Impact of enzymatic hydrolysis on the quantification of total urinary concentrations of chemical biomarkers.

Human exposure to consumer and personal care products chemicals such as phenols, including parabens and other antimicrobial agents, can be assessed through biomonitoring by quantifying urinary concentrations of the parent chemical or its metabolites, often after hydrolysis of phase II conjugates. Developing suitable analytical methods for the concurrent quantification of multiple exposure biomarkers is challenging because optimal conditions for the hydrolysis of such conjugates (e.g., O-glucuronides, N-glucuronides, sulfates) may differ depending on the biomarker. We evaluated the effectiveness of seven commercial hydrolytic enzymes to simultaneously hydrolyze N-glucuronides (using the antibacterial triclocarban as example compound) and other conjugates (using select phenols and parabens as examples) by using on-line solid phase extraction-high performance liquid chromatography-isotope dilution-tandem mass spectrometry. Incubation (30 min, 55 °C) with a genetically engineered ß-glucuronidase (IMCS, ≥15 units/µL urine) hydrolyzed N-glucuronide triclocarban, but did not fully hydrolyze the conjugates of phenols and parabens. By contrast, incubation (4 h, 37 °C) with solid ß-glucuronidase (Helix pomatia, Type H-1, ≥30 units/µL urine) or liquid ß-glucuronidase/arylsulfatase (Helix pomatia, 30 units/µL urine [i.e., 30 µL/100 µL urine]) in the presence of 100 µL methanol for 100 µL urine completely hydrolyzed N-glucuronide triclocarban and the conjugates of several phenols and parabens, without cleaving the ester bond of the parabens to form p-hydroxybenzoic acid. These results highlight the relevance of method validation procedures that include optimizing the hydrolysis of phase II urinary conjugates (e.g., enzyme type and amount used, reaction time, temperature) to quantify accurately and concurrently multiple exposure biomarkers for biomonitoring purposes.

Prevalence of substandard and falsified artemisinin-based combination antimalarial medicines on Bioko Island, Equatorial Guinea.

INTRODUCTION: Poor-quality artemisinin-containing antimalarials (ACAs), including falsified and substandard formulations, pose serious health concerns in malaria endemic countries. They can harm patients, contribute to the rise in drug resistance and increase the public's mistrust of health systems. Systematic assessment of drug quality is needed to gain knowledge on the prevalence of the problem, to provide Ministries of Health with evidence on which local regulators can take action. METHODS: We used three sampling approaches to purchase 677 ACAs from 278 outlets on Bioko Island, Equatorial Guinea as follows: convenience survey using mystery client (n=16 outlets, 31 samples), full island-wide survey using mystery client (n=174 outlets, 368 samples) and randomised survey using an overt sampling approach (n=88 outlets, 278 samples). The stated active pharmaceutical ingredients (SAPIs) were assessed using high-performance liquid chromatography and confirmed by mass spectrometry at three independent laboratories. RESULTS: Content analysis showed 91.0% of ACAs were of acceptable quality, 1.6% were substandard and 7.4% falsified. No degraded medicines were detected. The prevalence of medicines without the SAPIs was higher for ACAs purchased in the convenience survey compared with the estimates obtained using the full island-wide survey-mystery client and randomised-overt sampling approaches. Comparable results were obtained for full island survey-mystery client and randomised overt. However, the availability of purchased artesunate monotherapies differed substantially according to the sampling approach used (convenience, 45.2%; full island-wide survey-mystery client, 32.6%; random-overt sampling approach, 21.9%). Of concern is that 37.1% (n=62) of these were falsified. CONCLUSION: Falsified ACAs were found on Bioko Island, with the prevalence ranging between 6.1% and 16.1%, depending on the sampling method used. These findings underscore the vital need for national authorities to track the scale of ineffective medicines that jeopardise treatment of life-threatening diseases and value of a representative sampling approach to obtain/measure the true prevalence of poor-quality medicines.

Urinary Concentrations of the Antibacterial Agent Triclocarban in United States Residents: 2013-2014 National Health and Nutrition Examination Survey.

