Direct mass spectrometry analysis of exhaled human breath in real-time.

The molecular composition of exhaled human breath can reflect various physiological and pathological conditions. Considerable progress has been achieved over the past decade in real-time analysis of exhaled human breath using direct mass spectrometry methods, including selected ion flow tube mass spectrometry, proton transfer reaction mass spectrometry, extractive electrospray ionization mass spectrometry, secondary electrospray ionization mass spectrometry, acetone-assisted negative photoionization mass spectrometry, atmospheric pressure photoionization mass spectrometry, and low-pressure photoionization mass spectrometry. Here, recent developments in direct mass spectrometry analysis of exhaled human breath are reviewed with regard to analytical performance (chemical sensitivity, selectivity, quantitative capabilities) and applications of the developed methods in disease diagnosis, targeted molecular detection, and real-time metabolic monitoring.

Rapid analysis of untreated food samples by gel loading tip spray ionization mass spectrometry.

Rapid, efficient, versatile, easy-to-use, and non-expensive analytical approaches are globally demanded for food analysis. Many ambient ionization approaches based on electrospray ionization (ESI) have been developed recently for the rapid molecular characterization of food products. However, those approaches mainly suffer from insufficient signal duration for comprehensive chemical characterization by tandem MS analysis. Here, a commercially available disposable gel loading tip is used as a low-cost emitter for the direct ionization of untreated food samples. The most important advantages of our approach include high stability, and durability of the signal (> 10 min), low cost (ca. 0.1 USD per run), low sample and solvent consumption, prevention of tip clogging and discharge, operational simplicity, and potential for automation. Quantitative analysis of sulfapyridine, HMF (hydroxymethylfurfural), and chloramphenicol in real sample shows the limit-of-detection 0.1 µg mL-1, 0.005 µg mL-1, 0.01 µg mL-1; the linearity range 0.1-5 µg mL-1, 0.005-0.25 µg mL-1, 0.01-1 µg mL-1; and the linear fits R2 ≥ 0.980, 0.991, 0.986. Moreover, we show that tip-ESI can also afford sequential molecular ionization of untreated viscous samples, which is difficult to achieve by conventional ESI. We conclude that tip-ESI-MS is a versatile analytical approach for the rapid chemical analysis of untreated food samples.

Online Sequential Determination of Organic/Inorganic Lead Speciation in PM2.5 Using Electrochemical Mass Spectrometry.

The information regarding the occurrence and abundance of lead (Pb) in PM2.5 is useful for the evaluation of air pollution status and tracing the pollution source. Herein, electrochemical mass spectrometry (EC-MS) for sequential determination of Pb species in PM2.5 samples without sample pretreatment has been developed using the combination of online sequential extraction with mass spectrometry (MS) detection. Four kinds of Pb species including water-soluble Pb compounds, fat-soluble Pb compounds, water/fat-insoluble Pb compounds, and a water/fat-insoluble Pb element were sequentially extracted from PM2.5 samples, in which water-soluble Pb compounds, fat-soluble Pb compounds, and water/fat-insoluble Pb compounds were extracted sequentially by elution using H2O, CH3OH, and EDTA-2Na as the eluent respectively, while the water/fat-insoluble Pb element was extracted by electrolysis using EDTA-2Na as the electrolyte. The extracted water-soluble Pb compounds, water/fat-insoluble Pb compounds, and water/fat-insoluble Pb element were transformed into EDTA-Pb in real time for online electrospray ionization mass spectrometry analysis, while the extracted fat-soluble Pb compounds were directly detected by electrospray ionization mass spectrometry. The advantages of the reported method include the obviation of sample pretreatment, high speed of analysis (<60 min/sample), low detection limit (0.16 pg), low sample consumption (30 µg), and high accuracy (>90%), which indicates the potential of this method for the rapid quantitative species detection of metals in environmental particulate matter samples.

Polarity-Specific Profiling of Metabolites in Single Cells by Probe Electrophoresis Mass Spectrometry.

