Lukas Bregy

Drug Pharmacokinetics Determined by Real-Time Analysis of Mouse Breath†

Noninvasive, real-time pharmacokinetic (PK) monitoring of ketamine, propofol, and valproic acid, and their metabolites was achieved in mice, using secondary electrospray ionization and high-resolution mass spectrometry. The PK profile of a drug influences its efficacy and toxicity because it determines exposure time and levels. The antidepressant and anaesthetic ketamine (Ket) and four Ket metabolites were studied in detail and their PK was simultaneously determined following application of different sub-anaesthetic doses of Ket. Bioavailability after oral administration vs. intraperitoneal injection was also investigated. In contrast to conventional studies that require many animals to be sacrificed even for low-resolution PK curves, this novel approach yields real-time PK curves with a hitherto unmatched time resolution (10 s), and none of the animals has to be sacrificed. This thus represents a major step forward not only in animal welfare, but also major cost and time savings.

Effects of CPAP therapy withdrawal on exhaled breath pattern in obstructive sleep apnoea

Background Obstructive sleep apnoea (OSA) is highly prevalent and associated with cardiovascular and metabolic changes. OSA is usually diagnosed by polysomnography which is time-consuming and provides little information on the patients phenotype thus limiting a personalised treatment approach. Exhaled breath contains information on metabolism which can be analysed by mass spectrometry within minutes. The objective of this study was to identify a breath profile in OSA recurrence by use of secondary-electrospray-ionization-mass spectrometry (SESI-MS). Methods Patients with OSA effectively treated with CPAP were randomised to either withdraw treatment (subtherapeutic CPAP) or continue therapeutic CPAP for 2 weeks. Exhaled breath analysis by untargeted SESI-MS was performed at baseline and 2 weeks after randomisation. The primary outcome was the change in exhaled molecular breath pattern. Results 30 patients with OSA were randomised and 26 completed the trial according to the protocol. CPAP withdrawal led to a recurrence of OSA (mean difference in change of oxygen desaturation index between groups +30.3/h; 95% CI 19.8/h,40.7/h, p<0.001) which was accompanied by a significant change in 62 exhaled features (16 metabolites identified). The panel of discriminating mass-spectral features allowed differentiation between treated and untreated OSA with a sensitivity of 92.9% and a specificity of 84.6%. Conclusion Exhaled breath analysis by SESI-MS allows rapid and accurate detection of OSA recurrence. The technique has the potential to characterise an individuals metabolic response to OSA and thus makes a comprehensible phenotyping of OSA possible. Trial registration number NCT02050425 (registered at ClinicalTrials.gov).

Detection and Quantification of Benzothiazoles in Exhaled Breath and Exhaled Breath Condensate by Real-Time Secondary Electrospray Ionization-High-Resolution Mass Spectrometry and Ultra-High Performance Liquid Chromatography

2-Subtituted benzothiazoles are widely used industrial chemicals whose occurrence in environmental samples has been shown to be ubiquitous. However, knowledge about human exposure to these compounds and their excretion route is still scarce. Here, we demonstrate for the first time the detection of benzothiazole derivatives in exhaled breath. Real-time analysis of breath was carried out by means of secondary electrospray ionization coupled to high-resolution mass spectrometry. This coupling allowed not only the detection of these compounds in breath with a sensitivity in the pptv range but also their robust identification by comparing tandem high-resolution mass spectra from breath and standards. For further confirmation, benzothiazoles were also determined in exhaled breath condensate samples by means of ultra high-performance liquid chromatography. This approach strengthened the identification as a result of excellent matches in retention times and also allowed quantification. An estimated total daily exhalation of ca. 20 μg day(-1) was calculated for the six benzothiazole derivatives found in breath.

Real-Time High-Resolution Tandem Mass Spectrometry Identifies Furan Derivatives in Exhaled Breath

The identification of chemical compounds in exhaled human breath is promising in the search for new biomakers of diseases. However, the analytical techniques used nowadays are not capable of achieving a robust identification, especially in real-time analysis. In this work, we show that real-time high-resolution tandem mass spectrometry (HRMS/MS) is suitable for the identification of biomarkers in exhaled breath. Using this approach, we identified a number of furan derivatives, compounds found in the exhalome whose nature and origin are not yet clearly understood. It is also shown that the combination of HRMS/MS with UHPLC allowed not only the identification of the furan derivatives but also the proper separation of their isomeric forms.

Differentiation of oral bacteria in in vitro cultures and human saliva by secondary electrospray ionization - mass spectrometry.

