Thermal Desorption Methodology for Volatile Organic Compounds (VOCs) Identification: Possible Application in Invasive Fungal Infections A. Vicent Claramunta*, A. Paškevičiusb , J. Švedienėb , E. Gudeliūnaitė b, S. Kiverytėc , R. Petrauskaitėc, L. Griškevičiusc, A. Zolumskisa, E. Naujalisa Center for Physical Sciences and Technology, Savanorių Ave. 231, LT-02300 Vilnius, Lithuania. b Nature Research Center, Akademijos 2, LT-08412 Vilnius, Lithuania. c Vilnius University Hospital Santariškių Klinikos and Vilnius University, Santariškių 2, LT-08661 Vilnius, Lithuania * Email:
[email protected] a
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Introduction Almost 2000 volatile organic compounds (VOCs) found in healthy humans. Identification of unusual compounds or differences in their levels could be used as biomarkers for several diseases, including invasive fungal infections. Invasive pulmonary Aspergillosis (IA) is a major cause of mortality in patients with weak immunity system. IA diagnosis is still very difficult in early stages. Aspergillus fumigatus fungi emits a characteristic VOCs profile, distinctive from other pathogenic molds. Breath analysis could be used as non-invasive methodology for IA diagnosis. Thermal Desorption has been used for VOC analysis among other techniques. A home-made sampling system is being developed for detection of VOC from Aspergillus fungi in-vitro and in-vivo. For in-vitro samples a study of different parameters was performed; firstly focusing on separation for identification and the improvement of the intensity in the key components.
Sample Fungi samples were prepared in the Nature Research Center in Lithuania. 20 ml flasks containing 5 ml of matrix and the fungal culture. For all the samples at least 1 control sample was prepared. Several different Aspergillus species in different matrixes were prepared. Once sporulation started, the air inside the flasks was collected.
Fungi Aspergillus fumigatus. “Microcosmos” by Brandon Broll
Methods • Desorption tubes (Markes Tenax TA) were connected to an air pump (AMETEK Alpha-2) and VOC’s produced by fungi were collected through a syringe with a 0.22 μm filter to avoid the release of spores to the environment. • Flasks were placed into a water bath (35.5 oC) and air was pumped at 25 ml/min during 60 min. A total of 1.5 l of air trapped in the sorbent tubes was then run in our Thermal desorption system (ATD 400 Perkin Elmer) followed by GC-HRMS (Agilent 6890N coupled with Waters MS AutoSpec Premier) analysis. Parameters Pump
Thermal Desorption
GC-HRMS Oven temperatures
35oC; 5 min
Flow
25 ml/min
Carrier gas
Helium
Sampling time
60 min
Desorption flow
60 ml/min
10oC/min; 120oC; 5 min
Desorption time
5 min
15oC/min; 220oC; 10 min
Oven Temp.
320oC
Gas flow
1 ml/min
Outlet split
10 ml/min MS range (full scan)
50-220 u
Results Comparing with “Control” sample we could identify at least 12 different compounds coming from fungi. 2-Pentyl Furan and Bergamotene could be considered as biomarkers for IA.
3: Bergamotene 4: Unknown 5: β-Farnesene 6: Oxalic acid
Conclusions and Future work • Thermal desorption seems a good an easy way for fungal VOC detection during in-vitro analysis. • SIM analysis should be performed to increase sensitivity. • In-vivo analysis should be performed and correlate with in-vitro results. • Single use breath sampling needs to be developed for patients with IA risk.
Fungi Samples
1: 2-Pentyl Furan 2: 2-Hexyl Furan
References
Chambers et al. “Novel diagnosis: progress toward a breath test for invasive Aspergillus Fumigatus” Medical Mycology. April 2011, 49(Suppl. 1), S54-S61. Sethi et al. “Clinical Application of Volatile Organic Compounds Analysis for Detecting Infectious Diseases. Clin. Microbiol. Rev. 2013, 26(3):462 Moularat et al. “Detection of fungal development in a closed environment through the identification of specific VOC: Demonstration of a specific VOC fingerprint for fungal development” Science of total Environment 407 (2008) 139-146 Lin et al. “ Identification of Unique Volatile compounds of Aspergillus Fumigatus for Potential diagnostic Breath Test by HS-SPME and GC-MS” J Immunol Tech Infect Dis 2013, 2:3 Koo et al. “A breath Secondary Metabolite Signature to Diagnose Invasive Aspergillosis” Clinical Infectious Diseases October 22, 2014
Chromatogram for Aspergillus sample overlapped with “Control” (Subtracted masses 81 and 119 u)