Sensitive analysis of metabolites in a single cell is of fundamental significance for the better understanding of biological variability, differential susceptibility in disease therapy, and cell-to-cell heterogeneity as well. Herein, polarity-specific profiling of metabolites in a single cell was implemented by probe electrophoresis mass spectrometry (PEMS), which combined electrophoresis sampling of metabolites from a single cell and nanoelectrospray ionization-mass spectrometry (nanoESI-MS) analysis of the sampled metabolites. Enhanced extraction of either negatively or positively charged metabolites from a single cell was achieved by applying a DC voltage offset of +2.0 and -2.0 V to the probe, respectively. The experimental data demonstrated that PEMS features high throughput (≥200 peaks) and high sensitivity (≥10-times signal enhancement for [Choline + H]+, [Glutamine + H]+, [Arginine + H]+, etc.) in comparison with direct nanoESI-MS analysis. The biological effects of CdSe quantum dots (QDs) and γ-radiation on Allium cepa cells were investigated by PEMS, which revealed that CdSe QDs lead to the increase of intracellular amines while γ-radiation causes the decrease of intracellular acids. Therefore, this work provides an alternative platform to probe novel insights of cells by sensitive analysis of polarity-specific metabolites in a single cell.

Formation of protonated water-hydrogen clusters in an ion trap mass spectrometer at room temperature.

Protonated water-hydrogen clusters [H+(H2O)n·m(H2)] present an interesting model for fundamental water research, but their formation and isolation presents considerable experimental challenges. Here, we report the detection of [H+(H2O)n·m(H2)] (2 ≤ n ≤ 3, m ≤ 2) clusters alongside protonated water clusters H+(H2O)n (2 ≤ n ≤ 3) in a linear ion trap mass spectrometer under two different experimental conditions: (1) when water vapor was ionized by +5.5 kV ambient corona discharge in front of the mass spectrometer inlet; (2) when isolated H+(H2O)n clusters were exposed to H2 gas inside the linear trap. Chemical assignment of [H+(H2O)n·m(H2)] clusters was confirmed using reference experiments with isotopically labeled water and deuterium. Also, the formation of H2 gas in the corona discharge area was indicated by a flame test. Overall, our findings clearly indicate that [H+(H2O)n·m(H2)] clusters can be produced at room temperature through the association of protonated water clusters H+(H2O)n with H2 gas, without any cooling necessary. A mechanism for the formation of the protonated water-hydrogen complexes was proposed. Our results also suggest that the association of water ions with H2 gas may play a notable role in corona discharge ionization processes, such as atmospheric pressure chemical ionization, and may be partially responsible for the stabilization of reactive radical species occasionally reported in corona discharge ionization experiments.

Applications of direct analysis in real time mass spectrometry in food analysis: A review.

RATIONALE: Direct analysis in real time (DART) combined with mass spectrometry (MS) detection has become one of the most broadly used analytical approaches for the direct molecular characterization of food samples with regard to their chemical quality, safety, origin, and authentication. The major advantages of DART-MS for food analysis include high chemical sensitivity and specificity, high speed and throughput of analysis, simplicity, and the obviation of tedious sample preparation and solvents. METHODS: The recent applications of DART coupled with different mass analyzers, including quadrupole, ion trap, Orbitrap, and time of flight, are discussed. In addition, sample pretreatment methods that have been coupled with DART-MS are discussed. RESULTS: We summarize the applications of DART-MS in food science and industry published in the period from 2005 to this date. The applications and analytical characteristics are systematically categorized across the three major types of foods: solid foods, liquid foods, and viscous foods. CONCLUSIONS: DART-MS has proved its high suitability for the direct, rapid, and high-throughput molecular analysis of very different food samples with minimal or no sample preparation, thus offering a high-speed alternative to liquid chromatography/mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS) approaches that are traditionally employed in food analysis.

Rapid and sensitive detection of acetone in exhaled breath through the ambient reaction with water radical cations.

