Internal Extractive Electrospray Ionization Mass Research Articles

Machine Learning-Based Integrated Multiomics Characterization of Colorectal Cancer Reveals Distinctive Metabolic Signatures.

The metabolic signature identification of colorectal cancer is critical for its early diagnosis and therapeutic approaches that will significantly block cancer progression and improve patient survival. Here, we combined an untargeted metabolic analysis strategy based on internal extractive electrospray ionization mass spectrometry and the machine learning approach to analyze metabolites in 173 pairs of cancer samples and matched normal tissue samples to build robust metabolic signature models for diagnostic purposes. Screening and independent validation of metabolic signatures from colorectal cancers via machine learning methods (Logistic Regression_L1 for feature selection and eXtreme Gradient Boosting for classification) was performed to generate a panel of seven signatures with good diagnostic performance (the accuracy of 87.74%, sensitivity of 85.82%, and specificity of 89.66%). Moreover, seven signatures were evaluated according to their ability to distinguish between cancer and normal tissues, with the metabolic molecule PC (30:0) showing good diagnostic performance. In addition, genes associated with PC (30:0) were identified by multiomics analysis (combining metabolic data with transcriptomic data analysis) and our results showed that PC (30:0) could promote the proliferation of colorectal cancer cell SW480, revealing the correlation between genetic changes and metabolic dysregulation in cancer. Overall, our results reveal potential determinants affecting metabolite dysregulation, paving the way for a mechanistic understanding of altered tissue metabolites in colorectal cancer and design interventions for manipulating the levels of circulating metabolites.

Combination of Internal Extractive Electrospray Ionization Mass Spectrometry and Statistical Analysis for High-Throughput Molecular Differentiation of Rehmannia glutinosa Samples.

Rehmannia glutinosa (Gaert.) Libosch. ex Fisch. et Mey. is a perennial herb of the Scrophulariaceae family, which has long enjoyed a good reputation in China, and has a wide range of pharmacological effects and clinical applications. The place of origin is an important factor affecting the chemical composition of R. glutinosa, resulting in different pharmacological effects. Herein, internal extractive electrospray ionization mass spectrometry (iEESI-MS) combined with statistical techniques was established for high-throughput molecular differentiation of different R. glutinosa samples. Dried and processed R. glutinosa samples from four different places of origin were analyzed by iEESI-MS with high throughput (>200 peaks) and rapidness (<2 min/sample) without sample pretreatment. Clear separation models created by OPLS-DA were then established for distinguishing the places of origin of dried and processed R. glutinosa by using the obtained MS data. In addition, the molecular differences between the pharmacological effects of dried and processed R. glutinosa were also investigated by OPLS-DA, and 31 different components were screened out. This work provides a promising method for evaluating the quality of traditional Chinese medicines and studying the biochemical mechanism of processing.

Liver Metabolic Dysregulation Induced by Polypropylene Nano- and Microplastics in Nile Tilapia Using Internal Extractive Electrospray Ionization Mass Spectrometry.

Understanding the metabolic disorders induced by nano- and microplastics in aquatic organisms at the molecular level could help us understand the potential toxicity of nano- and microplastics more thoroughly and provide a fundamental scientific basis for regulating the usage and management of plastic products. In this research, the effect of polypropylene nanoplastics (PP-NPs) and microplastics (PP-MPs) on metabolites in the tilapia liver was comprehensively investigated by internal extractive electrospray ionization mass spectrometry (iEESI-MS). A partial least-squares discriminant analysis (PLS-DA) and a one-component analysis of variance (ANOVA) were used for selecting 46 differential metabolites, including phospholipids, amino acids, peptides, carbohydrates, alkaloids, purines, pyrimidines, and nucleosides. Pathway enrichment analysis showed significant effects on glycerophospholipid metabolism, arginine and proline metabolism, and aminoacyl-tRNA biosynthesis after tilapia were exposed to PP-N/MPs. Dysregulation of these metabolites is mainly reflected in the possible induction of hepatitis, oxidative stress, and other symptoms. The application of iEESI-MS technology without sample pretreatment to the study of metabolic disorders in aquatic organisms under the interference of nano- and microplastics provides a promising analytical method for environmental toxicology research.

Metabolite Fingerprinting for Identification of Panax ginseng Metabolites Using Internal Extractive Electrospray Ionization Mass Spectrometry.

