Mass spectrometric monitoring of exhaled breath condensate proteome of a patient after lung transplantation

Analysis of samples of exhaled breath condensate (EBC) is a promising noninvasive method for the control of the status of the human respiratory system. In the present work, the proteome of the EBC samples received from a patient with lung dystrophy at different times before and after bilateral lung transplantation is analyzed by ion cyclotron resonance mass spectrometry. Qualitative protein composition of EBC samples obtained during the first month correlates with the clinical data on the acceptance of the transplanted lungs (allograft). Fifteen months after the lung surgery, the protein spectrum was similar to the normal composition of EBC proteins. This result agrees with the medical conclusion about normal lung functioning. The results suggest that the mass spectrometric monitoring of the protein spectra of EBC may be a tool for noninvasive pulmonological diagnostics.     

Selected ion flow tube mass spectrometry of exhaled breath condensate headspace

Collection of exhaled breath condensate (EBC) is a relatively simple noninvasive method of breath analysis; however, no data have been reported that would relate concentration of volatile compounds in EBC to their gaseous concentrations in exhaled air. The aim of the study was to investigate which volatile compounds are present in EBC and how their concentrations relate to results of direct breath analysis. Thus, samples of EBC were collected in a standard way from several subjects and absolute levels of several common volatile breath metabolites (ammonia, acetone, ethanol, methanol, propanol, isoprene, hydrogen cyanide, formaldehyde and acetaldehyde) were then determined in their headspace using selected ion flow tube mass spectrometry (SIFT-MS). Results are compared with those from on-line breath analyses carried out immediately before collecting the EBC samples. It has been demonstrated that SIFT-MS can be used to quantify the concentrations of volatiles in EBC samples and that, for methanol, ammonia, ethanol and acetone, the EBC concentrations correlate with the direct breath levels. However, the EBC concentrations of isoprene, formaldehyde, acetaldehyde, hydrogen cyanide and propanol do not correlate with direct breath measurements. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Determination of N epsilon-(carboxymethyl)lysine in exhaled breath condensate using isotope dilution liquid chromatography/electrospray ionization tandem mass spectrometry

In order to identify new biomarkers for pulmonary diseases in exhaled breath condensate (EBC) it was the aim of this study to develop an analytical method for the identification and quantification of N epsilon-(carboxymethyl)lysine (CML) in EBC. As detection by liquid chromatography with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) offers the advantage of structurally related detection with the necessary specificity required for the identification of a substance, it was the method chosen for the determination of the non-volatile compound. Specific mass transitions and comparison of retention times with standards under given conditions were used for the unequivocal identification of CML in EBC of healthy subjects. Synthesis of isotopically labelled CML was performed and used as an internal standard for an accurate determination. It was possible to identify the advanced glycation end-product CML in 8 out of 10 healthy subjects. The concentration range determined in the quantifiable examined samples ranged between 35 and 110 pg/mL. EBC samples from 11 patients with different diseases such as diabetes and chronic obstructive pulmonary disease were also measured. In one patient with pneumonia a concentration of 1509 pg CML/mL EBC could be detected. This is the first time that CML has been identified and determined in EBC. The developed LC/ESI-MS/MS method could be used to address the utility of CML as a biomarker in pulmonary diseases.     

A sensitive chemiluminescence method for the determination of H2O2 in exhaled breath condensate

In this paper, a novel flow injection chemiluminescence (FI-CL) method is proposed for the determination of picomolar L(-1) levels of hydrogen peroxide (H(2)O(2)) in exhaled breath condensate (EBC). This method is based on the oxidation of a low concentration of luminol (10(-7) M) by H(2)O(2) at a low concentration level (< 10(-8) M) in an alkaline medium catalyzed by a complex, K(5)[Cu(HIO(6))(2)] (DPC), which is not interfered by other metal ions or horseradish peroxidase (HRP). Under the optimum conditions, H(2)O(2) was determined over the range of 1.0 x 10(-10) to 1.0 x 10(-8) mol L(-1) with a detection limit of (3sigma) of 4.1 x 10(-11) mol L(-1). The relative standard deviation (RSD) was 3.2% for 5 nmol L(-1) H(2)O(2) (n = 7). The proposed method offers the advantages of ultra-sensitivity, selectivity, simplicity and rapidity for H(2)O(2) determination. It was successfully applied to directly determine trace amounts of H(2)O(2) (nmol L(-1)) in human's EBC of both rheum and healthy volunteers. A statistically significant difference was found between patients with rheum (n = 11) and control subjects without rheum (n = 11).     

