Confirmatory Testing by Mass Spectrometry in Forensic Context

Summary

This video explains the process of electrospray ionization mass spectrometry (ESI-MS), highlighting its role in analytical chemistry and forensic toxicology. The technique involves applying a high voltage to a liquid to produce ions for mass spectrometry. The video outlines the use of the Thermo Q Exactive LC-MS-MS system, demonstrating calibration, cleaning procedures, and the importance of sensitivity checks. Key steps such as preparing the syringe with calibration solutions, setting the flow rate, and using software for real-time data visualization are detailed. It also covers equipment maintenance, including capillary and tubing replacement, ensuring the machine's optimal performance.

Takeaways

• 😀 ESI-MS (Electrospray Ionization Mass Spectrometry) generates ions for mass spectrometry by applying a high voltage to a liquid, creating an aerosol or gas.
• 😀 Mass spectrometry is used to measure the mass-to-charge ratio of ions and produces a mass spectrum plot showing relative absorbance against mass-to-charge ratio.
• 😀 The Thermo Q Exactive Quadrupole Orbitrap LC-MS-MS system is used for this process, operating in full scan mode where all ions are passed through the quadrupole and sent to the Orbitrap for detection.
• 😀 In the Orbitrap, ions are trapped in orbital motion around an electrode, and their frequency of rotation is related to their mass-to-charge ratio.
• 😀 Positive calibration is required before running samples, involving the observation of protonated analyte molecules in the mass spectra.
• 😀 To prevent cross-contamination, separate tubes and capillaries are used for calibration solutions and sample runs, and the syringe is cleaned before use.
• 😀 Calibration involves drawing the positive calibration solution into a syringe, attaching it to the mass spectrometer, and setting the flow rate to 10 microliters.
• 😀 The software program Tune is used to visualize real-time data and switch the machine status from standby to 'on' for calibration.
• 😀 The mass spectrometer settings for calibration include parameters such as scan time, scan range, resolution, and polarity, with a focus on using the highest resolution for easier interpretation.
• 😀 Cleaning the equipment involves sonication of the capillary cover with methanol, followed by drying with nitrogen gas, ensuring proper maintenance between sample runs.

Q & A

What technique is being used in this lab for mass spectrometry?

The technique being used is Electrospray Ionization Mass Spectrometry (ESI-MS), which generates ions by applying a high voltage to a liquid to produce an aerosol or gas for mass spectrometry analysis.

How does mass spectrometry work in general?

Mass spectrometry works by measuring the mass-to-charge ratio of ions. The results are presented in a mass spectrum, a plot of relative absorbance as a function of the mass-to-charge ratio.

What type of mass spectrometer is used in this lab?

The lab uses the Thermo Q Exactive quadrupole Orbitrap LC-MS-MS mass spectrometer.

What is the purpose of running the mass spectrometer in full scan mode?

In full scan mode, all ions are passed through the quadrupole, collected, stabilized, and sent to the Orbitrap for detection. This allows for the analysis of all ions present in the sample.

What happens in the Orbitrap during the mass spectrometry process?

In the Orbitrap, ions are trapped in an orbital motion around an electrode, with the frequency of their rotation related to their mass-to-charge ratio. Ions with different mass-to-charge ratios will oscillate at different frequencies.

What is the purpose of the calibration step before running samples?

Calibration is required to ensure the mass spectrometer is properly tuned. The positive calibration helps to observe protonated analyte molecules, and it is crucial for accurate mass measurements.

How is the syringe prepared for calibration solution?

The syringe is cleaned by rinsing it three times with deionized water. After cleaning, it is filled with the positive calibration solution up to the 200 microliter mark and then attached to the tubing connecting it to the mass spectrometer.

What is the role of the software program 'Tune' in the mass spectrometry process?

'Tune' is used to visualize the data in real time as it is acquired. It also allows the user to load predetermined calibration methods and ensure that all parameters are correctly set before running the samples.

What is the significance of the 'calibration check' method?

The 'calibration check' method serves as both a calibration and sensitivity check. It ensures that the mass spectrometer has a stable flow and sufficient sensitivity for accurate analysis before any other samples are run.

How is the equipment cleaned after a run?

The equipment is cleaned by first removing the cover and placing it in a beaker with methanol for sonication. The capillary is unscrewed and placed in a 'dirty' beaker. A clean capillary is then inserted into the instrument, and the cover is dried with nitrogen gas after sonication before reattaching it to the instrument.