Ginseng, a kind of functional food and medicine with high nutritional value, contains various pharmacological metabolites that influence human metabolic functions. Therefore, it is very important to analyze the composition and metabolites of ginseng. However, the analysis of active metabolites in ginseng samples usually involves various experimental steps, such as extraction, chromatographic separation, and characterization, which may be time-consuming and laborious. In this study, an internal extractive electrospray ionization mass spectrometry (iEESI-MS) method was developed to analyze active metabolites in ginseng samples with sequential sampling and no pretreatment. A total of 44 metabolites, with 32 ginsenosides, 6 sugars, and 6 organic acids, were identified in the ginseng samples. The orthogonal partial least-squares discriminant analysis (OPLS-DA) score plot showed a clear separation of ginseng samples from different origins, indicating that metabolic changes occurred under different growing conditions. This study demonstrated that different cultivation conditions of ginseng can be successfully discriminated when using iEESI-MS-based metabolite fingerprints, which provide an alternative solution for the quality identification of plant drugs.

Highly sensitive quantification of ultratrace As(V) via iEESI-MS.

Arsenate (As(V)) is a fatal poison and is widely distributed in the environment. The quick and accurate determination of As(V) is of great importance. Herein, we developed a novel competitive coordination strategy for the quantification of ultratrace As(V) using online internal extractive electrospray ionization mass spectrometry (iEESI-MS). Our strategy demonstrates great success for direct ultratrace As(V) detection in broad types of samples, from solids to liquids, such as food, water and biological samples.

Metabolic-Dysregulation-Based iEESI-MS Reveals Potential Biomarkers Associated with Early-Stage and Progressive Colorectal Cancer.

The application of rapid and accurate diagnostic methods can improve colorectal cancer (CRC) survival rates dramatically. Here, we used a non-targeted metabolic analysis strategy based on internal extractive electrospray ionization mass spectrometry (iEESI-MS) to detect metabolite ions associated with the progression of CRC from 172 tissues (45 stage I/II CRC, 41 stage III/IV CRC, and 86 well-matched normal tissues). A support vector machine (SVM) model based on 10 differential metabolite ions for differentiating early-stage CRC from normal tissues was built with a good prediction accuracy of 92.6%. The biomarker panel consisting of lysophosphatidylcholine (LPC) (18:0) has good diagnostic potential in differentiating early-stage CRC from advanced-stage CRC. We showed that the down-regulation of LPC (18:0) in tumor tissues is associated with CRC progression and related to the regulation of the epidermal growth factor receptor. Pathway analysis showed that metabolic pathways in CRC are related to glycerophospholipid metabolism and purine metabolism. In conclusion, we built an SVM model with good performance to distinguish between early-stage CRC and normal groups based on iEESI-MS and found that LPC (18:0) is associated with the progression of CRC.

Internal extractive electrospray ionization mass spectrometry for investigating the phospholipid dysregulation induced by perfluorooctanoic acid in Nile tilapia.

Direct profiling of endogenous biomolecules in tissue samples is considered as a promising approach to investigate metabolic-related toxicity in organisms induced by emerging pollutants. Herein, we report the application of internal extractive electrospray ionization mass spectrometry (iEESI-MS) to direct phospholipid profiling in the liver and spleen tissues of Nile tilapia exposed to perfluorooctanoic acid (PFOA). Combining positive and negative ion detection modes, 130 phospholipid signals were directly detected and identified by iEESI-MS in the tissues of Nile tilapia, including phosphatidyl cholines (PCs), sphingomyelins (SMs), phosphatidic acids (PAs), phosphatidyl ethanolamines (PEs), phosphatidyl glycerols (PGs), phosphatidyl inositols (PIs) and phosphatidyl serines (PSs). With the help of partial least squares discriminant analysis (PLS-DA) and one-way analysis of variance (ANOVA), several phospholipid signals showed a significant difference in the tissue of Nile tilapia between the control group and PFOA exposure groups. In addition, pathway analysis revealed that PFOA has a significant metabolic impact on the glycerophospholipid metabolism in Nile tilapia. Without complex sample preparation, iEESI-MS was applied to direct phospholipid profiling in the liver and spleen tissues of Nile tilapia treated with PFOA, which provided a promising methodology for investigating environmental toxicity and phospholipid-dysregulation caused by emerging pollutants in aquatic organisms.