Factors influencing level of hydrogen peroxide in exhaled breath condensate

Hydrogen peroxide (H₂O₂) in exhaled breath condensate (EBC) has been proposed as a marker for oxidative stress in the airways. The aim of the present study was to evaluate the measurement of H₂O₂ in EBC with or without use of a nose clip, and the influence of mouth rinsing, sampling time and storage.An elevated H₂O₂ level was seen during nasal breathing compared to mouth breathing with nose clip (3.4 pmol/s vs. 2.1 pmol/s, p = 0.02). Breathing through the mouth, using a nose clip, was therefore practiced in all experiments. The H₂O₂ levels were increased when mouth rinsing was performed using an acid buffer (1.4 pmol/s vs. 1.9 pmol/s, p = 0.03). 15 min sampling time decreased the H₂O₂ output by almost 50% compared with 2 min sampling time (1.2 vs. 0.6 pmol/s, p = 0.03). When samples were left unattended for 15 min no change in H₂O₂ concentration in the EBC was seen.We found no significant differences in H₂O₂ levels between samples stored for 4 weeks at − 80 °C and samples analysed directly; however, a significant decrease in the levels was seen for samples stored for 4 weeks at − 20 °C.In conclusion, the method of EBC collection and storage plays an important role in reducing variability within and between individuals.     

Ion trap liquid chromatography/tandem mass spectrometry analysis of leukotriene B4 in exhaled breath condensate

The objective of this study is the measurement of leukotriene B7 (LTB4), a potent inflammatory mediator, in exhaled breath condensate by using liquid chromatography/mass spectrometry (LC/MS and LC/MS/MS). Condensation of exhaled breath is a non-invasive method to collect airway secretions. Deuterated (d4)-LTB4 was used as internal standard. The MS and MS/MS behavior of LTB4 and LTB4-d4 was studied by electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive and negative ion polarity mode. Preliminary results show that monitoring negative ions in ESI mode has the best sensitivity for both LTB4 and LTB4-d4. Therefore, negative ESI was chosen, and the [M-H]- ions at m/z 335 and 339 were selected for quantification. The lower limit of quantification for LTB4, expressed as the lowest point of the calibration curve, was 100 pg/mL. Using this technique, we measured LTB4 in exhaled breath condensate in two healthy subjects, four asthmatic patients on anti-inflammatory treatment, and four asthmatic patients who were not on anti-inflammatory drugs. Exhaled LTB4 concentrations were detected only in asthmatic patients who were not on anti-inflammatory therapy. This method is potentially useful for non-invasive assessment of airway inflammation, but the sensitivity of the technique needs to be improved.     

An optimized sampling and GC-MS analysis method for benzene in exhaled breath, as a biomarker for occupational exposure

Benzene is known to be toxic and carcinogenic: therefore, in case of exposure to benzene vapours, a reliable biological monitoring procedure is needed, particularly in the field of occupational hygiene. The determination of the concentration of benzene in the exhaled air 8 h after the exposure has been demonstrated to be a significant biomarker, even for low concentrations of airborne benzene vapours. This work presents a sampling and analysis method that optimizes previously described procedures: in the sampling phase, a double-step sample collection in Tedlar bags is used, in order to remove the breath moisture and to standardise the sample volumes. The analytical phase uses a cryogenic trap for the concentration of the air samples to be injected in the GC-MS, without the need for trapping materials, significantly reducing time and costs of the analysis and improving sensitivity. The presented method has been successfully applied to the biological monitoring of a mixed population (occupationally exposed and not exposed subjects, smokers and non-smokers), with a lower detection limit of 1.5 ng of benzene per litre of exhaled air, that is 1/200 of the biological exposure index recommended by the American Conference of Governmental Hygienists.     

Real-time, in vivo monitoring and pharmacokinetics of valproic acid via a novel biomarker in exhaled breath

Extractive electrospray ionization mass spectrometry is shown to allow real-time, in vivo drug monitoring and pharmacokinetic measurement in a non-invasive, pain-free manner as demonstrated by the mass spectral measurement of a novel exhaled breath biomarker for valproic acid, a medication used to control epilepsy.     

