Mass Spectrometry in Clinical Chemistry David Hardy What is Mass - PowerPoint PPT Presentation

Quadrupole Mass Filters 1 • Ions enter quadrupole region – Because of RF voltage and DC offset the polarity of each pair of rods continually changes – Ion in quadrupole is alternately repelled and attracted to given rod – Ion follows helical path through quadrupole – For given RF and DC voltage settings only certain m/z ions have stable trajectory to detector – the rest collide with rods – By changing values of the voltages different m/z ions can be focussed onto the detector • Quadrupole mass filter transmits one m/z ratio at once

Quadrupole Mass Filters 2

Quadrupole Mass Filters 3

Quadrupole Mass Filters 4

Quadrupole Mass Filters 5

Quadrupole Mass Filters 6 • Resolution – Discrimination between ions of similar m/z ratio – Variation of RF and DC voltage with fixed ratio allows mass range to be scanned – Variation of ratio alters resolution • Running the voltages closer to apices of regions of stability leads to higher resolution • Ion counts fall with increasing ratio – Better resolution \ fewer extraneous ions transmitted – Ions have more energy \ more for given m/z ratio lost to collisions with rods

Quadrupole Mass Filters 7

Quadrupole Ion Traps 1 • Two end caps and central toroidal ring electrode • Unlike quadrupole mass filter QIT can be used to store a range of m/z ratios and then eject them sequentially

Quadrupole Ion Traps 2

Quadrupole Ion Traps 3 • RF voltage is applied to trap. • DC voltage may or may not be applied to end caps – How voltages are applied determines how trap works • Keep all ions • Keep one m/z ratio • Ions adopt figure of 8 trajectory – By altering RF (+/- DC) voltages ion trajectory become unstable and ions leave trap by end plate hole • Variation of voltages allows sequential exit of different m/z ratio ions

Quadrupole Analysers 2 • Advantages – Compact (esp. QIT) – Cheap – Robust • Disadvantages – Poorer resolution than sector instruments

Time-of-Flight Analysers 1 • All ions are given same k.e. by accelerating potential (ca. 3 keV) • Ions drift freely down “drift tube” to reach detector 1- 2 m away • Different ions have different masses and different velocities – K.e. = mv 2 /2 • Ions of different masses separated by taking different times to reach detector – From knowing start time, m/z can be determined from time to reach detector

Time-of-Flight Analysers 2

Time-of-Flight Analysers 3 • Some issues with ions of same m/z having slightly different k.e. • Performance of TOF improved by including “reflectron” (“ion mirror”) – Device for reflecting ions – Faster moving ions with same m/z penetrate reflectron more than slower ones so that they are re- focused on reflection – Additionally by reflecting ions back to drift again drift path and resolution increased

Time-of-Flight Analysers 4

Detectors • Photographic plate • Faraday cage • Electron multiplier • Photomultiplier • Charge collectors

Photographic Plate • Original detector • Ions of same m/z hit plate at same point • Intensity of spot relates to relative abundance of ions • Little used now except for Mattauch-Herzog geometry (plane-focussing instrument)

Faraday Cage • Collection cylinder for ions • Ions enter cylinder and discharge generating current • Current amplified and measured – Current proportional to ion abundance • Limited sensitivity

Electron Multiplier 1 • Useful for positive or negative ions • Ions strike high voltage conversion dynode generating secondary particles of opposite charge – Positive ions liberate electrons; negative ions positive ions • Secondary particles collide with walls of electron multiplier cascading out more electrons which cascade out even more electrons in further collisions • Amplified current measured as related to ion count • Sensitive • Allows rapid scanning

Electron Multiplier 2

Photomultiplier 1 • Detects positive or negative ions • Two conversion dynodes (+ve and –ve), phosphor screen and photomultiplier – Negative ions hit positive conversion dynode – Positive ions hit negative dynode • Secondary particles from conversion dynode hit phosphor ejecting photons • Photons detected by photomultiplier • Current proportional to ion count

Photomultiplier 2

Mass Spectrum Acquisition • Three basic types of scanning – Full scan • Detect all ions in given m/z range – Continuum, multichannel analysis (MCA) and centroid variations – Selected Ion Monitoring (SIM) • Set to detect only one m/z – Selected Reaction Monitoring (SRM) • Tandem MS experiment – see later • Detect specific fragment ion from specific precursor ion • Can cycle between a number pairs of ions – Multiple Reaction Monitoring (MRM)

MCA vs Centroid Data 16July02PT009 1 (1.117) Cn (Cen,2, 80.00, Ht); Sm (SG, 2x0.75); Sb (33,10.00 ); Sb (33,10.00 ) Neutral Loss 102ES+ 222 1.46e7 100 Centroid 172 % 186 185 260 188 223 212 238 261 174 176 227 162 221 242 259 158 191 228 232 246 269 272 283 293 295 298 203 209 215 192 206 154 157 163 169 183 202 247250 274 277 281 287 0 16July02PT009 1 (1.117) Sm (SG, 2x0.75); Sb (33,10.00 ); Sb (33,10.00 ) Neutral Loss 102ES+ 222 1.48e7 100 Multichannel Analysis 172 % 186 260 185 188 223 212 238 261 174 227 162 0 m/z 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300