Triclocarban is widely used as an antibacterial agent in personal care products, and the potential for human exposure exists. We present here the first nationally representative assessment of exposure to triclocarban among Americans ≥6 years of age who participated in the 2013-2014 National Health and Nutrition Examination Survey. We detected triclocarban at concentrations above 0.1 µg/L in 36.9% of 2686 urine samples examined. Triclocarban was detected more frequently in adolescents and adults than in children, and in non-Hispanic black compared to other ethnic groups. In univariate analysis, log-creatinine, sex, age, race, and body surface area (BSA) were significantly associated with the likelihood of having triclocarban concentrations above the 95th percentile. In multiple regression models, persons with BSA at or above the median (≥1.86 m2) were 2.43 times more likely than others, and non-Hispanic black and non-Hispanic white were 3.71 times and 2.23 times more likely than "all Hispanic," respectively, to have urinary concentrations above the 95th percentile. We found no correlations between urinary concentrations of triclocarban and triclosan, another commonly used antibacterial agent. Observed differences among demographic groups examined may reflect differences in physiological factors (i.e., BSA) as well as use of personal care products containing triclocarban.

Microplasma Ionization of Volatile Organics for Improving Air/Water Monitoring Systems On-Board the International Space Station.

Low molecular weight polar organics are commonly observed in spacecraft environments. Increasing concentrations of one or more of these contaminants can negatively impact Environmental Control and Life Support (ECLS) systems and/or the health of crew members, posing potential risks to the success of manned space missions. Ambient plasma ionization mass spectrometry (MS) is finding effective use as part of the analytical methodologies being tested for next-generation space module environmental analysis. However, ambient ionization methods employing atmospheric plasmas typically require relatively high operation voltages and power, thus limiting their applicability in combination with fieldable mass spectrometers. In this work, we investigate the use of a low power microplasma device in the microhollow cathode discharge (MHCD) configuration for the analysis of polar organics encountered in space missions. A metal-insulator-metal (MIM) structure with molybdenum foil disc electrodes and a mica insulator was used to form a 300 µm diameter plasma discharge cavity. We demonstrate the application of these MIM microplasmas as part of a versatile miniature ion source for the analysis of typical volatile contaminants found in the International Space Station (ISS) environment, highlighting their advantages as low cost and simple analytical devices. Graphical Abstract ᅟ.

Electrothermal Vaporization Sample Introduction for Spaceflight Water Quality Monitoring via Gas Chromatography-Differential Mobility Spectrometry.

In the history of manned spaceflight, environmental monitoring has relied heavily on archival sampling. However, with the construction of the International Space Station (ISS) and the subsequent extension in mission duration up to one year, an enhanced, real-time method for environmental monitoring is necessary. The station air is currently monitored for trace volatile organic compounds (VOCs) using gas chromatography-differential mobility spectrometry (GC-DMS) via the Air Quality Monitor (AQM), while water is analyzed to measure total organic carbon and biocide concentrations using the Total Organic Carbon Analyzer (TOCA) and the Colorimetric Water Quality Monitoring Kit (CWQMK), respectively. As mission scenarios extend beyond low Earth orbit, a convergence in analytical instrumentation to analyze both air and water samples is highly desirable. Since the AQM currently provides quantitative, compound-specific information for air samples and many of the targets in air are also common to water, this platform is a logical starting point for developing a multimatrix monitor. Here, we report on the interfacing of an electrothermal vaporization (ETV) sample introduction unit with a ground-based AQM for monitoring target analytes in water. The results show that each of the compounds tested from water have similar GC-DMS parameters as the compounds tested in air. Moreover, the ETV enabled AQM detection of dimethlsilanediol (DMSD), a compound whose analysis had proven challenging using other sample introduction methods. Analysis of authentic ISS water samples using the ETV-AQM showed that DMSD could be successfully quantified, while the concentrations obtained for the other compounds also agreed well with laboratory results.

Quality of artemisinin-based combination formulations for malaria treatment: prevalence and risk factors for poor quality medicines in public facilities and private sector drug outlets in Enugu, Nigeria.

