving this goal. In this work, 4 reversed phase LC columns coupled with a high field quadrupole orbitrap mass spectrometer (Q Exactive HF) were thoroughly compared using complex lipid standard mixture and rat plasma and liver samples. A good separation of all lipids was achieved in 24 min of gradient. The columns compared include C30 and C18 functionalization on either core–shell or totally porous silica particles, with size ranging from 1.7 to 2.6 μm. Accucore C30 column showed the narrowest peaks and highest theoretical plate number, and excellent peak capacity and retention time reproducibility (<1% standard deviation). As a result, it resulted in 430 lipid species identified from rat plasma and rat liver samples with highest confidence. The high resolution offered by the up-front RPLC allowed discrimination of cis/trans isomeric lipid species, and the high field orbitrap mass spectrometer afforded the clear distinction of isobaric lipid species in full scan MS and the unambiguous assignment of sn-positional isomers for lysophospholipids in MS/MS. Taken together, the high efficiency LC separation and high mass resolving MS analysis are very promising tools for untargeted lipidomics analysis.
The confident identification and in-depth profiling of molecular lipid species remain to be a challenge in lipidomics analysis. In this work, an off-line two-dimensional mixed-mode and reversed-phase liquid chromatography (RPLC) method combined with high-field quadrupole orbitrap mass spectrometer (Q Exactive HF) was developed to profile lipids from complex biological samples. In the first dimension, 22 different lipid classes were separated on a monolithic silica column with elution order from neutral to polar lipids. A total of 13 fractions were collected and run on a RPLC C30 column in the second dimension for further separation of the lipid molecular species based on their hydrophobicity, with the elution order being determined by both the length and degree of unsaturation in the fatty-acyl chain. The method was applied to analyze lipids extracted from rat plasma and rat liver. Fatty acid methyl ester analysis by gas chromatography-mass spectrometry was used to identify the fatty acyls from total lipid extracts, which provided a more confident identification of the lipid species present in these samples. More than 800 lipids were identified in each sample and their molecular structures were confidentially confirmed using tandem mass spectrometry (MS/MS). The number of lipid molecular species identified in both rat plasma and rat liver by this off-line two-dimensional method is approximately twice of that by one-dimensional RPLC-MS/MS employing a C30 column. This off-line two-dimensional mixed-mode LC-RPLC-MS/MS method is a promising technique for comprehensive lipid profiling in complex biological matrices.
Changes in diacylglycerols, monoacylglycerols, and free fatty acid composition of subcutaneous fat of six Iberian hams during the dry-cured process were investigated. In addition, an analytical method for simultaneous quantification of diacylglycerols, monoacylglycerols, and free fatty acid by solid-phase extraction-gas chromatography was developed. The different molecular species of free fatty acids, monoacylglycerols, and diacylglycerols and 1,2- and 1,3-isomers of diacylglycerols have been described for the first time in this type of sample. A logarithmic increase of the 1,3-diacylglycerol profile throughout the processing time has been found, reaching a balance value of 62% around 500 days. The formation of diacylglycerol isomers takes place, although the 1,3-/1,2-diacylglycerol ratio increases during the process to 1.65 due to isomerization of the 1,2-form toward the 1,3-form. The profiles of monoacyl- and diacylglycerols and free fatty acids follow the same trend. The experimental values of free fatty acid are greater than theoretical prediction, probably due to phospholipid and monoacylglycerol hydrolysis.
INTRODUCTION:
Bile reflux may cause for lung allograft rejection, yet there are no studies that determine (i) the relationship between gastric and lung bile concentrations, (ii) whether bile is present in lungs of nontransplant patients, (iii) the relationship between gastric dysmotility and lung bile, (iv) the impact of reflux therapies on lung bile, and (v) whether lung bile worsens outcomes in nontransplant patients. This study will address these gaps in the literature.
METHODS:
We prospectively recruited lung transplant (LTX) patients and nontransplant patients with respiratory symptoms (RP) and collected paired gastric and lung samples. Bile concentration and composition of samples was assessed using liquid chromatography–mass spectrometry. Bile results were compared with clinical parameters, including the presence of esophagitis, gastric dysmotility, and/or pathologic gastroesophageal reflux.
RESULTS:
Seventy patients (48 RP and 22 LTX) were recruited. Overall, 100% of gastric and 98% of bronchoalveolar lavage samples contained bile. The mean gastric bile concentrations in RP and LTX patients were 280 ± 703 nmol/L and 1,004 ± 1721 nmol/L, respectively (P = 0.02). There was no difference in lung bile concentrations between RP (9 ± 30 nmol/L) and LTX (11 ± 15 nmol/L, P = 0.7). Patients with delayed gastric emptying had higher lung bile concentrations (15.5 ± 18.8 nmol/L) than patients with normal gastric emptying (4.8 ± 5.7 nmol/L, P = 0.05) independently of reflux burden. Proton pump inhibitor use increased the proportion of unconjugated gastric bile acids. High lung bile concentrations were associated with an increased risk of hospitalization and longer hospital stays in RP patients (P < 0.05).
DISCUSSION:
Lung bile is almost universally present in symptomatic patients, and higher concentrations are associated with poorer respiratory outcomes.
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