The peppermint breath test: a benchmarking protocol for breath sampling and analysis using GC-MS.

Exhaled breath contains hundreds of volatile organic compounds (VOCs) which offer the potential for diagnosing and monitoring a wide range of diseases. As the breath research field has grown, sampling and analytical practices have become highly varied between groups. Standardisation would allow meta-analyses of data from multiple studies and greater confidence in published results. Washout of VOCs from ingestion into the blood and subsequently breath could provide data for an initial assessment of inter-group performance. The Peppermint Initiative has been formed to address this task of standardisation. In the current study we aimed to generate initial benchmark values for thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) analysis of breath samples containing peppermint-derived VOCs using data from three independent European research groups. Initially, headspace analysis of peppermint oil capsules was performed to determine compounds of interest. Ten healthy participants were recruited by each three groups across Europe. The standard Peppermint protocol was followed. In brief, each participant provided a baseline breath sample prior to taking a peppermint capsule, with further samples collected at 60, 90, 165, 285 and 360 min following ingestion. Sampling and analytical protocols were different for each group, in line with their usual practice. Samples were analysed by TD-GC-MS and benchmarking values determined for the time taken for detected peppermint VOCs to return to baseline values. Sixteen compounds were identified in the capsule headspace, and all were confirmed in breath following ingestion of the peppermint capsules. Additionally, 2,3-dehydro-1,8-cineole was uniquely found in the breath samples, with a washout profile that suggested it was a product of metabolism of peppermint compounds. Five compounds (α-pinene, ß-pinene, eucalyptol, menthol and menthone) were quantified by all three groups. Differences were observed between the groups, particularly for the recovery of menthone and menthol. The average time taken for VOCs to return to baseline was selected as the benchmark and were 377, 423, 533, 418 and 336 min for α-pinene, ß-pinene, eucalyptol, menthone and menthol respectively. We have presented an initial set of easy-to-measure benchmarking values for assessing the performance of TD-GC-MS systems for the analysis of VOCs in breath. These values will be updated when more groups provide additional data.

A sampler prototype for the simultaneous collection of exhaled air and breath condensate.

Exhaled air and breath condensate contain a large number of health biomarkers, such as volatile and semi-volatile organic compounds, proteins and lipids. Nowadays, the collection of breath samples is carried out by commercial or lab-made sampling systems that collect only one type of sample (e.g. gaseous or condensate phase), thus limiting the diagnostic capability of breath tests. This work presents a portable prototype optimized for the simultaneous collection of gaseous exhaled breath and exhaled breath condensate within five minutes. The system is fully portable and has a total weight of about 1 Kg. An illustrative determination of ethanol, isoprene, acetone, isopropyl alcohol, 1-propanol, 2-butanone, 2-pentanone, toluene and xylenes in breath, and cortisol and 8-iso-prostaglandin F2α in breath condensate is discussed.

Determination of carbonyl compounds in exhaled breath by on-sorbent derivatization coupled with thermal desorption and gas chromatography-tandem mass spectrometry.

A reliable method for the determination of carbonyl compounds in exhaled breath based on on-sorbent derivatization coupled with thermal desorption and gas chromatography-tandem mass spectrometry is described. The analytical performances were optimized for a mixture of C2-C9 aldehydes and C3-C9 ketones, particularly interesting for clinical applications, by using an internal standard and applying a 23 full factorial design. A volume of sample (250 ml) was loaded at 50 ml min-1 into a Tenax GR sorbent tube containing 130 nmol of O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride. All compounds showed a limit of detection lower than 200 pptv. The yield of the derivatization procedure was normalized by adding to the sample a known amount of 6D-acetone as an internal standard. This allowed halving the relative standard deviation to 10% and 15% for the mono-and di-carbonyl compounds, respectively, thus improving reliability. The optimized method was applied to the determination of carbonyl compounds in 12 breath samples collected from four patients suffering from heart failure during hospitalization.

Determination of volatile organic compounds in exhaled breath of heart failure patients by needle trap micro-extraction coupled with gas chromatography-tandem mass spectrometry.

The analytical performances of needle trap micro-extraction (NTME) coupled with gas chromatography-tandem mass spectrometry were evaluated by analyzing a mixture of twenty-two representative breath volatile organic compounds (VOCs) belonging to different chemical classes (i.e. hydrocarbons, ketones, aldehydes, aromatics and sulfurs). NTME is an emerging technique that guarantees detection limits in the pptv range by pre-concentrating low volumes of sample, and it is particularly suitable for breath analysis. For most VOCs, detection limits between 20 and 500 pptv were obtained by pre-concentrating 25 ml of a humidified standard gas mixture at a flow rate of 15 ml min-1. For all compounds, inter- and intra-day precisions were always below 15%, confirming the reliability of the method. The procedure was successfully applied to the analysis of exhaled breath samples collected from forty heart failure (HF) patients during their stay in the University Hospital of Pisa. The majority of patients (about 80%) showed a significant decrease of breath acetone levels (a factor of 3 or higher) at discharge compared to admission (acute phase) in correspondence to the improved clinical conditions during hospitalization, thus making this compound eligible as a biomarker of HF exacerbation.

Comparison of sampling bags for the analysis of volatile organic compounds in breath.

Nalophan, Tedlar and Cali-5-Bond polymeric bags were compared to determine the most suitable type for breath sampling and storage when volatile organic compounds are to be determined. Analyses were performed by thermal desorption gas chromatography mass spectrometry. For each bag, the release of contaminants and the chemical stability of a gaseous standard mixture containing eighteen organic compounds, as well as the CO2 partial pressure were assessed. The selected compounds were representative of breath constituents and belonged to different chemical classes (i.e. hydrocarbons, ketones, aldehydes, aromatics, sulfurs and esters). In the case of Nalophan, the influence of the surface-to-volume ratio, related to the bag's filling degree, on the chemical stability was also evaluated. Nalophan bags were found to be the most suitable in terms of contaminants released during storage (only 2-methyl-1,3-dioxalane), good sample stability (up to 24 h for both dry and humid samples), and very limited costs (about 1 € for a 20 liter bag). The (film) surface-to-(sample) volume ratio was found to be an important factor affecting the stability of selected compounds, and therefore we recommended to fill the bag completely.