BACKGROUND: Artemisinin-based combination therapies are recommended by the World Health Organisation (WHO) as first-line treatment for Plasmodium falciparum malaria, yet medication must be of good quality for efficacious treatment. A recent meta-analysis reported 35% (796/2,296) of antimalarial drug samples from 21 Sub-Saharan African countries, purchased from outlets predominantly using convenience sampling, failed chemical content analysis. We used three sampling strategies to purchase artemisinin-containing antimalarials (ACAs) in Enugu metropolis, Nigeria, and compared the resulting quality estimates. METHODS: ACAs were purchased using three sampling approaches--convenience, mystery clients and overt, within a defined area and sampling frame in Enugu metropolis. The active pharmaceutical ingredients were assessed using high-performance liquid chromatography and confirmed by mass spectrometry at three independent laboratories. Results were expressed as percentage of APIs stated on the packaging and used to categorise each sample as acceptable quality, substandard, degraded, or falsified. RESULTS: Content analysis of 3024 samples purchased from 421 outlets using convenience (n=200), mystery (n=1,919) and overt (n=905) approaches, showed overall 90.8% ACAs to be of acceptable quality, 6.8% substandard, 1.3% degraded and 1.2% falsified. Convenience sampling yielded a significantly higher prevalence of poor quality ACAs, but was not evident by the mystery and overt sampling strategies both of which yielded results that were comparable between each other. Artesunate (n=135; 4 falsified) and dihydroartemisinin (n=14) monotherapy tablets, not recommended by WHO, were also identified. CONCLUSION: Randomised sampling identified fewer falsified ACAs than previously reported by convenience approaches. Our findings emphasise the need for specific consideration to be given to sampling frame and sampling approach if representative information on drug quality is to be obtained.

A Repeat Random Survey of the Prevalence of Falsified and Substandard Antimalarials in the Lao PDR: A Change for the Better.

In 2003, a stratified random sample survey was conducted in the Lao People's Democratic Republic (Laos) to study the availability and quality of antimalarials in the private sector. In 2012, this survey was repeated to allow a statistically valid analysis of change through time. The counterfeit detection device 3 (CD-3) was used to assess packaging quality in the field and HPLC and mass spectroscopy analysis chemical analysis performed. The availability of oral artesunate monotherapies had significantly decreased from 22.9% (22) of 96 outlets in southern Laos in 2003 to 4.8% (7) of 144 outlets in 2012 (P < 0.0001). All the samples collected in the 2012 survey contained the correct active pharmaceutical ingredients (APIs) in contrast to the 21 (84%) falsified artesunate samples found in the 2003 survey. Although none of the medicines found in 2012 survey had evidence for falsification, 25.4% (37) of the samples were outside the 90-110% pharmacopeial limits of the label claim, suggesting that they were substandard or degraded. Results obtained from this survey show that patients are still exposed to poorly manufactured drugs or to ineffective medicines such as chloroquine. The quality of artemisinin-based combination therapies (ACTs) used in Laos needs to be monitored, since falsified ACTs would have devastating consequences in public health.

Quality of antimalarials at the epicenter of antimalarial drug resistance: results from an overt and mystery client survey in Cambodia.

Widespread availability of monotherapies and falsified antimalarials is thought to have contributed to the historical development of multidrug-resistant malaria in Cambodia. This study aimed to document the quality of artemisinin-containing antimalarials (ACAs) and to compare two methods of collecting antimalarials from drug outlets: through open surveyors and mystery clients (MCs). Few oral artemisinin-based monotherapies and no suspected falsified medicines were found. All 291 samples contained the stated active pharmaceutical ingredient (API) of which 69% were considered good quality by chemical analysis. Overall, medicine quality did not differ by collection method, although open surveyors were less likely to obtain oral artemisinin-based monotherapies than MCs. The results are an encouraging indication of the positive impact of the country's efforts to tackle falsified antimalarials and artemisinin-based monotherapies. However, poor-quality medicines remain an ongoing challenge that demands sustained political will and investment of human and financial resources.

Desorption atmospheric pressure photoionization and direct analysis in real time coupled with travelling wave ion mobility mass spectrometry.

RATIONALE: Ambient mass spectrometry (MS) is a tool for screening analytes directly from sample surfaces. However, background impurities may complicate the spectra and therefore fast separation techniques are needed. Here, we demonstrate the use of travelling wave ion mobility spectrometry in a comparative study of two ambient MS techniques. METHODS: Desorption atmospheric pressure photoionization (DAPPI) and direct analysis in real time (DART) were coupled with travelling wave ion mobility mass spectrometry (TWIM-MS) for highly selective surface analysis. The ionization efficiencies of DAPPI and DART were compared. Test compounds were: bisphenol A, benzo[a]pyrene, ranitidine, cortisol and α-tocopherol. DAPPI-MS and DART-TWIM-MS were also applied to the analysis of chloroquine from dried blood spots, and α-tocopherol from almond surface, and DAPPI-TWIM-MS was applied to analysis of pharmaceuticals and multivitamin tablets. RESULTS: DAPPI was approximately 100 times more sensitive than DART for bisphenol A and 10-20 times more sensitive for the other compounds. The limits of detection were between 30-290 and 330-8200 fmol for DAPPI and DART, respectively. Also, from the authentic samples, DAPPI ionized chloroquine and α-tocopherol more efficiently than DART. The mobility separation enabled the detection of species with low signal intensities, e.g. thiamine and cholecalciferol, in the DAPPI-TWIM-MS analysis of multivitamin tablets. CONCLUSIONS: DAPPI ionized the studied compounds of interest more efficiently than DART. For both DAPPI and DART, the mobility separation prior to MS analysis reduced the amount of chemical noise in the mass spectrum and significantly increased the signal-to-noise ratio for the analytes.