The levels of acetone and other ketones in exhaled human breath can be associated with various metabolic conditions, e.g. ketosis, lung cancer, dietary fat loss and diabetes. In this study, ketones in breath samples were charged through the reaction with water radical cations to form [M + H2O]˙+ ions, which were detected by mass spectrometry. Our experimental data indicate that under the optimized experimental conditions, the limit of detection for acetone using our approach is 0.14 ng L-1 (∼0.06 ppb). The linear dynamic range of detection spans four orders of magnitude. The developed approach was applied to real-time semi-quantitative analysis of acetone in the exhaled breath of human volunteers, revealing significantly higher levels of acetone in the breath of smokers compared to non-smokers. The developed approach features the obviation of sample collection, easy operation, high speed of analysis (10 s per run), high sensitivity, and spectral interpretation, which indicates the potential of ambient corona discharge ionization mass spectrometry as a selective, sensitive and noninvasive technique for the determination of exhaled ketones in clinical diagnosis including lung cancer, diabetes, etc.

Selective detection of phospholipids in human blood plasma and single cells for cancer differentiation using dispersed solid-phase microextraction combined with extractive electrospray ionization mass spectrometry.

Phospholipids in microvolume biofluid samples (≤0.5 µL), including human plasma and single cells, were selectively captured by dispersed magnetic Fe3O4@TiO2 nanocomposite particles (40 µg). A suspension containing Fe3O4@TiO2 nanoparticles was loaded into a glass capillary (i.d. 0.75 mm) by capillary force. The supernatant solution was discarded, while the Fe3O4@TiO2 particles were retained inside the capillary by using an external magnetic field (ca. 1.3 T). The phospholipids on the surface of Fe3O4@TiO2 nanoparticles were directly analyzed using internal extractive electrospray ionization mass spectrometry (iEESI-MS) by pumping ≤1 µL of extraction solution of methanol containing 1.5% ammonia (w/w) through the capillary tube toward the ESI tip. A single sample analysis was accomplished within 4 min. Phospholipids in blood plasma samples from 59 patients with ovarian cancer and 43 healthy controls, and 28 patients with pancreatic cancer and 23 healthy controls were studied. Based on the orthogonal partial least squares discriminant analysis (OPLS-DA), the cancer patients were confidently discriminated from the healthy controls. Phospholipids in single human cells (MV4-11 and NB4) were determined, showing the sensitivity for single cell analysis. Therefore the results demonstrated that rapid cancer differentiation is achieved using this approach through the detection of trace phospholipids in microvolume blood and cell samples with high sensitivity, high specificity, low sample consumption, and high throughput.

Comparative study of alterations in phospholipid profiles upon liver cancer in humans and mice.

Comparative studies of molecular alterations upon cancer between mice and humans are of great importance in order to determine the relevance of research involving mouse cancer models to the development of diagnostic and therapeutic approaches in clinical practice as well as for the mechanistic studies of pathology in humans. Herein, using molecular fingerprinting by internal extractive electrospray ionization mass spectrometry (iEESI-MS), we identified 50 differential signals in mouse liver tissue and 62 differential signals in human liver tissue that undergo significant intensity alterations (variable importance in the project (VIP) >1.0) upon liver cancer, out of which only 27 were common in both mouse and human tissues. Out of the 27 common differential signals, six types of phospholipids were also identified to undergo significant alterations in human serum upon liver cancer, including PC(34:2), PC(36:4), PC(38:6), PC(36:2), PC(38:4) and PC(42:9). Statistical analysis of the relative intensity distribution of these six identified phospholipids in serum allowed confident determination of liver cancer in humans (sensitivity 91.0%, specificity 88.0%, and accuracy 90.0%). Our results indicate that, despite the significant difference in the overall alterations of phospholipid profiles upon liver cancer between humans and mice, the six identified 'core' differential phospholipids of liver cancer found in the liver tissues of both humans and mice as well as in human serum show high potential as a minimal panel for the rapid targeted diagnosis of liver cancer with high accuracy, sensitivity and specificity using direct mass spectrometry (MS) analysis.

Validation of Breast Cancer Margins by Tissue Spray Mass Spectrometry.

