An exposome atlas of serum reveals the risk of chronic diseases in the Chinese population.

Although adverse environmental exposures are considered a major cause of chronic diseases, current studies provide limited information on real-world chemical exposures and related risks. For this study, we collected serum samples from 5696 healthy people and patients, including those with 12 chronic diseases, in China and completed serum biomonitoring including 267 chemicals via gas and liquid chromatography-tandem mass spectrometry. Seventy-four highly frequently detected exposures were used for exposure characterization and risk analysis. The results show that region is the most critical factor influencing human exposure levels, followed by age. Organochlorine pesticides and perfluoroalkyl substances are associated with multiple chronic diseases, and some of them exceed safe ranges. Multi-exposure models reveal significant risk effects of exposure on hyperlipidemia, metabolic syndrome and hyperuricemia. Overall, this study provides a comprehensive human serum exposome atlas and disease risk information, which can guide subsequent in-depth cause-and-effect studies between environmental exposures and human health.

Concentric Hybrid Nanoelectrospray Ionization-Atmospheric Pressure Chemical Ionization Source for High-Coverage Mass Spectrometry Analysis of Single-Cell Metabolomics.

High-coverage mass spectrometry analysis of single-cell metabolomics remains challenging due to the extremely low abundance and wide polarity of metabolites and ultra-small volume in single cells. Herein, a novel concentric hybrid ionization source, nanoelectrospray ionization-atmospheric pressure chemical ionization (nanoESI-APCI), is ingeniously designed to detect polar and nonpolar metabolites simultaneously in single cells. The source is constructed by inserting a pulled glass capillary coaxially into a glass tube that acts as a dielectric barrier layer. Benefitting from the integrated advantages of nanoESI and APCI, its limit of detection is improved by one order of magnitude to 10 pg mL-1 . After the operational parameter optimization, 254 metabolites detected in nanoESI-APCI are tentatively identified from a single cell, and 82 more than those in nanoESI. The developed nanoESI-APCI is successively applied to study the metabolic heterogeneity of human hepatocellular carcinoma tissue microenvironment united with laser capture microdissection (LCM), the discrimination of cancer cell types and subtypes, the metabolic perturbations to glucose starvation in MCF7 cells and the metabolic regulation of cancer stem cells. These results demonstrated that the nanoESI-APCI not only opens a new avenue for high-coverage and high-sensitivity metabolomics analysis of single cell, but also facilitates spatially resolved metabolomics study coupled with LCM.

Z-Ligustilide Combined with Cisplatin Reduces PLPP1-Mediated Phospholipid Synthesis to Impair Cisplatin Resistance in Lung Cancer.

Lung cancer is a malignant tumor with one of the highest morbidity and mortality rates in the world. Approximately 80-85% of lung cancer is diagnosed as non-small lung cancer (NSCLC), and its 5-year survival rate is only 21%. Cisplatin is a commonly used chemotherapy drug for the treatment of NSCLC. Its efficacy is often limited by the development of drug resistance after long-term treatment. Therefore, determining how to overcome cisplatin resistance, enhancing the sensitivity of cancer cells to cisplatin, and developing new therapeutic strategies are urgent clinical problems. Z-ligustilide is the main active ingredient of the Chinese medicine Angelica sinensis, and has anti-tumor activity. In the present study, we investigated the effect of the combination of Z-ligustilide and cisplatin (Z-ligustilide+cisplatin) on the resistance of cisplatin-resistant lung cancer cells and its mechanism of action. We found that Z-ligustilide+cisplatin decreased the cell viability, induced cell cycle arrest, and promoted the cell apoptosis of cisplatin-resistant lung cancer cells. Metabolomics combined with transcriptomics revealed that Z-ligustilide+cisplatin inhibited phospholipid synthesis by upregulating the expression of phospholipid phosphatase 1 (PLPP1). A further study showed that PLPP1 expression was positively correlated with good prognosis, whereas the knockdown of PLPP1 abolished the effects of Z-ligustilide+cisplatin on cell cycle and apoptosis. Specifically, Z-ligustilide+cisplatin inhibited the activation of protein kinase B (AKT) by reducing the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3). Z-ligustilide+cisplatin induced cell cycle arrest and promoted the cell apoptosis of cisplatin-resistant lung cancer cells by inhibiting PLPP1-mediated phospholipid synthesis. Our findings demonstrate that the combination of Z-Ligustilide and cisplatin is a promising approach to the chemotherapy of malignant tumors that are resistant to cisplatin.