Plasma-spray ionization (PLASI): a multimodal atmospheric pressure ion source for liquid stream analysis.

A new ion generation method, named plasma-spray ionization (PLASI) for direct analysis of liquid streams, such as in continuous infusion experiments or liquid chromatography (LC), is reported. PLASI addresses many of the analytical limitations of electrospray ionization (ESI) and has potential for real time process stream analysis and reaction monitoring under atmospheric conditions in non-ESI friendly scenarios. In PLASI-mass spectrometry (MS), the liquid stream is pneumatically nebulized and partially charged at low voltages; the resultant aerosol is thus entrained with a gaseous plasma plume from a distal glow discharge prior to MS detection. PLASI-MS not only overcomes ESI-MS limitations but also generates simpler mass spectra with minimal adduct and cluster formation. PLASI utilizes the atomization capabilities of an ESI sprayer operated below the ESI threshold to generate gas-phase aerosols that are then ionized by the plasma stream. When operated at or above the ESI threshold, ionization by traditional ESI mechanisms is achieved. The multimodal nature of the technique enables readily switching between plasma and ESI operation. It is expected that PLASI will enable analyzing a wide range of analytes in complex matrices and less-restricted solvent systems, providing more flexibility than that achievable by ESI alone.

An effective approach for coupling direct analysis in real time with atmospheric pressure drift tube ion mobility spectrometry.

Drift tube ion mobility spectrometry (DTIMS) has evolved as a robust analytical platform routinely used for screening small molecules across a broad suite of chemistries ranging from food and pharmaceuticals to explosives and environmental toxins. Most modern atmospheric pressure IM detectors employ corona discharge, photoionization, radioactive, or electrospray ion sources for efficient ion production. Coupling standalone DTIMS with ambient plasma-based techniques, however, has proven to be an exceptional challenge. Device sensitivity with near-ground ambient plasma sources is hindered by poor ion transmission at the source-instrument interface, where ion repulsion is caused by the strong electric field barrier of the high potential ion mobility spectrometry (IMS) inlet. To overcome this shortfall, we introduce a new ion source design incorporating a repeller point electrode used to shape the electric field profile and enable ion transmission from a direct analysis in real time (DART) plasma ion source. Parameter space characterization studies of the DART DTIMS setup were performed to ascertain the optimal configuration for the source assembly favoring ion transport. Preliminary system capabilities for the direct screening of solid pharmaceuticals are briefly demonstrated.

A tiered analytical approach for investigating poor quality emergency contraceptives.

Reproductive health has been deleteriously affected by poor quality medicines. Emergency contraceptive pills (ECPs) are an important birth control method that women can use after unprotected coitus for reducing the risk of pregnancy. In response to the detection of poor quality ECPs commercially available in the Peruvian market we developed a tiered multi-platform analytical strategy. In a survey to assess ECP medicine quality in Peru, 7 out of 25 different batches showed inadequate release of levonorgestrel by dissolution testing or improper amounts of active ingredient. One batch was found to contain a wrong active ingredient, with no detectable levonorgestrel. By combining ultrahigh performance liquid chromatography-ion mobility spectrometry-mass spectrometry (UHPLC-IMS-MS) and direct analysis in real time MS (DART-MS) the unknown compound was identified as the antibiotic sulfamethoxazole. Quantitation by UHPLC-triple quadrupole tandem MS (QqQ-MS/MS) indicated that the wrong ingredient was present in the ECP sample at levels which could have significant physiological effects. Further chemical characterization of the poor quality ECP samples included the identification of the excipients by 2D Diffusion-Ordered Nuclear Magnetic Resonance Spectroscopy (DOSY 1H NMR) indicating the presence of lactose and magnesium stearate.

Electro-thermal vaporization direct analysis in real time-mass spectrometry for water contaminant analysis during space missions.