Example of EI Use • GC/MS for organic acids – Organic acids converted to trimethylsilyl esters – Esters separated by capillary GC column – As compounds elute they enter EI source, are ionised, fragmented and the fragments detected to give mass spectrum – Current in detector is plotted vs. time to create chromatogram (“total ion chromatogram”) – Mass spectrum for each chromatogram peak can be inspected and likely compounds identified by library matching

Library Matching 1

Library Matching 2

Tandem Mass Spectrometry 1 • Abbreviated MS/MS or MS 2 – TMS should not be used!!! • Essentially two mass analyser in series (tandem) • Placed between analysers is a collision cell – Ions from first analyser collide with Ar in cell and fragment (collision-induced dissociation) – Fragments then analysed by second analyser • Allow greater range of investigations

Tandem Mass Spectrometry 2 • Ion source usually of soft type to form ions with little fragmentation • Commonly ESI and ACPI sources used • Commonest instruments are “triple quadrupoles” – Generic name for class of instruments – 1 st and 3 rd quadrupoles (Q1, Q3) have RF and DC voltages to effect ion separation – Middle quadrupole (q2)is RF only • In absence of DC voltage time averaged voltage experienced by ions is zero • RF only focuses ions for transmission to next stage

Tandem Mass Spectrometry 3 • Generic triple quadruple MS/MS Detector 2ns Analyser (Q3) Ion source Collision Cell (q2) 1 st Analyser (Q1)

Tandem Mass Spectrometry 4

Tandem Mass Spectrometry 5 • Types of MS/MS experiments – Simple MS scan using one quadrupole – Neutral Loss • Also (rarely) Neutral Gain – Precursor ion scan (“parents of”) – Product ion scan (“daughters of”)

Neutral Loss Experiment • Ion transmitted by Q1 • Collision induced dissociation in collision cell causes ion to lose a neutral molecule and form a smaller ion • Smaller ion transmitted by Q3 • By scanning Q1 and Q3 at the same time with an offset equal to the neutral fragment mass only ions that lose the neutral fragment are detected – Increased sensitivity as background signal reduced

Precursor Ion Experiment • Aim to find all precursor ions that generate a given product ion • Q1 scanned to transmit ions to collision cell • Ions fragment and fragments transmitted to Q3 • Q3 static for specific m/z of product ion of interest • Only ions generating specific product ion are detected – Increased sensitivity

Amino Acids 1 • Amino acids measured as butyl esters – butyl esters lose butyl formate in collision cell (mass 102 Da) NH 3 + CID R BuO NH 2 + R O BuO 2 CH – Scanning MS1 and MS2 together but with MS2 lagging 102 behind MS1 only those species losing 102 Da fragments are detected

Amino Acids 2 • Phenylalanine butyl ester (m/z 222) • ions ---> MS1 ---> Col cell ---> MS2 --> detector scan 120 - 300 scan 18 - 198 222 -----> -102 ------> 120 ---> – when MS1 transmits m/z 222 MS2 is set to transmit m/z 120 – only ions of m/z 222 losing 102 Da fragment detected

Amino Acids 3 • NL 102 generic experiment – Some amino acids are better detected by other scans • Basic amino acids NL 119 (butyl formate + ammonia) • Glycine NL 56 • Arginine NL 161

Normal Amino Acid Spectrum 05Jan01IMD027 1 (1.125) Sm (SG, 2x1.00); Sb (33,10.00 ) 2: Neutral Loss 102ES+ 172.1 7.97e5 100 222.1 227.1 % 188.2 146.2 142.9 238.0 186.1 260.2 191.3 242.1 209.2 211.9 174.3 162.1 228.1 246.3 163.1 192.3 132.3 0 m/z 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280

Phenylketonuria IM D 018 1 (1.021) 2: N eutral Loss 102E S + 222.0 1.70e7 100 % 227.0 172.1 188.0 191.1 260.1 173.9 242.0 246.1 162.1 209.0 0 m /z 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

MS/MS Spectra For Normal vs. PKU Endo 172.0 100 188.0 227.0 260.0 Normal Subject 222.0 174.0 % 191.0 238.0 242.0 146.0 246.0 209.0 161.9 185.0 176.0 228.0 260.9 205.9 192.0 211.9 247.0 159.9 162.9 277.1 0 m/z 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 222.0 100 Phe Tyr PKU Patient % 172.0 227.0 188.0 260.0 174.0 191.0 246.0 242.0 161.9 209.0 0 m/z 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

Tyrosinaemia 26OctIMD007 1 (1.031) Sm (SG, 2x1.00); Sb (33,10.00 ) 2: Neutral Loss 102ES+ 238.0 6.27e5 100 % 172.0 226.9 242.0 185.0 222.0 185.9 146.0 260.1 191.1 220.9 162.0 209.0 203.0 0 m/z 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

