Summary of Study ST002543

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, where it has been assigned Project ID PR001638. The data can be accessed directly via it's Project DOI: 10.21228/M8ZX4D This work is supported by NIH grant, U2C- DK119886.


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Study IDST002543
Study TitleGC/MS analysis of hypoxic volatile metabolic markers in the MDA-MB-231 breast cancer cell line
Study SummaryHypoxia in disease describes persistent low oxygen conditions, observed in a range of pathologies, including cancer. In the discovery of biomarkers in biological models, pathophysiological traits present a source of translatable metabolic products for the diagnosis of disease in humans. Part of the metabolome is represented by its volatile, gaseous fraction; the volatilome. Human volatile profiles, such as those found in breath, are able to diagnose disease, however accurate volatile biomarker discovery is required to target reliable biomarkers to develop new diagnostic tools. Using custom chambers to control oxygen levels and facilitate headspace sampling, the MDA-MB-231 breast cancer cell line was exposed to hypoxia (1% oxygen) for 24 hours. The maintenance of hypoxic conditions in the system was successfully validated over this time period. Targeted and untargeted gas chromatography mass spectrometry approaches revealed four significantly altered volatile organic compounds when compared to control cells. Three compounds were actively consumed by cells: methyl chloride, acetone and n-Hexane. Cells under hypoxia also produced significant amounts of styrene. This work presents a novel methodology for identification of volatile metabolisms under controlled gas conditions with novel observations of volatile metabolisms by breast cancer cells.
University of York
Last NameIssitt
First NameTheo
AddressBiology Dept. University of York, Personal
Submit Date2023-03-31
Num Groups4
PublicationsT. Issitt et al., Volatile compounds in human breath: critical review and meta-analysis Journal of Breath Research, Volume 16, Number 2 (2022)
Analysis Type DetailGC-MS
Release Date2023-04-21
Release Version1
Theo Issitt Theo Issitt application/zip

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Combined analysis:

Analysis ID AN004190
Analysis type MS
Chromatography type GC
Chromatography system HP GCD 1800B
Column Agilent PoraBOND Q (25m x 0.32mm x 0.5um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent/HP 5972 MSD
Units pg/hr/ug and g/hr for media


MS ID:MS003937
Analysis ID:AN004190
Instrument Name:Agilent/HP 5972 MSD
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:Calibration was performed using standard gases (BOC Specialty Gases, Woking, UK). Linear regression of calibration curves confirmed strong, positive linear relationships between observed compound peak areas and moles of gas injected for each VOC (r2 > 0.9 in all cases). For compounds not purchased in gaseous state (BOC Specialty gases, as above), 1–2 mL of compound in liquid phase was injected neat into butyl sealed Wheaton-style glass vials (100 mL) and allowed to equilibrate for 1 h. 1 mL of headspace air was then removed from neat vial headspace using a gas tight syringe (Trajan, SGE) and injected into the headspace of a second 100 mL butyl sealed Wheaton-style glass vial. This was then repeated, and 1 mL of the 2nd serial dilution vial was injected into the GCMS system with 29 mL of lab air to give ppb concentrations. This was performed for methanethiol (MeSH (SPEXorganics, St Neots, UK)), isoprene (Alfa Aesar, Ward Hill, MA, USA), acetone (Sigma-Aldrich, Burlington, MA, USA), 2- & 3-methyl pentane and n-hexane (Thermo Scientific, Waltham, MA, USA). Reported compounds detected by the GC/-MS were confirmed by matching retention times and mass–charge (m/z) ratios with known standards. Equation 1: [VOC](ppt)=(CF x 〖10〗^12 x Peak area x Calibration slope)/n Equation 1 outlines the approach to calculating VOC concentrations in parts-per-trillion-by-volume, or pptv. Here Peak area refers to the combined peak areas for the mass-charge ratios identified in Table 1. Multiplying Peak areas by their associated calibration curves (Calibration Slope) generate molar amounts which, when divided by the number of moles of headspace air injected (n), generate a unitless (moles compound/moles of air) ratio. Pptv concentrations are then obtained by multiplying this unitless ratio by 1x1012. For clarity, part-per-billion-by-volume values would be obtained by multiplying the unitless ratios by 1x109, or one billion. Sample VOC concentrations were then normalised to CFC-11 concentrations (240 parts-per-trillion-by-volume (pptv)) through multiplication by a “correction factor”, or CF, Equation 1). CFC-11 was used as an internal standard, since atmospheric concentrations of CFC-11 are globally consistent and stable (Redeker et al., 2007). Quantification of Styrene was done as above but normalisation to CFC-11 was not possible under flushed, hypoxic conditions. NEGATIVE VALUES IN DATA SHOW CONSUMPTION OVER TIME. VARIATION IN SCALE BETWEEN MEDIA SAMPLES ARE DUE TO NORMALISATION OF CELLULAR DATA TO PROTEIN. AS DESCRIBED, MEDIA VALUES ARE SUBTRACTED FROM CELLULAR DATA PRIOR TO NORMALISATION AND EXPRESSED AS PG/HR/UG.