Potential of Orbitrap mass spectrometry for application in ... · disorders like maple syrup urine...
Transcript of Potential of Orbitrap mass spectrometry for application in ... · disorders like maple syrup urine...
MSc Chemistry
Analytical Sciences
Literature Thesis
Potential of Orbitrap mass spectrometry for
application in the newborn screening program
by
Bram Grob
July 2015
Supervisor:
dr. H. Lingeman
Clinical Chemistry - Metabolic Unit
VU University Medical Center
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Table of content
Abbreviations ........................................................................................................ 3
Abstract ............................................................................................................... 4
Introduction .......................................................................................................... 6
1. Newborn screening – history and introduction TQMS ........................................... 6
2. Criteria for inclusion of a disease into the NBS program ....................................... 7
3. Sampling, Results and second tier testing .......................................................... 9
4. Cost-effectiveness ..........................................................................................10
5. Some disadvantages of NBS ............................................................................11
6. Research aim ................................................................................................11
Metabolomics, Orbitrap MS and applications ............................................................13
1. Potential new techniques for NBS .....................................................................13
2. Orbitrap MS – theory and description of operation ..............................................14
3. Orbitrap MS - characteristics of performance .....................................................18
4. Orbitrap MS - applications ...............................................................................21
Discussion ...........................................................................................................26
Conclusions .........................................................................................................30
Acknowledgement ................................................................................................31
References ..........................................................................................................32
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Abbreviations
APCI Atmospheric pressure chemical ionization
CSF Cerebrospinal fluid
CID Collision induced dissociation
C8 Octanoylcarnitine
C10 Decanoylcarnitine
DBS Dried blood spots
DIMS Direct infusion mass spectrometry
ESI Electrospray ionization
FT-ICR Fourier transform ion cyclotron resonance
FWHM Full width at half maximum
HCD Higher energy collisional dissociation
1H-NMR 1H-nuclear magnetic resonance
HR-MS High-resolution mass spectrometry
LC Liquid chromatography
MCADD Medium-chain acyl-Coenzyme A dehydrogenase deficiency
MRM Multiple reaction monitoring
MS Mass spectrometry
M/z Mass-to-charge
NBS Newborn screening
PKU Phenylketonuria
RF Radio frequency
TQMS Tandem quadrupole mass spectrometry
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Abstract
The newborn screening (NBS) is a population examination program, testing neonates in
the first weeks after birth on a selection of, mostly treatable, inherited disorders which
are asymptomatic at this early stage. Direct treatment following early diagnosis prevents
the development of severe symptoms, such as irreversible mental or motor retardation.
The current targeted approach of the tandem quadrupole mass spectrometry- (TQMS)
based method limits the number of simultaneously measurable primary and secondary
targets. Furthermore, the measurement of metabolite masses at unit resolution without a
liquid chromatography (LC) separation might lead to false elevation of signals by equal
mass compounds. The aim of this literature study was to examine whether application of
Orbitrap mass spectrometry (MS) could lead to improved analytical performance of the
current NBS by reducing the numbers of false positives and negatives, and the
simultaneous measurement of confirmative secondary targets.
Orbitrap MS is a relatively new technology, and able to perform untargeted
measurements at high resolution and (mass) accuracy, which might solve the drawbacks
of TQMS. For two NBS included diseases, it was shown that the Orbitrap MS and the
TQMS approach showed similar capacities in primary biomarker based discrimination
between the positive and negative group. Possible secondary biomarkers were discovered
by the untargeted method applied with Orbitrap MS improving the differentiation
between positives and negatives, which appears to be a major benefit of this technology.
Furthermore, both techniques showed comparable characteristics in terms of precision,
accuracy, specificity and the lower limit of quantification for drug analysis. There are
some considerations however, this highly sensitive technique can also be influenced by
matrix induced ion suppression during direct infusion, moreover, measurements at high
resolution might increase the scan cycle times.
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The Orbitrap MS appears to perform comparable to TQMS in terms of discrimination
between true positives and negatives by primary target screening. However, the ability of
the simultaneous measurement of secondary targets by Orbitrap MS might lead to
improved analytical performances of the current TQMS-based NBS.
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Introduction
1. Newborn screening – history and introduction TQMS
Newborn screening (NBS) is a population test performed in the first weeks after the birth,
examining the existence of inherited disorders which are asymptomatic under normal
conditions in the neonatal period. Most of the examined disorders are treatable, if
diagnosed quickly after the birth. Early diagnosis improves the patients quality of life,
moreover, it could reduce costs compared to clinical symptom based diagnoses. In the
beginning of the 1960s, NBS was initiated by testing neonates for phenylketonuria (PKU).
