Development of an Ultra High Pressure Development of an Ultra High - - PowerPoint PPT Presentation

Development of an Ultra High Pressure Development of an Ultra High Pressure Liquid Chromatography Liquid Chromatography-

• Tandem Mass

Tandem Mass Spectrometry screening method for X Spectrometry screening method for X-

• Linked Adrenoleukodystrophy.

Linked Adrenoleukodystrophy.

Marcus Williams Marcus Williams Senior Biomedical Scientist Senior Biomedical Scientist Biochemical Genetics & Newborn Screening Laboratory Biochemical Genetics & Newborn Screening Laboratory Southmead Southmead Hospital Hospital Bristol Bristol

I ntroduction I ntroduction

n n X

X-

• ALD

ALD

– – Most common of the Most common of the peroxisomal peroxisomal disorders affecting disorders affecting somewhere in the region of 1:17000 individuals. somewhere in the region of 1:17000 individuals.

n n Affects the nervous system, adrenal cortex and

Affects the nervous system, adrenal cortex and testis. testis.

n n Wide Phenotypic variation

Wide Phenotypic variation

n n BMT most affective intervention in severe forms

BMT most affective intervention in severe forms

n n Narrow therapeutic window

Narrow therapeutic window

n n Current diagnosis is by plasma VLCFA analysis and

Current diagnosis is by plasma VLCFA analysis and confirmed by DNA mutation analysis. confirmed by DNA mutation analysis.

n n Newborn screening would greatly improve

Newborn screening would greatly improve prognosis prognosis

Project aims Project aims

1. 1.

Look at C26:0 Look at C26:0 lysophosphatidylcholine lysophosphatidylcholine (C26:0 (C26:0-

• lyso

lyso-

• pc) as a marker for X

pc) as a marker for X-

• ALD

ALD in newborn screening bloodspots in newborn screening bloodspots

2. 2.

Optimise MS conditions Optimise MS conditions

3. 3.

Optimise LC conditions Optimise LC conditions

4. 4.

Validate method Validate method

MS parameters MS parameters

Expected fragmentation of lyso pc’s

I nitial MRM transitions C26:0-lyso-pc

636 > 104 636 > 184

C26:0-lyso-pc D4

640 > 104 640 > 184

Chromatography

Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 % 100 06031102 MRM of 4 Channels ES+ TIC 2.63e5

3.60 3.40

I nternal Standard

Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 % 100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 % 100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 % 100 13031103 Sm (SG, 2x5) MRM of 4 Channels ES+ TIC 2.66e4

2.77 2.63

13031103 Sm (SG, 2x5) MRM of 4 Channels ES+ 640 > 104 (C26 IS) 1.15e4

2.77

13031103 Sm (SG, 2x5) MRM of 4 Channels ES+ 640 > 184 (C26 IS) 1.50e4

2.76 2.63

Standard

STD

Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 % 100 0.50 1.00 1.50 2.00 2.50 3.00 3.50 % 100 0.50 1.00 1.50 2.00 2.50 3.00 3.50 % 100 13031102 Sm (SG, 2x5) MRM of 4 Channels ES+ 636 > 104 (C26) 2.21e4

2.79

13031102 Sm (SG, 2x5) MRM of 4 Channels ES+ 636 > 184 (C26) 2.47e4

2.79 2.64

13031102 Sm (SG, 2x5) MRM of 4 Channels ES+ TIC 4.70e4

2.79 2.64

636>104 636>184 TIC

Standard curve

1 2 3 4 5 6 7 8 200 400 600 800 1000 1200 1400 1600 1800 C26:0-lyso-pc Standard concentration (nmol/L) C26:0-lyso-pc response ratio

The Standard curve for C26:0-lyso-pc up to 1560 nmol/L (n= 11) Pearson’s correlation coefficient, r= 0.999986

Recovery experiment

0.05 0.1 0.15 0.2 0.25 500 1000 1500 2000 2500 3000 3500 C26:0-lyso-pc spiked sample concentraion (nmol/L) C26:0-lyso-pc response ratio n=12

The mean response of each of the 11 spiked samples (r= 0.99700325)

3.9 96 10 6.2 191 9 9.9 383 8 12.9

765

7 16.6 1500 6 27.0 3000 5 74.1 6000 4 99.5 12000 3 172.1 24500 2 349.3 49000 1

Signal / noise ratio C26:0-lyso-pc concentration (pmol/ L) Sample

Limit of detection Limit of detection experiment experiment

Summary Summary

n n C26:0

C26:0-

• lyso

lyso-

• pc is a putative marker for X

pc is a putative marker for X-

• ALD

ALD

n n UPLC

UPLC-

• MS/MS method

MS/MS method is suitable is suitable for high for high throughput screening throughput screening

n n ‘

‘Dilute and shoot Dilute and shoot’ ’ bloodspot extraction bloodspot extraction procedure procedure

n n Analysis time of 3.5 minutes

Analysis time of 3.5 minutes

n n Further work to include establishing a

Further work to include establishing a reference range in neonates reference range in neonates