Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Summary

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Abstract

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

Introduction

With fast development of modern analytical techniques, hundreds of volatile organic compounds (VOCs) have been identified in human exhaled breath¹. These VOCs mostly result from alveolar air (~350 mL for a healthy adult) and anatomical dead space air (~150 mL)², which are affected by body metabolism^3,4,5,6,7,8 and environmental pollution⁹, respectively. As a result, if identified, these VOCs are promis....

Protocol

Caution: Please consult all relevant material safety data sheets (MSDS) before use. Please use appropriate personal protective equipment, e.g., lab coat, gloves, goggles, full length pants and closed-toe shoes).

1. Set up the Sec-nanoESI source

1. Set up a Sec-nanoESI source according to the SESI process, i.e., the breath gas is introduced to intersect an electrospray plume and ionized by the charged droplets (Figure 1). The sources built in individual labs depend on the interface of the mass spectrometer used^24,25. Here, set ....

Results

Figure 3 shows the breath fingerprints in the mass range of m/z 50-750 recorded under both positive and negative ion detection modes. 291 peaks (peak intensity > 5.0x10⁴) and 173 peaks (peak intensity > 3.0x10⁴) have been observed in background-subtracted breath fingerprints in the positive and negative ion detection modes, respectively. To identify peaks in the mass spectra, please refer to prior publications for details<.......

Discussion

Constructing the Sec-nanoESI source based on a commercial nanoESI source, the ionization efficiency is higher than that of using an ESI source³⁰. In addition, the ionization efficiency is further improved in a closed chamber, as it isolates the process from the ambient background air, and at the same time facilitates the mixing between the gas sample and the spray plume. By using a Sec-nanoESI, less parameters need to be optimized compared to an ESI source, making it easier for installation, appli.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ultrapure water	Merck Millipore, USA	MPGP04001	Resistance >18.2 MΩ·cm
Formic acid	Sigma-Aldrich, USA	F0507	Corrosive to the respiratory tract.
Nitrogen gas	Guangzhou Shiyuan Gas Co. Ltd., China	N.A.a	Purity >99.99%
Q Exactive hybrid quadrupole-orbitrap mass spectrometer	Thermo Scientific, USA	02634L(S/N)	Beware of high voltage and high temperature
NanoESI source	Thermo Scientific, USA	ES002373(S/N); ES071(P/N)	Beware of high voltage and high temperature
Nano LC pump	Thermo Scientific, USA	5041.0010A(P/N)	/
Xcalibur software (Version 3.0)	Thermo Scientific, USA	BRE0008596	/
Dino-Lite Digital Microscope	Tech Video System (SuZhou) Co.Ltd., China	CQ401833R(S/N)	/
Nafion tubing	Perma Pure LLC, USA	ME60	/
PTFE tubing (I.D. 4 mm)	Dongguan Hongfu Insulating Material Co. Ltd., China	N.A.	Beware of the possible loss of polar compounds
Mass flow controller	Line-Tech, Korea	M15122007 (S/N)	/
Flow meter	Yuyao Industrial Automation Meter Factory, China	40784	/
aN.A.: not available.