SEG- 2050 Application Note for The Analytical Scientist 11-13 Neonatal Screening using Shimadzu LCMS- 8040 Introduction The development of electrospray tandem mass spectrometry (LCMS/MS) in recent years has facilitated the introduction of expanded newborn screening programmes in many countries. This technology has increased the capacity to test newborns for rare metabolic disorders during the neonatal period. LCMS/MS can identify and quantify several acylcarnitines, amino acids, adenosine, deoxyadenosine and succynilacetone 1,2 and is able to detect more than 40 inborn 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 diseases. In collaboration with Dr. G. la Marca (Meyer Children s Hospital, Metabolomic Unit, Florence, Italy), we have developed a method for identifying and quantifying rare metabolic disorders using Shimadzu s LCMS- 8040. All data has been collected and evaluated using Shimadzu s LabSolution and Neonatal softwares. The LCMS/MS tandem mass spectrometer LCMS- 8040 of Shimadzu Sample preparation and mass spectrometry condition (Fig.1) Dried blood spots (DBS) were generated by dispensing 25 µl of blood. The blood spots were dried overnight. A small disc (3.2 mm, 3.4 µl) of a DBS was punched and deposited in a microwell plate. The sample was extracted by dispensing 300 µl of an extraction solution consisting of a mixture of methanol (200 µl) and aqueous solution of 3 mmol/l hydrate hydrazine (100 µl). Internal standards, stable heavy isotope analogs of several amino acids, carnitine, acylcarnitines, succylacetone, adenosine and deoxyadenosine were also present in the extract solution. The sample was shacked for 25 minutes at room temperature, transferred to a new plate and sent to be dried under Nitrogen flow. The sample was then eluted with 260 µl of Acetonitrile/Water (70/30; 0.1% Formic Acid).
Fig.1 Method developed for Neonatal Screening The extracted sample was injected into the Shimadzu LCMS- 8040. Mass spectral data for the amino acids 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 spectra for adenosine, deoxyadenosine, succynilacetone, glycine, arginine, citruline, ornithine and methionine were acquired through multiple- reaction monitoring (MRM) in positive mode. All compounds screened are reported in Table 1. 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 500-2500 µmol/l for amino acids, and in the range of 7.6-152 µmol/l for acylcarnitines. Spiked samples with different concentrations of analyte are used as daily control quality test. Compound Type ISTD Group m/z Event C0 Target 1 162.1 2:Precursor(+) C0 IS ISTD 1 171.2 2:Precursor(+) C5 Target 4 246.2 2:Precursor(+) C5 IS ISTD 4 255.2 2:Precursor(+) C6 Target 4 260.2 2:Precursor(+) C8 Target 5 288.2 2:Precursor(+) C8 IS ISTD 5 291.2 2:Precursor(+) C6 IS Target 4 255.2 2:Precursor(+) C2 Target 6 204.1 2:Precursor(+) C2 IS ISTD 6 207.1 2:Precursor(+) C3 Target 7 218.1 2:Precursor(+) C3 IS ISTD 7 221.1 2:Precursor(+) C4 Target 8 232.2 2:Precursor(+) C4 IS ISTD 8 235.2 2:Precursor(+)
C10 Target 5 316.1 2:Precursor(+) C10 IS Target 5 291.2 2:Precursor(+) C10:1 Target 5 314.1 2:Precursor(+) C10:1 IS Target 5 291.2 2:Precursor(+) C12 Target 9 344.3 2:Precursor(+) C12 IS Target 9 381.3 2:Precursor(+) C12:1 Target 9 342.3 2:Precursor(+) C12:1 IS Target 9 381.3 2:Precursor(+) C14 Target 9 372.3 2:Precursor(+) C14 IS ISTD 9 381.3 2:Precursor(+) C14:1 Target 9 370.3 2:Precursor(+) C14:1 IS Target 9 381.3 2:Precursor(+) C16 Target 10 400.3 2:Precursor(+) C16 IS ISTD 10 403.3 2:Precursor(+) C16:1 Target 10 398.3 2:Precursor(+) C16:1 IS Target 10 403.3 2:Precursor(+) C18 Target 10 428.4 2:Precursor(+) C18 IS Target 10 403.3 2:Precursor(+) C18:1 Target 10 426.4 2:Precursor(+) C18:1 IS Target 10 403.3 2:Precursor(+) C18:2 