Fully automatized newborn screening for acylcarnitines, amino acids and steroids by LC-MS
A more secure and efficient screening technique for foracylcarnitines, amino acids and steroids in newborns.
In Switzerland, 84’000 newborns are screened by dried blood spot analysis for innate errors every year [1-3]. However, traditional methods in this field are laborious as they are based on punching and offline extraction. Even more critically, sometimes sample confusion is caused electrostatic effects, which direct punched samples into the wrong tube or well. A method using steroids, acylcarnitines and amino acid based on the DBS-500 is presented here.
Classical DBS punching versus online CAMAG DBS-500. The classical workflow includes a semi-automatic punch-out step whereas the automated DBS-500 allows direct extraction of blood spots into the mass spectrometer.
A fully automated dried blood spot sampler, the DBS-MS 500,(CAMAG, Muttenz BL, CH) and a 6410 triple quadrupole mass spectrometer (+ESI) equipped with a 1100 series quaternary HPLC pump (Agilent, Santa Clara CA, USA) were used. Solvents and additives were provided by Sigma-Aldrich (Buchs SG, CH). Standards for amino acids and acylcarnitines were provided by Chromsystems (München, D). The method was validated according to ICH guidelines [4].
Extraction conditions An optimum extraction is achieved by a mixture of methanol / water 7:3, which is the best compromise of extracting the analytes, extracting low amounts of suppressant and minimum interference with the chromatographic separation.
Chromatography Most compounds are separated by MRM only. Deoxycorticosteron and 17 OHP, which have the same MRM, are separated by chromatography.
Validation The averaged CV of the amino acids and acylcarnitines was 4.2 % within one day and 7.2% for several days using the automated method. This was comparable to the established routine method, which was found to be 5.6% CV for one day and 6.3% CV for several days.
It has been shown that the new direct elution method has significant advantages compared to the traditional method. The combination of the 25 markers allows fully automated screening of several diseases in less than 2.5 minutes per sample.
This new direct extraction approach significantly improves not only newborn screening programs, but also opens new avenues for dried blood spot analysis in other fields such as screening for doping, therapeutic drug monitoring, or point of care analytics.
[1] Dried Blood Spots: Applications and Techniques ISBN: 9781118054697 [2] Neugeborenscreening Kinderspital Zürich http://www.neoscreening.ch [3] Fingerhut et al. Rapid Commun. Mass Spectrom. 2014, 28, 965–973 [4] ICH Validation Guidelines http://www.ich.org
Research by: Christian Berchtold[1], Irene Wegner[1], Stefan Gaugler[2], Tamara von Däniken[3], Markus Wyss[2], Ralph Fingerhut[3], Götz Schlotterbeck[1]
[1] University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Institute of Chemistry and Bioanalytics, 4132 Muttenz, Switzerland [2] CAMAG Chemie-Erzeugnisse & Adsorptionstechnik AG, Sonnenmattstrasse 11, 4132 Muttenz, Switzerland [3] University Children`s Hospital, Children’s Research Center, Swiss Newborn Screening Laboratory Steinwiesstrasse 75, 8032 Zürich, Switzerland
