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

Mass Spectrometry in Clinical Chemistry David Hardy

What is Mass Spectrometry?

• The separation of ions on the basis of mass

to charge ratio (m/z)

• Gas phase technique requiring high vacuum

– Non-volatile compounds require conversion to volatile derivatives

Uses and Problems

• Uses

– Structural elucidation

• Masses and fragmentation patterns

– Identification

• Fragmentation characteristic of compound
• Library searches/peak matching
• Problems

– Isomeric/isobaric interference (esp. with soft ionisation methods)

Instrumentation

• Components of mass spectrometer

– High vacuum system

• Rotary backing pump plus turbomolecular or diffusion pump

– Inlet and ion source (forms ions) – Analyser (separates ions) – Detector (records ions) – Computer control

• Data acquisition
• Data manipulation

Pre-analysis Considerations

• Volatile compounds

– Analyse without modification

• Non-volatile compounds

– Convert to volatile species

• Alkylsilyl deriviatives (Me3Si (TMS); tBuMe2Si

(tBDMS)

• Esters
• More unusual species (hydrazones, etc)

Ion Sources

• Electron impact (EI)
• Chemical ionisation (CI)
• Atmospheric pressure ionisation (API)

– Electrospray (ESI) – Atmospheric pressure chemical ionisation (APCI)

• Fast atom bombardment (FAB) and Liquid

secondary ion mass spectrometry (LSIMS)

• Plasma desorption (PD)
• Inductively coupled plasma (ICP)
• Matrix-assisted laser desorption (MALDI)

Ion Nomenclature

• Molecular ion

– Ion formed by loss of electron from molecule

• Pseudomolecular ion

– Ion formed from molecule by gain of small ion (e.g. H+, OH-)

• Precursor ion (a.k.a. “parent ion”)

– Ion from which a smaller fragment ion has formed

• Usually molecular or pseudomolecular ion
• Product ion (a.k.a. “daughter ion”)

– Fragment ion from larger ion

Electron Impact 1

• Fast electron stream ionised molecules

– e-(fast) + M à M.+ + 2 e-(slow)

• Very energetic

– Transfer of electron k.e. to ion may lead to bond dissociation

• Molecular ion may not be seen if easily

fragmented

• Many product ions formed

Electron Impact 2

Electron Impact 3

• Fragments may be useful for structure

determination

– Fragments may be ions, neutrals or radicals – Certain fragments or losses may be characteristic of functional groups (e.g. loss of 18 (H2O) suggests OH group)

Oxalic Acid (bis TMS ester)

O O O Si Si O Mw = 234

Chemical Ionisation 1

• Uses EI source with a “cage” to trap ions

with a reagent gas (CH4 or NH3)

• Electrons ionise reagent gas which reacts to

form reactive ions (e.g. CH3

+ and CH5 +

from methane)

Chemical Ionisation 2

Chemical Ionisation 3

• Different types of compound react

differently with reagent gas ions

– Generally proton transfer

• M + CH5

+ à [M+H]+ + CH4

– Saturated hydrocarbons

• RH + CH5

+ à R+ + CH4 + H2

– Polar molecules

• M + CH3

+ à [M+CH3]+

Chemical Ionisation 4

• Mass spectrum shows few fragments

usually

– Main peak is [M+H]+ pseudomolecular ion – Soft ionisation technique

Chemical Ionisation 5

Electrospray 1

• Stream of solution sprayed out of capillary at high

voltage (ca. 3 – 5 kV)

• Charged droplets formed by spray (“Taylor cone”)
• Solvent evaporated by stream of warm N2
• As droplet shrinks, charge density increases until

analyte ions ejected

– Ions may be formed by proton transfer in spray or from reactions prior to analysis (i.e. may already be in solution) – Pseudomolecular [M+H]+ ions formed

• Solvent pumped away and ions admitted to mass

spectrometer

Electrospray 2

Electrospray 3

Electrospray 4

Electrospray 5

• Soft ionisation technique; little fragmentation
• Large molecules may be protonated more

than once

– Peaks seen for same compound with different m/z ratios – “Deconvolution” of peaks allows representation

