Received on November 23, 2007
Accepted on May 6, 2008

Lipids, Lipoproteins, and Cardiovascular Risk Factors

Potential of Sterol Analysis by Liquid Chromatography–Tandem Mass Spectrometry for the Prenatal Diagnosis of Smith-Lemli-Opitz Syndrome

¹ Institute of Mass Spectrometry, School of Medicine, Swansea University, Swansea, UK
² The School of Pharmacy, University of London, London, UK
³ Thermo Fisher Scientific, Hemel Hempstead, UK
⁴ Children's Hospital Oakland Research Institute, Oakland, CA

^* To whom correspondence should be addressed. E-mail: w.j.griffiths{at} swansea.ac.uk.

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: 3-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.