PKU is an inborn error of metabolism, discovered in 1934, with an incidence of more than
1 in 13,000 births in the USA.[1] Due to an impaired intra cellular enzyme phenylalanine
hydroxylase, phenylalanine is accumulated at the cellular level and subsequently
elevated expressed in blood. In patients suffering from PKU, phenylalanine levels in blood
of more than 20-fold higher than normal controls can be observed.[1] Exposure to high
levels of phenylalanine causes neurotoxic effects, resulting in mental retardation of which
the development is initiated after the birth. This process can be overcome if patients are
treated with a phenylalanine-free diet from this early stage.[1] Therefore, a method
using dried blood spots (DBS) was setup to determine blood phenylalanine levels, based
on its bacterial growth inhibition.[2] Population-based NBS was successfully initiated in
the USA with this assay, and screening on PKU is nowadays included in screening
programs of all developed countries. In the following decades, tests for other treatable
disorders like maple syrup urine disease, galactosemia, homocystinuria, congenital
hypothyroidism, congenital toxoplasmosis, hemoglobinopathies, congenital adrenal
hyperplasia, biotinidase deficiency, medium-chain acyl-Coenzyme A dehydrogenase
deficiency (MCADD) and cystic fibrosis were added to the NBS program.[3]
Until the 1990s the NBS program was based on several tests for the diagnosis of the
included diseases. Tandem quadrupole mass spectrometry (TQMS) was already in use in
clinical laboratories at this time for mass based measurement of drugs and metabolites.
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Therefore, TQMS was proposed for application in the NBS program since for many of the
screened diseases metabolite quantities were measured and this technique allows for the
measurement of different analytes simultaneously.[4] Prior to a TQMS analysis, it is
required to ionize the analytes, which made TQMS still a rather laborious technique as
cleaning of the first ion source types was needed after every sample. The invention of
electrospray ionization (ESI) allowed for the injection of a liquid phase, containing the
sample metabolites, directly in the TQMS without the need of cleaning the source. ESI-
TQMS was suitable to perform rapidly (2-3 minutes for 1 sample) highly sensitive and
selective analyses, with low reagent costs.[5] The introduction of this technique in NBS
was a major step towards in the change from several tests, to determine many diseases,
to ‘one test’.[6],[7] Even though separately, many of the screened disorders have a low
incidence, as a group the total amount of affected newborns is considerable, for instance
around 1 on 800 births in the West Midlands, UK.[8] The ability of determining several
diseases in one run by TQMS was a reason for inclusion of additional disorders in
screening programs, the so called expanded NBS. In 2010, nearly all developed countries
perform expanded NBS, including up to ~40 TQMS detectable inherited metabolic
diseases, depending on the particular screening program.[9]
2. Criteria for inclusion of a disease into the NBS program
In 1968, 5 years after the start of NBS in the USA, the World Health Organization
published a report written by Wilson and Jungner with principal criteria for screening.[10]
These criteria, known as the principal Wilson and Jungner criteria, are widely accepted as
general guidelines in deciding whether a disease should be included in a population
screening program. The first criteria describes that the progression of a disease should
be known without diagnosis prior to symptom exposure, which is derived from the cost
effectiveness of a screening program. For instance diseases which expose symptoms
prior to irreversible damaging of organs are often not considered to be cost-effective to
be included in a screening program. Furthermore, the second criterion described for the
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admittance to a screening program is the requirement of proven therapy for a disease,
preventing irreversible damage. Since a screening program such as NBS delivers a large
amount of samples, of which the major part has a negative outcome, the third criteria is
the availability of a simple and inexpensive test. This test should be highly sensitive and
specific. The high sensitivity is required to obtain a minimal amount of false negatives,
the high specificity on the other hand, is requested to reduce the amount of false
positives. The fourth criterion is the possibility to confirm or disprove measured positives
after screening with a second tier test. Furthermore, medical care should be available for
managing treatment and possibilities to perform tests for families when a genetic disease
is observed. The last criterion described by Wilson and Jungner is of a different category,
mentioning the need of acceptance of the screening program by the subjected
population. This criterion is vital for including diseases in the NBS program, as negative
acceptance could lead to reduced participation of the society, subsequently making NBS
less effective.
Even though the Wilson and Jungner criteria are still used as the classical criteria, some
countries started with inclusion of untreatable disorders after the start of expanded NBS.
The Health Council of the Netherlands published a report in 2015 with revised
recommendations for the minister of health, welfare and sports.[11] In this report
different categories are described to verify whether a disease should be included in the
NBS program. In these categories a difference is made between disorders with a
substantial improved health perspective for the patient if detected by NBS, and disorders
with less impact on health or less effective proven interventions. Diseases of this latter
category could still be added to the program after an assessment of pros, cons and the
efficiency of NBS determination of these diseases compared to usual diagnosis. In the
Netherlands, untreatable disorders are excluded from the NBS program. For these
disorders NBS could deliver positive effects such as shortening of the diagnostic route,
decreasing the possibility of wrong diagnosis or treatment and adapting of the patient
and family lives to the particular disease. However, the Dutch Health Council has the
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opinion that a minor part of the pros of screening untreatable diseases concerns the
affected patient. Table 1 shows the current TQMS screened biomarkers for the diseases
included in the NBS program of the Netherlands.[12]
Table 1: TQMS screened diseases and biomarkers of the Dutch NBS program, including
abbreviations corresponding to the disease. [12] Def.: deficiency
Disease Abbreviation Biomarker
Carnitine transporter (OCTN2) def. OCTN2 Free carnitine (C0)
Glutaric aciduria type I GA-I Glutarylcarnitine (C5DC)
Isovaleric academia IVA Isovalerylcarnitine (C5)
C2/C5 ratio (C2: acetylcarnitine)
Long-chain hydroxyacylCoA
dehydrogenase deficiency
LCHADD C16-hydroxyacylcarnitine
(C16OH)
Maple syrup urine disease MSUD Leucine, Valine
Medium-chain acylCoA
dehydrogenase deficiency
MCADD Octanoylcarnitine (C8)
C8/C10 ratio (C10: decanoylcarnitine)
3-methylcrotonyl-CoA-
carboxylase1 def.
