Neonatal Screening using Shimadzu LCMS-8040

Transcription

1 PO-CON1355E Neonatal Screening using Shimadzu LCMS-8040 HPLC 2013 BIOM-31 Stefano Zaza 1 ; G. La Marca 2 ; S.Malvagia 2 1 Shimadzu Italy, Milano, Italy 2 Mass Spectrometry and Pharmacology Laboratory, A. Meyer Children s Hospital Pediatric Neurology Unit and Laboratories, University of Florence, Italy

2 1. Introduction The development of electrospray tandem mass spectrometry (LCMSMS) in recent years has facilitated the introduction of expanded newborn screening programmes in many countries. This techonology has increased the capacity to test newborns for rare metabolic disorders during the neonatal period. LCMSMS can identify and quantify several acylcarnitines, amino acids, adenosine, deoxyadenosine, succynilacetone 1, 2 and is able to detect more than 40 inborns errors of metabolism in a single test, allowing a change from the concept one spot, one test, one disease to one spot, one test, many disease. In collaboration with Dr. G. la Marca (Meyer Children s Hospital, Metabolomic Unit, Florence, Italy), we are developing a method for identify and quantify rare metabolic disorders using Shimadzu LCMS All data were collected and elaborated using Shimadzu LabSolution software and Shimadzu Neonatal Software. 2. Method Sample Preparation Dried blood spot (DBS) were generated by dispensing 25 µl of blood. The blood spots were allowed to dry overnight. A small disc (3.2 mm) of a DBS was punched and deposited in a 96 microwell plate. The sample was extracted by dispensing 300 µl of an extraction solution consisting of a mixture of methanol and aqueous solution of 3 mmol/l hydrate hydrazine at an approximate relative volume/volume ratio of 66.6% and 33.3%, respectively. Internal standards, stable heavy isotope analogues of several amino acids, carnitine, acylcarnitines, succylacetone, adenosine and deoxyadenosine, were also present in extract solution. The extracted sample was injected into Shimadzu LCMS Mass spectral data for the aminoacids were acquired through a neutral loss scan of 46 Da in positive mode (CE-15V) ; mass spectral data for the acylcarnitines were acquired through a precursor ion scan of 85 m/z in positive mode (CE-25V). Mass spectral for adenosine, deoxyadenosine, succynilacetone, glycine, arginine, citruline, ornithine, methionine were acquired through multiple-reaction monitoring (MRM) in positive mode. The percentage of each analyte recovered was determined through comparison with an internal standard for each analyte. The Standard Concentrations were in the range of µmol/l for amino acids, and in the range µmol/l for acylcarnitines. Spiked samples with different concentrations of analyte are used as daily control quality test. 2

3 Compound Type m/z Event C0 Target :Precursor(+) C0 IS ISTD :Precursor(+) C5 Target :Precursor(+) C5 IS ISTD :Precursor(+) C6 Target :Precursor(+) C8 Target :Precursor(+) C8 IS ISTD :Precursor(+) C6 IS Target :Precursor(+) C2 Target :Precursor(+) C2 IS ISTD :Precursor(+) C3 Target :Precursor(+) C3 IS ISTD :Precursor(+) C4 Target :Precursor(+) C4 IS ISTD :Precursor(+) C10 Target :Precursor(+) C10 IS ISTD :Precursor(+) C10:1 Target :Precursor(+) C10:1 IS ISTD :Precursor(+) C12 Target :Precursor(+) C12 IS ISTD :Precursor(+) C12:1 Target :Precursor(+) C12:1 IS ISTD :Precursor(+) C14 Target :Precursor(+) C14 IS ISTD :Precursor(+) C14:1 Target :Precursor(+) C14:1 IS ISTD :Precursor(+) C16 Target :Precursor(+) C16 IS ISTD :Precursor(+) C16:1 Target :Precursor(+) C16:1 IS ISTD :Precursor(+) C18 Target :Precursor(+) C18 IS ISTD :Precursor(+) C18:1 Target :Precursor(+) C18:1 IS ISTD :Precursor(+) C18:2 Target :Precursor(+) C18:2 IS ISTD :Precursor(+) C3DC Target :Precursor(+) C3DC IS ISTD :Precursor(+) C4OH Target :Precursor(+) C4OH IS ISTD :Precursor(+) C5:1 Target :Precursor(+) C5:1 IS ISTD :Precursor(+) C5DC Target :Precursor(+) C5DC IS ISTD :Precursor(+) C5OH Target :Precursor(+) C5OH IS ISTD :Precursor(+) C12OH Target :Precursor(+) C12OH IS ISTD :Precursor(+) C14OH Target :Precursor(+) C14OH IS ISTD :Precursor(+) C16OH Target :Precursor(+) C16OH IS ISTD :Precursor(+) C18OH Target :Precursor(+) C18OH IS ISTD :Precursor(+) C18:1OH Target :Precursor(+) 3