Carbon Quantum Dots Derived from Herbal Medicine as Therapeutic Nanoagents for Rheumatoid Arthritis with Ultrahigh Lubrication and Anti-inflammation.

As a typical chronic inflammatory joint disease with swelling and pain syndromes, rheumatoid arthritis (RA) is closely related to articular lubrication deficiency and excessive proinflammatory cytokines in its progression and pathogenesis. Herein, inspired by the dual effects of joint lubrication improvement and anti-inflammation to treat RA, two novel potential therapeutic nanoagents have been developed rationally by employing herbal medicine-derived carbon quantum dots (CQDs), i.e., safflower (Carthamus tinctorius L.) CQDs and Angelica sinensis CQDs, yielding ultrahigh lubrication and anti-inflammation bioefficacy. In vitro experimental results show that the two nanoagents display excellent friction reduction due to their good water solubility and spherical structure. Using RA rat models, it is indicated that the nanoagents significantly relieved swelling symptoms and inhibited the expression of related inflammatory cytokines, including IL-1, IL-6, and TNF-α, indicating their extraordinary anti-inflammation bioefficacy. Thus, combining the lubricating and anti-inflammation bioefficacy of CQDs derived from herbal medicine is an attractive strategy to develop new nanoagents for RA treatment.

Screening strategy for 1210 exogenous chemicals in serum by two-dimensional liquid chromatography-mass spectrometry.

Humans are at risk of exogenous exposure to exogenous chemicals. Challenges exist for the comprehensive monitoring of residues with different physical and chemical properties in serum. Here, an on-line two-dimensional liquid chromatography (2D-LC) - high resolution mass spectrometry system (HRMS) was developed, expanding the range of the partition coefficient in octanol/water of the residue analysis from -8 to 12. A high-coverage serum residue screening strategy was further designed by integrating 2D-LC system with HRMS full MS/data independent acquisition and automatic spectral library searching. This strategy enables to simultaneously screen 1210 pesticides, veterinary/human drugs, other chemical pollutants and their metabolites in serum with a single analysis. Method validation showed 92% and 81% of 1022 residues spiked in serum could be detected at 50 ng/mL and 5 ng/mL, respectively. The developed method was applied to the analysis of 24 separately pooled serum samples, 58 suspect residues were found, some of them were detected at high frequencies over than 50%. Among them, 4,6-Dinitro-O-cresol and probable carcinogenic folpet are highly toxic, and cimaterol is banned in China. Collectively, this study developed a 2D-LC-HRMS -based screening strategy for screening pesticides, veterinary/human drugs, and other chemical pollutants in serum, it is helpful for studying the effect of exogenous exposures on human health.

Steroid profiling for the diagnosis of congenital adrenal hyperplasia by microbore ultra-performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: A rapid and accurate measurement approach for 17α-hydroxyprogesterone (17-OHP) and related steroids in amount/volume-limited clinic samples is of importance for precise newborn diagnosis of congenital adrenal hyperplasia (CAH) and its subtypes in clinic. METHODS: Sixteen steroids (17-OHP, androstenedione, cortisol, tetrahydro-11-deoxycortisol, pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, 21-deoxycortisol, 11-deoxycortisol, dehydroepiandrosterone, testosterone, aldosterone, 17α-hydroxypregnenolone, dihydrotestosterone and 18-hydroxycorticosterone) were included in the panel of high-throughput microbore ultra-performance liquid chromatography-tandem mass spectrometry. Samples were collected from 126 normal subjects and 65 patients including different subtypes of CAH. RESULTS: The method was validated with satisfactory analytical performance in linearity, repeatability, recovery and limit of detection. Reference intervals for 16 steroids were established by quantifying the level of steroids detected in normal infants. The applicability of the method was tested by differentiating steroid metabolic characteristics between normal infants and infants with CAH, as well as between infants with different CAH subtypes. The relevance of 17-OHP, 21-deoxycortisol, and 17-OHP/11-deoxycortisol for 21-hydroxylase deficiency screening was demonstrated. The level of 11-deoxycorticosterone, 11-deoxycortisol, progesterone and androstenedione can be used for the diagnosis of different rare subtypes of CAH. CONCLUSION: This study provides a strategy for highly efficient steroid analysis of amount/volume-limited clinic samples and holds great potential for clinical diagnosis of CAH.