3-hydroxy-3-methylglutaryl2-
CoA lyase def.
Multiple CoA carboxylase def.3
1 3-MCC
2 HMG
3 MCD
3-hydroxyisovalerylcarnitine (C5OH)
Phenylketonuria PKU Phenylalanine (PHE)
PHE/tyrosine ratio
Tyrosinemia type I TYR-I Succinylacetone
Very long chain acylCoA
dehydrogenase deficiency
VLCADD Tetradecenoylcarnitine (C14:1)
C14:1/C2 ratio
3. Sampling, Results and second tier testing
In the Netherlands, collection of blood samples on filter paper for the NBS program is
performed between 72 and 168 hours after the birth. All the spots on the card should be
completely filled with blood collected from a heel prick. During collection the nurse should
wear gloves and collect the blood without touching the skin. This to prevent
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contamination of the blood samples with for instance a lotion, which could affect the
screenings results. After collection, the bloodspots should naturally dry before sending
the card to the screening laboratory.[12]
For every screened disease, cut-off values are determined to discriminate between
positives and negatives. In general, cut-off values are set to obtain a minimal number of
false negatives.[7] Positive screens are usually repeated, and if this result is confirmed, a
second tier test is performed in the same bloodspot, if such a test is available for this
disorder. If the additional test also shows a positive result, a more sensitive and specific
conformational test is required in a new sample to finally approve or disprove the positive
screened disorder. A conformational test is often more laborious, however, the false
positive rate is minimal or zero. Disproval of a positive screen is a false positive of the
screened disorder. Although the primary objective of cut-off values is to obtain a minimal
number of false negatives, a minimal number of false positives is also preferred.[13]
4. Cost-effectiveness
One of the most important objectives of NBS is the rapid diagnosis of diseases with
clinically severe perspectives, the subsequent increased quality of life of the affected
patients and the reduced parental stress. However, for population based screening
programs, such as the NBS, the cost-effectiveness is another important aspect. Several
parts of the process are generating costs, like for instance the required personnel,
analytical and medical equipment, reagents, second tier tests, and treatment and
medical follow-up of true positive screened patients. On the other hand, diagnosing
patients suffering from metabolic diseases by their clinical symptoms, without NBS, can
also be expensive. For instance diagnostic routes can be long and the chance of
irreversibly damaged organs leading to a lifelong healthcare-dependency is present. In a
study of Venditti et al. a cost-effectiveness study was performed on NBS for MCADD
indicating that costs would remain underneath a certain accepted range.[14]
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5. Some disadvantages of NBS
Even though (expanded) NBS programs in different countries yields considerable
advantages such as the prevention of many newborns from the effects of a selection
inherited disorders by diagnosis at early stage, negative aspects have also been
observed. One of these disadvantages is the time schedule of screening and the
turnaround time for patients with diseases which can be fatal in the first days of life. For
instance: a risk of sudden death is present in the first 48 hours after the birth of
newborns with carnitine-acylcarnitine translocase deficiency.[15] Another drawback was
reported of NBS programs including diseases without severe, or not completely
disentangled clinical perspectives. Bleicher et al. described that screening on such
diseases can be less beneficial for the screened patient compared to the advantages of
NBS.[16] Furthermore, it is possible to obtain false negatives if the acquired results are
not clearly representative for the disease. This occurred for instance with neonates
suffering from tyrosinemia type I.[17] Contradictory, as described in paragraph 3, false
positives might be encountered with NBS in addition to true diagnoses. False positive
screens cause additional work for a metabolic laboratory due to the subsequent
confirmative tests, moreover, it unnecessary disrupts family lives and cause parental
stress. Nevertheless, the intensity of parental stress is showing a decrease for diseases
picked up by NBS compared with (later) clinical diagnosis.[13]
6. Research aim
Current TQMS-based NBS programs are designed to obtain a minimal number of false
negatives.[7] A major drawback of this approach is the considerable number of false
positives, causing unnecessary parental stress[13], additional workload and healthcare
costs. At present, TQMS measurements are obtained in unit resolution, which might lead
to elevated signals by interfering compounds of a slightly different mass. Furthermore,
high sensitivity and selectivity is obtained via a targeted approach during TQMS.
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Recently, other analytical techniques have been applied during metabolic studies, such as
1H-nuclear magnetic resonance (1H-NMR) spectroscopy and high-resolution mass
spectrometry (HR-MS).[18], [19] The Orbitrap mass spectrometer, a relatively new
technique to perform HR-MS, is based on a different mass spectrometric mechanism
compared to TQMS, which results in altered analytical characteristics.[20] Orbitrap MS
allows for the performance of untargeted high accurate mass scans, and therefore may
be a technique of interest for NBS.
The aim of this literature study is to investigate whether Orbitrap MS could improve the
analytical performance of TQMS-based NBS in terms of reducing false positive and
negative numbers by enhanced resolution and simultaneous determination of secondary
targets.
