Neonatal Screening using Shimadzu LCMS-8040

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  • Published: Oct 24, 2013
  • Author: Shimadzu Europa GmbH
  • Categories: Base Peak
thumbnail image: 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 succynilacetone1,2 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.


LCMS-8040

The LCMS-8040 tandem mass spectrometer from Shimadzu


Sample preparation and mass spectrometry condition (Figure 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).


Figure 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.


Table 1. 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 (Figure 2) was reprocessed using Shimadzu Neonatal Software, which calculated the concentration of each compound automatically (Figure 3).


Figure 2. Example of mass spectrum acquired with a High-level quality control


Figure 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.

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