High-throughput single cell metabolomics and cellular heterogeneity exploration by inertial microfluidics coupled with pulsed electric field-induced electrospray ionization-high resolution mass spectrometry.

Single cell metabolomics can obtain the metabolic profiles of individual cells and reveal cellular heterogeneity. However, high-throughput single-cell mass spectrometry (MS) analysis under physiological conditions remains a great challenge due to the presence of complex matrix and extremely small cell volumes. Herein, a serpentine channel microfluidic device which was designed to achieve continuous cell separation and inertial focusing, was coupled with a pulsed electric field-induced electrospray ionization-high resolution MS (PEF-ESI-HRMS) to achieve high-throughput single cell analysis. The pulsed square wave electric field was applied to realize on-line cell disruption and induce electrospray ionization. Single cells were analyzed under near-physiological conditions at a throughput of up to 80 cells min-1. More than 900 features were detected and approximately 120 metabolites were tentatively identified from a single cell. Further, by continually analyzing more than 3000 MCF7 and HepG2 cells, discrimination of different cancer cells based on their individual metabolic profiles was achieved by using the principal component analysis. The PEF-ESI-HRMS method was also applied for the analysis of single yeast cells, and more than 40 metabolites were annotated. This method is versatile and has good robustness, which is promising for high-throughput single cell metabolomics analysis.

Medium-chain triglyceride production in Nannochloropsis via a fatty acid chain length discriminating mechanism.

Depending on their fatty acid (FA) chain length, triacylglycerols (TAGs) have distinct applications; thus, a feedstock with a genetically designed chain length is desirable to maximize process efficiency and product versatility. Here, ex vivo, in vitro, and in vivo profiling of the large set of type-2 diacylglycerol acyltransferases (NoDGAT2s) in the industrial oleaginous microalga Nannochloropsis oceanica revealed two endoplasmic reticulum-localized enzymes that can assemble medium-chain FAs (MCFAs) with 8-12 carbons into TAGs. Specifically, NoDGAT2D serves as a generalist that assembles C8-C18 FAs into TAG, whereas NoDGAT2H is a specialist that incorporates only MCFAs into TAG. Based on such specialization, stacking of NoDGAT2D with MCFA- or diacylglycerol-supplying enzymes or regulators, including rationally engineering Cuphea palustris acyl carrier protein thioesterase, Cocos nucifera lysophosphatidic acid acyltransferase, and Arabidopsis thaliana WRINKLED1, elevated the medium-chain triacylglycerol (MCT) share in total TAG 66-fold and MCT productivity 64.8-fold at the peak phase of oil production. Such functional specialization of NoDGAT2s in the chain length of substrates and products reveals a dimension of control in the cellular TAG profile, which can be exploited for producing designer oils in microalgae.

Comprehensive metabolite quantitative assay based on alternate metabolomics and lipidomics analyses.