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Metabolomics, Orbitrap MS and applications
1. Potential new techniques for NBS
In the last decennia several new omics-termed scientific fields appeared, one of these
research topics is metabolomics. In contrast to metabolic investigation in which targeted
metabolites are measured, metabolomics typically studies the entire representation of
metabolites in an organism by identification and quantification. The obtained results are
useful to examine fluxes of normal and disordered metabolism.[21] Due to next
generation sequencing techniques, the amount of known metabolic disorders and less
severe phenotype presentations is quickly increasing. By a metabolomics approach, it
may be possible to verify the corresponding differences in metabolism. In the last years,
1H-NMR spectroscopy, and the relatively new HR-MS technique ion trap mass
spectrometry, has been applied to metabolomics studies, showing the analytical potency
of these techniques for this field.[19], [22], [23]
1H-NMR is an analytical technique which can measure a major part of proton-containing
molecules, covering almost all metabolites. Measurements for metabolic investigation can
be performed in all body fluids of interest, e.g. plasma, urine and cerebrospinal fluid
(CSF).[22] This technique requires minimal sample preparation, it is non-destructive, and
is applicable to metabolite identification. Furthermore, it is possible to acquire unbiased
quantitative measurements, reaching typical limits of detection at the lower micro molar
level.[24] This relatively high detection limit is a drawback of 1H-NMR since metabolites
at lower concentrations are excluded from the analysis, although these could be of
interest. On the contrary, HR-MS applications are more sensitive, reaching limits of
detection at the femtomole level.[25] HR-MS is compatible with all body fluids described
for 1H-NMR, in general after some sample preparation steps. In general, mass
spectrometry (MS) is destructive for the sample, however, usually a minimal sample
volume is required. TQMS can be applied for quantification of the metabolites and
determination of their chemical structures.[26] Nevertheless, TQMS scans are obtained in
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unit resolution, therefore HR-MS such as ion trap technology is required if a more
accurate approach of the exact mass is needed.[27] The mass range of metabolites is
mainly <1000 Dalton, masses measured at unit resolution in this relatively low mass
range are often corresponding with several compounds. Therefore, the application of unit
resolution masses might lead to poor identification of a metabolite. Subsequently, it can
influence the quantification of a certain metabolite if simultaneously several compounds
with the same unit-mass are detected. However, by the application of a liquid
chromatography (LC) separation and unique analyte specific mass transitions this
influence is reduced, allowing for the performance of selective and sensitive TQMS
measurements. Since highly accurate masses are obtained by the application of HR-MS
such as ion trap MS, the corresponding number of chemical structures is considerable
reduced.[28] Due to this mass accuracy, it is possible to perform highly selective and
sensitive measurements, without the required preliminary separation or fragmentation
necessary with TQMS. Since ion trap MS is currently an important analytical tool for the
‘omics’ research fields, it might also show potential for other research or diagnostically
applications, like for instance the NBS.
Ion trap based MS techniques would have a major potential for application in NBS since
the required sample volume is lower, detection levels are decreased and improved high-
throughput compatibility compared to 1H-NMR. Orbitrap MS is a relative affordable ion
trap technique, therefore, this type of mass spectrometer could be a technique of interest
for NBS.
2. Orbitrap MS – theory and description of operation
In the Orbitrap mass spectrometer, several equivalent principles of longer existing
techniques are combined, such as the Kingdon trap, the Paul trap and the Fourier
transform ion cyclotron resonance mass analyzer.[20] The Orbitrap mass spectrometer is
commercially available since 2005. Nowadays, several Orbitrap variants have been
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developed. In this brief introduction for Orbitrap MS, the main elements of an Orbitrap
mass spectrometer are described according to the schematic in figure 1.
Fig. 1: Schematic of an initial experimental Orbitrap mass spectrometer. On the left side, ionization
of the analytes is performed. The ions are transported by several quadrupoles to the storage
quadrupole, from this point the ions can be introduced via transfer lenses into the Orbitrap mass
analyzer. (obtained from Hu et al. [20])
Two frequently applied, liquid chromatography compatible, ionization sources for Orbitrap
analyses are atmospheric pressure chemical ionization (APCI) and ESI.[29], [30] The
latter source is often used for metabolic investigations.[31], [32] After ionization,
transport of ions with different mass-to-charge (m/z) ratios to the transport quadrupole
is performed via a radio frequency- (RF) lens. The RF-lens and transport quadrupole
transfers ions with different m/z ratios from atmospheric pressure into high vacuum.[20],
[30] The transport quadrupole can operate as mass filter, by selecting a certain m/z
ratio, or inversely a broad m/z range could be transported.[33] In the storage
quadrupole, the ions velocity is reduced by collision with an inert gas and by the
application of electrical fields. At this stage, ions are accumulated and focused towards
the exit position, prior to the Orbitrap analysis. Subsequently, after pulsed opening of
lens 1, a strong electrical field towards the Orbitrap mass analyzer is created. Spatial
focused ion bundles are ejected in short periods (100-200 ns), after opening the lens to
the Orbitrap mass analyzer. Acceleration of the ions is performed by a deflection lens
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system, followed by injection of the ions into the Orbitrap analyzer. During this
movement ions with a higher m/z ratio reach the Orbitrap later compared with lower m/z
ratios.[20], [30] The Orbitrap analyzer consists of an inner and outer electrode with oval
shapes, as displayed in figure 2, creating an electrical field. The velocity of the entered
ion bundles, in combination with the applied electrical field, causes the initiation of a
trajectory around the inner electrode.[20] All injected ions show an equal amplitude
during this movement. However, due to the injection at a point offset from the equator of
both electrodes (z=0), an oscillation movement of the ions around this point over the z-
axis can be initiated during administering a specific electrical field. The unique frequency
of this axial oscillation is directly related with the m/z ratio of the ions. Circulating groups
of a certain m/z ratio will form a thin ring shape, perpendicular to the z-axis, in which the
ions are homogeneous spread over the collective orbit. The m/z dependent movement of
these thin ion bands around z=0, is closely associated with a swung pendulum.
