Methodology of targeted mass spectrometry By Prof. Moustafa Rizk Prof. of Clinical Pathology Faculty of Medicine, University of Alexandria 4/3/2015

Definitive identification of samples eluting from GC or HPLC columns is possible when an MS is used as a detector. The coupled techniques, GC/MS and LC/MS, have powerful analytic capabilities with widespread clinical applications.

Why Use Mass Spectrometry ? Advantages of GC or LC/MS/MS Analysis Specificity mass-based (m/z) vs. non-specific (UV-VIS, fluorescence), ELS (evap.-light scattering),etc. Selectivity ability to discriminate against matrix, background, other chemical species Sensitivity Speed Throughput Short analysis times and multiple compounds in single analysis. Specificity Older generation techniques such as UV and Fluorescence are non-specific – they cannot provide structural information or convincing evidence of analyte identity Modern, robust, easy to operate MS and MS/MS systems allow far greater confidence in quantitation and also vast amounts of qualitative information MS allows far more analytical confidence Non-specific techniques, such as UV and Fluorescence, are unable to provide proof of the analyte identity

Mass Spectrometry for Small Molecule Quantitative and Qualitative (Identification) Applications Pharma Applications (Discovery, Development, Production) DMPK, ADME, Microdosing, Bioavailability, Tissue Imaging Degradant Analysis Forensics Opiates, Benzodiazepines, THC, other drugs of abuse Food Testing Pesticides, Nitrofuranes, Mycotoxins, other contaminants Amino Acids, Vitamins, other food constituents Environmental Pesticides, Perchlorate, HAAs, PPCPs, other contaminants Clinical Research Studying Inborn Errors of Metabolism Research into Therapeutic Drug Efficacy It should be mentioned that this point that the API 3200 system can be used for a wide range of applications including DMPK and ADME studies for pharmaceutical analysis, Forensic drug testing for Benzodiazapines, Opiates, THC or other drugs of abuse, pesticides, malachite green, nitrofurane, chloramphenicol, mycotoxins or other food contaminants, vitamins, amino acids or other food constituents, Perchlorate, Halo Acetic acids, pesticides, pharmaceutical personal care of other environmental contaminants or research into inborn errors of metabolism or therapeutic drugs.

How Mass Spectrometry Works – The Basics sample is introduced into the instrument ions are sorted by m/z the signal is detected and counted the results are displayed m/z intensity ionized volatilized The sample in an MS is first volatilized and then ionized to form charged molecular ions and fragments that are separated according to their mass-to-charge (m/z) ratio; the sample is then measured by a detector, which gives the intensity of the ion current for each species.

These steps take place in the four basic components that are standard in all MSs 1-The sample inlet 2-Ionization source 3-Mass analyzer 4-Ion detector. Ultimately, molecule identification is based on the formation of characteristic fragments.

General Principles

Sample Introduction Direct infusion is commonly used to interface a GC or LC with an MS; however, the challenge of introducing a liquid sample from an LC column into an MS was a significant barrier until recent technological advances in ionization techniques.

Interface

General Principles

Ionization If a quantity of energy is supplied to a molecule equivalent to the ionisation energy of the molecule, a molecule ion is formed M+ There are several ways of doing this Electron Ionization (EI) Atmospheric pressure Ionisation (API) Electrospray Ionisation(ESI) Atmospheric pressure Chemical Ionisation (APCI) Atmospheric Pressure Photo-Ionization (APPI) New dual sources (ESI/APCI) or (APCI/APPI)

Electron Ionization The most common form of ionization used in GC/MS is electron ionization (EI). This method requires a source of electrons in the form of filament to which an electric potential is applied, typically at 70 electron volts. The most common form of ionization used in GC/MS is electron ionization (EI). This method requires a source of electrons in the form of a filament to which an electric potential is applied, typically at 70 electron volts.The molecules in the source are bombarded with high energy electrons, resulting in the formation of charged molecular ions and fragments. Molecules break down into characteristic fragments according to their molecular Structure . The ions formed and their relative proportions are reproducible and can be used for qualitative identification of the compound. Electron bombardment breaks cocaine into fragments, with number and size quantified. Unlike the illustrative glass tumbler, the result of mass fragmentation of cocaine or other chemical compounds is both predictable and reproducible, especially with electron ionization. The molecules in the source are bombarded with high energy electrons, resulting in the formation of charged molecular ions and fragments. Molecules break down into characteristic fragments according to their molecular structure .

