Carbon fiber ionization mass spectrometry coupled with solid phase microextraction for analysis of Benzo[a]pyrene.

Solid phase micro-extraction (SPME) is an effective technique that can be used to selectively enrich trace analytes of interest from complex samples. Owing to its high sensitivity and high selectivity, mass spectrometry (MS) has been widely used as the detection tool to confirm the analytes enriched on SPME fibers. Generally, thermal desorption or solvent desorption is used to desorb analytes from SPME fibers for MS analysis. A straightforward ionization method called carbon fiber ionization (CFI), which uses a single carbon fiber (diameter: ∼10 µm) as ionization emitter in MS, has been demonstrated lately. Analytes adsorbed on the carbon fiber, which is placed close (∼5 mm) to the inlet of a mass spectrometer, can be readily ionized through corona discharge and detected by the mass spectrometer. One unique feature regarding this approach over other existing ambient ionization methods is that no additional electric contact is applied directly on the carbon fiber. Nevertheless, on the basis of the electric field provided by the mass spectrometer, corona discharge can readily occur for ionizing analytes on the carbon fiber. Carbon fiber has high affinity toward polycyclic aromatic hydrocarbons due to its graphite-like surface structure. We herein explore a hyphenated-technique by combining carbon-fiber based SPME with CFI-MS for extraction of benzo[a]pyrene (BaP), a carcinogen, from aqueous samples. After BaP are adsorbed on a carbon fiber through SPME, the SPME carbon fiber can be readily placed in front of the mass spectrometer for MS analysis. The ions at m/z 252 derived from BaP adsorbed on the carbon fiber can be immediately acquired by the mass spectrometer without the requirement of applying heating or solvent. The limit of detection of BaP using the developed method was as low as ∼60 pM. It is also feasible to detect BaP from complex serum sample. The feasibility of using the approach for quantitative analysis of BaP was also demonstrated. The linear dynamic range toward BaP was 0.2-5 nM. The extraction efficiency using this approach for aqueous samples was ∼91%.

Detection of pesticide residues on intact tomatoes by carbon fiber ionization mass spectrometry.

Trace and toxic pesticide residues may still remain on crops after harvest. Thus, maximum residual levels (MRLs) of pesticides on crops have been regulated. To determine whether the remaining pesticide residue level is below MRL, time-consuming sample pretreatment is needed prior to analysis of crop samples by suitable analytical tools. By elimination of sample pretreatment steps, a high-throughput method can be developed to determine the presence of pesticide residues directly on intact crops. Carbon fiber ionization mass spectrometry (CFI-MS) is effective in determining analytes with different polarities in solid, liquid, and vapor phases in open air. Moreover, the vapor derived from solid or liquid samples possessing high vapor pressure can be readily detected by CFI-MS. The setup of CFI-MS is straightforward. A carbon fiber (diameter of ~ 10 µm and length of ~ 1 cm) is placed close (~ 1 mm) to the inlet of the mass spectrometer applied with a high voltage (- 4.5 kV). No direct electrical contact applied on the carbon fiber is required. When placing the sample with certain vapor pressure underneath the carbon fiber, analyte ions derived from the sample can be readily detected by the mass spectrometer. Given that most pesticides possess a certain vapor pressure (~ 1.33 × 10-5-~ 1.33 × 10-4 Pa), we herein develop a qualitative and quantitative analysis method to determine pesticide residues on intact fruits such as tomato based on CFI-MS without requiring any sample pretreatment. Atrazine, ametryn, carbofuran, chlorpyrifos, isoprocarb, and methomyl were selected as model samples. Low limits of detection (at nM range) were achieved for the model pesticides using the current approach. Moreover, we demonstrated that the precision and accuracy of quantitative analysis of ~ 5% and ~ 2%, respectively, could be achieved using this approach. Graphical Abstract ᅟ.

Ultrasonication-assisted spray ionization-based micro-reactors for online monitoring of fast chemical reactions by mass spectrometry.

