TANDEM MASS SPECTROSCOPY

Transcription

1 TANDEM MASS SPECTROSCOPY 1

2 MASS SPECTROMETER TYPES OF MASS SPECTROMETER PRINCIPLE TANDEM MASS SPECTROMETER INSTRUMENTATION QUADRAPOLE MASS ANALYZER TRIPLE QUADRAPOLE MASS ANALYZER TIME OF FLIGHT MASS ANALYSER MASS DETECTOR APPLICATIONS 2

3 MASS SPECTROMETRY Separate and measures ions based on their mass-to-charge (m/z) ratio. Operate under high vacuum (keeps ions from bumping into gas molecules) Key specifications are resolution,mass measurement,accuracy and sensitivity. Several kinds exist: for bioanalysis, quadrapole,time of flight (TOF) and ion traps are most used. 3

4 TYPES OF MASS SPECTROSCOPY GC-MS - Gas Chromatography MS separates volatile compounds in gas column LC-MS - Liquid Chromatography MS separates delicate compounds in HPLC column MS-MS - Tandem Mass Spectrometry separates compound fragments by magnetic field LC/LC-MS/MS-Tandem LC and Tandem MS Separates by HPLC 4

5 PRINCIPLE: The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass to charge ratios. A sample is loaded onto the MS instrument, and undergoes vaporization. The components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of charged particles (ions) The ions are separated according to their mass to charge ratio in an analyzer by electromagnetic fields The ions are detected, usually by a quantitative method The ion signal is processed into mass spectra 5

6 Mass Spectroscopy Principles Sample + _ Ionizer Mass Analyzer Detector 6

7 INSTRUMENTATION Introduce sample to the instrument Generate ions in the gas phase Separate ions on the basis of differences in m/z with a mass analyzer Detect ions 7

8 MS vs. MS/MS Inlet Ionize Mass Analyze Detect Identification Separation MS Inlet Ionize Mass Analyze Fragment Mass Analyze Detect MS1 Collision Cell MS2 MS/MS 8

9 TANDEM MASS SPECTROSCOPY An instrument used in medical laboratories consisting of two mass spectrometers in series connected by a chamber known as a collision cell. The sample to be examined is essentially sorted and weighed in the first mass spectrometer, then broken into pieces in the collision cell, and a piece or pieces sorted and weighed in the second mass spectrometer. Tandem mass spectrometry is used in new born screening to detect molecules such as amino acids and fatty acids. 9

10 may be abbreviated as Tandem MS or Tandem mass spectrometry MS/MS A tandem mass spectrometer consists of at least two analyzers separated by a collision cell into which an inert gas (typically argon or xenon) is admitted to collide with the selected sample ions and bring about their fragmentation. The process is known as collision induced dissociation (CID). 10

11 Tandem mass spectrometry is used to produce structural information about a compound by fragmenting specific sample ions inside the mass spectrometer and identifying the resulting fragment ions. This information can then be pieced together to generate structural information regarding the intact molecule. Tandem mass spectrometry also enables specific compounds to be detected in complex mixtures on account of their specific and characteristic fragmentation patterns 11

12 Components of Tandem Mass Ionization Source Spectrometer ESI APPI APCI MALDI Mass Spectrometer Quadrupole Magnetic Sector Collision Cell Argon Xenon Mass Spectrometer Quadrupole Magnetic Sector Time-of-flight Detector MS1 Collision cell MS2 12

13 INSTRUMENTATION 13

14 Tandem mass spectrometers like other mass spectrometers have 3 main parts: ionization source. analyzer. detector. A sample introduced into the ionization source (typically by HPLC) becomes ionized. This makes the sample components easier to manipulate. The ions are removed into the analyzer where they are separated according to their mass -to-charge ratios (m/z). The separated ions are then fragmented. The fragments are detected and the signal created by the detected fragment ions is sent to a data system where the m/z ratios are stored together with their relative abundance. This data is presented in the format of a m/z spectrum.. Most MS/MS the ionization sources use an Atmospheric pressure ionization (API) technique 14

15 Sample introduction Sample can be inserted directly into the ionization source, or can undergo some type of chromatography en route to the ionization source. This latter method of sample introduction usually involves: high pressure liquid chromatography (HPLC), gas chromatography (GC) or capillary electrophoresis (CE) separation column. Methods of sample ionization The best ionization method to use depends on the type of sample under investigation and the mass spectrometer. Ionization methods most commonly used in tandem mass spectrometry include: The first 3 ionization techniques are commonly adapted for small molecular weight compounds. 15