The development of a direct analysis in real time-mass spectrometry (DART-MS) method and first prototype vaporizer for the detection of low molecular weight (∼30-100 Da) contaminants representative of those detected in water samples from the International Space Station is reported. A temperature-programmable, electro-thermal vaporizer (ETV) was designed, constructed, and evaluated as a sampling interface for DART-MS. The ETV facilitates analysis of water samples with minimum user intervention while maximizing analytical sensitivity and sample throughput. The integrated DART-ETV-MS methodology was evaluated in both positive and negative ion modes to (1) determine experimental conditions suitable for coupling DART with ETV as a sample inlet and ionization platform for time-of-flight MS, (2) to identify analyte response ions, (3) to determine the detection limit and dynamic range for target analyte measurement, and (4) to determine the reproducibility of measurements made with the method when using manual sample introduction into the vaporizer. Nitrogen was used as the DART working gas, and the target analytes chosen for the study were ethyl acetate, acetone, acetaldehyde, ethanol, ethylene glycol, dimethylsilanediol, formaldehyde, isopropanol, methanol, methylethyl ketone, methylsulfone, propylene glycol, and trimethylsilanol.

Ion mobility and liquid chromatography/mass spectrometry strategies for exhaled breath condensate glucose quantitation in cystic fibrosis studies.

RATIONALE: Cystic fibrosis related diabetes (CFRD) is an important complication of cystic fibrosis (CF) because it causes acceleration in the decline in lung function. Monitoring concentrations of key metabolites such as glucose in airway lining fluid is necessary for improving our understanding of the biochemical mechanisms linking diabetes and CF. Targeted-metabolomic strategies for glucose quantitation in exhaled breath condensate (EBC) from healthy individuals are presented. METHODS: Three different electrospray ionization mass spectrometry (ESI-MS)-based methods were developed for EBC sample interrogation and glucose quantitation without derivatization. Two methods utilized ultra-high-performance liquid chromatography (UHPLC) coupled to either time-of-flight (TOF) MS or triple quadrupole (QqQ) tandem MS (MS/MS). A third approach involved direct-infusion traveling wave ion mobility spectrometry (TWIMS) with TOF-MS detection. UHPLC/QqQ-MS/MS was used for urea quantitation as the EBC dilution marker. Matrix effects were mitigated using isotopically labeled glucose and urea as internal standards. RESULTS: All the developed methods allowed glucose and urea quantitation in EBC with high accuracy and precision. The UHPLC/TOF-MS and UHPLC/QqQ-MS/MS methods provided similar analytical figures of merit. UHPLC/QqQ-MS/MS provided the highest sensitivity and the lowest limit of detection (LOD) of 1.5 nM in EBC for both glucose and urea. The TWIMS-TOF-MS-based method provided the highest sample throughput capability; however, the glucose LOD was ~3-fold higher than with the two chromatographic methods. CONCLUSIONS: Mass spectrometric methods for the quantitative analysis of trace EBC glucose levels are reported and compared for the first time. The analytical figures of merit demonstrate the applicability of these methods to metabolite analysis of airway samples for CF and CFRD research.

Metabolic Profiling of Human Blood by High Resolution Ion Mobility Mass Spectrometry (IM-MS).

A high resolution ion mobility time-of-flight mass spectrometer with electrospray ionization source (ESI-IM-MS) was evaluated as an analytical method for rapid analysis of complex biological samples such as human blood metabolome was investigated. The hybrid instrument (IM-MS) provided an average ion mobility resolving power of ~90 and a mass resolution of ~1500 (at m/z 100). A few µL of whole blood was extracted with methanol, centrifuged and infused into the IM-MS via an electrospray ionization source. Upon IM-MS profiling of the human blood metabolome approximately 1,100 metabolite ions were detected and 300 isomeric metabolites separated in short analyses time (30 minutes). Estimated concentration of the metabolites ranged from the low micromolar to the low nanomolar level. Various classes of metabolites (amino acids, organic acids, fatty acids, carbohydrates, purines and pyrimidines etc) were found to form characteristic mobility-mass correlation curves (MMCC) that aided in metabolite identification. Peaks corresponding to various sterol derivatives, estrogen derivatives, phosphocholines, prostaglandins, and cholesterol derivatives detected in the blood extract were found to occupy characteristic two dimensional IM-MS space. Low abundance metabolite peaks that can be lost in MS random noise were resolved from noise peaks by differentiation in mobility space. In addition, the peak capacity of MS increased six fold by coupling IMS prior to MS analysis.

Metabolic profiling of Escherichia coli by ion mobility-mass spectrometry with MALDI ion source.

