Regio-specific lipid fingerprinting of edible sea cucumbers using LC/MS.

Sea cucumbers are a rich source of bioactive compounds and are gaining popularity as nutrient-rich seafood. They are consumed as a whole organism in Pacific regions. However, limited data are available on the comparison of their lipid composition and nutritional value. In this study, untargeted liquid chromatography/mass spectrometry was applied to comprehensively profile lipids in the skin, meat, and intestinal contents of three color-distinct edible sea cucumbers. Multivariate principal component analysis revealed that the lipid composition of the intestinal contents of red, black, and blue sea cucumbers differs from that of skin, and meats. Polyunsaturated fatty acids (PUFAs) are abundant in the intestinal contents, followed by meats of sea cucumber. Lipid nutritional quality assessments based on fatty acid composition revealed a high P:S ratio, low index of atherogenicity, and high health promotion indices for the intestinal contents of red sea cucumber, suggesting its potential health benefits. In addition, hierarchical cluster analysis revealed that the intestinal contents of sea cucumbers were relatively high in PUFA-enriched phospholipids and lysophospholipids. Ceramides are abundant in black skin, blue meat, and red intestinal content samples. Overall, this study provides the first insights into a comprehensive regio-specific profile of the lipid content of sea cucumbers and their potential use as a source of lipid nutrients in food and nutraceuticals.

Sex-Specific Effect of Ethanol on Colon Content Lipidome in a Mice Model Using Nontargeted LC/MS.

Consumption of alcohol has widespread effects on the human body. The organs that are most significantly impacted are the liver and digestive system. When alcohol is consumed, it is absorbed in the intestines and processed by the liver. However, excessive alcohol use may affect gut epithelial integrity, microbiome composition, and lipid metabolism. Despite past studies investigating the effect of ethanol on hepatic lipid metabolism, the focus on colonic lipid metabolism has not been well explored. In this study, we investigated the sex-specific effect of ethanol on the colonic content lipidome in a mouse model using nontargeted liquid chromatography-mass spectrometry. Comprehensive lipidome analysis of colonic flush samples was performed using ethanol-fed (EF) and pair-fed (PF) mice of each sex. Partial least-squares discriminant analysis revealed that ethanol altered colonic lipid composition largely in male mice compared with female mice. A significant increase in free fatty acids, ceramides, and hexosylceramides and decreased phosphatidylglycerols (PG) was observed in the EF group compared to the PF group in male mice. Phosphatidylethanolamine (PE) levels were increased significantly in the EF group of both sexes compared to the PF group. The volcanic plot shows that PG (O-15:1/15:0) and PE (O-18:2/15:0) are common markers that are increased in both sexes of the EF group. In addition, decreased fatty acid esters of hydroxy fatty acids (FAHFA) were observed specifically in the EF group of female mice. Overall, a significant variation in the mice colonic content lipidome between the EF and PF groups was observed. Target pathways, such as sphingolipid metabolism in males, FAHFA in females, and PE metabolism in both sexes, were suggested. This study provides new insight into the sex-dependent lipid change associated with alcohol-induced gut-microbiota dysfunction and its potential health impacts.

Dissecting new lipids and their composition in herbal tea using untargeted LC/MS.

Herbal teas and beverages have gained global attention because they are rich in natural bioactive compounds, which are known to have diverse biological effects, including antioxidant and anticarcinogenic properties. However, the lipidomic profiles of herbal teas remain unclear. In this study, we applied an untargeted lipidomics approach using high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry to comprehensively profile, compare, and identify unknown lipids in four herbal teas: dokudami, kumazasa, sugina, and yomogi. A total of 341 molecular species from five major classes of lipids were identified. Multivariate principal component analysis revealed distinct lipid compositions for each of the herbs. The fatty acid α-linolenic acid (FA 18:3) was found to be abundant in kumazasa, whereas arachidonic acid (FA 20:4) was the most abundant in sugina. Interestingly, novel lipids were discovered for the first time in plants; specifically, short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs) with 4-hydroxy phenyl nonanoic acid as the structural core. This study provides insight into the lipidomic diversity and potential bioactive lipid components of herbal teas, offering a foundation for further research into their health-promoting properties and biological significance.

Lysosomal membrane integrity in fibroblasts derived from patients with Gaucher disease.

