Principles of Mass Spectrometry PDF

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Summary

Mass Spectrometry (MS) Detection- Specialized chromatograph detector that provides very sensitive detection and specific ID. - The only analytical detector that gives us a positive molecular ID of compounds. Chromatography ID is based on retention time and is not directly identified. Samples that el...

Description

MLSC-3111, Clinical Biochemistry II Mass Spectrometry (MS) Detection -

Specialized chromatograph detector that provides very sensitive detection and specific ID. The only analytical detector that gives us a positive molecular ID of compounds. Chromatography ID is based on retention time and is not directly identified. Samples that elute at a given point are just assumed to be the analyte that is known to elute at said point. MS directly and specifically identifies analytes without assumptions based on analyte behaviour with a SP and MP.

Clinical Applications of MS -

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This is a high-quality technique that is used to detect drugs/drug metabolites in patient samples. Generally, the samples are urine. High resolution MS can detect drugs such as designer fentanyl drugs. Also used for neonatal screening of metabolites (IEMs). Can also determine the amino acid composition of proteins. This is used to stage cancers via cancer markers. Proteomics, investigations of protein products encoded by genes. Used in microorganism detection and ID. The gas eluate from GC or liquid eluate from HPLC can be introduced into a MS system. The sample that has been separated by GC/HPLC will then undergo detection/ID by MS. Can also be used alone to detect proteins.

MS Definition -

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A process which involves conversion of a sample into gaseous ions, with or without fragmentation, which are then characterized by their mass to charge ratios and relative abundances. MS generates multiple ions from a sample then separates the ions according to their mass to charge ratio (m/z) and records the relative abundance (how much there is) of each ion type. MS works by ionizing neutral compounds to generate charged molecules and charged molecule fragments that can be detected according to their m/z ratio and plotting them as a mass spectrum. Mass, measures the molecular mass of compounds. Spectrometer, uses detection devices similar to spectrophotometry. Mass Spectrum, the plot of the relative abundance of each ion plotted as a function of its m/z ratio. Every compound produces a unique mass spectrum based upon their m/z ratio. It’s their specific mass spectrum that is detected by MS to ID the analyte. Mass Analysis, process by which a mixture of ionic species is identified according to their mass-to-charge ratio.

MLSC-3111, Clinical Biochemistry II -

m/z Ratio, the quantity formed by dividing the mass number of an ion by its charge. If the ratio is 1, this is indicative of mass. The number of ions produced relates to the concentration of compounds The MS is attached to a digital reference library. When it reads a pattern from a sample, it will compare the pattern to pre-logged patterns in the library until it finds one that matches to ID it.

Principles of MS -

Sample Ionization, produces ions from the sample or charging a molecule. This can occur by knocking off 1 or more electrons to give the analyte ion a positive charge. However, knocking off electrons may degrade the analyte so adding protons through chemical ionization will change the charge without damaging the analyte. Acceleration, ions are accelerated to high speeds to give all ions the same kinetic energy. Therefore, only the mass is left as an independent variable. Deflection, charged ions are deflected by a magnetic field according to their mass. As mass decreases and charge increases, so will the strength of deflection. Detection, the beam of ions passing through the analyzer is detected electronically.

Components of MS -

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Ionization Device, produces the ions from the sample. The ions are formed through several different processes. Positive ions are then directed through a vacuum, electric or magnetic field to enter the mass analyzer. Mass Analyzer, separates ions based on their m/z ratio for analysis. Ion Detector, Detector records ions after separation and produces the “mass spectrum” display based upon ion intensity as a function of m/z.

Ionization Techniques -

Hard Ionization, the formation of gas-phase ions with an extensive fragmentation of molecules. Used in electron impact ionization in GC. Soft Ionization, the formation of gas-phase ions without an extensive fragmentation of molecules. Seen in chemical ionization in GC or MALDI in LC.

Electron Impact Ionization -

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Hard ionization. Gaseous molecules are bombarded by electrons in a beam emitted from a heated filament. Produces large fragmentation of molecules. The ion beam is equal to 70 electron volts. It is very highly energized and can knock off electrons as well as transfer its extra energy to the ion. The transfer of energy causes the large fragmentation. Electron beam breaks apart molecules that react differently based on their m/z ratio. Graphically, it creates the molecular fingerprint based on the amount of abundance

MLSC-3111, Clinical Biochemistry II fragments found. Fragment occurrence is based on its likely frequency to be created when the main molecule hits the beam. Gas Phase ionization -

Soft ionization. Reagent gas is first introduced into the ion source and the 70 ev beam ionizes the reagent gas molecules. Sample will then enter the source and collide with reagent ions causing a charge transfer of protons from reagent to sample. Sample ions become positively charged and results in little fragmentation of molecules.