Maple Syrup Urine Disease 15JulyIMD007 1 (1.135) Sm (SG, 2x0.75); Sb (33,10.00 ) 2: Neutral Loss 102ES+ 188.1 8.87e7 100 % 172.0 189.1 174.0 227.1 0 m/z 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280

Citrullinaemia NL 102 IMD212 1 (1.021) 2: Neutral Loss 102ES+ 260.0 4.08e6 100 171.9 215.0 227.1 % 191.1 188.0 174.0 222.0 145.9 242.0 209.0 162.0 241.0 215.4 185.0 245.9 260.9 228.1 237.9 216.1 192.0 175.9 206.1 277.1 0 m/z 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

Citrullinaemia NL 119 IMD212 1 (1.021) 3: Neutral Loss 119ES+ 232.0 4.96e6 100 % 221.5 222.0 233.0 189.1 0 m/z 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

Normal Blood Spot NL 119 M K001 1 (1.120) Sm (SG, 2x0.75); S b (33,10.00 ) 3: Neutral Loss 119E S+ 221.6 1.45e6 100 % 203.1 238.1 0 m /z 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250 255 260 265 270

Acyl carnitines 1 • Butyl esters of acylcarnitines – prepared in same way as amino acids – esters all fragment to form an ion of m/z 85 O CH 2 + CID R O O CO 2 H O Bu RCO 2 H Me 3 N C 4 H 8 Me 3 N+ – by fixing MS2 to transmit m/z 85 but scanning MS1 only ions forming a m/z 85 fragment will be detected.

Acyl carnitines 2 • Butyl esters of free carnitine (m/z 218) and acetylcarnitine (m/z 260) • ions ---> MS1 ---> Col cell ---> MS2 --> detector scan 215 - 550 transmit 85 218 -----> -133 ------> 85 ---> 260 -----> -175 ----> 85 --->

Normal Bloodspot Acyl Carnitine Spectrum 05Jan01IMD027 1 (1.125) Sm (SG, 2x1.00); Sb (33,10.00 ) 1: Parents of 85ES+ 263.1 8.60e5 100 218.3 221.1 % 260.4 459.2 347.3 456.4 274.3 482.4 483.5 302.4 340.9 313.4 374.0 0 m/z 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

Normal Plasma Acyl Carnitine Spectrum MK002 1 (1.123) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 260 1.94e6 100 218 % 263 221 459 347 372 457 482 342 426 370 274 288 399 0 m/z 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

Post-mortem Specimen 2 M a y0 0 IM D 0 0 6 1 (1 .1 0 6 ) S m (S G , 2 x0 .7 5 ); S b (3 3 ,1 0 .0 0 ) 1 : P a re nts o f 8 5 E S + 2 6 0 .3 8 .6 3 e 6 1 0 0 2 1 8 .1 % 2 6 3 .3 2 2 1 .2 4 5 9 .6 2 8 8 .3 3 0 4 .3 4 8 2 .4 3 4 7 .0 4 5 6 .3 2 7 4 .0 0 m /z 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 5 2 0 5 4 0

Propionic Acidaemia 16MAR01IMD011 1 (1.126) Sm (SG, 2x1.00); Sb (33,10.00 ) 1: Parents of 85ES+ 274.4 3.29e7 100 260.4 % 218.4 482.4 459.4 221.3 318.3 0 m/z 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

Glutaric Aciduria Type 1 15FebIMD007 1 (1.117) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 218.3 1.92e6 100 388.5 260.4 % 459.5 263.4 221.3 274.3 347.3 332.4 288.3 482.7 0 m/z 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

HMG CoA Lyase Deficiency IM D 224 1 (1.022) Sm (SG , 2x0.75); S b (33,10.00 ) 1: P arents of 85ES + 218.2 2.00e6 100 221.2 % 260.2 261.2 288.4 318.3 459.5 347.4 356.6 300.4 0 m /z 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

MCADD 25JanIM D 011 1 (1.117) S m (S G , 2x0.75); S b (33,10.00 ) 1: P arents of 85ES + 221.3 6.13e5 100 218.3 263.3 344.5 % 260.4 259.8 264.4 459.8 316.3 510.6 347.1 370.4 288.2 342.2 274.2 228.3 237.3 297.6 0 m /z 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

LCHADD IMD202 1 (1.021) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 218.2 x4 2.28e6 100 260.3 221.3 261.2 % 456.4 459.6 482.4 472.2 484.5 500.3 498.5 454.5 460.5 263.4 426.5 400.6 428.1444.5 288.3 501.8 398.4 201.0 514.3 347.4 416.4 274.3 0 m /z 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

VLCADD 23NovIMD012 1 (1.035) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 263.4 4.03e5 100 218.4 221.3 459.3 % 426.4 260.2 456.6 428.4 482.4 347.4 454.3 484.3 423.9 288.2 480.9 316.4 429.6 227.8 274.1 400.7 302.0 372.3 370.4 512.5 0 m/z 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520

CPT-1 Deficiency 16M arIM D 003 1 (1.119) S m (S G , 2x0.75); S b (33,10.00 ) 1: P arents of 85E S + 218.3 4.82e6 100 % 260.3 263.2 221.3 459.7 347.4 274.1 0 m /z 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540