Gaucher disease (GD) is a recessively inherited lysosomal storage disorder characterized by a deficiency of lysosomal glucocerebrosidase (GBA1). This deficiency results in the accumulation of its substrate, glucosylceramide (GlcCer), within lysosomes. Here, we investigated lysosomal abnormalities in fibroblasts derived from patients with GD. It is noteworthy that the cellular distribution of lysosomes and lysosomal proteolytic activity remained largely unaffected in GD fibroblasts. However, we found that lysosomal membranes of GD fibroblasts were susceptible to damage when exposed to a lysosomotropic agent. Moreover, the susceptibility of lysosomal membranes to a lysosomotropic agent could be partly restored by exogenous expression of wild-type GBA1. Here, we report that the lysosomal membrane integrity is altered in GD fibroblasts, but lysosomal distribution and proteolytic activity is not significantly altered.Key words: glucosylceramide, lysosome, Gaucher disease, lysosomotropic agent.

Regiospecific analysis of lipidome in the brain from mammals of land and aquatic habitats-by liquid chromatography-mass spectrometry.

The brain is a complex organ demonstrated by the occurrence of specific types of functional lipids. Despite some studies focusing on providing the animal brain lipid signature, there are limited studies focusing on the comprehensive and regiospecific characterization of multiple animal brain lipidome. Herein we characterized about 294 lipid molecular species from six different lipid classes in different portions of the brain after fixation from mammals of different habitats, fully-aquatic (n = 6), semi-aquatic (n = 6), and terrestrial (n = 4), using liquid chromatography-mass spectrometry. The untargeted brain lipid profiling revealed a significant difference in total lipid levels between fully-aquatic, semi-aquatic, and terrestrial mammals. The polyunsaturated fatty acids and cholesterol esters are abundant in brain tissue of semi-aquatic followed by fully-aquatic mammals whereas phosphatidylethanolamines are profoundly high in terrestrial species. The regiospecific analysis revealed a predominance of sphingolipids in all the groups but no significant differences were observed between the different portions of the brain such as the cerebellum, cortex, pons, spinal cord, and thalamus. Interestingly the multivariate analysis showed almost the same lipid compositions in the spinal cord and thalamus of terrestrial mammals. Overall, this is the first report to compare the comprehensive brain-lipidome among different mammalian groups inhabiting three distinct habitats. These results indicate that the brain lipid composition is specific to the animal habitat.

Mass spectrometric approaches in discovering lipid biomarkers for COVID-19 by lipidomics: Future challenges and perspectives.

Coronavirus disease 2019 (COVID-19) has emerged as a global health threat and has rapidly spread worldwide. Significant changes in the lipid profile before and after COVID-19 confirmed the significance of lipid metabolism in regulating the response to viral infection. Therefore, understanding the role of lipid metabolism may facilitate the development of new therapeutics for COVID-19. Owing to their high sensitivity and accuracy, mass spectrometry (MS)-based methods are widely used for rapidly identifying and quantifying of thousands of lipid species present in a small amount of sample. To enhance the capabilities of MS for the qualitative and quantitative analysis of lipids, different platforms have been combined to cover a wide range of lipidomes with high sensitivity, specificity, and accuracy. Currently, MS-based technologies are being established as efficient methods for discovering potential diagnostic biomarkers for COVID-19 and related diseases. As the lipidome of the host cell is drastically affected by the viral replication process, investigating lipid profile alterations in patients with COVID-19 and targeting lipid metabolism pathways are considered to be crucial steps in host-directed drug targeting to develop better therapeutic strategies. This review summarizes various MS-based strategies that have been developed for lipidomic analyzes and biomarker discoveries to combat COVID-19 by integrating various other potential approaches using different human samples. Furthermore, this review discusses the challenges in using MS technologies and future perspectives in terms of drug discovery and diagnosis of COVID-19.

Determination of short-chain fatty acids by N,N-dimethylethylenediamine derivatization combined with liquid chromatography/mass spectrometry and their implication in influenza virus infection.