Compared with radial frequency based ion trap techniques such as the Kingdon trap, the
axial frequency in Orbitrap MS is not affected by chemical characteristics of the ions.
Therefore, this frequency is applied for calculation of the m/z ratios, leading to highly
accurate mass measurements and the high-resolution feature of this technique.[34]
Recording and multiplying of these signals and frequencies is performed at the equatorial
position, at which point the outer electrode is circular opened. The ion signals, measured
in the outer electrode as a function of time, are Fourier-transformed into frequencies and
subsequently converted to m/z ratios.[20]
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Fig. 2: Overview of the Orbitrap mass analyzer. Introduction of the ions occurs orthogonal to the z-
axis at a point deviated from the center (z=0), producing an oscillating movement. Moreover, the
ions velocity under influence of electrical fields produced by the inner- and outer electrode result in
a unique orbit around the inner electrode. Finally, homogeneous spread equal m/z ratio ions
circulate in a collective orbit perpendicular to the z-axis, oscillating around z=0 at an unique m/z
dependent frequency. (obtained from Hu et al. [20])
In more recent Orbitrap mass spectrometers, additional or improved devices such as an
octopole collision cell and a C-trap are often encased, as shown in figure 3. The C-trap,
for instance, is an advanced iontrap, comparable with the described storage quadrupole.
The performance of the Orbitrap after injection with a storage quadrupole can be
restricted, since the capacity to trap high amounts of ions is limited for this device. Due
to a different construction of the C-trap, the space-charge capacity has been improved.
This leads to improved injection conditions, and subsequently, advanced Orbitrap
performances for an increased number of ions.[35] Furthermore, an octopole collision
cell, in figure 3 the higher energy collisional dissociation (HCD) cell, can be applied to
obtain improved TQMS spectra. Collision induced dissociation (CID) is basically also
possible in the C-trap device. However, several configurations can be applied differently
in the octopole compared to the C-trap. For instance the pressure and type of collision
gas is adjustable, without influencing the Orbitrap parameters. Furthermore, the octopole
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allows for the administration of a higher collision energy compared to for instance the C-
trap.[36]
Fig. 3: Schematic of a Thermo Scientific Q Exactive Plus Orbitrap mass spectrometer, compared to
preliminary Orbitrap mass spectrometers, this instrument included improved devices such as the
‘C-trap’ ion trap. Furthermore, a HCD cell is enclosed into this model. (obtained from
http://planetorbitrap.com, 2015 [37])
The Orbitrap is, compared to techniques such as TQMS, an affordable high sensitive mass
spectrometer obtaining higher mass accuracy and resolution.[35] Therefore, this is an
valuable technique for analyses of complex samples, for instance for the proteomics and
metabolomics research fields.
3. Orbitrap MS - characteristics of performance
Sensitive mass based measurements of analytes in complex samples, such as bio fluids,
demand highly sensitive and selective MS strategies.[28] During TQMS methods, this can
be achieved by measuring unique mass transitions of precursor ions to an analyte-
specific fragment after CID.[7] Although, technically TQMS allows for the performance of
untargeted mass screening without fragmentation, the highly sensitive and selective
characteristics of this technique are mainly based on the fragmentation strategy.
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In high-resolution techniques such as Fourier transform ion cyclotron resonance- (FT-
ICR) and Orbitrap MS, high sensitivity and selectivity is also reached during untargeted
analysis.[38] While FT-ICR technology is an expensive technique, Orbitrap MS has
become more affordable. The Orbitrap mass spectrometer possesses improved
characteristics such as mass accuracy and resolving power, compared to for instance
TQMS. Mass accuracy for Orbitrap MS is described in parts per million (ppm), and is
calculated by dividing the mass error (exact analyte mass minus the measured mass) by
the exact mass, multiplied by 106.[39] A mass accuracy below 5 ppm can be reached by
Orbitrap mass analyzers, and even below 1 ppm under certain calibrated conditions.[40]
Application of HR-MS, with a mass accuracy below 5 ppm, facilitates the opportunity of
simultaneously quantification and qualification during a single measurement.[31]
Furthermore, high accurate measurements reduce the amount of possible chemical
structures which can influence the quantification. Another important parameter in MS is
the (mass) resolution, which is the capacity of the instrument of acquiring separated
signals of ions with a minimal mass difference.[33] The resolution is closely related with
the data acquisition time, increasing with a longer acquisition time, and also depends on
the height of the m/z ratio. In figure 4, an example is given of an insecticide, Pirimicarb,
measurement at different resolution levels.[41] Orbitrap MS can reach a mass resolution
up to 150,000.[20] Together, the high mass accuracy and resolution characteristics can
lead to a reduction of false positive and negative altered signals during
measurements.[42]
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Fig. 4: Example of a mass scan of Pirimicarb, an insecticide, obtained at a resolution of 15,000 and
80,000. In contrast to the low resolution measurement, a matrix component is separated from the
analyte peak at high resolution. (obtained from Thermo Scientific [41])
Other parameters that show the performance of a mass spectrometer are the mass range
and dynamic range. The dynamic range of a mass spectrometer is the ratio of the
minimal and maximal detectable signal. Both the number of ions detectable by the
detector and the digitization ability of certain signals influence the dynamic range value.
