Transcript Analytical Mass Spectrometry TECHNIQUES, APPLICATION

Measuring phenylalanine
R Neil Dalton
The WellChild Trust Laboratory
King’s College, London
Evelina Children’s Hospital at Guy’s Hospital
NSPKU, Skipton, 26th February 2004
Measuring phenylalanine
Phenylalanine
The bad boy of phenylketonuria?
Measuring phenylalanine
Phenylalanine is an essential amino acid
One of the building blocks of all proteins
Essential that the blood level of phenylalanine is
maintained at an optimum concentration
Optimum concentration range relatively wide, but in
phenylalanine hydroxylase deficiency needs to be
routinely monitored
Measuring phenylalanine
phenylalanine 3D structure
Measuring phenylalanine
phenylalanine structure
Measuring phenylalanine
Collection of the blood spot is the most important
step in obtaining an accurate measurement of
phenylalanine in the blood
How do we actually measure it?
Measuring phenylalanine
Guthrie bacterial inhibition assay
Measuring phenylalanine
Thin layer chromatography
Measuring phenylalanine
spotting the samples
Measuring phenylalanine
chromatography
Measuring phenylalanine
methods
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fluorimetry
enzymatic (PAL, PDH)
high pressure liquid chromatography
Electrospray mass spectrometry-mass spectrometry
Measuring phenylalanine
Electrospray (John Fenn)
Measuring phenylalanine
Electrospray Ionisation
Measuring phenylalanine
Electrospray Ionisation
Measuring phenylalanine
Electrospray Ionisation
Measuring phenylalanine
Electrospray Ionisation
Measuring phenylalanine
Electrospray Ionisation
Measuring phenylalanine
mass spectrometry-mass spectrometry
IONS
Q1
Q2
Q3
MS1
Collision Cell
MS2
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Measuring phenylalanine
product ion scan
Q1
IONS
Phe+
Q2
Q3
Collision Cell
MS2
Q1 - selection of phenylalanine ion
Q2 – fragment the phenylalanine ions
Q3 – look for fragments of the phenylalanine
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Measuring phenylalanine
product ion scan
Measuring phenylalanine
fragmentation
Measuring phenylalanine
Q1
IONS
Phe+
Q2
Q3
Collision Cell
x+
Q1 - selection of phenylalanine ion m/z 166
Q2 – fragmentation of phenylalanine ion
Q3 - selection of specific fragment ion m/z 120
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Measuring phenylalanine
optimal phenylalanine control
Checking in
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Samples posted
in are received
at the laboratory
and are logged
in to the
computer.
Punching out spot
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A special
machine
punches out the
spots
Measuring phenylalanine
Sample Preparation
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Punch 3mm diameter blood spot into deep-well
(2ml) microtitre plate
Add 150ml methanol/water (83:17) containing d5phe and d4-tyr (5.6mmol/l)
Seal plate with lid and mix for 20min at 300
cycles/min
Place on Gilson autosampler at RT for injection
Mixing and shaking
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Samples are
then put onto
plates and
mixed.
Measuring phenylalanine
Sample delivery & MSMS acquisition
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Mobile phase
acetonitrile/water (1:1) with formic acid (0.025%)
Flow rate
50ml/min
Sample volume
2ml
Acquisition time
55s
Cycle time
66s
 Precursor product pairs
phenylalanine (166/120)
tyrosine (182/136)
d5-phenylalanine (171/125)
d4-tyrosine (186/140)
Measuring phenylalanine
calibration
Measuring phenylalanine
poor phenylalanine control
results
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Sample results
are then
checked by the
laboratory staff.
Lab team
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Our hard
working
biochemistry
team.
Reported results
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Our dietitians
have both a
phoned and
faxed report of
all the day’s
phenylalanine
samples
received
Phoning parents
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With all the results
available, the
dietitian phones
the families,
discusses both the
child’s well being
dietary changes if
needed and often a
bit of a social chitchat.
Measuring phenylalanine
A multi-disciplinary team of parents/individual,
health care professionals, and leading edge
analytical technology are involved in maintaining
optimal blood phenylalanine
Collection of the blood spot is the most important
step in obtaining an accurate measurement of
phenylalanine in the blood
Acknowledgements
Charles Turner
Sue Bird
The WellChild Trust
Charitable Foundation Guy’s & St Thomas’ Hospitals
Guy’s and St Thomas’ NHS Trust