The detection of bacterial-specific volatile metabolites may be a valuable tool to predict infection. Here we applied a real-time mass spectrometric technique to investigate differences in volatile metabolic profiles of oral bacteria that cause periodontitis. We coupled a secondary electrospray ionization (SESI) source to a commercial high-resolution mass spectrometer to interrogate the headspace from bacterial cultures and human saliva. We identified 120 potential markers characteristic for periodontal pathogens Aggregatibacter actinomycetemcomitans (n = 13), Porphyromonas gingivalis (n = 70), Tanerella forsythia (n = 30) and Treponema denticola (n = 7) in in vitro cultures. In a second proof-of-principle phase, we found 18 (P. gingivalis, T. forsythia and T. denticola) of the 120 in vitro compounds in the saliva from a periodontitis patient with confirmed infection with P. gingivalis, T. forsythia and T. denticola with enhanced ion intensity compared to two healthy controls. In conclusion, this method has the ability to identify individual metabolites of microbial pathogens in a complex medium such as saliva.

119 Exhaled breath analysis by real-time mass spectrometry in patients with pulmonary fibrosis

Introduction: Idiopathic pulmonary fibrosis (IPF) is recognized as a distinct clinical disorder. However, despite major endeavors to understand the pathogenesis, the diagnosis of IPF remains elusive [1]. Metabolic profiling of biopsied tissue specimens has been shown promise to gain insights into IPF pathogenesis [2]. Thus we hypothesized that analysis of exhaled metabolites may also provide further insights. Methods: 21 patients with IPF and 21 matched controls were recruited for this exploratory study. Exhaled breath analysis was performed by Secondary Electrospray Ionization-Mass Spectrometry (SESI-MS) [3]. Significant differences in exhaled metabolite levels in IPF were subsequently sought. A two-sample t-test followed by estimation of false discovery rate in multiple comparisons was used for this purpose. Statistical significance was set at p < 0.05. Finally, we attempted to predict IPF vs control in a leaveone-out-cross-validation (LOOCV) using a set of 15 metabolites per sample predicted. Results: Fig. 1A shows an example of six replicate exhalations of one subject for one particular metabolite with the molecular formula C16H23NO5 (m/z 310.1640). Fig.1B shows the same compound exhaled in a selected group of controls (left-hand-side) and IPF patients (right-hand-side). The graph suggests that this particular compound is exhaled in higher concentrations in IPF patients than in controls. Fig. 1C shows the ROC curve obtained as a result of the LOOCV. The obtained area under the curve was 0.8 (0.61-0.91). Conclusions: Analysis of exhaled breath by real time mass spectrometry shows promise to help in the diagnostic process of IPF. Breath analysis complements ongoing efforts to characterize IPF at the metabolic level using more invasive approaches (e.g. biopsy). Ongoing in-depth structural identification of altered breath metabolites is likely to provide insights on IPF pathogenesis. These findings will require further validation in larger and independent cohorts.

Real-Time Quantification of Amino Acids in the Exhalome by Secondary Electrospray Ionization–Mass Spectrometry: A Proof-of-Principle Study

BACKGROUND Amino acids are frequently determined in clinical chemistry. However, current analysis methods are time-consuming, invasive, and suffer from artifacts during sampling, sample handling, and sample preparation. We hypothesized in this proof-of-principle study that plasma concentrations of amino acids can be estimated by measuring their concentrations in exhaled breath. A novel breath analysis technique described here allows such measurements to be carried out in real-time and noninvasively, which should facilitate efficient diagnostics and give insights into human physiology. METHODS The amino acid profiles in 37 individuals were determined by ion-exchange HPLC in blood plasma and simultaneously in breath by secondary electrospray ionization coupled to high-resolution mass spectrometry. Participants were split into training and test sets to validate the analytical accuracy. Longitudinal profiles in 3 individuals were additionally obtained over a 12-h period. RESULTS Concentrations of 8 slightly volatile amino acids (A, V, I, G, P, K, F, Orn) could be determined in exhaled breath with a CV of <10%. Exhalome validation studies yielded high accuracies for each of these amino acids, on average only 3% less compared to plasma concentrations (95% CI ±13%). Higher variations were found only for amino acids with a low plasma concentration. CONCLUSIONS This study demonstrates for the first time that amino acids can be quantified in the human breath and that their concentrations correlate with plasma concentrations. Although this noninvasive technique needs further investigation, exhalome analysis may provide significant benefits over traditional, offline analytical methods.