Metabolomics-based precision medicine is facing several obstacles including cross-platform data comparison issue and the lack of metabolome benchmark values of healthy population, one of main reasons is the shortage of comprehensive metabolome quantitation methods. Here, we developed an alternate reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) method to quantitatively determine metabolites and lipids. Assisted by a wide set of reference standards and real samples, up to 397 multiple reaction monitoring (MRM) transitions (239 for positive and 158 for negative ion modes) and 1080 MRM transitions (607 for positive and 473 for negative ion modes) were defined respectively in the metabolomic and lipidomic analyses with more than 1000 metabolites and lipids being quantified. Among them, 144 analytes including amines, amino acids, benzenoids, peptides, nucleobases and related, bile acids, carboxylic acids, fatty acids, hormones, indoles and others were absolutely quantified, while carnitines, lyso-phosphatidylcholines, lyso-phosphatidylethanolamines, free fatty acids, sphingomyelins, phosphatidylcholines (PCs), alkyl and alkenyl substituted PCs, phosphatidylethanolamines (PEs), alkyl and alkenyl substituted PEs and triacylglycerols were semiquantified. The developed method was validated to have good analytical characteristics. Analytical results of standard reference material 1950 human plasma had a good agreement with literature data. As a proof of application, this method was used to study serum metabolic pattern changes of patients with hyperuricemia and nonalcoholic fatty liver. This alternate RPLC-MS method for quantitative metabolites and lipids analysis can further be used to provide technology and large-scale data support for precision medicine and life sciences.

[Advances in application of molecularly imprinted polymers to the detection of polar pesticide residues].

Polar pesticides can be primarily classified as fungicides, herbicides, and insecticides; their rich variety and low cost have led to their extensive utilization in agriculture. However, the overuse of polar pesticides can lead to environmental contamination, such as water or soil pollution, which can also increase the risk of pesticide exposure among human life directly, or indirectly through contact with animal and plant-derived food. There are considerable differences in the physical and chemical properties of polar pesticides, as well as their trace amounts in complex food and environmental samples, posing immense challenges to their accurate detection. As a kind of artificially prepared selective adsorbent, molecularly imprinted polymers (MIPs) possess specific recognition sites complementary to template molecules in terms of the spatial structure, size, and chemical functional groups. With many advantages such as easy preparation, low cost, as well as good chemical and mechanical stability, MIPs have been widely applied in sample pretreatment and the analysis of polar pesticide residues. MIPs are typically used as adsorption materials in solid phase extraction (SPE) methods, including magnetic solid phase extraction (MSPE), dispersed solid phase extraction (DSPE), and stir bar sorptive extraction (SBSE). To rapidly detect polar pesticide residues with high sensitivity, MIPs are also used in the preparation of fluorescent sensors and electrochemical sensors. Furthermore, MIPs can be employed as the substrate in surface-enhanced Raman spectroscopy and as the substrate for the ion source in mass spectrometry for polar pesticide residue analysis. Thus far, various molecularly imprinted materials have been reported for the efficient separation and analysis of polar pesticide residues in various complex matrices. However, there is no review that summarizes the recent advances in MIPs for the determination of polar pesticides. This review introduces imprinting strategies and polymerization methods for MIPs, and briefly summarizes some new molecular imprinting strategies and preparation technologies. The application of MIPs in recent years (particularly the last five years) to the detection of polar pesticide residues including neonicotinoids, organophosphorus, triazines, azoles, and urea is then systematically summarized. Finally, the future development direction and trends for MIPs are proposed considering existing challenges, with the aim of providing reference to guide future research on MIPs in the field of polar pesticide residue detection.

Facile Synthesis of Antibody-Coupled Polydopamine-Coated Magnetic Graphene Oxide Composites for Efficient Immunopurification and Metabolomics Analysis of Mitochondria.