Molecular differentiation of Panax notoginseng grown under different conditions by internal extractive electrospray ionization mass spectrometry and multivariate analysis

Panax notoginseng is a highly valuable and widely used herb in traditional Chinese medicine. The quality and efficacy of Panax notoginseng grown under different conditions can greatly vary due to the differences in chemical composition. The analysis of chemical composition in Panax notoginseng typically involves various experimental steps including extraction, chromatographic separation and characterization, which can be time- and labor-consuming. Therefore, the efficient quality assessment and control of Panax notoginseng requires the development of more rapid methods for the chemical characterization and classification of Panax notoginseng. In this study, a method based on internal extractive electrospray ionization mass spectrometry (iEESI-MS) was developed to characterize chemical components of Panax notoginseng samples under different growth conditions (e.g., place of origin, soil quality, growth season) at the speed of 0.5 min per sample, without sample pretreatment and chromatographic separation. A total of 35 chemical components, including sugars, saponins, organic acids, etc., were identified in Panax notoginseng samples. Clear separation was observed in the multivariate analysis of the iEESI-MS data from Panax notoginseng samples grown under different conditions. The difference in the content of sucrose, fructose, Rg1, Rf, Rb1, Noto-R1, malonyl-Rb1, malonyl-Rg1, malonyl-Rf, Rd, Re, linoleic acid, palmitic acid and malic acid can be used as key characteristic indicators to discriminate origin, commercial specifications, and cultivation conditions of Panax notoginseng samples. The results of our study indicate the high power of iEESI-MS for the rapid molecular characterization and classification of Panax notoginseng under different growth conditions, which can be used for the quality assessment of traditional herbal medicines as well as in pharmaceutical and clinical analysis.

Evaluation of the phytotoxicity of nano-particles on mung beans by internal extractive electrospray ionization mass spectrometry.

The wide application of nano-particles (NPs) has raised a serious concern over their impact on plants. However, evaluation of the effects of NPs on plant metabolism by direct detection of chemicals inside solid tissues presents a challenge. In this study, we report on a direct ionization method in mass spectrometry, internal extractive electrospray ionization (iEESI), for the direct evaluation of phytotoxicity of three different NPs (including CdTe quantum dots (CdTe QDs), gold nano-particles (Au NPs), and silver nano-particles (Ag NPs)) both on surfaces and inside solid tissues from the mung bean seeds (Vigna radiata) that were cultured in aqueous solutions of three NPs at 50 μg mL-1. The results showed that NPs could stimulate the biological accumulation of trigonelline and the decomposition of polysaccharides/oligosaccharides to glucose and maltose within 21 h of culture. To the best of our knowledge, this is the first study to apply internal extractive electrospray ionization mass spectrometry (iEESI-MS) for the direct measurement of solid tissue samples to evaluate the phytotoxicity of NPs on mung bean sprouts. Our study lays a solid foundation for further examination of other NPs-induced damaging effects such as apoptosis/necrosis, helping us to understand the phytotoxicity of NPs on plants.

Direct analysis of metabolites in the liver tissue of zebrafish exposed to fiproles by internal extractive electrospray ionization mass spectrometry.

Exploring the metabolic disturbance of fipronil and its derivatives in aquatic organisms may provide a more comprehensive understanding of the impact of fipronil on the ecological environment. In this work, internal extractive electrospray ionization mass spectrometry (iEESI-MS) was used to directly analyze metabolites in the liver tissue of zebrafish exposed to fipronil and its three derivatives. Partial least squares-discriminant analysis (PLS-DA) revealed that 32 signals were considered as differential signals in zebrafish after the exposure treatment of fipronil and its derivatives, including phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs), phosphatidylethanolamines (PEs), fatty acids and so on. The pathway analysis result showed that both fipronil and its derivatives have a significant impact on the glycerophospholipid metabolism of zebrafish. Besides, the intensities of PC signals in the liver samples of each group showed such a trend: mixed fiprole exposed group > fipronil sulfone exposed group ≈ fipronil sulfide exposed group > fipronil exposed group > fipronil desulfinyl exposed group > control group, indicating that mixed exposure of fipronil and its derivatives exhibited more significant metabolic disturbance in zebrafish. Taken together, iEESI-MS is applied to environmental toxicology and investigating the metabolic disturbance induced by fipronil and its derivatives in aquatic organisms, providing a new analytical method for this field.

Quantification of Trace Organophosphorus Pesticides in Environmental Water via Enrichment by Magnetic-Zirconia Nanocomposites and Online Extractive Electrospray Ionization Mass Spectrometry.

