Environmental Engineering Reference
In-Depth Information
based on achieving separate enzymatic reactions, followed by their combination
and purification by liquid-liquid extraction (LLE) and solid phase extraction (SPE)
to remove the salts and detergents prior to mass spectrometry analysis, several
technical modifications were carried out focusing on simplifying the sample prep-
aration processes. In the last 6 years, to simplify the sample preparation procedures
and to reduce the running costs, several technical modifications, i.e. the conjunction
of mass spectrometry with separation techniques such as capillary electrophoresis
(CE) and high-performance liquid chromatography (HPLC), were carried out. An
attempt in this direction belongs to Marca et al. [
54
] who proposed a robust method
suitable for large-scale studies (screening) on dried blood spot samples, based on
five reaction mixtures from separate reactions, followed by their combination,
centrifugation and analysis on an LC MS/MS. The simplicity of their study emerges
from the only 4 min of analysis time, as well as from the supernatant analysis
without further manual purification after enzyme reaction. Two years later, in 2010
a similar approach [
55
], aimed to eliminate the off-line LLE and SPE steps, based
on high sensitivity, specificity and throughput, involved the combination of ultra-
high-performance liquid chromatography (UHPLC) and multiplexed sample intro-
duction. With the same objective of simplifying the analysis procedures, multiplex
approaches have been developed and piloted in newborn screening (NBS) studies.
Simplified newborn screening protocol based on high-throughput hexaplex assay
for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe and MPS I diseases have
been reported three years ago [
56
]. The protocol based on a dual-channel TLX-2
system (two TurboFlow and two analytical columns) coupled with a TSQ Quantum
Ultra AM mass spectrometer eliminated the use of the organic compounds from the
screening of LSDs by MS/MS and reduced the need for large amounts of
consumables.
There were still several multiplex studies based on LLE and SPE purification
steps, such as the screening of the three enzymes involved in Pompe, Fabry and
MPS-I diseases [
57
], and the study based on 4 + 1 multiplex MS/MS assay [
58
]. In
2013, it was reported a most comprehensive and robust multiplex approach using up
to nine lysosomal enzymes. Besides the Gaucher, Pompe, Krabbe, Fabry and
Niemann-Pick A/B enzymes, they added to the multiplex assay the MPS-I,
MPS-II, MPS-IVA and MPS-VI enzymes [
59
].
Since mass spectrometry methods have been developed to screen newborns for
LDSs, and now they are currently feasible to diagnose Pompe, Fabry, Gaucher,
Krabbe, Niemann-Pick A/B, as well as mucopolysaccharidosis I diseases, this
methodology could also discriminate between affected patients and controls of
various ages. In 2013 a study employed tandem mass spectrometry (MS/MS) assays
to screen for Pompe, Fabry, Gaucher, Krabbe, and Niemann-Pick A/B diseases, as
well as MPS I, in a total of 218 infants, children, and adults, from which 205 were
control patients and 13 affected patients [
60
]. A distinction between the enzyme
activity in non-affected infants, children, and adults of any age and the enzyme
activity in patients suffering from Pompe, Fabry, Gaucher, and Krabbe diseases
could be observed. Also, as compared to enzyme activities in newborns, in indi-
viduals older than 18 years those were significantly lower (about 50 %), the GLA
