Determination of peppermint compounds in breath by needle trap micro-extraction coupled with gas chromatography-tandem mass spectrometry.

Breath analysis is an alternative approach for disease diagnosis and for monitoring therapy. The lack of standardized procedures for collecting and analysing breath samples currently limits its use in clinical practice. In order to overcome this limitation, the 'Peppermint Consortium' was established within the breath community to carry out breath wash-out experiments and define reference values for a panel of compounds contained in the peppermint oil capsule. Here, we present a needle trap micro-extraction technique coupled with gas chromatography and tandem mass spectrometry for a rapid and accurate determination of alpha-pinene, beta-pinene, limonene, eucalyptol, menthofuran, menthone, menthol and menthyl acetate in mixed breath samples. Detection limits between 1 and 20 pptv were observed when 25 ml of a humidified standard gas mixture were loaded into a needle trap device at a flow rate of 10 ml min-1. Inter- and intra-day precisions were lower than 15%, thus confirming the reliability of the assay. Our procedure was used to analyse breath samples taken from a nominally healthy volunteer who was invited to swallow a 200 mg capsule of peppermint oil. Six samples were collected at various times within 6 h of ingestion. Analyte concentrations were not affected by the sampling mode (i.e. mixed vs. end-tidal fraction), whereas respiratory rate and exhalation flow rate values slightly influenced the concentration of the target compounds in breath samples.

Using labelled internal standards to improve needle trap micro-extraction technique prior to gas chromatography/mass spectrometry.

When working with humid gaseous samples, the amount of water vapour collected in a needle trap along with volatile analytes may vary from sample to sample and decrease during the storage. This has a major impact on desorption efficiency and recovery. We propose the addition of a labelled internal standards to nullify the effect of variable humidity on the analytical performance of needle trap micro-extraction combined with gas chromatography mass spectrometry. Triple-bed (Divinylbenzene/Carbopack X/Carboxen 1000) and single-bed (Tenax GR) needles were tested with standard gaseous mixtures prepared at different relative humidity levels (85%, 50% and 10%). The standard mixtures contained twenty-five analytes representative of breath and ambient air constituents, including hydrocarbons, ketones, aldehydes, aromatics, and sulphurs, in the concentration range 0.1-700 ppbv. The two needles showed different behaviours, as recovery was independent of humidity for single-beds, whereas a low recovery (10-20%) was observed when triple-beds trapped very volatile compounds at low humidity (e.g. pentane and ethanol, 10% relative humidity. Triple-beds showed an almost quantitative recovery (>90%) of all the analytes at 50% and 85% relative humidity. This big difference was probably due to the reduced action of water vapour pressure during the desorption step. The addition of 6D-acetone and 8D-toluene to the sorbent material before gas sampling and the normalization of raw data nullified this effect, thereby lowering the variations of analyte recovery at different humidity levels down to 20%. Internal standards were also exploited to limit within 10-20% alterations in peak areas of very volatile compounds during needle storage at room temperature. This variation may results from a loss of water vapour either retained from the sorbent material and/or condensed on triple-bed needle walls. After normalization, the inter- and intra-day precision were halved to 5% and 10% in the case of single-beds, respectively, and to 15% and 20% with three-beds. The addition of an internal standard to the sorbent helps to keep the overall analytical procedure under control and improves the reliability of needle trap micro-extraction for the analysis of volatile organic compounds at ultra-trace levels.

Evaluation of needle trap micro-extraction and solid-phase micro-extraction: Obtaining comprehensive information on volatile emissions from in vitro cultures.

Volatile organic compounds (VOCs) emitted from in vitro cultures may reveal information on species and metabolism. Owing to low nmol L-1 concentration ranges, pre-concentration techniques are required for gas chromatography-mass spectrometry (GC-MS) based analyses. This study was intended to compare the efficiency of established micro-extraction techniques - solid-phase micro-extraction (SPME) and needle-trap micro-extraction (NTME) - for the analysis of complex VOC patterns. For SPME, a 75 µm Carboxen®/polydimethylsiloxane fiber was used. The NTME needle was packed with divinylbenzene, Carbopack X and Carboxen 1000. The headspace was sampled bi-directionally. Seventy-two VOCs were calibrated by reference standard mixtures in the range of 0.041-62.24 nmol L-1 by means of GC-MS. Both pre-concentration methods were applied to profile VOCs from cultures of Mycobacterium avium ssp. paratuberculosis. Limits of detection ranged from 0.004 to 3.93 nmol L-1 (median = 0.030 nmol L-1 ) for NTME and from 0.001 to 5.684 nmol L-1 (median = 0.043 nmol L-1 ) for SPME. NTME showed advantages in assessing polar compounds such as alcohols. SPME showed advantages in reproducibility but disadvantages in sensitivity for N-containing compounds. Micro-extraction techniques such as SPME and NTME are well suited for trace VOC profiling over cultures if the limitations of each technique is taken into account.

Vacuum ultraviolet absorption spectroscopy in combination with comprehensive two-dimensional gas chromatography for the monitoring of volatile organic compounds in breath gas: A feasibility study.

Vacuum ultraviolet (VUV) absorption spectroscopy was recently introduced as a new detection system for one, as well as comprehensive two-dimensional gas chromatography (GC×GC) and successfully applied to the analysis of various analytes in several matrices. In this study, its suitability for the analysis of breath metabolites was investigated and the impact of a finite volume of the absorption cell and makeup gas pressure was evaluated for volatile analytes in terms of sensitivity and chromatographic resolution. A commercial available VUV absorption spectrometer was coupled to GC×GC and applied to the analysis of highly polar volatile organic compounds (VOCs). Breath gas samples were acquired by needle trap micro extraction (NTME) during a glucose challenge and analysed by the applied technique. Regarding qualitative and quantitative information, the VGA-100 is compatible with common GC×GC detection systems like FID and even TOFMS. Average peak widths of 300ms and LODs in the lower ng range were achieved using GC×GC-VUV. Especially small oxygenated breath metabolites show intense and characteristic absorption patterns in the VUV region. Challenge responsive VOCs could be identified and monitored during a glucose challenge. The new VUV detection technology might especially be of benefit for applications in clinical research.