Subsequently, the mass range covers the measurable m/z ratios with the mass analyzer,
for Orbitrap MS with an upper limit approximately at 4000.[33] Hu et al. described a
dynamic range of 102 – 104 during Orbitrap measurements of reserpine, a drug, obtained
with certain parameters as electrospray ionization at 150ºC, 3000V and 5 µL/min, and an
ion collection period of 70 ms in a storage quadrupole.[20] In a publication of Marakov a
detection level in de attomole range was described.[35] In figure 5, an overview is
displayed of several mass spectrometric technique investment costs versus the achieved
average resolutions. In this overview, Orbitrap MS is presented at the higher resolution
level together with Magnetic Sector MS and FT-ICR at the high-end.
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Fig. 5: Comparison of several mass spectrometric technologies, based on the investment costs
(American dollars) versus the resolution (FWHM). (obtained from http://www.chromacedemy.com,
2015 [43])
4. Orbitrap MS - applications
In the last years, Orbitrap MS has been used in several fields of interest such as
environmental studies, drug metabolism, metabolite specific applications and especially
for proteomics and metabolomics studies.[25], [31], [32], [44]–[47] In a study of Dénes
et al., a direct (chip based) infusion Orbitrap MS method was developed for amino acid
and acylcarnitine analysis in dried blood spots for the diagnosis and screening of inborn
metabolic disorders.[25] This type of DBS analysis is worldwide applied during NBS
programs by application of ESI-TQMS. The aim of this study was to investigate whether
the application of HR-MS could improve the sensitivity and selectivity to reduce the
number of false positives.
Direct infusion of 5 µL of filtered methanolic DBS extracts, containing stable isotope
labeled standards at a certain concentration, was performed with a TriVersa NanoMate
ion source. The ionization was switched from the positive to negative mode during data
acquisition. Signals were determined at a resolution of 50,000 – 100,000 full width at
half maximum (FWHM) and with a mass accuracy range of 1.0 ppm.
Measurements of DBS samples resulted in the detection of amino acids and acylcarnitines
that are relevant for NBS. Other metabolite groups like for instance organic- and fatty
acids and carbohydrates were also observed. Comparison of this new method with the
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conventional (ESI-TQMS) method was established by analyses of two well known NBS
diseases, PKU and MCADD. In general, Orbitrap MS measured concentrations
phenylalanine (marker PKU), octanoylcarnitine and decanoylcarnitine (resp. C8 and
C10:1; markers MCADD) were lower compared to ESI-TQMS, as illustrated in figure 6.
The lack of a derivatization step, which may hydrolyze proteins into amino acids, could
explain the higher phenylalanine levels obtained with ESI-TQMS (butyl esterification
required). Furthermore, metabolites with a similar molecular weight and fragmentation
properties are possibly discriminated by the Orbitrap method. Although, obtained
concentrations with the high-resolution method are possibly more accurate, the
separation between the patient and normal concentration range is comparable.[25]
Fig. 6: Comparison of box charts for markers of PKU (a, b, e, f) and MCADD (c, d, g, h) markers in
healthy and affected patients, obtained with TQMS (left) and Orbitrap MS (right). Markers:
phenylalanine (a, e), phenylalanine/tyrosine ratio (b, f), octanoylcarnitine (C8; c, g), and
decenoylcarnitine (C10:1; d, h) (obtained from Dénes et al. [25])
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An improvement of the Orbitrap- over the ESI-TQMS-method is the simultaneous
measurement of additional metabolites showing abnormal levels of true patients. For
instance, patients suffering from PKU also show elevated levels of phenylpyruvic acid and
phenyllactic acid, even the separation between the healthy and disordered group seems
to be larger with these metabolites in comparison to phenylalanine. Orbitrap MS could
also be beneficial for the diagnosis of MCADD since the corresponding confirmatory test is
the measurement of urinary organic acids.[25] In contrast with ESI-TQMS, these
metabolites are also detectable in DBS samples with negative mode Orbitrap MS, shown
in figure 7 for octenedioic acid and hexanoylglycine measured in the MCADD and healthy
group.