• f species with effectively m/z for one charge
• Allows determination of molecular weight

Electrospray 6

APCI 1

• Similar source to ESI but higher flow rate (ca. 2

mL/min)

• Discharge pin placed in the solvent spray

– Causes solvent molecules to ionise and behave like reagent gas – Solvent ions transfer protons to analyte

• Soft ionisation technique

– Pseudomolecular ion formed – Little fragmentation

APCI 2

Fast Atom Bomdardment

• Fast Ar (or Xe) atoms formed by

– Accelerating Ar+ or Xe+ ions by electric field then

• Colliding with Ar or Xe atoms

– Ar+

(fast) + Ar(slow) à Ar(fast) + Ar+ (slow)

• Or firing through electron cloud

– Ar+

(fast) + e- à Ar(fast)

• Atoms fired at compound in matrix and knock analyte

ions out

– Glycerol or m-nitrobenzylic acid commonly used for positive ions – Me2NH and Me3N used for negative ions

• Fast atoms cause little ionisation – essentially just ion

displacement

LSIMS

• Similar to FAB but uses Cs+ ions instead of Ar atoms

– Analyte ions emitted secondary to primary Cs+ ion collisions – Better sensitivity than atom beam for high MW compounds – Charge accumulation at surface can be problematic

• Use of matrix in FAB and LSIMS can complicate

spectrum

– FAB and LSIMS not “soft” ionisation techniques

• Pseudomolecular ions and fragments formed
• Exact pattern depends on amount of analyte
• Fragments may be masked by matrix ions
• Useful for polar compounds up to 10 kDa

Plasma Desorption

• Fission fragments of 252Cf impact on sample

deposited on Al/nylon mesh

– High energy impact

• Fission fragments have energy of several MeV

– Shockwave of impact ejects neutrals and ions – Useful for masses up to 10 kDa

• Rarely used, replaced by MALDI

MALDI 1

• Analyte dissolved in matrix containing dye
• Laser irradiation causes ion formation/ejection

– Complex process; not well understood – Energy from laser pulse absorbed by matrix – Clusters of matrix/analyte desorbed from surface – Proton transfer from matrix to analyte produces pseudomolecular ions; matrix species evaporate off

• Transfer can occur at any stage of process
• Exact mechanism unknown
• Useful for analytes up to 100 kDa

– Has been extended to 300 kDa

• Multiply charged ions may be formed from larger

molecules

MALDI 2

Mass Analysers

• Aim to separate (“resolve”) ions of different m/z ratios
• Sector instruments

– Magnetic fields (magnetic sectors) – Electric and magnetic sectors (double focussing instruments)

• Quadrupoles

– Quadrupole mass filters – Quadrupole ion traps

• Time-of-flight

Resolution 1

• Two peaks are resolved if the valley between

them is 10% of the smaller peak intensity

– Sometimes 10% is applied to sector instruments and 50% to quadrupoles

• Commonest definition

– Resolution = m1/(m2 – m1) – m1 is mass of lighter peak; m2 mass of heavier peak

Resolution 2

Magnetic Sectors 1

• Ions accelerated from source by potential,V
• On entering magnetic field, B, ions follow a

circular path of radius, r

• For ions of given m/z ratio

– m/z = r2B2/2V

• If r is fixed (using flight tube)

– Only one m/z will exit magnetic sector for given B

• Varying B allows all ions to be transmitted sequentially
• If r is not fixed

– All m/z will exit but at different points

• All ions focussed onto plane, e.g. photographic plate

(“dispersive instruments”)

Magnetic Sectors 2

Magnetic Sectors 3

• Problem

– For a given m/z ratio there is a spread of kinetic energies

• Thermal effects and collisions in the source ensure that

there is a spread of velocities

• Different k.e. for same m/z lead to “energy dispersion”
• This dispersion may be exacerbated by the journey through

the field – “angular dispersion”

• Inaccuracies in determined m/z ratio may result with loss
• f resolution
• Solution

– Include electric field (sector)