As a vital hub, a mitochondrion houses metabolic pathways that play important roles in cellular physiology. Aberrant metabolites occurring in mitochondria are closely associated with the emergence and progression of various mitochondria-related diseases. Therefore, a simple and versatile approach to efficiently purify intact mitochondria is urgently needed to precisely and comprehensively characterize the composition and abundance of the mitochondrial metabolome in different physiological and pathological states. In this work, novel immunoaffinitive magnetic composites MagG@PD@Avidin@TOM20 were prepared to achieve highly selective isolation of intact mitochondria from three different hepatocytes (LO2, HepG2, and Huh7). The prepared composites inherit combined merits, including strong magnetic responsiveness, excellent stability, and specific and high affinity between antibody TOM20 and mitochondrial outer membrane protein. These mitochondria attached on MagG@PD@Avidin@TOM20 were characterized by the western blot and fluorescence microscopy to confirm their purity and integrity, which are vital for reliable mitochondrial metabolic analysis. Subsequently, ultrahigh-performance liquid chromatography-high-resolution mass spectrometry-based untargeted metabolomics analysis was conducted to characterize the metabolomes in the immunopurified mitochondria and whole cells. Notably, the metabolite profiles of whole cells and mitochondria including itaconic acid, acetylcarnitine, malic acid, etc., were significantly different. These data underscore the importance of determining metabolites at the mitochondrial level, which would supplement us new knowledge at the subcellular level.

Lipid Profiling of 20 Mammalian Cells by Capillary Microsampling Combined with High-Resolution Spectral Stitching Nanoelectrospray Ionization Direct-Infusion Mass Spectrometry.

Studies of cellular metabolism can provide profound insights into the underlying molecular mechanisms and metabolic function. To date, the majority of cellular metabolism studies based on chromatography-mass spectrometry (MS) require population cells to obtain informative metabolome. These methods are not only time-consuming but also not suitable for amount-limited cell samples such as circulating tumor cells, stem cells, and neurons. Therefore, it is extremely essential to develop analytical methods enabling to detect metabolome from tens of cells in a high-throughput and high-sensitivity way. In this work, a novel platform for rapid and sensitive detection of lipidome in 20 mammalian cells was proposed using capillary microsampling combined with high-resolution spectral stitching nanoelectrospray ionization direct-infusion MS. It can be used to collect cells rapidly and accurately via the capillary microprobe, extract lipids directly in a 96-well plate using a spray solvent, and detect more than 500 lipids covering 19 lipid subclasses within 3 min. This novel platform was successfully applied to study the lipid features of different cancer cell types and subtypes as well as target cells from tissue samples. This study provides a strategy for determining the lipid species with rich information in tens of cells and demonstrates great potential for clinical applications.

Recent development of nanoparticle-assisted metabolites analysis with mass spectrometry.

Metabolomics systematically studies the changes of metabolites in biological systems in the temporal or spatial dimensions. It is a challenging task for comprehensive analysis of metabolomics because of diverse physicochemical properties and wide concentration distribution of metabolites. Used as enrichment sorbents, chemoselective probes, chromatographic stationary phases, MS ionization matrix, nanomaterials play excellent roles in improving the selectivity, separation performance, detection sensitivity and identification efficiency of metabolites when mass spectrometry is employed as the detection technique. This review summarized the recent development of nanoparticle-assisted metabolites analysis in terms of assisting the pretreatment of biological samples, improving the separation performance and enhancing the MALDI-MS detection.

Rational Synthesis of Aptamer-Functionalized Polyethylenimine-Modified Magnetic Graphene Oxide Composites for Highly Efficient Enrichment and Comprehensive Metabolomics Analysis of Exosomes.

Exosomes, which are phospholipid bilayer nanovesicles, can transfer their content to recipient cells, playing a crucial role in intercellular communication. Exosomes have emerged as promising cancer biomarkers. However, a convenient, efficient, and economical approach for their isolation and comprehensive analysis is still technically challenging. In this study, aptamer-based immunoaffinitive magnetic composites, MagG@PEI@DSP@aptamer, were prepared to achieve the convenient capture, efficient enrichment, and mild release of exosomes. The constructed composites contain three segments: a PEI-modified magnetic graphene scaffold, an aptamer CD63 sequence, and a cleavable cross-linker in between. Notably, the binding capacity of MagG@PEI@DSP for an aptamer is 93 nmol/mg, and per milligram MagG@PEI@DSP@aptamer could capture 450 µg exosomes. Moreover, the released exosomes from MagG@PEI@DSP@aptamer composites were intact and well-dispersed. The prepared composites were then applied to profile the metabolite composition of exosomes secreted by breast cancer cells MCF-7, and the number of detected features was obviously increased when compared to that obtained by the traditional ultracentrifugation method (4528 vs 3710 and 3967 vs 3785 in the positive and negative ionization modes). Besides, the exosomes secreted by MCF-7 and normal breast cells MCF-10A were isolated from cell culture medium with MagG@PEI@DSP@aptamer, and their metabolic profiles were then comprehensively analyzed; in total, 119 metabolites in MCF-7 and MCF-10A were identified. Compared with exosomes from MCF-10A, 43 and 42 metabolites were upregulated and downregulated, respectively, in those from MCF-7. These data showed that the prepared MagG@PEI@DSP@aptamer composites can be used to effectively capture exosomes and further for metabolomics analysis.

