The overall goal of this experiment is to characterize chemical composition of exhaled breath using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry. This method can help to answer key questions in the medical field such as early diagnosis of diseases, therapeutic check monitoring, and environmental exposure. The main advantage of the technique is the measurement can be performed in real time.
The technique is also highest output, sensitive, specific, and user friendly. The implications of this technique have been extend to the diagnosis of lung cancer. Because chance of bio marker candidates have been find in exhaled breath of lung caner patients at different stages.
Generally, individuals new to this method will struggle because the ionization source is commonly co-made. Visual demonstration is critical, as there is no standard protocol for this method yet. To begin, set up a secondary nano ESI source according to the secondary electrospary ionization process in which breath gas is introduced to intersect an electrospray plume and ionized by the charged droplets.
The sources for the individual labs depend on the mass spectrometer used. Here, the secondary nano ESI source is based on the commercial nano ESI source. And attached it to a bench drop quarter pull orbit trap mass spectrometer.
The main body of the source is a cubic stainless steel chamber with an inlet to introduce the nano ESI capillary into the chamber. Two stainless steel tubes are installed on each side of the chamber for gas delivery. Two quartz windows are equipped on the top and bottom of the chamber to check the position of the tip of the nano ESI capillary and nano ESI spray either by eyes or a digital microscope.
Finally, the chamber is welded to the sweep cone of the mass spectrometer. Note that the design may change depending on the particular geometry of the atmospheric pressure interface of the mass spectrometer used in individual labs. Calibrate the mass spectrometer in both positive and negative ion detection modes according to manufacturers instructions.
By applying calibration, mass spectrometer parameters, such as lens potentials and detection conditions are optimized to give good sensitivity and peak shape at a specified resolution value. Perform complete calibrations by using the commercial ESI source. The mass resolution of 70, 000 is used here.
Mass calibration can be performed with any compatible sources, including customized ones. Set the temperature of the ion transfer tube of the mass spectrometer to greater than 100 degrees Celsius. Though the highest temperature can be set at 350 degrees Celsius, it may result in the decomposition of some compounds.
For the ESI solvent and flow rate, select the appropriate ESI solvent on the basis of properties of solvent and targeted compounds. In this experiment use water containing 0.1%Formic Acid, for high ionization efficiency. Set the flow rate of the ESI solvent in the range of 0 1.5 microliters per minute, to 200 nanoliters per minute.
Optimize the secondary nano ESI source parameters, mainly nano ESI voltage and nano ESI capillary tip position. The voltage commonly ranges from 2.0 4.5 kilovolts. Use 2.5 kilovolts here.
Apply pure gas to the source to reduce the influence of volatile organic compounds or VOCs from indoor air. High purity nitrogen is used here and delivered at 0.8 liters per minute. With the presence of pure gas, the normalized intensity level observed in a mass spectrum should be greater than 1 x 10 to the 5th, and the variation of the TIC should be less than 10%in both positive and negative ion detection modes.
When performing measurement, inhale the indoor air and perform a normal exhalation to breathe out all the air in the lungs at a constant flow rate. Monitor the exhalation flow rate either by a manometer or a flow meter visible to the subject. Connect the inlet of the flow meter to nafion tubing to remove the water vapor in the exhaled breath.
Then, connect the outlet of the flow meter to PTFE tubing to deliver the breath gas. In this experiment, the subject exhaled at 0.4 liters per minute, as controlled by a flow meter. It takes less than 30 seconds for one exhalation measurement.
Perform four to six replicated measurements. To minimize confounding effects, have participants abstain from eating, drinking, and brushing their teeth at least 30 minutes prior to the measurements. During the measurement, keep checking if the iron intensity exceeds the linear detection limit of the instrument or not.
The saturation of signal can lead to an artifact peak that does not result from a compound in the sample. By inhaling through the nose, part of ambient VOCs and particles would be removed. However, it is noteworthy that compounds in the nasal passages may also be detected.
Use software to record chromatagrams and mass spectra. Because this is direct MS analysis, and no chromatagraphic separation is performed, the TIC actually indicates the time trace of all the signals detected in the mass spectra. And the EIC shows a time trace of a specified compound.
Shown here are representative results of exhaled breath measurements in real time. The time trace of endol, a typical endogenous compound, shows six replicated measurements in less than seven minutes. For all the measurements, reproducible background subtracted breath fingerprints were obtained in positive ion detection mode.
291 compounds have been observed. The compounds are most likely aldehydes, ketones, and alkynes. In the case of breath fingerprints in negative ion detection mode, 173 compounds are presented, and mainly result from fatty acids.
After watching this video, you should have a good understanding of how to set up a homemade CESI source and perform real time process analysis. Once mastered, the measurement can be performed in minutes. If it is performed properly.
While attending this procedure, it is important to remember to avoid interference of the compounds from the sampling tubing and endo air. Know this measure can provide insights into human exhaled breath, and can also be applied to other systems such as model alkylize, ion models, and in retro-cell systems. Following this procedure, other measures like GCMS, HPRCMS, can be performed to answer additional questions, like for photo confirmation, quantity of analysis.
After it's development, the technique paved the ways for researchers in the field of medicine to explore with this diagnosis, treatment effects in the patients.