Double Focussing Analysers 1

• In a radial electric field of intensity, E,

– the ions move in a trajectory where centrifugal electrostatic forces balance

• zE = mv2/r or
• r = mv2/zE

– Trajectory depends on k.e. rather than just mass – Electric sector is a k.e. analyser

• Electric sectors also suffer dispersion

Double Focussing Analysers 2

Double Focussing Analysers 3

• If electric (E) and magnetic (B) sectors with

same dispersion are combined the dispersions cancel out

• Order of sectors defines “geometry”

– EB Nier-Johnson (forward) geometry – BE reverse Nier-Johnson (reverse) geometry

Double Focussing Analysers 4

Double Focussing Analysers 5

Double Focussing Analysers 6

Double Focussing Analysers 7

• Advantage

– Correction of energy dispersion focuses ion beam

• Increased resolution
• Useful for isotope ratio work
• Disadvantage

– Size – Cost

Quadrupole Analysers 1

• Quadrupole mass filters

– Four rods arranged precisely with DC and RF alternating voltages applied to pairs

• Quadrupole ion traps

– Effectively the circular equivalent of above

• Resolution typically 1000

Quadrupole Mass Filters 1

• Ions enter quadrupole region

– Because of RF voltage and DC offset the polarity of each pair

• f 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. = mv2/2

• 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
• f 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

• ut 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

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 m/z 100 % 100 % 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+ 1.46e7 222 172 162 158 157 154 169 163 186 185 174 176 183 188 212 191 209 203 192 202 206 215 221 260 223 238 227 228 232 242 246 259 247250 261 274 272 269 277 281 287 283 293 295 298 16July02PT009 1 (1.117) Sm (SG, 2x0.75); Sb (33,10.00 ); Sb (33,10.00 ) Neutral Loss 102ES+ 1.48e7 222 172 162 186 185 174 188 212 260 223 238 227 261 Multichannel Analysis Centroid

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 MS2

– 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 – 1st and 3rd 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

Collision Cell (q2)

• Generic triple quadruple MS/MS

2ns Analyser (Q3) 1st Analyser (Q1) Ion source Detector

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
• ffset 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 measured as butyl esters

– butyl esters lose butyl formate in collision cell (mass 102 Da) – Scanning MS1 and MS2 together but with MS2 lagging 102 behind MS1 only those species losing 102 Da fragments are detected

Amino Acids 1

NH3+ O R BuO BuO2CH R NH2+ CID

• 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 2

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

130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 m/z 100 % 05Jan01IMD027 1 (1.125) Sm (SG, 2x1.00); Sb (33,10.00 ) 2: Neutral Loss 102ES+ 7.97e5

172.1 146.2 142.9 132.3 162.1 163.1 222.1 188.2 186.1 174.3 191.3 209.2 192.3 211.9 227.1 238.0 228.1 260.2 242.1 246.3

Normal Amino Acid Spectrum

120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 m /z 100 % IM D 018 1 (1.021) 2: N eutral Loss 102E S + 1.70e7

222.0 172.1 162.1 188.0 173.9 191.1 209.0 227.0 260.1 246.1 242.0

Phenylketonuria

Normal Subject PKU Patient

Phe Tyr

MS/MS Spectra For Normal vs. PKU

120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 m/z 100 % 26OctIMD007 1 (1.031) Sm (SG, 2x1.00); Sb (33,10.00 ) 2: Neutral Loss 102ES+ 6.27e5

238.0 172.0 146.0 162.0 226.9 185.0 222.0 185.9 191.1 220.9 209.0 203.0 242.0 260.1

Tyrosinaemia

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

188.1 172.0 174.0 189.1 227.1

Maple Syrup Urine Disease

120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 m/z 100 % IMD212 1 (1.021) 2: Neutral Loss 102ES+ 4.08e6

260.0 171.9 145.9 162.0 215.0 191.1 188.0 174.0 185.0 175.9 209.0 192.0 206.1 227.1 222.0 215.4 216.1 242.0 241.0 237.9 228.1 245.9 260.9 277.1

Citrullinaemia NL 102

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

232.0 221.5 189.1 222.0 233.0

Citrullinaemia NL 119

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 m /z 100 % M K001 1 (1.120) Sm (SG, 2x0.75); S b (33,10.00 ) 3: Neutral Loss 119E S+ 1.45e6

221.6 203.1 238.1

Normal Blood Spot NL 119

• Butyl esters of acylcarnitines

– prepared in same way as amino acids – esters all fragment to form an ion of m/z 85 – by fixing MS2 to transmit m/z 85 but scanning MS1 only ions forming a m/z 85 fragment will be detected.