16 1) Electrospray Ionization (ESI): is an API technique suited for analysis of polar molecules ranging from less than 100 Da to more than 1,000,000 Da in molecular mass. During electrospray ionization the sample is dissolved in a volatile polar solvent and pumped through a narrow stainless steel capillary (~100 µm diameter) at a flow rate between 1 µl/min and 1 ml/min. The electrospray is generated by applying a high voltage (~3000 V) to the tip of the capillary. This creates a strong electric field that causes the solvent and sample to elute from the tip of the capillary as an aerosol of highly charged droplets. The charged ions eventually become freed from the solvent then pass through an orifice (called a sampling cone) into a chamber that is under vacuum from which it is then directed through a small aperture into the analyzer section where molecules are separated according to molecular mass. 2)Nanospray ionization :is a special type of ESI that uses very low flow rates (30 to 1000 nl/min) and small sample volumes (1-4 µl) at concentrations of 1 to 10 µmol/l are used. 16

17 . 3) Atmospheric Pressure Chemical Ionization (APCI) uses heat to vaporize the column eluate and a corona discharge to ionize the solvent molecules and produce analyte ions by a variety of chemical ionization methods. 4) Atmospheric Pressure Photoionization (APPI) like APCI uses heat to vaporize the column eluate but uses UV light and chemicals added to the solvent to produce analyte ions through charge and proton transfer reactions. 5) Matrix assisted-lasar desorption/ionization (MALDI) uses laser light to ionize the sample. Typically a sample is pre-mixed with a special matrix compound for consistent and reliable results. The matrix compound allows the laser light to excite the sample causing it to ionize and move from the surface of the mixture and into the mass spectrometer. MALDI is also a "soft" ionization method and is mainly adapted to high molecular weight compounds like proteins. 17

18 Quadrupole Mass Analyzer 18

19 Quadrupole Mass Analyzer A quadrupole mass filter consists of four parallel metal rods with different charges Two opposite rods have an applied + potential and the other two rods have a - potential The applied voltages affect the trajectory of ions traveling down the flight path For given dc and ac voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path 19

20 Another type of mass analyzer, called a quadrupole mass filter, consists of four parallel poles or rods. Scanning is accomplished by systematically changing the field strengths, thereby changing the m/z value that is transmitted through the analyzer. Quadrupole mass spectrometers provide lower resolution than double focusing instruments but its cost is less. 20

21 Triple quadrupole mass spectrometer is a tandem mass spectrometer consisting of two quadrupole mass spectrometers in series, with a (non mass-resolving) radio frequency (RF) only quadrupole between them to act as a collision cell for collision-induced dissociation The first (Q1) and third (Q3) quadrupoles serve as mass filters, whereas the middle (Q2) quadrupole serves as a collision cell. This collision cell is an RF only quadrupole (non-mass filtering) using an inert gas such as Ar, He, or N2 gas to provide collision-induced dissociation of a selected precursor ion that is selected in Q1. Subsequent fragments are passed through to Q3 where they may be filtered or scanned. 21

22 Time-of-flight mass analyzers: seperate ions by virtue of their different flight times over a known distance. These ions are accelerated so that ions of like charge have equal kinetic energy and then are directed into a flight tube. Since kinetic energy is equal to 1/2 mv2, where m is the mass of the ion and v is the ion velocity, the lower the ion's mass, the greater the velocity and shorter its flight time. The travel time from the ion source through the flight tube to the detector, measured in microseconds, can be transformed to the m/z value through the relationships described above. 22

23 MS Detectors This process involves secondary emission In this single electron is bombarded to release electrons If electrical potential applied between metal plate, the emitted electron induce secondary emission of electrons collected by metal anode. Need constant and regular calibration 23

24 Mass Detectors Electron Multiplier (Dynode) 24

25 APPLICATIONS Tandem mass spectrometry (MS/MS) coupled with HPLC (high pressure liquid chromatography) is the analytical technique of choice for most assays used during new drug discovery. Mass spectrometers are used in industry and academia for both routine assays and research purposes. Some applications are: Biotechnology: Biotechnology & Pharmaceutical To determine chemical structure of drugs and drug metabolites. Detection/quantification of impurities, drugs and their metabolites in biological fluids and tissues. Analysis of liquid mixtures Clinical Biochemistry and Toxicology: Fingerprinting, herbal drugs/tracing source of natural products or drugs. The analysis of proteins, peptides, oligo nucleotides, neonatal screening, haemoglobin analysis drug testing 25

26 TANDEM MASS SPECTROSCOPY IS USED IN BIO CHEMICAL STUDIES AND NEW BORN SCREENING DETECTION OF IMPURITIES 1.Analysis of liquid mixtures 2.Fingerprinting 3.Nutraceuticals/herbal drugs/tracing source of natural products or drugs 4.The analysis of proteins, peptides, oligonucleotides 26

27 ORGANIC SPECTROSCOPY BY WILLIAM KEMP INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS BY SKOOG 27

28 THANK YOU 28