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SESI-MS SUPER SESI coupled with Thermo Fisher Scientific-Orbitrap

Secondary electro-spray ionization (SESI) is an ambient ionization technique for the analysis of trace concentrations of vapors, where a nano-electrospray produces charging agents that collide with the analyte molecules directly in gas-phase. In the subsequent reaction, the charge is transferred and vapors get ionized, most molecules get protonated (in positive mode) and deprotonated (in negative mode). SESI works in combination with mass spectrometry or ion-mobility spectrometry.


The fact that trace concentrations of gases in contact with an electrospray plume were efficiently ionized was first observed by Fenn and colleagues when they noted that tiny concentrations of plasticizers produced intense peaks in their mass spectra.[1] However, it was not until 2000 when this problem was reframed as a solution, when Hill and coworkers used an electrospray to ionize molecules in the gas phase,[2] and named the technique Secondary Electrospray Ionization. In 2007, the almost simultaneous works of Zenobi[3] and Pablo Sinues[4] applied SESI to breath analysis for the first time, marking the beginning of a fruitful field or research.[5] With sensitivities in the low pptv range (10−12), SESI has been used in other applications, where the detection of low volatility vapors is important.

Detecting low volatility species in the gas phase is important because larger molecules tend to have higher biological significance. Low volatility species have been overlooked because it is technically difficult to detect them, as they are in very low concentration, and they tend to condensate in the inner piping of instruments. However, as this problem is solved, and new instruments are able to handle larger and more specific molecules, the ability to perform on-line, real time analysis of molecules naturally released in the air, even at minute concentrations, is attracting attention to this ionization technique.

Principle of operation

Secondary electrospray ionization mechanism diagram

In the early days of SESI, two ionization mechanisms were under debate.: the droplet-vapor interaction model postulates that vapors are adsorbed in the electrospray ionization (ESI) droplets, and then reemitted as the droplet shrinks, just as regular liquid phase analytes are produced in electrospray ionization; on the other hand, the ion-vapor interaction model postulates that molecules and ions or small clusters collide, and the charge is transferred in this collision. Currently available commercial SESI sources operate at high temperature so as to better handle low volatility species.[6] In this regime, nanodroplets from the electrospray evaporate very quickly to form ion clusters in equilibrium. This results in ion-vapor reactions dominating the majority of the ionization region. As charging ions originate from nano-droplets, and no high energy ions are involved at any point of the ionization process nor the creation of ionizing agents, fragmentation in SESI is remarkably low, and the resulting spectra are very clean. This allows for a very high dynamic range, where low intensity peaks are not affected by more abundant species.[7]

Some related techniques are laser ablation electrospray ionization, proton-transfer-reaction mass spectrometry and selected-ion flow-tube mass spectrometry.


Real-time breath analysis

The main feature of SESI is that it can detect minuscule concentrations of low volatility species in real time, with molecular masses as high as 700 Da, falling in the realm of metabolomics. These molecules are naturally released by living organisms, and are commonly detected as odors, which means that they can be analyzed non-invasively. SESI, combined with High Resolution Mass Spectrometry, provides time-resolved, biologically relevant information of living systems, where the system does not need to be interfered with. This allows to seamlessly capture the time evolution of their metabolism and their response to controlled stimuli.

SESI has been widely used for breath gas analysis for biomarker discovery, and in vivo pharmacokinetic studies:

Biomarker discovery

Biomarker discovery

Bacterial infection

It has been widely reported the identification of bacteria by their volatile organic compound fingerprint. SESI-MS has proven to be a robust technique for the identification of bacteria from cell cultures and infections in vivo from breath samples, after the development of libraries of vapor profiles.[8][9][10][11] Other studies include: In vivo differentiation between critical pathogens Staphylococcus aureus and Pseudomonas aeruginosa.[12] or differential detection among antibiotic resistant S. aureus and its non-resistant strains.[13] Bacterial infection detection from other fluids such as saliva have also been reported.[14]

Respiratory diseases

Many chronic respiratory diseases lack of an appropriate method of monitoring and differentiation among disease stages. SESI-MS has been used to diagnose and distinguish exacerbations from breath samples in chronic obstructive pulmonary disease.[15][16] Metabolic profiling of breath samples has accurately differentiated healthy individuals from idiopathic pulmonary fibrosis[17] or obstructive sleep apnea patients.[18]


SESI-MS is being studied as a non-invasive detection system of cancer biomarkers in breath. A preliminary study differentiates patients suffering from breast neoplasia.[19]


Volatiles released from the skin can be detected by sampling the ambient gas surrounding it, providing a fast method for detecting metabolic changes in fatty acids composition patterns.[20][21]