Alternate reversed-phase and hydrophilic interaction liquid chromatography coupled with mass spectrometry for broad coverage in metabolomics analysis.

Because of the greatly different physicochemical properties of metabolites, comprehensive metabolite profiling analysis has always been a challenging task. Reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) have been used to the analysis of nonpolar metabolites and polar metabolites, respectively. In this work, an alternate HILIC/RPLC-mass spectrometry (MS) approach was developed for the comprehensive and high-throughput analysis of polar and nonpolar metabolites. HILIC and RPLC are respectively performed on two ultra-high performance LC (UHPLC) systems, and coupled to one mass spectrometer to acquire the data. When HILIC gradient elution is running RPLC is in a washing and equilibration state, and vice versa. As a result, the total analysis time was reduced by about one third to 25.4 min. Two hundred and eight representative standards including at least twelve types of commonly met metabolites, SRM 1950 plasma, serum, urine and liver tissue samples were used to test the established alternate HILIC/RPLC-MS method. The results demonstrated that the method possessed high metabolite coverage. The developed method was validated to have good linearity and repeatability. As an example of application, 61 significantly changed metabolites in the colon cancer tissues were defined by this established method.

Synthesis of metal-organic framework-5@chitosan material for the analysis of microcystins and nodularin based on ultra-performance liquid chromatography-tandem mass spectrometry.

Microcystins (MCs) and nodularin (NOD) are tumor promoters produced by cyanobacteria and present in surface water. In this work, a novel mesoporous metal-organic framework-5@chitosan (MOF-5@CS) material was synthesized and applied for the enrichment of MCs and NOD in water and fish samples. The mesoporous MOF-5@CS material was firstly synthesized via a one-step hydrothermal method, and the chitosan was combined with MOF-5 via chemical bonding assembly. As a new adsorbent, the as-synthesized material was found having a large specific surface area and good thermal stability. Under the optimized conditions, MCs and NOD were enriched by the MOF-5@CS material and detected by ultra-performance liquid chromatography-tandem mass spectrometry. The limit of detection of the new method for MCs and NOD were in the range of 0.0018-0.077 ng/mL. The value of relative standard deviation for repeatability were 2.69-6.30%, and the recovery of the analytes ranged from 84.36% to 118.51%. Compared with other reported method for MCs and NOD detection in complex matrices, better adsorption performance for MCs and NOD were obtained by our new method, and the sensitivity of MCs-RR and NOD were improved nearly 20 times and 30 times, respectively.

Untargeted Defining Protein-Metabolites Interaction Based on Label-Free Kinetic Size Exclusion Chromatography-Mass Spectrometry.