Short-chain fatty acids (SCFAs) are the end products of the fermentation of complex carbohydrates by the gut microbiota. Although SCFAs are recognized as important markers to elucidate the link between gut health and disease, it has been difficult to analyze SCFAs with mass spectrometry technologies due to their poor ionization efficiency and high volatility. Here, we present a novel and sensitive method for the quantification of SCFAs, including C2-C6 SCFAs and their hydroxy derivatives, by liquid chromatography/tandem mass spectrometry (LC-MS/MS) upon N,N-dimethylethylenediamine (DMED) derivatization with a run time of 10 min. Moreover, the quantification method of DMED-derivatized SCFAs in intestinal contents using isotope-labeled internal standards was also established. The method validation was performed by analyzing spiked intestinal samples; the limits of detection and quantification of SCFAs with this method were found to be 0.5 and 5 fmol, respectively; the recovery was greater than 80% and good linearity (0.9932 to 0.9979) of calibration curves was obtained over the range from 0.005 to 5000 pmol/µL; the intraday and interday precisions were achieved in the range of 1-5%. Furthermore, the validated method was applied to analyze SCFAs in the cecum and colon contents of mice infected with the influenza virus. The results showed that the concentration of most of the SCFAs tested here decreased significantly in a time-dependent manner after the infection, suggesting a possibility that SCFAs in intestinal samples could be used as severe disease markers. Overall, we here successfully developed a simple, fast, and sensitive method for SCFA analysis by LC-MS/MS combined with DMED derivatization. The method for the quantification of SCFAs will be a useful tool for both basic research and clinical studies.

Detection and characterization of lipids in eleven species of fish by non-targeted liquid chromatography/mass spectrometry.

Fish is an important nutrition source because its lipids, which are rich in ω-3 fatty acids, are beneficial for human health. However, studies focusing on their detection, composition, and nutritional value are limited. In this study, we applied a non-targeted lipidomic approach based on ultra-high performance liquid chromatography coupled with linear-ion trap-Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap-MS) to comprehensively profile, compare, and detect unknown lipids in eleven types of dietary fish. A total of 287 molecular species from five major lipid classes were characterized by MS/MS analysis. Multivariate principal component analysis revealed the distinct lipid composition in shishamo smelt and Japanese sardine compared to other fish types. The assessment of nutritional indices based on the levels of free fatty acid suggested that among the eleven fish types, shishamo smelt is highly beneficial for health. Further, lipids such as N-acyl lysophosphatidylethanolamine were detected and characterized for the first time in fish fillets. Hierarchical cluster correlations indicated the predominance of glycerophospholipids (GPs) and sphingolipids in sardine, whereas fatty acyls and triacylglycerols (TAGs) were predominant in shishamo smelt. The high levels of polyunsaturated fatty acid-enriched GPs and TAGs in dietary fish endow it with great potential as a health-promoting food for human consumption. This study offers a comprehensive analysis of lipids and their compositions in fish fillets, demonstrating their potential use in the nutritional assessment of functional foods.

Simple and Sensitive Method for the Quantitative Determination of Lipid Hydroperoxides by Liquid Chromatography/Mass Spectrometry.

Lipid hydroperoxides (LOOH) are the initial products of the peroxidation of unsaturated lipids and play a crucial role in lipid oxidation due to their ability to decompose into free radicals and cause adverse effects on human health. Thus, LOOHs are commonly considered biomarkers of oxidative stress-associated pathological conditions. Despite their importance, the sensitive and selective analytical method for determination is limited, due to their low abundance, poor stability, and low ionizing efficiency. To overcome these limitations, in this study, we chemically synthesized eight fatty acid hydroperoxides (FAOOH), including FA 18:1-OOH, FA 18:2-OOH, FA 18:3-OOH, FA 20:4-OOH, FA 20:5-OOH, FA 22:1-OOH, FA 22:6-OOH as analytes, and FA 19:1-OOH as internal standard. Then, they were chemically labeled with 2-methoxypropene (2-MxP) to obtain FAOOMxP by one-step derivatization (for 10 min). A selected reaction monitoring assisted targeted analytical method was developed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). The MxP-labelling improved the stability and enhanced the ionization efficiency in positive mode. Application of reverse-phase chromatography allowed coelution of analytes and internal standards with a short analysis time of 6 min. The limit of detection and quantification for FAOOH ranged from 0.1-1 pmol/µL and 1-2.5 pmol/µL, respectively. The method was applied to profile total FAOOHs in chemically oxidized human serum samples (n = 5) and their fractions of low and high-density lipoproteins (n = 4). The linoleic acid hydroperoxide (FA 18:2-OOH) and oleic acid hydroperoxide (FA 18:1-OOH) were the most abundant FAOOHs in human serum and lipoproteins. Overall, our validated LC-MS/MS methodology features enhanced detection and rapid separation that enables facile quantitation of multiple FAOOHs, therefore providing a valuable tool for determining the level of lipid peroxidation with potential diagnostic applications.