Non-invasive drug monitoring


To study pharmacokinetics, it is necessary a robust technique because of the complex nature of the samples' matrix, be it plasma, urine, or breath.[22] Recent studies show that secondary electrospray ionization (SESI) is a powerful technique to monitor drug kinetics via breath analysis.[23][24] Because breath is naturally produced, several datapoints can be readily collected. This allows for the number of collected data-points to be greatly increased.[25] In animal studies, this approach SESI can reduce animal sacrifice while yielding pharmacokinetic curves with unmatched time resolutions.[24][25] In humans, SESI-MS non-invasive analysis of breath can help study the kinetics of drugs at a personalized level.[23][26][27] Monitoring exogenously introduced species allows tracking their specific metabolic pathway, which reduces the risk of picking confounding factors.

In-vivo metabolomics

Time-resolved metabolic analysis

Introducing known stimuli, such as specific metabolites isotopically labeled compounds, or other sources of stress triggers metabolic changes which can be easily monitored with SESI-MS. Some examples if this include: cell culture volatile compounds profiling;[28] and metabolic studies for plant[29] or trace human metabolic pathways.[30][31][32]

Other applications

Other applications developed with SESI-MS include:

  • Detection of illicit drugs;[3]
  • Detection of explosives;[33][34]
  • Food quality control monitoring.[35][36]