O O O Bu R O Me3N+ C4H8 Me3N RCO2H CID CH2+ CO2H

Acyl carnitines 1

• 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 --->

Acyl carnitines 2

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % 05Jan01IMD027 1 (1.125) Sm (SG, 2x1.00); Sb (33,10.00 ) 1: Parents of 85ES+ 8.60e5

263.1 218.3 221.1 260.4 459.2 347.3 274.3 302.4 340.9 313.4 456.4 374.0 482.4 483.5

Normal Bloodspot Acyl Carnitine Spectrum

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % MK002 1 (1.123) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 1.94e6

260 218 221 263 459 347 342 288 274 372 370 457 426 399 482

Normal Plasma Acyl Carnitine Spectrum

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 m /z 1 0 0 % 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 + 8 .6 3 e 6

2 6 0 .3 2 1 8 .1 2 2 1 .2 2 6 3 .3 4 5 9 .6 2 8 8 .3 2 7 4 .0 3 0 4 .3 3 4 7 .0 4 5 6 .3 4 8 2 .4

Post-mortem Specimen

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

274.4 260.4 218.4 221.3 482.4 459.4 318.3

Propionic Acidaemia

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % 15FebIMD007 1 (1.117) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 1.92e6

218.3 388.5 260.4 221.3 263.4 274.3 347.3 332.4 288.3 459.5 482.7

Glutaric Aciduria Type 1

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m /z 100 % IM D 224 1 (1.022) Sm (SG , 2x0.75); S b (33,10.00 ) 1: P arents of 85ES + 2.00e6

218.2 221.2 260.2 261.2 288.4 318.3 300.4 459.5 347.4 356.6

HMG CoA Lyase Deficiency

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m /z 100 % 25JanIM D 011 1 (1.117) S m (S G , 2x0.75); S b (33,10.00 ) 1: P arents of 85ES + 6.13e5

221.3 218.3 263.3 260.4 259.8 228.3 237.3 344.5 264.4 316.3 288.2 274.2 297.6 342.2 459.8 347.1 370.4 510.6

MCADD

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m /z 100 % IMD202 1 (1.021) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 2.28e6

x4 218.2 201.0 260.3 221.3 261.2 456.4 454.5 426.5 400.6 263.4 288.3 274.3 398.4 347.4 416.4 428.1444.5 459.6 482.4 472.2 460.5 484.5 500.3 498.5 501.8 514.3

LCHADD

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 m/z 100 % 23NovIMD012 1 (1.035) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 4.03e5

263.4 218.4 221.3 260.2 227.8 459.3 426.4 347.4 288.2 274.1 316.4 302.0 423.9 400.7 372.3 370.4 456.6 428.4 454.3 429.6 482.4 480.9 484.3 512.5

VLCADD

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

218.3 260.3 221.3 263.2 459.7 347.4 274.1

CPT-1 Deficiency

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m /z 100 % IM D 252 1 (1.021) S m (SG , 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES + 1.13e6

221.2 218.3 302.4 260.2 243.2 261.3 263.2 288.2 459.5 347.2 316.4 456.7 356.3 482.6

Isovaleric Acidaemia

302 459 260 263 347 456

What is the diagnosis?

Wrong!

• PAR 85 experiment not specific to

acylcarnitines

– acylcarnitines are detected because they form a m/z 85 fragment – other species forming m/z 85 fragments will also be detected – possible diagnostic problems if other species has same mass as an acylcarnitine

Why?