Current methods for the intraoperative determination of breast cancer margins commonly suffer from the insufficient accuracy, specificity and/or low speed of analysis, increasing the time and cost of operation as well the risk of cancer recurrence. The purpose of this study is to develop a method for the rapid and accurate determination of breast cancer margins using direct molecular profiling by mass spectrometry (MS). Direct molecular fingerprinting of tiny pieces of breast tissue (approximately 1 × 1 × 1 mm) is performed using a home-built tissue spray ionization source installed on a Maxis Impact quadrupole time-of-flight mass spectrometer (qTOF MS) (Bruker Daltonics, Hamburg, Germany). Statistical analysis of MS data from 50 samples of both normal and cancer tissue (from 25 patients) was performed using orthogonal projections onto latent structures discriminant analysis (OPLS-DA). Additionally, the results of OPLS classification of new 19 pieces of two tissue samples were compared with the results of histological analysis performed on the same tissues samples. The average time of analysis for one sample was about 5 min. Positive and negative ionization modes are used to provide complementary information and to find out the most informative method for a breast tissue classification. The analysis provides information on 11 lipid classes. OPLS-DA models are created for the classification of normal and cancer tissue based on the various datasets: All mass spectrometric peaks over 300 counts; peaks with a statistically significant difference of intensity determined by the Mann-Whitney U-test (p < 0.05); peaks identified as lipids; both identified and significantly different peaks. The highest values of Q2 have models built on all MS peaks and on significantly different peaks. While such models are useful for classification itself, they are of less value for building explanatory mechanisms of pathophysiology and providing a pathway analysis. Models based on identified peaks are preferable from this point of view. Results obtained by OPLS-DA classification of the tissue spray MS data of a new sample set (n = 19) revealed 100% sensitivity and specificity when compared to histological analysis, the "gold" standard for tissue classification. "All peaks" and "significantly different peaks" datasets in the positive ion mode were ideal for breast cancer tissue classification. Our results indicate the potential of tissue spray mass spectrometry for rapid, accurate and intraoperative diagnostics of breast cancer tissue as a means to reduce surgical intervention.

Water Radical Cations in the Gas Phase: Methods and Mechanisms of Formation, Structure and Chemical Properties.

Water radical cations, (H2O)n+•, are of great research interest in both fundamental and applied sciences. Fundamental studies of water radical reactions are important to better understand the mechanisms of natural processes, such as proton transfer in aqueous solutions, the formation of hydrogen bonds and DNA damage, as well as for the discovery of new gas-phase reactions and products. In applied science, the interest in water radicals is prompted by their potential in radiobiology and as a source of primary ions for selective and sensitive chemical ionization. However, in contrast to protonated water clusters, (H2O)nH+, which are relatively easy to generate and isolate in experiments, the generation and isolation of radical water clusters, (H2O)n+•, is tremendously difficult due to their ultra-high reactivity. This review focuses on the current knowledge and unknowns regarding (H2O)n+• species, including the methods and mechanisms of their formation, structure and chemical properties.

Chemical Profiling of Bulk Alloys Using Micro-Electrochemical Probe Mass Spectrometry.

Micro-electrochemical probe mass spectrometry (µECP-MS) is demonstrated as a method for the direct profiling of chemical composition of bulk alloy samples without tedious sample pretreatment. The spatial distribution of Zn and Cu components of a Cu/Zn alloy sample was successively identified by scanning the electrolysis potential from -0.6 V to 0.6 V. The lateral resolution of alloy chemical profiling was ≤10 µm, and the depth resolution was ≤0.5 nm. Besides metal components, the method also allows the simultaneous detection of organic molecules on the sample surface. The limit of detection for Rhodamine B, Zn, and Cu depositions was 4.47, 9.58, and 24.25 ag per µm2, respectively. The method is particularly useful for high-throughput (<2 min per single run) quality monitoring of industrial parts and conductive materials of irregular geometries, such as alloy, microchips, solder side, etc.

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.

Generating Supercharged Protein Ions for Breath Analysis by Extractive Electrospray Ionization Mass Spectrometry.