A total of 15 representative organophosphorus pesticides (OPPs), a class of water pollutants causing serious global concerns, have been sensitively quantified by internal extractive electrospray ionization mass spectrometry (iEESI-MS) after enrichment with Fe3O4-ZrO2 innovatively synthesized in our lab by a one step coprecipitation method. For the premium enrichment, the amounts of Fe3O4-ZrO2, pH value, adsorption time, type and volume of desorption solvent, and shaking time of desorption were systematically optimized. Under optimized conditions, the proposed method provided low limits of detection (LODs) of 0.14-16.39 ng L-1 with relative standard deviations (RSDs) of less than 8.7%. A wide linear response range of about 4 orders of magnitude was achieved with linear coefficients (R2) of 0.9921-0.9999 for all the analytes tested. The present method also provided good recoveries (85.4-105.9%) with acceptable precision (RSDs < 7.2%) in spiked environmental water samples. Furthermore, multiple analytes including dimethoate, omethoate, etc. were simultaneously detected in a single sample run, which was accomplished within 1 min, resulting in significantly improved analytical throughput for quantitative analysis of bulk amounts of samples. The experimental results demonstrated that Fe3O4-ZrO2-iEESI-MS provided advantages, including high sensitivity, high speed, and reasonable selectivity for the detection of OPPs, showing potential applications in environmental water sample analysis and environmental science.

Sequential Detection of Lipids, Metabolites, and Proteins in One Tissue for Improved Cancer Differentiation Accuracy.

Traditionally, molecular information on metabolites, lipids, and proteins is collected from separate individual tissue samples using different analytical approaches. Herein a novel strategy to minimize the potential material losses and the mismatch between metabolomics, lipidomics, and proteomics data has been demonstrated based on internal extractive electrospray ionization mass spectrometry (iEESI-MS). Sequential detection of lipids, metabolites, and proteins from the same tissue sample was achieved without sample reloading and hardware alteration to MS instrument by sequentially using extraction solutions with different chemical compositions. With respect to the individual compound class analysis, the sensitivity, specificity, and accuracy obtained with the integrative information on metabolites, lipids, and proteins from 57 samples of 13 patients for lung cancer prediction was substantially improved from 54.0%, 51.0%, and 76.0% to 100.0%, respectively. The established method is featured by low sample consumption (ca. 2.0 mg) and easy operation, which is important to minimize systematic errors in precision molecular diagnosis and systems biology studies.

Capturing hemoglobin on graphene sheet from sub-microliter whole blood for quantitative characterization by internal extractive electrospray ionization mass spectrometry

A disposable blood sampler, which is consisted of a sub-microliter whole blood collector and a graphene filter, loading graphene sheet to selectively capture hemoglobin from sub-microliter whole blood, was developed for both qualitative and quantitative characterization hemoglobin by internal extractive electrospray ionization mass spectrometry (iEESI-MS). The blood collector was elegantly fabricated in syringe-like fashion for precisely sampling tiny amounts (1.0 μL - 2%) of whole blood, which was immediately diluted by water inside the syringe and was then pressed through the graphene filter placed between the waste outlet and the syringe reservoir to capture the hemoglobin in the blood sample. Then the graphene with hemoglobin was directly eluted by a charged (+2.5 kV) solution (mathanol/water/formic acid, 48/48/4, v/v/v) to produce the hemoglobin ions for mass spectrometric analysis. Low detection-of-limit (19.3 mg L−1 (89.5 picomol)), acceptable linear response range (300–1500 mg L−1, R2 = 0.998), relative standard deviation (0.5–6.5%, n = 3), low sample consumption (≤1.0 μL) and a relatively high speed (≤4 min per sample, including the sample loading) were achieved, demonstrating that the graphene based iEESI-MS was an alternative choice for direct detection of hemoglobin in whole blood with minimal sample consumption.

Selective Enrichment of Phosphopeptides and Phospholipids from Biological Matrixes on TiO2 Nanowire Arrays for Direct Molecular Characterization by Internal Extractive Electrospray Ionization Mass Spectrometry.