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  2. ^ Wu, C.; Siems, W. F.; Hill, H. H. (2000-01-15). "Secondary electrospray ionization ion mobility spectrometry/mass spectrometry of illicit drugs". Analytical Chemistry. 72 (2): 396–403. doi:10.1021/ac9907235. ISSN 0003-2700. PMID 10658336.
  3. ^ a b "Zenobi Group, Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich".
  4. ^ "Botnar Professorship, University Children's hospital Basel, UKBB".
  5. ^ "Zurich Exhalomics project, University of Zurich, UZH". Archived from the original on 2021-05-20. Retrieved 2019-05-29.
  6. ^ "Fossil Ion Technology, Málaga, Spain".
  7. ^ Martinez-Lozano Sinues, Pablo; Criado, Ernesto; Vidal, Guillermo (2012). "Mechanistic study on the ionization of trace gases by an electrospray plume". International Journal of Mass Spectrometry. 313: 21–29. Bibcode:2012IJMSp.313...21M. doi:10.1016/j.ijms.2011.12.010.
  8. ^ Ballabio, Claudia; Cristoni, Simone; Puccio, Giovanni; Kohler, Malcolm; Sala, Maria Roberta; Brambilla, Paolo; Martinez-Lozano Sinues, Pablo (2014). "Rapid identification of bacteria in blood cultures by mass-spectrometric analysis of volatiles". Journal of Clinical Pathology. 67 (8): 743–746. doi:10.1136/jclinpath-2014-202301. ISSN 0021-9746. PMID 24817704. S2CID 43907088.
  9. ^ Zhu, Jiangjiang; Bean, Heather D.; Jiménez-Díaz, Jaime; Hill, Jane E. (2013). "Secondary electrospray ionization-mass spectrometry (SESI-MS) breathprinting of multiple bacterial lung pathogens, a mouse model study". Journal of Applied Physiology. 114 (11): 1544–1549. doi:10.1152/japplphysiol.00099.2013. ISSN 8750-7587. PMC 3680826. PMID 23519230.
  10. ^ Zhu, Jiangjiang; Hill, Jane E. (2013). "Detection of Escherichia coli via VOC Profiling using Secondary Electrospray Ionization-Mass Spectrometry (SESI-MS)". Food Microbiology. 34 (2): 412–417. doi:10.1016/j.fm.2012.12.008. ISSN 0740-0020. PMC 4425455. PMID 23541210.
  11. ^ Ratiu, Ileana-Andreea; Ligor, Tomasz; Bocos-Bintintan, Victor; Buszewski, Bogusław (2017). "Mass spectrometric techniques for the analysis of volatile organic compounds emitted from bacteria". Bioanalysis. 9 (14): 1069–1092. doi:10.4155/bio-2017-0051. ISSN 1757-6180. PMID 28737423.
  12. ^ Zhu, Jiangjiang; Jiménez-Díaz, Jaime; Bean, Heather D; Daphtary, Nirav A; Aliyeva, Minara I; Lundblad, Lennart K A; Hill, Jane E (2013-07-18). "Robust detection of P. aeruginosa and S. aureus acute lung infections by secondary electrospray ionization-mass spectrometry (SESI-MS) breathprinting: from initial infection to clearance". Journal of Breath Research. 7 (3): 037106. Bibcode:2013JBR.....7c7106Z. doi:10.1088/1752-7155/7/3/037106. ISSN 1752-7155. PMC 4425453. PMID 23867706.
  13. ^ Bean, Heather D.; Zhu, Jiangjiang; Sengle, Jackson C.; Hill, Jane E. (2014). "Identifying methicillin-resistantStaphylococcus aureus(MRSA) lung infections in mice via breath analysis using secondary electrospray ionization-mass spectrometry (SESI-MS)". Journal of Breath Research. 8 (4): 041001–41001. Bibcode:2014JBR.....8d1001B. doi:10.1088/1752-7155/8/4/041001. ISSN 1752-7163. PMC 4443899. PMID 25307159.
  14. ^ Bregy, Lukas; Müggler, Annick R.; Martinez-Lozano Sinues, Pablo; García-Gómez, Diego; Suter, Yannick; Belibasakis, Georgios N.; Kohler, Malcolm; Schmidlin, Patrick R.; Zenobi, Renato (2015). "Differentiation of oral bacteria in in vitro cultures and human saliva by secondary electrospray ionization – mass spectrometry". Scientific Reports. 5 (1): 15163. Bibcode:2015NatSR...515163B. doi:10.1038/srep15163. ISSN 2045-2322. PMC 4609958. PMID 26477831.
  15. ^ Gaugg, Martin Thomas; Nussbaumer-Ochsner, Yvonne; Bregy, Lukas; Engler, Anna; Stebler, Nina; Gaisl, Thomas; Bruderer, Tobias; Nowak, Nora; Sinues, Pablo (2019). "Real-Time Breath Analysis Reveals Specific Metabolic Signatures of COPD Exacerbations". Chest. 156 (2): 269–276. doi:10.1016/j.chest.2018.12.023. PMID 30685334. S2CID 59304252.
  16. ^ Bregy, Lukas; Nussbaumer-Ochsner, Yvonne; Martinez-Lozano Sinues, Pablo; García-Gómez, Diego; Suter, Yannick; Gaisl, Thomas; Stebler, Nina; Gaugg, Martin Thomas; Kohler, Malcolm (2018). "Real-time mass spectrometric identification of metabolites characteristic of chronic obstructive pulmonary disease in exhaled breath". Clinical Mass Spectrometry. 7: 29–35. doi:10.1016/j.clinms.2018.02.003.
  17. ^ Kohler, M.; Zenobi, R.; Engler, A.; Bregy, L.; Gaugg, M. T.; Nussbaumer-Ochsner, Y.; Sinues, P. (2017-05-01). "119 Exhaled breath analysis by real-time mass spectrometry in patients with pulmonary fibrosis". Chest. 151 (5): A16. doi:10.1016/j.chest.2017.04.017. ISSN 0012-3692. S2CID 79732485.
