High throughput screening for X-linked adrenoleukodystrophy and other peroxisomal
disorders in dried blood spots by using Acquity UPLC-XEVO TQD
Taposh G, Bhaskar K, Rajesh PMN, Anil Kurup
Waters India, Applications Laboratory, Bangalore - 560058.
INTRODUCTION
Adrenoleukodystrophy (ALD) is a serious progressive, genetic disorder that affects
the adrenal glands, the spinal cord and the white matter of the nervous system. It was
first recognized in 1923 and has been known as Schilder’s disease and sudanophilic
leukodystrophy. In 1970, the name adrenoleukodystrophy was introduced; ‘adreno’
refers to the adrenal glands; ‘leuko’ refers to the white matter of the brain, and
‘dystrophy’ means abnormal growth or development. There is no relation with
“neonatal adrenoleukodystrophy” which belongs to the peroxisomal biogenesis
disorders of the Zellweger spectrum.
UPLC & XEVO TQD
Fig 6 Result table for linearity and unknown concentration of Lyso PC-22-0
Biochemistry
ALD is an inherited metabolic storage disease whereby a defect in a specific enzyme
results in the accumulation of very long-chain fatty acids (VLCFA) in all tissues of
the body. These VLCFA ( C26:0 to 20:0 Lyso PC) are harmful for cells and tissues.
For reasons that have not yet been resolved brain, spinal cord, testis and the adrenal
glands are primarily affected. In the central nervous system the buildup of VLCFA
eventually destroys the myelin sheath that surrounds the nerves causing neurologic
problems. VLCFA are toxic to adrenal gland cells and their malfunction causes
Addison’s disease (adrenal insufficiency).
Objective:
RESULTS AND DISCUSSION
To develop a simple rapid, novel, sensitive LC-MS/MS method for all the four
markers (C26:0 to 20:0 Lyso PC) from human dried blood spot.
Fig 2: Result table for linearity and unknown concentration of Lyso PC-26 –0
Fig 7: CCurve from 10.0-200.0ng/ml and chromatogram of Lyso PC 22-0
Fig 8 : Result table for linearity and unknown concentration of Lyso PC-20 –0
Figure 1: Chemical structure of Lyso PC 26 to 20
METHOD SUMMARY
MRM Conditions:
Compound
Parent m/z Daughter m/z Cone (V)
Lyso PC 26-0
Lyso PC 24-0
Lyso PC 22-0
Lyso PC 20-0
Lyso PC 26-0_D4
636.66
608.468
580.415
552.415
640.66
104
104
104
104
104
60
60
60
60
60
CE
30
30
30
30
30
Fig 9: Calibration curve from 10.0-200.0ng/ml and chromatogram of Lyso PC 20-0
Fig 3: Calibration curve from 10.0-200.0ng/ml and chromatogram of Lyso PC 26-0
CONCLUSION
1) The demonstrated quantification method is reliable, high sensitive, precise,
accurate and reproducible for DBS samples.
2) The four analytes were quantified from DBS spot in single run.
3) Phospholipids interference were effectively separated by focused gradient
on column with good reproducibility of the method.
MS Conditions:
MS System
: Xevo TQD
Mode
: ESI
Cone gas
: 170 L/Hr
Source Temp
: 150 ºC
Desolvation gas flow : 1000 L/hr
Cone gas flow
4) The four analytes are showing good linearity with r<0.99 regression
coefficient.
5) Opportunity to
compounds.
validate the method for routine analysis of Lyso PC
Fig 4: Result table for linearity and unknown concentration of Lyso PC-24 –0
REFERENCES
: 50 L/hr
1,
1. Hubbard WC Moser AB etal.(2009) 2009 Jul;97(3):212-20. Newborn
screening for X-linked adrenoleukodystrophy (X-ALD): validation of a
combined liquid chromatography-tandem mass spectrometric (LC-MS/MS)
method, Mol Genet Metab, doi: 10.1016/j.ymgme.2009.03.010. Epub 2009
Data processed through TargetLynx application manager.
Sample preparation: Punch out a blood spot from DBS card and place it in 96
well plate. Add water followed by ISTD (internal standard). Add Extraction solvent to
the well, slowly vortex the sample for 1 hr. Supernatant is used for the LC-MS/MS
analysis of the four molecules.
ACKNOWLEDGMENT
Fig 5: Calibration curve from 10.0-200.0ng/ml and chromatogram of Lyso PC 24-0
The authors would like to thanks Dr. Ritachristopher (HOD Neurobilogical
sciences, NIMHANS) and Ms. Archana (Research Scientist Neurobilogical
sciences, NIMHANS) for providing Reference standards, DBS card and Blood
samples.