Group IIA secreted phospholipase A2 controls skin carcinogenesis and psoriasis by shaping the gut microbiota.

Besides promoting inflammation by mobilizing lipid mediators, group IIA secreted phospholipase A2 (sPLA2-IIA) prevents bacterial infection by degrading bacterial membranes. Here, we show that, despite the restricted intestinal expression of sPLA2-IIA in BALB/c mice, its genetic deletion leads to amelioration of cancer and exacerbation of psoriasis in distal skin. Intestinal expression of sPLA2-IIA is reduced after treatment with antibiotics or under germ-free conditions, suggesting its upregulation by gut microbiota. Metagenome, transcriptome, and metabolome analyses have revealed that sPLA2-IIA deficiency alters the gut microbiota, accompanied by notable changes in the intestinal expression of genes related to immunity and metabolism, as well as in the levels of various blood metabolites and fecal bacterial lipids, suggesting that sPLA2-IIA contributes to shaping of the gut microbiota. The skin phenotypes in Pla2g2a-/- mice are lost (a) when they are cohoused with littermate WT mice, resulting in the mixing of the microbiota between the genotypes, or (b) when they are housed in a more stringent pathogen-free facility, where Pla2g2a expression in WT mice is low and the gut microbial compositions in both genotypes are nearly identical. Thus, our results highlight a potentially new aspect of sPLA2-IIA as a modulator of gut microbiota, perturbation of which affects distal skin responses.

Defining the kinetic effects of infection with influenza virus A/PR8/34 (H1N1) on sphingosine-1-phosphate signaling in mice by targeted LC/MS.

Influenza remains a world-wide health concern, causing 290,000-600,000 deaths and up to 5 million cases of severe illnesses annually. Noticing the host factors that control biological responses, such as inflammatory cytokine secretion, to influenza virus infection is important for the development of novel drugs. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite and has essential biological functions in inflammation. However, the kinetic effects of influenza virus infection on physiological S1P levels and their signaling in multiple tissues remain unknown. In this study, we utilized a mouse model intranasally infected with 50 or 500 plaque forming units (PFU) of A/Puerto Rico/8/34 (H1N1; PR8) virus to investigate how S1P levels and expression of its regulating factors are affected by influenza virus infection by the liquid-chromatography/mass spectrometry and real-time PCR, respectively. The S1P level was significantly high in the plasma of mice infected with 500 PFU of the virus than that in control mice at 6 day-post-infection (dpi). Elevated gene expression of sphingosine kinase-1 (Sphk1), an S1P synthase, was observed in the liver, lung, white adipose tissue, heart, and aorta of infected mice. This could be responsible for the increased plasma S1P levels as well as the decrease in the hepatic S1P lyase (Sgpl1) gene in the infected mice. These results indicate modulation of S1P-signaling by influenza virus infection. Since S1P regulates inflammation and leukocyte migration, it must be worth trying to target this signaling to control influenza-associated symptoms.

Docosahexaenoic Acid Esters of Hydroxy Fatty Acid Is a Novel Activator of NRF2.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with interesting physiological functions in mammals. Despite their structural diversity and links with nuclear factor erythroid 2-related factor 2 (NRF2) biosynthesis, FAHFAs are less explored as NRF2 activators. Herein, we examined for the first time the synthetic docosahexaenoic acid esters of 12-hydroxy stearic acid (12-DHAHSA) or oleic acid (12-DHAHOA) against NRF2 activation in cultured human hepatoma-derived cells (C3A). The effect of DHA-derived FAHFAs on lipid metabolism was explored by the nontargeted lipidomic analysis using liquid chromatography-mass spectrometry. Furthermore, their action on lipid droplet (LD) oxidation was investigated by the fluorescence imaging technique. The DHA-derived FAHFAs showed less cytotoxicity compared to their native fatty acids and activated the NRF2 in a dose-dependent pattern. Treatment of 12-DHAHOA with C3A cells upregulated the cellular triacylglycerol levels by 17-fold compared to the untreated group. Fluorescence imaging analysis also revealed the suppression of the degree of LDs oxidation upon treatment with 12-DHAHSA. Overall, these results suggest that DHA-derived FAHFAs as novel and potent activators of NRF2 with plausible antioxidant function.

