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LC–MS/MS-based quantification of cholesterol and related metabolites in dried blood for the screening of inborn errors of sterol metabolism

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Abstract

Smith–Lemli–Opitz syndrome (SLOS) is an inherited metabolic disease in the cholesterol biosynthesis pathway which is characterised by accumulation of 7- and 8-dehydrocholesterol and by reduced cholesterol concentrations in all tissues and body fluids. With this study, we developed a new, rapid, robust and high-throughput tandem mass spectrometric method as routine application for the selective SLOS screening and therapy monitoring in serum and dried blood. After protein precipitation of 10 μL serum or 4.7 mm dried blood spot, the sum of 7- and 8-dehydrocholesterol (DHC) was analysed by rapid chromatography combined with tandem mass spectrometry. Method comparison with GC–MS was performed for 46 serum samples. A comparison between serum and corresponding dried blood spots for DHC and cholesterol was performed with 40 samples from SLOS patients. Concentrations of DHC and cholesterol were analysed in 2 dried blood samples from newborns with SLOS and 100 unaffected newborns. Intra- and inter-assay variabilities ranged between 3.7 and 17.7 % for serum and dried blood spots. Significant correlations between the new LC–MS/MS method and GC–MS were determined for DHC (r = 0.937, p < 0.001) and for cholesterol (r = 0.946, p < 0.001). Significant coefficients of correlation between serum and dried blood spot samples above 0.8 were calculated for both analytes. A cut-off value of 5.95 for the ratio of DHC/cholesterol (multiplied by 1000) was found to distinguish newborns diagnosed with SLOS from normal newborns in a retrospective analysis after 5 years. The developed method enables a rapid quantification of the sum parameter 7- and 8-DHC in newborns and SLOS patients under therapy in serum as well as dried blood spot samples.

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Abbreviations

APPI:

Atmospheric pressure photo ionisation

CH:

Cholesterol

CI:

Confidence interval

7-DHC:

7-Dehydrocholesterol

8-DHC:

8-Dehydrocholesterol

DHCR7:

7-Dehydrocholesterol reductase

ESI:

Electrospray ionisation

GC–MS:

Gas chromatography mass spectrometry

LC–MS/MS:

Liquid chromatography tandem mass spectrometry

LOD:

Limit of detection

LLOQ:

Lower limit of quantification

SLOS:

Smith–Lemli–Opitz syndrome

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Acknowledgments

This publication is supported by LIFE—Leipzig Research Center for Civilization Diseases, Universität Leipzig. LIFE is funded by means of the European Union, by the European Regional Development Fund (ERDF) and by means of the Free State of Saxony within the framework of the excellence initiative.

Conflict of interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

  1. Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103, Leipzig, Germany

    S. Becker, J. Thiery & U. Ceglarek

  2. LIFE—Leipzig Research Center for Civilization Diseases, University Leipzig, Philipp-Rosenthal-Strasse 27, 04103, Leipzig, Germany

    S. Becker, J. Thiery & U. Ceglarek

  3. Department of Pediatrics, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, 39120, Magdeburg, Germany

    S. Röhnike, S. Empting & K. Mohnike

  4. Division of Inborn Metabolic Diseases, University Children’s Hospital, Im Neuenheimer Feld 430, 69121, Heidelberg, Germany

    D. Haas

  5. Hospital for Children and Adolescents, Centre of Paediatric Research (CPR), Department of Women and Child Health, University Hospitals, University of Leipzig, Liebigstraße 20 a, 04103, Leipzig, Germany

    S. Beblo & U. Mütze

  6. Center for Inborn Metabolic Disorders, Children’s Hospital, Jena University Hospital, Kochstr. 2, 07740, Jena, Germany

    R. A. Husain

Authors
  1. S. Becker
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  2. S. Röhnike
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  3. S. Empting
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  4. D. Haas
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  5. K. Mohnike
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  6. S. Beblo
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  7. U. Mütze
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  8. R. A. Husain
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  9. J. Thiery
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  10. U. Ceglarek
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Corresponding author

Correspondence to S. Becker.

Additional information

Published in the topical collection Lipidomics with guest editor Michal Holčapek.

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Becker, S., Röhnike, S., Empting, S. et al. LC–MS/MS-based quantification of cholesterol and related metabolites in dried blood for the screening of inborn errors of sterol metabolism. Anal Bioanal Chem 407, 5227–5233 (2015). https://doi.org/10.1007/s00216-015-8731-1

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  • DOI: https://doi.org/10.1007/s00216-015-8731-1

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