4 Compound Type m/z Event C18:1OH IS ISTD :Precursor(+) C14:2 Target :Precursor(+) C14:2 IS ISTD :Precursor(+) C16:1OH Target :Precursor(+) C16:1OH IS ISTD :Precursor(+) C10:2 Target :Precursor(+) C10:2 IS ISTD :Precursor(+) C4DC Target :Precursor(+) C4DC IS ISTD :Precursor(+) C6DC Target :Precursor(+) C6DC IS ISTD :Precursor(+) C10 OH Target :Precursor(+) C10 OH IS ISTD :Precursor(+) C8DC Target :Precursor(+) C8DC IS ISTD :Precursor(+) C18:2 OH Target :Precursor(+) C18:2 OH IS ISTD :Precursor(+) C8:1 Target :Precursor(+) C8:1 IS ISTD :Precursor(+) Ala Target 90 3:NLS(+) Ala IS ISTD 94 3:NLS(+) Val Target :NLS(+) Val IS ISTD :NLS(+) Xleu Target :NLS(+) Xleu IS ISTD :NLS(+) Met Target :NLS(+) Met IS ISTD :NLS(+) Tyr Target :NLS(+) Tyr IS ISTD :NLS(+) Asp Target :NLS(+) Asp IS ISTD :NLS(+) Glu Target :NLS(+) Glu IS ISTD :NLS(+) Phe Target :NLS(+) Phe IS ISTD :NLS(+) Gly Target 76.00> :MRM(+) Gly IS ISTD 78.00> :MRM(+) Cit MRM Target > :MRM(+) Cit MRM IS ISTD > :MRM(+) Arg MRM Target > :MRM(+) Arg MRM IS ISTD > :MRM(+) Arg Succ MRM Target > :MRM(+) Arg Succ MRM IS ISTD > :MRM(+) Orn Target > :MRM(+) Orn IS ISTD > :MRM(+) Met MRM Target > :MRM(+) Met MRM IS ISTD > :MRM(+) SuAC Target > :MRM(+) SuAC IS ISTD > :MRM(+) ADO Target > :MRM(+) ADO IS ISTD > :MRM(+) Deoxi ADO Target > :MRM(+) Deoxi ADO IS ISTD > :MRM(+) 4

5 Analysis Condition Run of 2.2 minutes in FIA Flow µl/min (A: Water+0.05% of Formic Acid, B: Acetonitrile, A/B: 30%/70%) 40 µl of sample injected Column Oven 30 C, Desolvatation Line 300 C, Heat 500 C, Nebulizing Gas 3 L/min, Drying Gas 20 L/min. All data collected were reprocessed using Shimadzu Neonatal Software, that calculated automatically the concentration of each compounds. dried blood spot (DBS) Dlameter of circle 3.2 mm 200 µl of MeOH labelled internal standards and 100 µl of hydrazine 3 mmol/l 25 min 37 C Evaporation under nitrogen flow 45 C ACN/Water 0.05 formic acids 70:30 Flow Injection Analysis (LC-MS/MS) Method develloped by Dr. Giancarlo LaMarca 5

6 Example of Results obtained by Neonatal Software 3. References 1-Progress in expanded newborn screening for metabolic conditions by LC-MS/MS in Tuscany: Update on methods to reduce false test (G. la Marca, S. Malvagia, B. Casetta, E. Pasquini, M.A. Donati, E. Zammarchi, JIMD Short Report 2008) 2-Neonatal screening for severe combined immunodeficiency caused by anadenosine deaminase defect: a rialable and inexpensive method using tandem mass spectrometry (Azzari, la Marca, Resti, J. Allergy Clin. Immunol., Vol. 127, Number ) First Edition: June, For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2013