Detection and Structural Characterization of SFAHFA Homologous Series in Mouse Colon Contents by LTQ-Orbitrap-MS and Their Implication in Influenza Virus Infection.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with promising physiological functions in mammals. We previously introduced a new type of lipids to this family called short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs), branching specific to the C2 carbon of a long-chain fatty acid (≥C20). In this study, we discovered a homologous series of SFAHFAs comprising C16-C26 hydroxy fatty acids esterified with short-chain fatty acids (C2-C5) in mouse colon contents. The detected SFAHFAs were characterized by high-resolution mass spectrometry with MSn analysis. The double-bond position of monounsaturated SFAHFAs was determined by the epoxidation reaction of samples with m-chloroperoxybenzoic acid and their MSn analysis. Further, the measurement of SFAHFA concentration in the colon contents of mice infected with influenza A/Puerto Rico/8/34 (H1N1; PR8) virus revealed a significant increase in their levels compared to native control. A strong correlation was observed between hydroxy fatty acid and SFAHFAs. Detection, characterization, and profiling of these new SFAHFA levels in relation with pandemic H1N1; PR8 influenza virus will contribute to the in-depth study of their function and metabolism.

Lipidomic analysis of non-esterified furan fatty acids and fatty acid compositions in dietary shellfish and salmon by UHPLC/LTQ-Orbitrap-MS.

Lipids such as furan fatty acids (F-acids) are the valuable minor bioactive components of food such as fatty fish and plants. They are reported to have positive health benefits, including antioxidant and anti-inflammatory activities. Despite their importance, limited studies are focusing on F-acid determination in dietary seafood. This study aimed to identify and profile non-esterified F-acids and free fatty acids in total lipid extract of seafood such as shellfish and salmon. The lipidomic analysis using liquid chromatography-linear trap quadrupole-orbitrap mass spectrometry led to identifying seven types of free F-acids in shellfish (n = 5) and salmon (n = 4). The identified F-acids were confirmed by their high-resolution masses and acquired mass spectra. The relative concentrations of F-acids in shellfish range from 0.01 to 10.93 mg/100 g of the fillet, and in salmon, 0.01 to 14.21 mg/100 g of the fillet. The results revealed the highest abundance of F-acids in Sakhalin surf clam, Japanese scallop, and a fatty salmon trout. Besides, relative levels of saturated, monounsaturated, and polyunsaturated fatty acids (PUFAs) in these seafoods were compared with each other, suggesting basket clams and salmon trout to have significantly higher levels of PUFAs. The dietary seafoods enriched with F-acids and PUFAs may have possible health benefits. Hence, the applied technique could be a promising tool for rapid detection and analysis of non-esterified fatty acids in food.

Chemical Labeling Assisted Detection and Identification of Short Chain Fatty Acid Esters of Hydroxy Fatty Acid in Rat Colon and Cecum Contents.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are novel endogenous lipids with important physiological functions in mammals. We previously identified a new type of FAHFAs, named short-chain fatty acid esterified hydroxy fatty acids (SFAHFAs), with acetyl or propyl esters of hydroxy fatty acids of carbon chains, C ≥ 20. However, sensitive determination of SFAHFAs is still a challenge, due to their high structural similarity and low abundance in biological samples. This study employs one-step chemical derivatization following total lipid extraction using 2-dimethylaminoethylamine (DMED) for enhanced detection of SFAHFAs. The labeled extracts were subjected to ultrahigh performance liquid chromatography coupled to linear ion trap quadrupole-Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap MS). Our results demonstrated that the detection sensitivities of SFAHFAs increased after DMED labeling, and is highly helpful in discovering six additional novel SFAHFAs in the cecum and colon contents of WKAH/HKmSlc rats fed with normal and high-fat diet (HFD). The identified DMED labeled SFAHFAs were characterized by their detailed MS/MS analysis, and their plausible fragmentation patterns were proposed. The concentrations of SFAHFAs were significantly reduced in the cecum of HFD group compared to the control. Hence, the proposed method could be a promising tool to apply for the enhanced detection of SFAHFAs in various biological matrices, which in turn facilitate the understanding of their sources, and physiological functions of these novel lipids.