Mass spectrum of the trimethylsilane derivative of 9-carboxytetrahydrocannabinol (marijuana metabolite).

Since most instruments use the same 70 eV potential, the fragmentation of molecules on different days and different instruments is remarkably similar, allowing the comparison of unknown spectra to spectra in a published reference library.

Atmospheric Pressure Ionization Unlike EI in GC/MS, most LC/MS ionization techniques are conducted at atmospheric pressure. Two types of ionization for LC/MS : Electrospray ionization (ESI) Atmospheric pressure chemical ionization (APCI).

Electrospray ionization (ESI) (Spraying a charged “mist”) Turbo Gas 1-Production of charged droplets. 3-Gas phase ion formation LC effluent 2-Droplet size reduction, and fission Thanks to its wide mass range and high sensitivity, ESI can be applied to a wide range of biological macromolecules in addition to small molecules and has become the most common ionization source for LC/MS. ESI involves passing the LC effluent through a capillary to which a voltage has been applied. The energy is transferred to the solvent droplets, which become charged. Evaporation of the solvent through heat and gas causes the droplets to decrease in size, which increases the charge density on the surface. Eventually, the Coulombic repulsion of like charges lead to the ejection of ions from the droplet. The individually charged molecules are drawn into the MS for mass analysis. Electrospray is a method of getting the solution phase ions into the gas phase so that they can be sampled by the mass spectrometer.

5kV LC Desolvation & Fission Droplet Formation Gas Phase Ion Orifice Plate 5kV LC Desolvation & Fission Nebulizing Gas Droplet Formation To MS Gas Phase Ion Generation A large voltage ( up to 6kV) is applied between the end of a capillary carrying the LC mobile phase and the mass spectrometer entrance. Ions (of the same polarity) are drawn out toward the counter electrode (curtain plate) pulling the mobile phase along. When the excess charge at the tip of the capillary overcomes surface tension, a droplet is formed Drying Gas Curtain Plate Curtain Gas

ESI: Droplet size reduction , fission Droplet size reduction occurs by the continual repetition of two processes: Desolvation (evaporation of neutral solvent and volatile buffers) Droplet fission caused by electric repulsion between like charges. ESI is adept at forming singly charged small molecule

Electrospray – Based on Ion Evaporation Theory The key is to get rid of the solvent before the ion enters the MS

Larger molecules such as proteins become multiply charged in ESI, and since MSs measure the m/z, even these large molecules can be observed in an instrument with a relatively small mass range A theoretical protein with a molecular weight of 10,000 can be multiply charged, which will generate numerous peaks. A mass spectrometer with a relatively small mass range can still detect the multiply charged ions since the m/z is reduced.

ESI: Types of Ions Formed Electrospray can operate in either positive or negative mode. Positive mode: Best suited to basic analytes that form a stable HCl salt. Negative mode: Best suited to acidic analytes that form stable Na salts. ESI is adept at forming singly charged small molecules, but larger molecules can also be ionized using this method. Larger molecules such as proteins become multiply charged in ESI, and since MSs measure the m/z, even these large molecules can be observed in an instrument with a relatively small mass range.

ESI: Pros and Cons Pros Soft ionization technique, resulting in little decomposition of labile analytes. Generally produces only molecular ions. Multi charged analytes easily produced, allowing proteins to be analyzed. Wide range of analytes Highly efficient ion production.

ESI: Pros and Cons Cons Lower flow rates concentration dependent Analyte must form solution phase ion. HCl or Na salt good indicator of suitability Ion Suppression Higher molecular weight analyte ions can suppress smaller analytes

Atmospheric Pressure Chemical Ionization (APCI) Corona discharge example - positive ion 1) EI on atmosphere cause e- removal from N2, O2 forming N2+•,O2+• 2) In a complex series of reactions N2+•,O2+• react with H2O, CH3OH forming H3O+ and CH3OH2+ as reagent ions for CI. 3) H3O+, CH3OH2+ donate protons to analyte forming [M+H]+ ESI and APCI also differ from EI in that they are “soft” ionization techniques that leave the molecular ion largely intact in the source. Many LC/MS techniques employ technologies after the source, in the mass analyzer, to fragment molecules and generate the “fingerprint” spectra used in identification. However, ionization techniques used in LC/MS produce fragments and therefore mass spectra that are somewhat less reproducible between instruments than EI used in GC/MS. This may prove to limit the utility of reference library spectra produced on other instruments.