• Pivaloylcarnitine and isovalerylcarnitine
• Valproylcarnitine and octanoylcarnitine

Isobaric Species

• Translocase deficiency patient - blood spot

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 m /z 1 0 0 % 2 0 A p r0 1 IM D 0 0 8 1 (1 .1 2 5 ) S m (S G , 2 x 1 .0 0 ); S b (3 3 ,1 0 .0 0 ) 1 : P a re n ts o f 8 5 E S + 7 .3 0 e 6

4 5 6 .5 2 6 3 .3 2 2 1 .2 2 6 0 .3 2 7 7 .2 3 4 7 .3 3 2 2 .2 4 5 4 .3 4 2 8 .4 3 7 4 .4 4 0 0 .6 4 5 9 .4 4 8 2 .6 4 7 0 .5 4 8 3 .8

Blood Spots Or Plasma? 1

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % MK001 1 (1.120) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 2.69e6

263.3 221.2 260.4 459.4 456.4 347.3 274.2 318.3 482.2 484.6

Normal Blood Spot Acyl Carnitine Spectrum

• Normal blood spot?

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % MK003 1 (1.122) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 3.48e6

263.3 221.3 260.3 459.5 456.5 347.4 428.5 482.6

Blood Spots or Plasma? 2

• But abnormal plasma!

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % M K005 1 (1.123) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 9.91e5

457 263 260 221 228 347 322 288 274 302 318 342 428 402 400 374 403 404 427 454 430 444 482 459 480 470

Blood Spots or Plasma? 3

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % MK002 1 (1.123) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 1.94e6

260 218 221 263 459 347 342 288 274 372 370 457 426 399 482

Normal Plasma Acylcarnitine Spectra

• Plasma appears to be a more sensitive

medium for detecting acylcarnitine abnormalities in most cases

– an exception is HMG CoA lyase deficiency where the reverse is true

• Send blood spots and plasma!!!

Blood Spots or Plasma? - 4

• MS/MS for acyl carnitines and amino acids is

diagnostically powerful

• Results can be obtained rapidly on small samples
• But problems:

– isomers not differentiated

• leucine/isoleucine not separately measurable this way
• interference from drugs etc

– not specific for MMA/PA or CPT-II/translocase – blood spots or plasma can be used but both ideally

Amino Acids & Acyl Carnitines - Comment

MRM For FK506 1

• Amino acid and acyl carnitine determination look for

classes of compounds using scans

• For specific compounds it is better to use MRM

acquisitions

– MRM allows specification of precursor and product ion pairs – Q1 stays set to transmit precursor ion – Q3 stays set to transmit product ion

• Quadrupoles dwell on set mass for longer \ better s/n ratio and

sensitivity compared to scan

– Can do this for one or more compounds

• If more than one Q1 and Q3 keep switching between settings to transmit

the different ion pairs

MRM For FK506 2

• FK506 assayed against ascomycin as internal

standard in presence of NH4

+ ions

– FK506 and ascomycin form ammoniated ions ([M+NH4]+)

• [FK506+NH4]+

m/z 809.4

• [Asco+NH4]+

m/z 821.4

– Collision induced dissociation causes both to fragment giving product ions of m/z 756.5 and 768.4 respectively

• MRM monitoring of following transitions gives allows

sensitive detection of FK506 and ascomycin

– FK506 809.4 > 756.5 – Asco 821.4 > 768.4

MRM For FK506 3

• By looking for specific ion pairs during elution a

total ion chromatogram can be formed

.2 .4 .6 .8 1 .0 1 .2 1 .4 1 .6 1 .8 2 .0 2 .2 2 .4 T im e 1 % ta c ro c a l_ 1 2 M R M

• f 2

C h a n n e ls E S + T IC 4 .1 7 e 4

1 .0 8

MRM For FK506 4

• Because only specific ions are monitored no

mass spectrum is formed; only specific precursor ions are recorded

809 810 811 812 813 814 815 816 817 818 819 820 821 822 m/z 100 % tacrocal_12 143 (1.092) MRM of 2 Channels ES+ 2.52e4

821 809