Fig. 7: Box charts for proposed (secondary) markers for PKU (a, b) and MCADD (c, d), obtained
with Orbitrap MS in healthy and affected patients. Markers: phenylpyruvic acid (a), phenyllactic
acid (b), octenedioic acid (c), and hexanoylglycine (d) (obtained from Dénes et al. [25])
In a study of Henry et al., LC-coupled to Orbitrap MS was compared to TQMS for the
quantitative analysis of drugs in plasma samples.[38] The examined drugs were divided
in 3 groups, requiring different sample preparation and separation procedures. Detection
with TQMS was performed in the SRM mode. Orbitrap MS was performed in a ‘high-
24
resolution full scan’ and ‘all-ion fragmentation’ mode with a m/z range of 100 to 2000
and a mass window of 5 ppm and a resolution of 50,000 FWHM (m/z 200). For both
techniques, ESI was applied in the positive ionization mode. Obtained analytical
characteristics such as precision, accuracy, specificity, equations of lower limit of
quantification calibration curves were relatively similar for both MS approaches. An
advantage of Orbitrap MS was the possibility to perform simultaneous quantitative, semi-
quantitative and qualitative measurements. Furthermore, the author described Orbitrap
MS as an attractive instrument for quantitative measurements in clinical laboratories,
since it is exchangeable with TQMS without required alteration of sample preparation or
chromatographic method.[38]
Besides LC coupled to Orbitrap MS, also direct infusion mass spectrometry (DIMS)
methods have been applied with this MS technique. Although DIMS is fast and relatively
easy due to the lack of a separation method, also some disadvantages have been
published.[31] Since no separation is applied, one of the drawbacks of such approaches
is ion suppression induced by matrix components, reducing the sensitivity and precision
of a method. The effect of matrix on the analyte signal was for instance examined during
a study of Madalinski et al. Measurements of a selection 14N-labeled metabolites spiked in
yeast cell extracts were performed at different cell concentrations.[31] In table 2 the
percentages are shown relative to metabolite signals measured in solvent
(water/methanol, 50/50, containing 0.1% formic acid). These results show that sample
matrix can substantial affect the intensity of the analyte signals, however the exact origin
of suppression was not investigated in this study. Furthermore, non separated samples
may cause formation of product, isotopic and adduct ions during the ionization-
evaporation process. For instance in the study of Madalinski et al. it was shown that
differentiating between γ-glutamyl-cysteine and cysteine in extracts of yeast cells is not
possible with DIMS, since fragmentation of the former into cysteine occurs during the
25
ionization process. Although, fragmentation in the source can also take place in LC-MS
methods, separation of these metabolites allows for the selective detection of cysteine.
Table 2: Results of a matrix effect determination for several metabolites at increasing
concentrations of yeast extracts. (obtained from Madalinski et al. [31])
In order to reach high resolution (100,000) during Orbitrap MS, a large scan period of 1 s
is required.[28] This scan time is relatively high for coupling a separation method,
therefore applied resolutions during LC-Orbitrap MS are frequently around 30,000 in
these methods.[48]
Even though TQMS allows for sensitive and selective measurements of many compounds
simultaneous[49], this method is only focused on targets of its multiple reaction
monitoring (MRM) program. Analytes which are not included, are not visible in this mode,
and addition of other interesting compounds for quantitative objectives can be time-
consuming due to method development or required validation. Moreover, additional mass
transitions cause a reduction in sensitivity of the method since cumulative dwell times for
individual compounds will decrease. An increased number of analytes in the MRM method
can also lead to high scan cycle times, and subsequent, a reduced number of measuring
points during acquiring data. Application of Orbitrap MS might therefore be an
improvement for quantitative methods addressing to the measurement of additional
analytes.[28]
26
Discussion
During NBS neonates are tested for a selection of, mostly treatable, disorders, which
display no symptoms in this period of live. Determination and treatment of these
diseases at early stage prevents the development of severe symptoms, and thereby
increases the patients quality of life.[1] Current TQMS-based NBS causes a relatively
high number of false positives, causing i.a. unnecessary parental stress. Additional
second tier tests are required to confirm or disprove a positive screened result.[7] For
some diseases a primary biomarker-based false positive result can be disproved by the
measurement of secondary biomarker concentrations. The analytical performance of
TQMS to measure many different types of primary and secondary biomarkers
simultaneous is limited, since high sensitivity and selectivity is achieved via a targeted
approach. HR-MS, such as the relatively new Orbitrap technology, can perform
untargeted high accurate (mass) measurements[20], allowing for the determination of
many primary and secondary biomarkers simultaneous. Furthermore, the high mass
accuracy obtained with this technique might lead to increased selectivity towards
biomarker measurements, compared to the TQMS-based method.
Orbitrap MS can detect accurate masses up to 5 digits behind the comma instead of
masses at unit resolution obtained with TQMS. In biological material, such as blood, urine
and CSF, hundreds of different metabolites are present, in which some metabolites have
a minimal mass difference. Consequently, if these metabolites are measured in unit
resolution following direct infusion, the examined metabolite signal might be falsely
elevated due to the simultaneous measurement of metabolites with nearly equal
masses.[25] During TQMS-based NBS, an incorrect elevated signal can lead to the
determination of false positives or negatives. Therefore, the research question of this
study was to theoretically investigate whether Orbitrap technology could improve the
current TQMS-based NBS. Based on the currently applied MRM-mode TQMS method
during NBS, false elevation of a signal should be only possible with equal mass
compounds showing a similar mass transition after CID.
27
As shown in the previous chapter, Dénes et al. obtained a reduction of concentrations
with Orbitrap MS compared to TQMS results, for PKU and MCADD biomarkers in DBS of
healthy and affected patients.[25] Separation between these groups was approximately
similar to the TQMS results. However, the measurement of secondary biomarkers showed
an improved separation between the healthy and patient group. In order to maintain the
sensitivity and low cycle times, currently targeted TQMS in the MRM-mode is restricted to
a certain number of mass transitions. The untargeted approach of the Orbitrap technique
is therefore a major improvement and allows the simultaneous measurement of primary
and secondary biomarkers. Furthermore, in contrast to TQMS, metabolites like
acylglycines, fatty- and dicarboxylic acids can be measured in DBS with Orbitrap MS.[25]
At present, the measurement of these metabolites is performed in urine by a gas
chromatography coupled to MS analysis. The difference in results obtained with TQMS
and Orbitrap MS during this study might not be exclusively caused by switching to
another MS technique. Additional parameters were altered, omitting of derivatization for
the Orbitrap analysis and the measurement of intact ionized molecules instead of mass
transitions after CID. Therefore additional studies, determining the influence of CID and
derivatization on Orbitrap measurements could clarify the currently reduced
concentrations compared to TQMS and the similar separation between the healthy and
patient group. Even though, results obtained by Orbitrap analysis of PKU and MCADD
biomarkers show similar discrimination between the positive and negative group
compared to TQMS, inclusion of other NBS disorders in a future study might show
improved differentiation of these groups.