MALDI Ionization -

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Soft ionization involving a laser and matrix compound (usually organic and can absorb UV light). Sample is combined with matrix on the stainless steel plate and placed into the analyzer. The laser is pointed at the sample and is ablated upon being hit with the laser. The sample becomes irradiated by the laser in the mixture and becomes volatile and positively ionized. The matrix serves as a proton donor in the ionization of the gaseous analyte.

Mass Analyzers -

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Beam-Type, ions make one trip through the instrument and strike the detector. o Magnetic Sensor, uses magnetic fields. o Time of Flight (TOF), uses a vacuum. Trapping Type, ions held in a spatially confined region of space by combinations of magnetic, electrical, or electrostatic fields.

Effects of Magnetic Fields on Ions -

The interaction results in a force on the charged particle that causes it to move in a curved path upon entering the magnetic field. When the charged particle leaves the magnetic field, it resumes moving in a straight path. The mass and charge of the particle determines the shaped of the curved path the ion takes and where it exits the magnetic field. Magnetic fields separate ions according to their momentum (product of mass x velocity). Ions are accelerated by a high voltage pulse which ensures that all ions are travelling at the same high speed when they enter the mass analyzer. A slit is placed to filter the ions of interest.

Magnetic Sector Mass Analyzer

MLSC-3111, Clinical Biochemistry II

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Magnetic Sector, the original beam type MS. Uses magnetic fields applied in a direction perpendicular to the direct of ion movement to separates ions. Ions have that have a constant kinetic energy, but different m/z ratios are brought into focus at the detector slit at different magnetic field strength. Changing the magnetic field strength during analysis allows differing ions to pass through the detector.

TOF MS Analyzer -

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Detects the time required for ions to travel through a vacuum flight tube and reach the detector based upon their velocity as per their m/z ratio. They are all accelerated initially to the same velocity but change as they enter the vacuum. Light ions are small and move faster, while bigger ones are heavier and move slower. Vacuums are meant to eliminate the interference from air molecules hitting the ions trying to pass through to the detector. Hitting air molecules would change the ions speed and path.

Inductively Coupled Plasma MS -

An elemental analysis capable of detecting most of the periodic table of elements at mg to ng levels per litre. It is used to detect and quantitate trace metals and replaces AAS MS. Inductively Coupled Plasma, ionization source that fully decomposes a sample into its constituent elements and transforms those elements into ions. The plasma is typically composed of argon gas and energy is coupled to it using an induction coil to form extremely hot “plasma”. Used to detect Iodine, Manganese, Copper, Selenium and Zinc. Also measures toxic metals like Arsenic, Cadmium, Mercury and Lead.

MS Detector -

Uses the same electron multipliers as seen in spectrophotometry. Uses a series of dynodes with increasing potentials linked together. Serves to amplify the signal strength over time.

GC-MS vs LC-MS

MLSC-3111, Clinical Biochemistry II

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GC-MS, sample from GC is directly injected into the MS. The sample is already in a gaseous phase and components separated via the GC column and MP. LC-MS, sample is in a liquid phase but needs to be in a gaseous phase for the MS. Requires an electrospray ionization technique, which uses a small-bore tube with a heated 4u nozzle at the MS inlet. The nozzle is charged with several kilovolts. Heat helps to evaporate LC solvent and the droplet becomes highly charged.

The Mass Spectrum -

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The plot of relative abundance vs m/z ratio of ions hitting the detector. Different molecules will create different mass spectra. Base Spectra, ion with highest abundance on the spectrum. Identified by the base peak (ion with the highest abundance). Molecular/Parent Ion, the original unfragmented molecule. Has the highest m/z ratio. Identified by the M peak. M+1 Peak, occasionally seen as a small peak 1 m/z higher than the M-Peak. Occurs in the rare circumstance where 13C appears (which can occur in a hydrocarbon sample), which can produce a small extra peak right after the M peak. When asked to determine the original compound, the molar mass of the compound must add up to the m/z ratio of the M-Peak constituent.

MS Interferences and Contaminants -

Enzymes, used for enzyme digestion of proteins. Keratins, from skin cells in household and lab dust. Can contaminate the sample. Solvents/Alkali/Metal Ions/PEG, used in sample pre-treatment techniques. Quaternary Ammonium Compounds, used in disinfectants like Conflikt. Plasticware, causes polymeric interference....