Supercharged protein ions produced by electrospray ionization are extremely efficient proton donors for secondary ionization. Here, by electrospraying the protein solutions containing 5% 1,2-butylene carbonate, the supercharged protein ions with unusually high proton density were produced as the primary ions for the ionization of exhaled breath samples in the extractive electrospray ionization mass spectrometry (EESI-MS), which resulted in the enhanced ionization efficiency for the breath analytes even with relatively low gas phase basicity. Moreover, the total number of metabolites detected in breath increased by about 260% in the mass range of 200-500 Da, owing to the substantial signal enhancement for breath metabolites, providing complementary and additional information to conventional SESI.

Floral volatiles identification and molecular differentiation of Osmanthus fragrans by neutral desorption extractive atmospheric pressure chemical ionization mass spectrometry.

RATIONALE: Floral volatiles are commonly present only at trace amounts and can be degraded or lost during vapor collection, which is often challenging from the analytical standpoint. Osmanthus fragrans Lour. is a widely cultivated plant known for the highly distinct fragrance of its flowers. The identification of specific volatile organic compounds (VOCs) and molecular differentiation of O. fragrans without any chemical pretreatment and VOC collection are important. METHODS: Twenty-eight VOCs released by the flowers from ten different cultivars of O. fragrans were identified using neutral desorption extractive atmospheric pressure chemical ionization mass spectrometry (ND-EAPCI-MS) without any chemical pretreatment or VOC collection. Chemical identification was performed by high-resolution MSn analysis and whenever possible was confirmed by the analysis of standards. RESULTS: According to our literature search, nine of the identified VOCs, 3-buten-2-one, cyclohexadiene, 2-methylfuran, 3-allylcyclohexene, cuminyl alcohol, hotrienol oxide, epoxy-linalool oxide, N-(2-hydroxyethyl) octanamide, and 3-hydroxy-dihydro-ß-ionone, have not been reported in O. fragrans in earlier studies. Confident differentiation between ten different cultivars of O. fragrans was achieved by the principal component analysis of the mass spectrometric results. CONCLUSIONS: The results of our ND-EAPCI-MS analysis substantially increase our knowledge about the chemistry of the O. fragrans floral fragrance and demonstrate the power of this technique for direct molecular profiling for plant recognition or in biotechnological applications.

Rapid detection of metal impurities on the surfaces of intact objects with irregular shapes using electrochemical mass spectrometry.

New approaches are demanded in daily life and industry for the rapid inspection of chemical impurities on various objects, particularly those with irregular shapes. Herein, an analytical strategy combining electrochemistry (EC) and mass spectrometry (MS) has been developed for the direct inspection of metal impurities on various objects which are commonly used in daily life and industry. An intact object (e.g., necklace, bearing, ring) was immersed in an electrolytic cell containing EDTA/acetonitrile/water solution at appropriate potentials to form metal ions. The formed metal ions were instantly chelated with specific ligands (e.g. ethylenediaminetetraacetic acid) and sampled for online electrospray ionization (ESI) with high-resolution MS detection. The unique feature of the method is that metal speciation information can be obtained even when just a metal impurity (e.g., Pb, Ni) is localized on a hard-to-reach tiny spot on the inner surface of objects with extremely irregular shapes. A single sample analysis requires less than 10 minutes, regardless of the object shape. The limit of detection is 0.05 ppb with sample consumption on the nanogram level. The experimental results demonstrate that the method is promising for the non-destructive quality and safety inspection of metal impurities on virtually any kinds of objects with high chemical sensitivity.

Molecular analysis of semen-like odor emitted by chestnut flowers using neutral desorption extractive atmospheric pressure chemical ionization mass spectrometry.