Microfluidics can be used to handle relatively small volumes of samples and to conduct reactions in microliter-sized volumes. Electrospray ionization can couple microfluidics with mass spectrometry (MS) to monitor chemical reactions online. However, fabricating microfluidic chips is time-consuming. We herein propose the use of a micro-reactor that is sustained by two capillaries and an ultrasonicator. The inlets of the capillaries were individually immersed to two different sample vials that were subjected to the ultrasonicator. The tapered outlets of the two capillaries were placed cross with an angle of ~60° close to the inlet of the mass spectrometer to fuse the eluents. On the basis of capillary action and ultrasonication, the samples from the two capillaries can be continuously directed to the capillary outlets and fuse simultaneously to generate gas phase ions for MS analysis through ultrasonication-assisted spray ionization (UASI). Any electric contact applied on the capillaries is not required. Nevertheless, UASI spray derived from the eluents can readily occur in front of the mass spectrometer. That is, a micro-reactor was created from the fusing of the eluent containing different reactants from these two UASI capillaries, allowing reactions to be conducted in situ. The solvent in the fused droplets was evaporated quickly, and the product ions could be immediately observed by MS because of the extreme rise in the concentration of the reactants. For proof of concept, pyrazole synthesis reaction and cortisone derivatization by Girard T reagent were selected as the model reactions. The results demonstrated the feasibility of using UASI-based micro-reactor for online MS analysis to detect reaction intermediates and products.

Carbon Fiber Ionization Mass Spectrometry for the Analysis of Analytes in Vapor, Liquid, and Solid Phases.

Various ionization methods in mass spectrometry (MS) are available for the analysis of analytes with different properties. Nonetheless, the use of a single ionization method to analyze mixtures containing analytes with different polarities and volatilities in different phases at atmospheric pressure remains a challenge. Exploring an ionization method that can ionize small organics and large biomolecules with different properties for MS analysis is advantageous. Carbon fiber ionization mass spectrometry (CFI-MS), which uses a carbon fiber bundle as the ion source, is useful for the analysis of small organics with low polarities. Voltage needs to be applied on the carbon fiber bundle to initiate corona discharge for ionization of analytes. In this study, we explore the suitability of using CFI-MS in the analysis of analytes in vapor, liquid, and solid phases using a single carbon fiber (length : ∼1 cm; diameter: ∼10 µm) as the ion source. Furthermore direct electric contact on the carbon fiber is not required. We demonstrate that CFI-MS is useful for analyzing not only small and low-polarity organics but also polar biomolecules, such as peptides and proteins. The limits of detection for analytes with high polarities such as dodecyl trimethylammonium bromide and bradykinin are estimated to be ∼16 and ∼53 pM, respectively. Ionization mechanisms, including corona discharge and electrospray, are involved in the ionization of analytes with the polarity from low to high. Furthermore, sesame oil containing aromatic volatiles and compounds with different polarities is used as a model sample to demonstrate the capability of the developed ionization method to provide comprehensive chemical information from a complex sample. In addition, the feasibility of using the developed method for quantitative analysis of nonpolar as well as medium and high polarity analytes is also demonstrated. The sensitivity of the developed method toward analytes with high polarity is higher than those with low polarity. The method precision was estimated to be ∼7.8%.

Inhibition of human alcohol and aldehyde dehydrogenases by aspirin and salicylate: assessment of the effects on first-pass metabolism of ethanol.