Discovery of Eicosapentaenoic Acid Esters of Hydroxy Fatty Acids as Potent Nrf2 Activators.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids with anti-inflammatory and anti-diabetic properties. Despite the possible link between endogenous FAHFA levels and nuclear factor erythroid 2-related factor 2 (Nrf2), their possible function as antioxidants and the mechanisms involved in this are unknown. Here, we investigate FAHFAs' plausible antioxidant potential with reference to their effect on the Nrf2 levels, oxidative stress, and lipid droplet oxidation in human hepatocytes (C3A). Six authentic FAHFAs were chemically synthesized and performed activity-based screening by reporter gene assay. Among them, eicosapentaenoic acid (EPA) esterified 12-hydroxy stearic acid (12-HSA) and 12-hydroxy oleic acid (12-HOA) FAHFAs showed less cytotoxicity compared to their free fatty acids and potent activators of Nrf2. To define their mode of action, relative levels of nuclear Nrf2 were determined, which found a higher amount of Nrf2 in nucleus of cells treated with 12-EPAHSA compared to the control. Furthermore, 12-EPAHSA increased the expression of Nrf2-dependent antioxidant enzyme genes (NQO1, GCLM, GCLC, SOD-1, and HO-1). Fluorescence imaging analysis of linoleic-acid-induced lipid droplets (LDs) in C3A cells treated with 12-EPAHSA revealed the strong inhibition of small-size LD oxidation. These results suggest that EPA-derived FAHFAs as a new class of lipids with less cytotoxicity, and strong Nrf2 activators with plausible antioxidant effects via the induction of cytoprotective proteins against oxidative stress, induced cellular damage.

A Simple and Efficient Method for Synthesis of sn-Glycero-Phosphoethanolamine.

An efficient three-step strategy for the convenient synthesis of Sn-glycero-3-phosphoethanolamine (GroPEtn) from a commercially available 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) is reported. Direct hydrolysis of DPPE produces a complex inseparable mixture, hence a protection and deprotection strategy is employed to prepare GroPEtn. The primary amine of DPPE is protected with a highly stable acid-labile trityl group, followed by strong base hydrolysis of N-trityl-DPPE gives N-trityl-GroPEtn. Further a mild, rapid, and efficient deprotection method is established using trifluoroacetic acid to remove N-trityl moiety, affords GroPEtn as a single product. This is the first semisynthetic approach and efficient method to produce GroPEtn with a total yield of 66% in three steps. GroPEtn did not show any cytotoxicity against human kidney (HK-2) cells and reporter gene assay for activation of Keap1-Nrf2-mediated antioxidant defense mechanism showed no significant effects.

Facile determination of sphingolipids under alkali condition using metal-free column by LC-MS/MS.

Extraction and analysis of sphingolipids from biological samples is a critical step in lipidomics, especially for minor species such as sphingoid bases and sphingosine-1-phosphate. Although several liquid chromatography-mass spectrometry methods enabling the determination of sphingolipid molecular species have been reported, they were limited in analytical sensitivity and reproducibility by causing significant peak tailing, especially by the presence of phosphate groups, and most of the extraction techniques are laborious and do not cover a broad range of sphingolipid metabolites. In this study, we developed a rapid single-phase extraction and highly sensitive analytical method for the detection and quantification of sphingolipids (including phosphates) comprehensively using liquid chromatography-triple quadruple mass spectrometry. After validating the reliability of the method, we analyzed the intestinal tissue sphingolipids of germ-free (GF) and specific pathogen-free (SPF) mice and found significantly higher levels of free sphingoid bases and sphingosine-1-phosphate in the GF condition as compared to the SPF condition. This method enables a rapid extraction and highly sensitive determination of sphingolipids comprehensively at low femtomolar ranges. Graphical abstract Diagrammatic comparision of sphingolipid (phosphates) analysis between conventional and this method.