Target 10 424.3 2:Precursor(+) C18:2 IS Target 10 403.3 2:Precursor(+) C3DC Target 7 248.1 2:Precursor(+) C3DC IS Target 7 221.1 2:Precursor(+) C4OH Target 8 248.1 2:Precursor(+) C4OH IS Target 8 235.2 2:Precursor(+) C5:1 Target 4 244.2 2:Precursor(+) C5:1 IS Target 4 255.2 2:Precursor(+) C5DC Target 5 276.1 2:Precursor(+) C5DC IS Target 5 291.2 2:Precursor(+) C5OH Target 4 262.2 2:Precursor(+) C5OH IS Target 4 255.5 2:Precursor(+) C12OH Target 9 360.3 2:Precursor(+) C12OH IS Target 9 381.3 2:Precursor(+) C14OH Target 9 388.3 2:Precursor(+) C14OH IS Target 9 381.3 2:Precursor(+) C16OH Target 10 416.3 2:Precursor(+) C16OH IS Target 10 403.3 2:Precursor(+) C18OH Target 10 444.4 2:Precursor(+) C18OH IS Target 10 403.3 2:Precursor(+) C18:1OH Target 10 442.4 2:Precursor(+) C18:1OH IS Target 10 403.4 2:Precursor(+) C14:2 Target 9 368.3 2:Precursor(+) C14:2 IS Target 9 381.3 2:Precursor(+) C16:1OH Target 10 414.3 2:Precursor(+) C16:1OH IS Target 10 403.3 2:Precursor(+)
C10:2 Target 5 312.2 2:Precursor(+) C10:2 IS Target 5 291.2 2:Precursor(+) C4DC Target 8 262.1 2:Precursor(+) C4DC IS Target 8 235.2 2:Precursor(+) C6DC Target 4 290.2 2:Precursor(+) C6DC IS Target 4 255.2 2:Precursor(+) C10 OH Target 5 332.2 2:Precursor(+) C10 OH IS Target 5 291.2 2:Precursor(+) C8DC Target 9 318.2 2:Precursor(+) C8DC IS Target 9 381.3 2:Precursor(+) C18:2 OH Target 10 440.4 2:Precursor(+) C18:2 OH IS Target 10 403.4 2:Precursor(+) C8:1 Target 5 285.2 2:Precursor(+) C8:1 IS Target 5 291.2 2:Precursor(+) Ala Target 11 90 3:NLS(+) Ala IS ISTD 11 94 3:NLS(+) Val Target 12 118.1 3:NLS(+) Val IS ISTD 12 126.1 3:NLS(+) Xleu Target 13 132.1 3:NLS(+) Xleu IS ISTD 13 135.1 3:NLS(+) Met Target 14 150.1 3:NLS(+) Met IS ISTD 14 153.1 3:NLS(+) Tyr Target 15 182.1 3:NLS(+) Tyr IS ISTD 15 188.1 3:NLS(+) Asp Target 16 134.1 3:NLS(+) Asp IS ISTD 16 137.1 3:NLS(+) Glu Target 17 148.1 3:NLS(+) Glu IS ISTD 17 151.3 3:NLS(+) Phe Target 2 166.1 3:NLS(+) Phe IS ISTD 2 172.1 3:NLS(+) Gly Target 18 76.00>30.10 1:MRM(+) Gly IS ISTD 18 78.00>32.10 1:MRM(+) Cit MRM Target 19 176.10>113.10 1:MRM(+) Cit MRM IS ISTD 19 178.10>115.10 1:MRM(+) Arg MRM Target 20 175.10>116.10 1:MRM(+) Arg MRM IS ISTD 20 180.10>121.10 1:MRM(+) Arg Succ MRM Target 20 291.10>176.20 1:MRM(+) Arg Succ MRM IS Target 20 180.10>121.10 1:MRM(+) Orn Target 21 133.10>70.10 1:MRM(+) Orn IS ISTD 21 135.10>72.10 1:MRM(+) Met MRM Target 22 150.10>104.10 1:MRM(+) Met MRM IS ISTD 22 153.10>107.10 1:MRM(+) SuAC Target 23 155.10>137.10 1:MRM(+) SuAC IS ISTD 23 159.20>141.10 1:MRM(+) ADO Target 24 268.20>136.10 1:MRM(+) ADO IS ISTD 24 269.20>136.10 1:MRM(+)
Deoxi ADO Target 25 252.20>136.10 1:MRM(+) Deoxi ADO IS ISTD 25 257.20>136.10 1:MRM(+) Table1. Compound screened in NBS method Analysis condition Run of 2.2 minutes in FIA Flow 0.070 µl/min (A: Water + 0.1% of Formic Acid, B: Acetonitrile, A/B: 30%/70%) 40 µl of sample injected Column Oven 30 C, DL 300 C, Heat Block 500 C, Nebulizing Gas 3 L/min, Drying Gas 20 L/min. All data collected (Fig. 2) was reprocessed using Shimadzu Neonatal Software, which calculated the concentration of each compound automatically (Fig. 3). Fig.2 Example of mass spectrum acquired with a High- level quality control
Fig. 3 Example of results obtained automatically with Neonatal Software References 1 Progress in expanded newborn screening for metabolic conditions by LCMS/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 reliable and inexpensive method using tandem mass spectrometry (Azzari, la Marca, Resti, J. Allergy Clin. Immunol., Vol. 127, Number 6 2011) Available in EU only. Shimadzu Europa GmbH Albert- Hahn- Str. 6 10, D- 47269 Duisburg, Germany tel. +49 203 76 87 0 fax +49 203 76 66 25 E- mail: shimadzu@shimadzu.eu Website: www.shimadzu.eu