Previous studies have reported that aspirin significantly reduced the first-pass metabolism (FPM) of ethanol in humans thereby increasing adverse effects of alcohol. The underlying causes, however, remain poorly understood. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), principal enzymes responsible for metabolism of ethanol, are complex enzyme families that exhibit functional polymorphisms among ethnic groups and distinct tissue distributions. We investigated the inhibition profiles by aspirin and its major metabolite salicylate of ethanol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and acetaldehyde oxidation by ALDH1A1 and ALDH2, at pH 7.5 and 0.5 mM NAD(+). Competitive inhibition pattern was found to be a predominant type among the ADHs and ALDHs studied, although noncompetitive and uncompetitive inhibitions were also detected in a few cases. The inhibition constants of salicylate for the ADHs and ALDHs were considerably lower than that of aspirin with the exception of ADH1A that can be ascribed to a substitution of Ala-93 at the bottom of substrate pocket as revealed by molecular docking experiments. Kinetic inhibition equation-based simulations show at higher therapeutic levels of blood plasma salicylate (1.5 mM) that the decrease of activities at 2-10 mM ethanol for ADH1A/ADH2 and ADH1B2/ADH1B3 are predicted to be 75-86% and 31-52%, respectively, and that the activity decline for ALDH1A1 and ALDH2 at 10-50 µM acetaldehyde to be 62-73%. Our findings suggest that salicylate may substantially inhibit hepatic FPM of alcohol at both the ADH and ALDH steps when concurrent intaking aspirin.

MDM2 is overexpressed and regulated by the eukaryotic translation initiation factor 4E (eIF4E) in human squamous cell carcinoma of esophagus.

BACKGROUND: We investigated the association between the increased eukaryotic translation initiation factor 4E (eIF4E) level and MDM2 overexpression in the esophageal cancer tissue and cells. METHODS: This was a retrospective study of specimens from esophageal cancer patients treated over a 5-year period in a Taiwan university hospital. The predictor variable was eIF4E level in esophageal tumors and CE48T/VGH and TE6 esophageal carcinoma cell lines. The main outcome variable was MDM2 overexpression. Appropriate descriptive and univariate statistics were computed, and a P value of <0.05 was considered statistically significant. RESULTS: There were two study sample groups. Immunohistochemistry analyses of the first sample group (51 esophageal tumors) revealed that 19 specimens demonstrated MDM2 elevation and 20 specimens had eIF4E overexpression. eIF4E elevation was evidenced by accumulation of the protein in the cytoplasm. There was a significant association between the eIF4E and MDM2 expression (P < 0.001). Western blot analysis and semiquantitative reverse transcriptase-polymerase chain reaction of the second specimen group (20 pairs of tumors and normal tissues) revealed the co-elevation of MDM2 and eIF4E (P = 0.008). There was no increased mdm2 transcript in most of the specimens. Without significant alterations in the mdm2 mRNA level and subcellular distribution, MDM2 protein was upregulated in CE48T/VGH cultured cells expressing ectopic eIF4E. Conversely, reduction of eIF4E by specific siRNA enabled TE6 cells synthesizing reduced amounts of MDM2. CONCLUSIONS: Our findings indicate that MDM2 protein levels are strongly associated with and regulated by eIF4E in a posttranscriptional mechanism in esophageal cancer.

Nuclear epidermal growth factor receptor (EGFR) interacts with signal transducer and activator of transcription 5 (STAT5) in activating Aurora-A gene expression.

Loss of the maintenance of genetic material is a critical step leading to tumorigenesis. It was reported that overexpression of Aurora-A and the constitutive activation of the epidermal growth factor (EGF) receptor (EGFR) are implicated in chromosome instability. In this study, we examined that when cells treated with EGF result in centrosome amplification and microtubule disorder, which are critical for genetic instability. Interestingly, the expression of Aurora-A was also increased by EGF stimulus. An immunofluorescence assay indicated that EGF can induce the nuclear translocation of EGFR. Chromatin immunoprecipitation (ChIP) and re-ChIP assays showed significant EGF-induced recruitment of nuclear EGFR and signal transducer and activator of transcription 5 (STAT5) to the Aurora-A promoter. A co-immunoprecipitation assay further demonstrated that EGF induces nuclear interaction between EGFR and STAT5. A small interfering (si)RNA knockdown assay also showed that EGFR and STAT5 are indeed involved in EGF-increased Aurora-A gene expression. Altogether, this study proposes that the nuclear EGFR associates with STAT5 to bind and increase Aurora-A gene expression, which ultimately may lead to chromosome instability and tumorigenesis. The results also provide a novel linkage between the EGFR signaling pathway and overexpression of Aurora-A in tumorigenesis and chromosome instability.