The specific interactions between protein and metabolites (PMIs) are closely related to many cellular processes and play a vital role in signal transduction and regulating material and energy metabolism. However, most of the available analytical strategies for PMIs involve chemical modification of metabolites or immobilization of protein, which has restricted current PMIs study mainly to lipid-protein and hydrophobic metabolites. In this work, a label-free online kinetic size exclusion chromatography-mass spectrometry (KSEC-MS) method combined with untargeted metabolomics was developed to define PMIs in a complex system. The metabolite mixture and target protein were injected into the SEC column sequentially without preincubation, and the separation results of KSEC were monitored by global metabolite profiling with mass spectrometry. The potential ligands in the metabolite mixture can be discovered if their migration patterns were affected by the target protein and the variation was positively correlated with the concentration of target protein. To verify this approach, carbonic anhydrase was first selected as a test protein, and acetazolamide as its known inhibitor was successfully defined. Furthermore, human serum albumin (HSA) as the common transport carrier of metabolites was selected as a target protein to demonstrate the usefulness of this approach. Multiple endogenous ligands of HSA were simultaneously defined from the extracted metabolites of human serum; most of them are polar metabolites rather than nonpolar lipids. This approach can provide a novel way for mapping and identifying unknown PMIs in a complex system, especially for polar metabolites-protein interactions.

Pseudotargeted Method Based on Parallel Column Two-Dimensional Liquid Chromatography-Mass Spectrometry for Broad Coverage of Metabolome and Lipidome.

Metabolite and lipid profilings usually need two liquid chromatography-mass spectrometry (LC-MS) methods because of a great polarity difference. A pseudotargeted metabolomics method as a novel emerging approach can integrate the advantages of nontargeted and targeted methods. Here, we aim to establish a comprehensive method for metabolome and lipidome by using a parallel column-based two-dimensional LC (PC-2DLC)-MS and pseudotargeted approach. To simultaneously extract as many polar metabolites and nonpolar lipids as possible, we systematically optimized the sample pretreatment process, and isopropanol/methanol (3:1, v/v) and isopropanol/water (7:3, v/v) were selected as the extraction and reconstitution solvents, respectively. The detected triglycerides significantly increased after the sample pretreatment optimization. Then PC-2DLC coupled with Triple TOF MS was applied to analyze a mixed sample from serum, urine, and liver tissue matrixes. The multiple reaction monitoring (MRM) transitions of the metabolome and lipidome were defined according to the "MRM-Ion Pair Finder" software and lipidomics MRM-transition database, respectively. After verification by QTRAP MS in the scheduled MRM mode, 1609 potential metabolites and lipids corresponding to 1294 MRM transitions, and 847 potential metabolites and lipids corresponding to 687 MRM transitions were detected in positive and negative ion modes, respectively. They range at about 30 orders of magnitude in octanol/water partition coefficient. The pseudotargeted 2DLC-MS method was validated to have good analytical characteristics. As a proof of applicability, sera from type 2 diabetic patients were investigated by the established method. The results indicated that the pseudotargeted 2DLC-MS method is reliable and repeatable and can be used in a metabolomics study.

Parallel derivatization strategy coupled with liquid chromatography-mass spectrometry for broad coverage of steroid hormones.

Steroid hormones are a type of crucial substances that mediate numerous vital physiological functions. The comprehensive detection of steroid hormones can help understand the physiopathologic mechanism of steroid hormone-related diseases. It is very difficult to determine steroid hormones in biological samples due to their low endogenous concentrations and poor ionization efficiency. In this study, an efficient and sensitive approach was developed for profiling steroid hormones by combining liquid-liquid extraction and parallel derivatization with liquid chromatography-tandem mass spectrometry. Methoxyamine and dansyl chloride were used to derivatize steroid hormones containing carbonyl and phenolic hydroxyl groups, respectively. Our established method achieved simultaneous analysis of carbonyl and phenolic hydroxyl-containing steroid hormones and could cover estrogens, androgens, corticoids and progestogens. Twenty-nine steroid hormones were detected at pg/mL levels with the sensitivity enhanced by three orders of magnitude after derivatization. The linearity (with linear range of 2-4 orders of magnitude), precision (less than 15%) and recovery (71.1-128.7%) were satisfactory for quantitative analysis of steroid hormones. Finally, the established method was successfully employed to the determination of steroid hormones in serum samples of healthy males and females as well as ovarian cancer patients. The results showed that this approach was suitable and reliable for routine test of steroid hormones containing carbonyl and phenolic hydroxyl groups.