Atmospheric Pressure Chemical Ionization Corona discharge needle LC Flow Atmospheric Pressure Chemical Ionization Heated nebulizer O2 or N2 Solvent molecule Ion of interest Gas 1 (GS1) Curtain Plate Curtain Gas(CUR) + . + 2. Corona discharge needle ionizes N2 or O2 in source + . 3. N2 or O2 pass charge to vaporized solvent + . 4. Vaporized, charged solvent passes charge to analyte + . + + . + 5. Ions enter Mass Analyzer aided by DP Another important ionization source is APCI, which is similar to ESI in that the liquid from LC is introduced directly into the ionization source. However, the droplets are not charged and the source contains a heated vaporizer to allow rapid desolvation of the drops. A high voltage is applied to a corona discharge needle, which emits a cloud of electrons to ionize compounds after they are converted to the gas phase. Orifice (DP) 1. Molecules in gas phase

ESI or APCI? - Which is better? For some applications, the choice is obvious… For analytes <1000 Da, you had to try both and see which one yielded the best sensitivity. Now, vendors are starting to offer “dual mode” sources to speed up method development… ESI tends to be an efficient ion source for polar compounds or for compounds that are ionized in solution, which include a high percentage of medically interesting compounds. ESI, along with APCI, allows an effective interface between a liquid chromatograph and a mass spectrometer. These have become the most widely used ion sources in clinical mass spectrometry. However, APCI is often a more efficient ion source than ESI for relatively nonpolar compounds.

General Principles

Mass Analyzer The quadrupole Ion trap The actual measuring of the m/z occurs when the gas phase ions pass into the mass analyzer. Two types of mass analyzers will be discussed: The quadrupole Ion trap

Quadrupole The electric field on the two sets of diagonally opposed rods allows only ions of a single selected m/z value to pass through the analyzer to the detector All other ions are deflected into the rods

This technique will generate a full scan mass spectrum This technique will generate a full scan mass spectrum. Alternatively, specific masses can be selected to monitor a few target analytes. This technique is called selected ion monitoring (SIM) A full scan provides more information than SIM since ions not specifically selected in SIM are not detected. Therefore, a full scan would be preferable for general unknown screening while SIM analysis is more suitable for target compound analysis.

Ion Trap The ion trap can be thought of as a modified quadrupole. A linear ion trap employs a stopping potential on the end electrodes to confine ions along the two-dimensional axis of the quadrupoles. In a three-dimensional ion trap, the four rods, instead of being arranged parallel to each other, form a three-dimensional sphere in which ions are “trapped.” In all ion traps, after a period of accumulation, the electric field adjusts to selectively destabilize the trapped ions, which are mass-selectively ejected from the cavity to the detector based on their m/z.21 The unique feature of ion trap MSs is that they trap and store ions generated over time, effectively concentrating the ions of interest and yielding a greater sensitivity.

Trapping the Ions Radial Trapping Axial Trapping Axial Trapping Ions can be trapped for a specific time (2-250ms) which allows increasing signal.

Triple Quads v. Ion Traps Triple Quadrupole Advantages Very sensitive. (SIM) Good for quantitation Some useful MS scanning modes Limitations No MSn Expensive Limited to unit mass resolution. Less sensitive in full scan mode. Advantages Higher full scan sensitivity Higher mass resolution MSn Limitations Not as good for quantitations. Space Charge Effects 1/3 cut-off rule. Cannot perform certain MS experiments.

MS/MS Tandem MSs (GC/MS/MS and LC/MS/MS) can be usedor greater selectivity and lower detection limits. A common form of MS/MS is to link three quadrupoles inseries; such an instrument is referred to as a triple quad. Generally , each quadrupole has a separate function. Following an appropriate ionization method, the first quadrupole (Q1) is used to scan across a preset m/z range and select an ion of interest. The second quadrupole (Q2) functions as a collision cell.

In a process called collision-induced dissociation (CID), the ions are accelerated to high kinetic energy and allowed to collide with neutral gas molecules (usually nitrogen,helium, or argon) to fragment the ions. The single ion passed through the first analyzer is called the precursor(or parent) ion while the ions formed during fragmentation of the precursor ions are called product (or daughter ) ions.

Curtain Gas™ interface General Set Up of a Triple Quadrupole Instrument Highest Selectivity and Sensitivity for Screening and Quantitation Turbo V™ source Curtain Gas™ interface Q1 LINAC® collision cell Q2 Q3 Q0 Ion production Ion filtering Ion filtering Ion transport Fragmentation Ion detection