Potential of sterol analysis by liquid chromatography-tandem mass spectrometry for the prenatal diagnosis of Smith-Lemli-Opitz syndrome.
BACKGROUND
Smith-Lemli-Opitz syndrome (SLOS), a severe disorder of cholesterol synthesis, is classically diagnosed prenatally by GC-MS analysis of sterols in amniotic fluid. Considering the current trend toward tandem mass spectrometry (MS/MS) methodologies, we developed prototype LC-MS/MS methods for accurate diagnosis of the disorder.


METHODS
3beta-Hydroxysterols in amniotic fluid are oxidized with cholesterol oxidase to their corresponding 3-ketones, which are then derivatized with Girard P (GP) hydrazine in a "one-pot" reaction. The resulting GP-hydrazones give an improved response in electrospray (ES)-MS/MS owing to the presence of a charged quaternary nitrogen and are analyzed by reversed-phase LC-ES-MS/MS. Both capillary and conventional LC-MS/MS formats are suitable, and the method is also applicable to paper-absorbed blood spots.


RESULTS
In a double-blind analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the ratio of 7 + 8-dehydrocholesterol (7 + 8-DHC) to cholesterol was <0.02 [range 0.00-0.02, mean (SD) 0.01 (0.007)] in all control samples (intraassay variation 5.91%) and >0.20 [0.20-1.13, 0.79 (0.35)] in SLOS (intraassay variation 4.56%), corresponding to a difference in ratios between the 2 groups of at least a factor of 10. The limit of quantification was equivalent to that of 2 nL amniotic fluid injected on-column.


CONCLUSIONS
We describe a proof-of-concept for the prenatal diagnosis of SLOS. Further developments will be necessary to automate sample handling and reduce chromatographic time for the methodology to be used in pre- and postnatal diagnosis.
Griffiths
William J
WJ
Institute of Mass Spectrometry, School of Medicine, Swansea University, Swansea, UK. w.j.griffiths@swansea.ac.uk
Wang
Yuqin
Y
Karu
Kersti
K
Samuel
Emmanuel
E
McDonnell
Shane
S
Hornshaw
Martin
M
Shackleton
Cedric
C
D000653:Amniotic Fluid / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D004311:Double-Blind Method; D005260:Female; D006801:Humans; D015394:Molecular Structure; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019082:Smith-Lemli-Opitz Syndrome / Q000175:diagnosis* / Q000196:embryology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013261:Sterols / Q000032:analysis* / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
D013261:Sterols