  18. ^ Schwarz, Esther I; Martinez-Lozano Sinues, Pablo; Bregy, Lukas; Gaisl, Thomas; Garcia Gomez, Diego; Gaugg, Martin T; Suter, Yannick; Stebler, Nina; Nussbaumer-Ochsner, Yvonne (2015-12-15). "Effects of CPAP therapy withdrawal on exhaled breath pattern in obstructive sleep apnoea". Thorax. 71 (2): 110–117. doi:10.1136/thoraxjnl-2015-207597. ISSN 0040-6376. PMID 26671307.
  19. ^ He, Jingjing; Sinues, Pablo Martinez-Lozano; Hollmén, Maija; Li, Xue; Detmar, Michael; Zenobi, Renato (2014-06-06). "Fingerprinting Breast Cancer vs. Normal Mammary Cells by Mass Spectrometric Analysis of Volatiles". Scientific Reports. 4 (1): 5196. Bibcode:2014NatSR...4E5196H. doi:10.1038/srep05196. ISSN 2045-2322. PMC 5381500. PMID 24903350.
  20. ^ Martínez-Lozano, Pablo (2009). "Mass spectrometric study of cutaneous volatiles by secondary electrospray ionization". International Journal of Mass Spectrometry. 282 (3): 128–132. Bibcode:2009IJMSp.282..128M. doi:10.1016/j.ijms.2009.02.017. ISSN 1387-3806.
  21. ^ Martínez-Lozano, Pablo; Mora, Juan Fernández (2009). "On-line detection of human skin vapors". Journal of the American Society for Mass Spectrometry. 20 (6): 1060–1063. doi:10.1016/j.jasms.2009.01.012. ISSN 1044-0305. PMID 19251441.
  22. ^ Casas-Ferreira, Ana María; Nogal-Sánchez, Miguel del; Pérez-Pavón, José Luis; Moreno-Cordero, Bernardo (January 2019). "Non-separative mass spectrometry methods for non-invasive medical diagnostics based on volatile organic compounds: A review". Analytica Chimica Acta. 1045: 10–22. Bibcode:2019AcAC.1045...10C. doi:10.1016/j.aca.2018.07.005. PMID 30454564. S2CID 53874283.
  23. ^ a b Gamez, Gerardo; Zhu, Liang; Disko, Andreas; Chen, Huanwen; Azov, Vladimir; Chingin, Konstantin; Krämer, Günter; Zenobi, Renato (2011). "Real-time, in vivo monitoring and pharmacokinetics of valproic acid via a novel biomarker in exhaled breath". Chemical Communications. 47 (17): 4884–6. doi:10.1039/c1cc10343a. ISSN 1359-7345. PMID 21373707.
  24. ^ a b Li, Xue; Martinez-Lozano Sinues, Pablo; Dallmann, Robert; Bregy, Lukas; Hollmén, Maija; Proulx, Steven; Brown, Steven A.; Detmar, Michael; Kohler, Malcolm; Zenobi, Renato (2015-06-26). "Drug Pharmacokinetics Determined by Real-Time Analysis of Mouse Breath". Angewandte Chemie International Edition. 54 (27): 7815–7818. doi:10.1002/anie.201503312. hdl:20.500.11850/102558. PMID 26015026.
  25. ^ a b Gaugg, Martin T; Engler, Anna; Nussbaumer-Ochsner, Yvonne; Bregy, Lukas; Stöberl, Anna S; Gaisl, Thomas; Bruderer, Tobias; Zenobi, Renato; Kohler, Malcolm; Martinez-Lozano Sinues, Pablo (2017-09-13). "Metabolic effects of inhaled salbutamol determined by exhaled breath analysis". Journal of Breath Research. 11 (4): 046004. Bibcode:2017JBR....11d6004G. doi:10.1088/1752-7163/aa7caa. hdl:20.500.11850/220016. ISSN 1752-7163. PMID 28901297.
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  27. ^ Tejero Rioseras, Alberto; Singh, Kapil Dev; Nowak, Nora; Gaugg, Martin T.; Bruderer, Tobias; Zenobi, Renato; Sinues, Pablo M.-L. (2018-06-05). "Real-Time Monitoring of Tricarboxylic Acid Metabolites in Exhaled Breath". Analytical Chemistry. 90 (11): 6453–6460. doi:10.1021/acs.analchem.7b04600. ISSN 0003-2700. PMID 29767961.
  28. ^ Tejero Rioseras, Alberto; Garcia Gomez, Diego; Ebert, Birgitta E.; Blank, Lars M.; Ibáñez, Alfredo J.; Sinues, Pablo M-L (2017-10-27). "Comprehensive Real-Time Analysis of the Yeast Volatilome". Scientific Reports. 7 (1): 14236. Bibcode:2017NatSR...714236T. doi:10.1038/s41598-017-14554-y. ISSN 2045-2322. PMC 5660155. PMID 29079837.
  29. ^ Barrios-Collado, César; García-Gómez, Diego; Zenobi, Renato; Vidal-de-Miguel, Guillermo; Ibáñez, Alfredo J.; Martinez-Lozano Sinues, Pablo (2016-02-04). "Capturing in Vivo Plant Metabolism by Real-Time Analysis of Low to High Molecular Weight Volatiles". Analytical Chemistry. 88 (4): 2406–2412. doi:10.1021/acs.analchem.5b04452. ISSN 0003-2700. PMID 26814403.
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  35. ^ Bean, Heather D.; Mellors, Theodore R.; Zhu, Jiangjiang; Hill, Jane E. (2015-04-22). "Profiling Aged Artisanal Cheddar Cheese Using Secondary Electrospray Ionization Mass Spectrometry". Journal of Agricultural and Food Chemistry. 63 (17): 4386–4392. doi:10.1021/jf5063759. ISSN 0021-8561. PMID 25865575.
  36. ^ Farrell, Ross R.; Fahrentrapp, Johannes; García-Gómez, Diego; Martinez-Lozano Sinues, Pablo; Zenobi, Renato (2017). "Rapid fingerprinting of grape volatile composition using secondary electrospray ionization orbitrap mass spectrometry: A preliminary study of grape ripening". Food Control. 81: 107–112. doi:10.1016/j.foodcont.2017.04.041. ISSN 0956-7135.

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