Rapid analysis of phosphopeptides and phospholipids in biological matrixes is of significant interest in multiple disciplines of life sciences. Herein, trace phospholipids in human plasma, whole blood, and undiluted human urine as well as phosphopeptides in protein digest were selectively captured on a homemade array of TiO2 nanowires for sensitive characterization by internal extractive electrospray ionization mass spectrometry (TiO2-iEESI-MS). Sequential release of captured chemicals from TiO2 array was achieved by tuning pH of the extraction solvent. A single sample analysis, including sample loading, chemical extraction and MS detection, was accomplished within 3 min. As far as the quantification of phospholipids, acceptable linearity ( R2 ≥ 0.9985) and relative standard deviations (RSDs ≤ 8.9%) were obtained within the range of 0.1-500 μg L-1 for LysoPC(14:0) and LysoPC(16:0) in raw urine samples. Limit of detection (LOD) ≤ 0.025 μg L-1 and recovery rates of 94.8-101.6% were obtained for these phospholipids. As far as the quantification of phosphopeptides, R2 ≥ 0.9994 and RSDs ≤ 9.2% within the range of 0.3-200 μg L-1 were obtained for two phosphopeptides in nonphosphopeptides mixtures. LODs ≤ 0.09 μg L-1 and recovery rates of 83.4-107.0% were obtained for these phosphopeptides. On the basis of the orthogonal partial least-squares discriminant analysis, TiO2-iEESI-MS patterns from the blood of 46 patients with ovarian cancer were confidently discriminated from the MS patterns of 46 healthy volunteers. Our results indicate the strong potential of TiO2-iEESI-MS approach for the selective detection of trace phosphopeptides and phospholipids in various biological matrixes with high sensitivity, high specificity, low sample consumption, and high throughput.

Fast quantification of fluoroquinolones in environmental water samples using molecularly imprinted polymers coupled with internal extractive electrospray ionization mass spectrometry.

In this study, a facile method based on molecularly imprinted polymers (MIPs) combined with internal extractive electrospray ionization tandem mass spectrometry (iEESI-MS/MS) was developed for the quantitative analysis of fluoroquinolones (FQs) in environmental water samples. FQ molecules in water samples were captured by the MIPs, which was retained on a 0.22 μm syringe filter. Then, an electrospray solution selected as the elution solution was employed to extract the FQs from the MIPs, getting an eluate of FQs for mass spectrometric interrogation. Under the optimized experimental conditions, low limits of detection (LODs, 0.015–0.026 μg L−1), with relative standard deviations (RSDs) less than 8.81% (n = 6) were obtained. The present method also provides good recoveries (91.14–103.60%) with acceptable precision (RSDs < 6.18%) and have no serious matrix effects for environmental water samples. The experimental results demonstrated that MIPs-iEESI-MS/MS has advantages including easy use, high speed (less than 3 min per sample) and high sensitivity for the analysis of FQs in environmental water samples, showing potential application in environmental science and water safety control.

Internal Extractive Electrospray Ionization Mass Spectrometry for Quantitative Determination of Fluoroquinolones Captured by Magnetic Molecularly Imprinted Polymers from Raw Milk

Antibiotics contamination in food products is of increasing concern due to their potential threat on human health. Herein solid-phase extraction based on magnetic molecularly imprinted polymers coupled with internal extractive electrospray ionization mass spectrometry (MMIPs-SPE-iEESI-MS) was designed for the quantitative analysis of trace fluoroquinolones (FQs) in raw milk samples. FQs in the raw milk sample (2 mL) were selectively captured by the easily-lab-made magnetic molecularly imprinted polymers (MMIPs), and then directly eluted by 100 µL electrospraying solvent biased with +3.0 kV to produce protonated FQs ions for mass spectrometric characterization. Satisfactory analytical performance was obtained in the quantitative analysis of three kinds of FQs (i.e., norfloxacin, enoxacin, and fleroxacin). For all the samples tested, the established method showed a low limit of detection (LOD ≤ 0.03 µg L−1) and a high analysis speed (≤4 min per sample). The analytical performance for real sample analysis was validated by a nationally standardized protocol using LC-MS, resulting in acceptable relative error values from −5.8% to +6.9% for 6 tested samples. Our results demonstrate that MMIPs-SPE-iEESI-MS is a new strategy for the quantitative analysis of FQs in complex biological mixtures such as raw milk, showing promising applications in food safety control and biofluid sample analysis.