Determination of hexavalent chromium in exhaled breath condensate and environmental air among chrome plating workers

Chromium speciation has attracted attention because of the different toxicity of Cr(III), which is considered relatively non-toxic, and Cr(VI), which can cross cell membranes mainly as a chromate anion and has been classified as a class I human carcinogen. The aims of the present study were to measure soluble Cr(VI) levels in environmental samples, to develop a simple method of quantifying Cr(VI) in exhaled breath condensate (EBC), and to follow the kinetics of EBC Cr(VI) in chrome plating workers.Personal air samples were collected from 10 chrome platers; EBC was collected from the same workers immediately after the work shift on Tuesday and before the work shift on the following Wednesday. Environmental and EBC Cr(VI) levels were determined by means of colorimetry and electrothermal absorption atomic spectrometry, respectively.The method of detecting Cr(VI) in environmental air was based on the extraction of the Cr(VI)-diphenylcarbazide (Cr(VI)-DPC) complex in 1-butanol, whereas EBC Cr(VI) was determined using a solvent extraction of Cr(VI) as an ion pair with tetrabutylammonium ion, and subsequent direct determination of the complex (Cr(VI)-DPC) in EBC.Kinetic data showed that airborne Cr(VI) was reduced by 50% in airway lining fluid sampled at the end of exposure and that there was a further 50% reduction after about 15 h. The persistence of Cr(VI) in EBC supports the use of EBC in assessing target tissue levels of Cr(VI).     

Determination of phenytoin in exhaled breath condensate using electromembrane extraction followed by capillary electrophoresis

Application of hollow fiber-based electromembrane extraction was studied for extraction and quantification of phenytoin from exhaled breath condensate (EBC). Phenytoin is extracted from EBC through a supported liquid membrane consisting of 1-octanol impregnated in the walls of a hollow fiber, and into an alkaline aqueous acceptor solution inside the lumen of the fiber. Under the obtained conditions of electromembrane extraction, that is, the extraction time of 15 min, stirring speed of 750 rpm, donor phase pH at 11.0, acceptor pH at 13.0, and an applied voltage of 15 V across the supported liquid membrane, an enrichment factor of 102-fold correspond to extraction percent of 25.5% was achieved. Good linearity was obtained over the concentration range of 0.001–0.10 µg/mL (r² = 0.9992). Limits of detection and quantitation were 0.001 and 0.003 µg/mL, respectively. The proposed method was successfully applied to determine phenytoin from EBC samples of patients receiving the drug. No interfering peaks were detected that indicating excellent selectivity of the method. The intra- and interday precisions (RSDs) were less than 14%.     

Design-of-experiment optimization of exhaled breath condensate analysis using a miniature differential mobility spectrometer (DMS)

Analytical instruments that can measure small amounts of chemicals in complicated biological samples are often useful as diagnostic tools. However, it can be challenging to optimize these sensors using actual clinical samples, given the heterogeneous background and composition of the test materials. Here we use gas chromatography-differential mobility spectrometry (GC/DMS) to analyze the chemical content of human exhaled breath condensate (EBC). Ultimately, this system can be used for non-invasive disease diagnostics. Many parameters can be adjusted within this instrument system, and we implemented a factorial design-of-experiments to systematically test several combinations of parameter settings while concurrently analyzing effects and interactions.We examined four parameters that affect sensitivity and detection for our instrument, requiring a 2⁴ factorial design. We optimized sensor function using EBC samples spiked with acetone, a known clinical biomarker in breath. Two outputs were recorded for each experiment combination: number of chemicals detected, and the amplitude of acetone signal. Our goal is to find the best parameter combination that yields the highest acetone peak while also preserving the largest number of other chemical peaks in the spectra. By optimizing the system, we can conduct further clinical experiments with our sensor more efficiently and accurately.     