Knowledge about the chemical composition of floral volatile organic compounds (VOCs) is valuable in biological studies as well as for the flavor, cosmetic, and fragrance industries. The flowers of Chinese chestnut (Castanea mollissima) emit a distinctive semen-like odor; however, the chemical composition and biological role for the semen-like odor of chestnut flowers remain scarcely studied. Herein, we report the floral VOCs and the pollinators of chestnut flowers. A fast method based on a neutral desorption (ND) device coupled to extractive atmospheric pressure chemical ionization mass spectrometry (EAPCI-MS) was developed for the rapid identification of VOCs from freshly collected chestnut flowers without any chemical pretreatment. Chemical identification was performed using high-resolution MS analysis in combination with tandem MS analysis and whenever possible was confirmed by the analysis of standard reference compounds. Twenty volatiles were identified, most of which are nitrogen-containing. Out of the identified volatiles, 1-pyrroline is known to have a semen-like odor and is probably also responsible for the semen-like odor of the chestnut flowers. Four nitrogenous VOCs of chestnut flowers, including 1-pyrroline, 1-piperideine, 2-pyrrolidone, and phenethylamine, were also common in other semen-like odor flowers such as Photinia serrulata, Castanopsis sclerophylla, and Stemona japonica, suggesting similar chemical origin. The main visitors of chestnut flowers were dipteran species, such as Eristalis tenax, Eristalis arvorum, Episyrphus balteatus, Lucilia sericata, Chrysomya megacephala, Chrysochus asclepiadeus, and Adalia bipunctata. Our results suggest that the chestnut flowers and other semen-like odor flowers may present a new type of sapromyophily. This study also indicates that ND-EAPCI-MS provides more sensitive and simpler detection of many VOCs (particularly nitrogen-containing VOCs) than GC-MS and therefore can be used to complement traditional approaches for the higher chemical coverage of VOCs analysis. Graphical abstract ᅟ.

Combination of Low-Temperature Electrosurgical Unit and Extractive Electrospray Ionization Mass Spectrometry for Molecular Profiling and Classification of Tissues.

Real-time molecular navigation of tissue surgeries is an important goal at present. Combination of electrosurgical units and mass spectrometry (MS) to perform accurate molecular visualization of biological tissues has been pursued by many research groups. Determination of molecular tissue composition at a particular location by surgical smoke analysis is now of increasing interest for clinical use. However, molecular analysis of surgical smoke is commonly lacking molecular specificity and is associated with significant carbonization and chemical contamination, which are mainly related to the high temperature of smoke at which many molecules become unstable. Unlike traditional electrosurgical tools, low-temperature electrosurgical units allow tissue dissection without substantial heating. Here, we show that low-temperature electrosurgical units can be used for desorption of molecules from biological tissues without thermal degradation. The use of extractive electrospray ionization technique for the ionization of desorbed molecules allowed us to obtain mass spectra of healthy and pathological tissues with high degree of differentiation. Overall, the data indicate that the described approach has potential for intraoperative use.

Charge-State-Dependent Variation of Signal Intensity Ratio between Unbound Protein and Protein-Ligand Complex in Electrospray Ionization Mass Spectrometry: The Role of Solvent-Accessible Surface Area.

Native electrospray ionization mass spectrometry (ESI-MS) is nowadays widely used for the direct and sensitive determination of protein complex stoichiometry and binding affinity constants ( Ka). A common yet poorly understood phenomenon in native ESI-MS is the difference between the charge-state distributions (CSDs) of the bound protein-ligand complex (PL) and unbound protein (P) signals. This phenomenon is typically attributed to experimental artifacts such as nonspecific binding or in-source dissociation and is considered highly undesirable, because the determined Ka values display strong variation with charge state. This situation raises serious concerns regarding the reliability of ESI-MS for the analysis of protein complexes. Here we demonstrate that, contrary to the common belief, the CSD difference between P and PL ions can occur without any loss of complex integrity, simply due to a change in the solvent-accessible surface area (ΔSASA) of the protein upon ligand binding in solution. The experimental CSD shifts for PL and P ions in ESI-MS are explained in relation to the magnitude of ΔSASA for diverse protein-ligand systems using a simple model based on the charged residue mechanism. Our analysis shows that the revealed ΔSASA factor should be considered rather general and be given attention for the correct spectral interpretation of protein complexes.

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.