Metabolic Effects of Clenbuterol and Salbutamol on Pork Meat Studied Using Internal Extractive Electrospray Ionization Mass Spectrometry

Direct mass spectrometry analysis of metabolic effects of clenbuterol and salbutamol on pork quality at the molecular level is incredibly beneficial for food regulations, public health and the development of new anti-obesity drugs. With internal extractive electrospray ionization mass spectrometry (iEESI-MS), nutrients including creatine, amino acids, L-carnitine, vitamin B6, carnosine and phosphatidylcholines in pork tissue were identified, without sample pretreatment, using collision-induced dissociation (CID) experiments and by comparison with authentic compounds. Furthermore, normal pork samples were clearly differentiated from pork samples with clenbuterol and salbutamol via principal component analysis (PCA). Correlation analysis performed on the spectral data revealed that the above-mentioned nutrients strongly correlated with pork quality, and the absolute intensity of phosphatidylcholines in normal pork was much higher than pork contaminated by clenbuterol and salbutamol. Our findings suggested that clenbuterol and salbutamol may render effects on the activity of carnitine acyltransferase I, hence the process that L-carnitine transports long-chain fatty acids into mitochondria and the formation of phosphatidylcholines might be affected. However, the underlying metabolic mechanisms of clenbuterol and salbutamol on carnitine acyltransferase I requires more comprehensive studies in future work.

Selective molecular characterization of particulate matter from gasoline cars using internal extractive electrospray ionization mass spectrometry

A facile method based on iEESI-MS has been established to selectively profile the polar chemical composition of vehicular PM.

Quantification of 1-hydroxypyrene in undiluted human urine samples using magnetic solid-phase extraction coupled with internal extractive electrospray ionization mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) are a group of ubiquitous environmental contaminants raising worldwide concerns due to their carcinogenic effects. In this study, 1-hydroxypyrene (1-OHP, the most widely used biomarker of internal dose of PAHs exposure) in undiluted human urine samples (10 mL) was selectively enriched by polypyrrole-coated Fe3O4 magnetite nanocomposites (termed as Fe3O4@Ppy, 1 mg) and then directly eluted by the electrospraying solvent (acetone/benzene/acetic acid (v/v/v, 90/10/1); 100 uL) biased with −3.5 kV to produce the deprotonated 1-OHP anions for mass spectrometric analysis. The method established here significantly improved the current performance for detection of urinary 1-OHP, providing the speed for a single sample analysis within 4 min, the limits of detection (LOD) of 0.0001 μg L−1, the linear response range of 0.001–5.000 μg L−1 (R2 = 0.9994), recovery rates of 90.6–96.1%, and relative standard deviation (RSD, n = 6) values between 2.9% and 8.0%. Human samples including raw human urine collected from 10 healthy volunteers (5 smokers and 5 nonsmokers) and 7 lung cancer patients have been successfully analyzed, showing that magnetic solid-phase extraction (MSPE) coupled with internal extractive electrospray ionization mass spectrometry (iEESI-MS) is an alternative strategy for high throughput quantitative detection of urinary 1-OHP for health risk assessment of PAHs exposure.

Direct Analysis of Phospholipids in Biological Tissues Using Internal Extractive Electrospray Ionization Mass Spectrometry

Abstract Phospholipids and their metabolites play an important role in a variety of cellular processes including cell-cell adhesion, cell growth and differentiation, apoptosis, phagocytosis as well as storage of energy. In this study, the phospholipid composition of cancer tissue and adjacent normal tissue from humans and animals were analyzed by internal extractive electrospray ionization mass spectrometry (iEESI-MS). Extractive solvent at high voltage (+5.5 kV) was injected into tissue samples using a fused silica capillary at a flow rate of 0.5–1.0 μL min−1, producing fine charged droplets containing analytes of tissue samples at the tip of the sample. Charged droplets were directly sampled to the atmospheric inlet of a mass spectrometer. Out of 21 different ratios of CH3OH-H2O solvent mixture, the ratio CH3OH-H2O (30:70, V/V) showed the optimal phospholipids extraction and visibility in MS. A large number of phospholipids from different tissue samples (such as cancer tissue and adjacent normal tissue of lung cancer, esophageal tissue, pork, beef, porcine heart and porcine lung) were obtained simultaneously by iEESI-MS analysis. The experimental results demonstrated that iEESI-MS was characterized by minimal sample pretreatment, low sample consumption, and rapid analysis (the analysis time per sample was less than 1 min), and the selectivity and sensitivity of iEESI-MS could be improved by choosing proper solvent. Importantly, the experimental results provided new information for further studies of phospholipids in biological tissues.