Alcohol in breath and blood: a selected ion flow tube mass spectrometric study

In this paper we compare the amounts of ethanol in breath and in blood after ingestion of whisky using analysis by selected ion flow tube mass spectrometry (SIFT-MS). Blood ethanol concentrations were also obtained using standard hospital forensic procedures for blood alcohol analyses. We demonstrate the quantitative nature of SIFT-MS analysis by correlating the observed alcohol content of the headspace above 5-mL amounts of venous blood and aqueous solution to which known trace amounts of alcohol have been added. This procedure provides a Henry's Law coefficient for ethanol in aqueous solution at 298 +/- 3 K of 209 +/- 7 mol/kg*bar. We also demonstrate that measurement of the ethanol concentration in the alveolar portion of a single breath using the SIFT-MS technique gives an accurate measure of blood alcohol and could obviate the need for blood samples in forensic processing. The storage performance of breath samples in Mylar bags with a volume greater than 1 L has been shown to maintain the mixture integrity for ethanol but not for some other species.     

High accuracy method for the application of isotope dilution to gas chromatography/mass spectrometric analysis of gases

A new isotope dilution mass spectrometry (IDMS) method for high-accuracy quantitative analysis of gases has been developed and validated by the analysis of standard mixtures of carbon dioxide in nitrogen. The method does not require certified isotopic reference materials and does not require direct measurements of the highly enriched spike. The relative uncertainty of the method is shown to be 0.2%. Copyright Crown copyright 2003. Reproduced with the permission of Her Majesty's Stationery Office.     

Determination of patterns of biologically relevant aldehydes in exhaled breath condensate of healthy subjects by liquid chromatography/atmospheric chemical ionization tandem mass spectrometry

A method for the simultaneous determination of several classes of aldehydes in exhaled breath condensate (EBC) was developed using liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS). EBC is a biological matrix obtained by a relatively new, simple and noninvasive technique and provides an indirect assessment of pulmonary status. The measurement of aldehydes in EBC represents a biomarker of the effect of oxidative stress caused by smoke, disease, or strong oxidants like ozone. Malondialdehyde (MDA), acrolein, alpha,beta-unsaturated hydroxylated aldehydes [namely 4-hydroxyhexenal (4-HHE) and 4-hydroxynonenal (4-HNE)], and saturated aldehydes (n-hexanal, n-heptanal and n-nonanal) were measured in EBC after derivatization with 2,4-dinitrophenylhydrazine (DNPH). Atmospheric pressure chemical ionization of the analytes was obtained in positive-ion mode for MDA, and in negative-ion mode for acrolein, 4-HHE, 4-HNE, and saturated aldehydes. DNPH derivatives were separated on a C18 column using variable proportions of 20 mM aqueous acetic acid and methanol. Linearity was established over 4-5 orders of magnitude and limits of detection were in the 0.3-1.0 nM range. Intra-day and inter-day precision were in the 1.3-9.9% range for all the compounds. MDA, acrolein and n-alkanals were detectable in all EBC samples, whereas the highly reactive 4-HHE and 4-HNE were found in only a few samples. Statistically significant higher concentrations of MDA, acrolein and n-hexanal were found in EBC from smokers.     

A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism

An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N¹-acetylspermidine (N¹AcSpd), N⁸-acetylspermidine (N⁸AcSpd), N¹-acetylspermine, N¹,N⁸-diacetylspermidine, and N¹,N¹²-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N¹AcSpd and N⁸AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with ¹³C₂-N¹AcSpd and ¹³C₂-N⁸AcSpd which have the ¹³C₂-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N¹-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N¹-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-¹⁵N₃]-N¹-acetylspermine and [1,4,8-¹⁵N₃]spermidine (¹⁵N₃-Spd), respectively; for SMO, [1,4,8,12-¹⁵N₄]spermine and ¹⁵N₃-Spd, respectively; and for SSAT, ¹⁵N₃-Spd and [1,4,8-¹⁵N₃]-N¹-acetylspermidine, respectively.     

A radioactive tracer dilution method to determine the mass of molten salt

A new technique for molten salt mass determination, termed radioactive tracer dilution, that uses ²²Na as a tracer was validated at bench scale. It has been a challenging problem to determine the mass of molten salt in irregularly shaped containers, where a highly radioactive, high-temperature molten salt was used to process nuclear spent/used fuel during electrochemical recycling (pyro-processing) or for coolant/fuel salt from molten salt reactors. A radioactive source with known activity is dissolved into the salt. After a complete mixture, a small amount of the salt is sampled and measured in terms of its mass and radioactivity. By finding the ratio of the mass to radioactivity, the unknown salt mass in the original container can be precisely determined.