Another advantage of Orbitrap MS, compared to the TQMS based NBS method, is the
possibility to perform high selective measurements without the need of fragmentation of
the analyte, as shown in the study of Dénes et al.[25] During the current TQMS method,
a derivatization step is included to obtain improved fragmentation properties for amino
acids and acylcarnitines. A reduction in sample preparation steps, by the absence of
derivatization, might lead to an improved representation of the real sample composition.
28
In the study of Henry et al., comparing Orbitrap MS with a TQMS application, similar
results were shown in terms of accuracy, precision, sensitivity and linearity.[38] This
study also describe the major advantage of Orbitrap MS of measuring additional analytes
simultaneous to the targets during full scan measurements. For some diseases, the ratio
between a primary and secondary target, e.g. octanoylcarnitine (C8) and
decanoylcarnitine (C10) for MCADD, is an additional evidence for diagnosis. The
measurement of biomarkers in DBS compared to e.g. plasma samples can be less reliable
due to for instance stability issues or incorrect filling of the spots, which might influence a
primary biomarker value. Obtained ratios are sometimes less affected compared to the
primary biomarker value. Therefore for certain diseases, obtaining of ratios by secondary
target measurement could possibly improve the diagnostic accuracy of the current
method, especially if primary target concentrations are affected.
Obtaining additional analytes by TQMS applied in the MRM mode is achieved by extra
mass transitions, affecting the methods analytical performances for instance by a
decreased number of data points if the cycle time increases. In contrast, Orbitrap MS in
full scan mode requires no alteration of the MS method. Although, Orbitrap MS can
perform high-resolution measurements, this also leads to elevated cycle times. In the
study of Madalinski et al., mass spectra of direct infused biological samples were
obtained at a resolution of 100,000 at m/z 400.[31] Acquired mass spectra were the
averages of 4 scans. In order to obtain an increased number of data points, measuring at
a lower resolution (50,000 – 100,000) as performed for DBS analysis in the study of
Dénes et al. might be advisable for a future application in NBS.[25]
Since the possibilities of injecting liquid phases into an Orbitrap mass spectrometer are
comparable to TQMS, this technique could relatively replace the current NBS.
Furthermore, in contrast to for instance FT-ICR HRMS, Orbitrap mass spectrometers are
becoming increasingly affordable.[43] The opportunity to obtain secondary targets, and
the subsequent reduction of required second tier tests, might lead to a reduction of costs
additional to the basic screening method. This in combination with the advantage of
29
measuring additional compounds in the full scan mode without concessions addressing
the analytical performance might show the added value of a future application of Orbitrap
MS during NBS, replacing TQMS.
Prior to a potential future implementation of Orbitrap MS, replacing current TQMS,
additional studies would be required. All screened disorders should be examined,
addressing the determination of positive and negative cut-off values of primary and
secondary targets, and the correlation with the presently applied methods. The
diagnostically value of new secondary biomarkers, like phenylpyruvic acid and
phenyllactic acid for PKU, need to be validated prior to implementation. Furthermore,
several parameters should be determined according to a validation procedure for new
analytical methods, such as the linearity, sensitivity, selectivity, accuracy, precision,
repeatability and reproducibility.
Since Orbitrap MS also can suffer from matrix induced ionization suppression, which
affect the analytical performance, it might be valuable to study the influence of this
effect. During studying the ion suppression, the performance of other ionization interface
types could be examined, such as the nanospray ionization applied during the study of
Dénes et al.[25]
30
Conclusions
The application of an untargeted Orbitrap HR-MS approach for NBS in DBS could improve
current TQMS based method by the simultaneously measurement of secondary
targets.[25], [38] This could lead to a reduced amount of required second tier tests to
confirm or disprove abnormal values. In terms of primary target-based separation
between the true positive and negative groups, both techniques currently seem to
perform comparable.[25]
Although, the simultaneous measurement of secondary targets by Orbitrap MS is a major
advantage compared to TQMS, additional studies are required to determine cut-off values
for primary and secondary biomarkers of NBS included diseases. Furthermore, studies
would be required to examine ion suppression effects and the analytical performance of
the new method.
31
Acknowledgement
This literature thesis is the final of my master study, and therefore the right moment to
thank some people who have helped me throughout this period.
I would like to express my gratitude for all the help I received during my literature study
from dr. Desirée Smith. I really appreciate all the effort you made in guiding me
throughout this study. Your feedback always helped me a lot to get more progression in
writing my thesis. Next, I like to thank dr. Henk Lingeman for his supervision and the
nice meetings during question and answer sessions.
I have followed this master study next to my daily work, therefore I want to thank prof.
dr. ir. Cornelis Jakobs, prof. dr. Henk Blom and prof. dr. Gajja Salomons for giving me
this opportunity. Furthermore, following this master study would not have been possible
without the help of all colleagues from the VUmc metabolic laboratory. Thank you all for
your flexibility, support and social talks during this time!
32
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