Comprehensive metabolome analysis using mass spectrometry (MS) often results in a complex mass spectrum and difficult data analysis resulting from the signals of numerous small molecules in the metabolome. In addition, MS alone has difficulty measuring isobars and chiral, conformational and structural isomers. When a matrix-assisted laser desorption ionization (MALDI) source is added, the difficulty and complexity are further increased. Signal interference between analyte signals and matrix ion signals produced by MALDI in the low mass region (<1500 Da) cause detection and/or identification of metabolites difficult by MS alone. However, ion mobility spectrometry (IMS) coupled with MS (IM-MS) provides a rapid analytical tool for measuring subtle structural differences in chemicals. IMS separates gas-phase ions based on their size-to-charge ratio. This study, for the first time, reports the application of MALDI to the measurement of small molecules in a biological matrix by ion mobility-time of flight mass spectrometry (IM-TOFMS) and demonstrates the advantage of ion-signal dispersion in the second dimension. Qualitative comparisons between metabolic profiling of the Escherichia coli metabolome by MALDI-TOFMS, MALDI-IM-TOFMS and electrospray ionization (ESI)-IM-TOFMS are reported. Results demonstrate that mobility separation prior to mass analysis increases peak-capacity through added dimensionality in measurement. Mobility separation also allows detection of metabolites in the matrix-ion dominated low-mass range (m/z < 1500 Da) by separating matrix signals from non-matrix signals in mobility space.

Resistive glass IM-TOFMS.

The design of a new ion mobility mass spectrometer (IM-MS) is presented. This new design features an ambient-pressure resistive glass ion mobility drift tube (RGIMS) coupled to a high-resolution time-of-flight mass spectrometer (TOFMS) by an enhanced interface that includes two segmented quadrupoles. The interface design demonstrates an increase in sensitivity while maintaining high resolving power typically achieved for ambient-pressure IMS drift tubes. Performance of the prototype instrument was evaluated and the analytical figures of merit for standard solutions as well as complex samples such as human blood were determined. For a 3 µM solution of caffeine, the peak was collected in 36 s and gave a response of 10 counts/s. The detection limit (defined as 1 count/s) was calculated to be 300 nM concentration of caffeine from the response rate from the 36 s run. Controlled fragmentation of caffeine was achieved through adjustment of voltages applied on the interface lenses. Over 300 tentative metabolites were detected in human blood along with 80 isomers/isobars with ion counts >5. Isotope ratios from extracted mass spectra of selected mobility peaks were used to identify selected metabolite compounds. High separation power for both IMS (resolving power, t(d)/Δt(w1/2), was 85) and MS (mass resolving power, m/Δm, maximum was 7000 with a mass accuracy between 2 and 10 ppm) was measured. Developed software for data acquisition, control and display allowed flexibility in instrument control, data evaluation and visualization.

Monitoring dynamic changes in lymph metabolome of fasting and fed rats by electrospray ionization-ion mobility mass spectrometry (ESI-IMMS).

Ambient pressure ion mobility time-of-flight mass spectrometry (IMMS) has recently emerged as a rapid and efficient analytical technique for applications to metabolomics. An important application of metabolomics is to monitor metabolome shifts caused by stress due to toxin exposure, nutritional changes, or disease. The research presented in this paper uses IMMS to monitor metabolic changes in rat lymph fluid caused by dietary stresses over time. Extracts of metabolites found in the lymph fluid collected from dietary stressed rats were subjected to analysis by electrospray (ESI) IMMS operated both in positive and negative ion detection mode. Metabolites detected were tentatively identified based on their mass to charge ratio (m/z). In one sample, 1180 reproducible tentative metabolite ions were detected in negative mode and 1900 reproducible tentative metabolite ions detected in positive mode. Only biologically reproducible ions, defined as metabolite ions that were measured in different rats under the same treatment, were analyzed to reduce the complexity of the data. A metabolite peak list including m/z, mobility, and intensity generated for each metabolome was used to perform principle component analysis (PCA). Dynamic changes in metabolomes were investigated using principle components PC1 and PC2 that described 62% of the variation of the system in positive mode and 81% of the variation of the system in negative mode. Analysis of variance (ANOVA) was performed for PC1 and PC2 and means were statistically evaluated. Profiles of intensities were compared for tentative metabolite ions detected at different times before and after the rats were fed to identify the metabolites that were changing the most. Mobility-mass correlation curves (MMCC) were investigated for the different classes of compounds.