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

2

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

3

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

4

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.

5

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.

6

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.

7

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

8

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

9

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

10

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.

12

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.

13

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

14

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

15

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

16

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]

17

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

18

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.

19

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]

20

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.

21

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

22

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])

23

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

References

[1] S. A. Berry, C. Brown, M. Grant, C. L. Greene, E. Jurecki, J. Koch, K. Moseley, R.

Suter, S. C. van Calcar, J. Wiles, and S. Cederbaum, “Newborn screening 50 years

later: access issues faced by adults with PKU.,” Genet. Med., vol. 15, no. 8, pp. 591–9, 2013.

[2] R. Guthrie and A. Susi, “A simple phenylalanine method for detecting

phenylketonuria in large populations of newborn infants,” Pediatrics, vol. 32, pp. 338–343, 1963.

[3] A. M. Comeau, C. Larson, and R. B. Eaton, “Integration of new genetic diseases

into statewide newborn screening: New England experience.,” Am. J. Med. Genet. C. Semin. Med. Genet., vol. 125C, no. 1, pp. 35–41, 2004.

[4] D. S. Millington, N. Kodo, D. L. Norwood, and C. R. Roe, “Tandem Mass-

Spectrometry: A New Method for Acylcarnitine Profiling With Potential for Neonatal

Screening for Inborn-Errors of Metabolism,” J. Inherit. Metab. Dis., vol. 13, no. 3,

pp. 321–324, 1990.

[5] D. H. Chace, T. a. Kalas, and E. W. Naylor, “Use of Tandem Mass Spectrometry for

Multianalyte Screening of Dried Blood Specimens from Newborns,” Clin. Chem., vol. 49, no. 11, pp. 1797–1817, 2003.

[6] M. S. Rashed, P. T. Ozand, M. P. Bucknall, and D. Little, “Diagnosis of inborn errors

of metabolism from blood spots by acylcarnitines and amino acids profiling using

automated electrospray tandem mass spectrometry.,” Pediatr. Res., vol. 38, no. 3, pp. 324–331, 1995.

[7] F. Rousseau, Y. Giguère, M. T. Berthier, D. Guérette, J. Girard, and M. Déry,

“Newborn Screening by Tandem Mass Spectrometry: Impacts, Implications and

Perspectives,” in Tandem Mass Spectrometry- Applications and Principles, J. Prasain, Ed. 2012, pp. 751–776.

[8] S. Sanderson, a Green, M. a Preece, and H. Burton, “The incidence of inherited

metabolic disorders in the West Midlands, UK.,” Arch. Dis. Child., vol. 91, no. 11, pp. 896–899, 2006.

[9] D. C. Lehotay, P. Hall, J. Lepage, J. C. Eichhorst, M. L. Etter, and C. R. Greenberg,

“LC-MS/MS progress in newborn screening,” Clin. Biochem., vol. 44, no. 1, pp. 21–31, 2011.

[10] J. M. Wilson and G. Jungner, “Principles and practice of mass screening for disease,” Bol. Oficina Sanit. Panam., vol. 65, no. 4, pp. 281–393, 1968.

[11] D. D. M. Braat, “Advies rapport neonatale screening: nieuwe aanbevelingen (Gezondheidsraad),” Den Haag, 2015.

[12] RIVM, Draaiboek neonatale hielprikscreening, 11.0 ed. 2005.

[13] S. E. Waisbren, S. Albers, S. Amato, M. Ampola, T. G. Brewster, L. Demmer, R. B.

Eaton, R. Greenstein, M. Korson, C. Larson, D. Marsden, M. Msall, E. W. Naylor, S.

Pueschel, M. Seashore, V. E. Shih, and H. L. Levy, “Effect of expanded newborn

33

screening for biochemical genetic disorders on child outcomes and parental

stress.,” JAMA, vol. 290, no. 19, pp. 2564–2572, 2003.

[14] L. N. Venditti, C. P. Venditti, G. T. Berry, P. B. Kaplan, E. M. Kaye, H. Glick, and C.

A. Stanley, “Acyl-CoA Dehydrogenase Deficiency : A Cost-Effectiveness Analysis,” Pediatrics, vol. 112, no. 5, pp. 1005–1015, 2003.

[15] R. S. Y. Lee, C. W. Lam, C. K. Lai, Y. P. Yuen, K. Y. Chan, C. C. Shek, A. Y. W.

Chan, and C. B. Chow, “Carnitine-acylcarnitine translocase deficiency in three

neonates presenting with rapid deterioration and cardiac arrest,” Hong Kong Med. J., vol. 13, no. 1, pp. 66–68, 2007.

[16] A. Bleicher, “Perils of Newborn Screening,” Sci. Am., vol. 307, no. 1, pp. 30–31, 2012.

[17] B. Wilcken, V. Wiley, J. Hammond, and K. Carpenter, “Screening newborns for

inborn errors of metabolism by tandem mass spectrometry.,” N. Engl. J. Med., vol.

348, no. 23, pp. 2304–2312, 2003.

[18] A. M. Weljie, J. Newton, P. Mercier, E. Carlson, and C. M. Slupsky, “Targeted

profiling: quantitative analysis of 1H NMR metabolomics data.,” Anal. Chem., vol. 78, no. 13, pp. 4430–4442, 2006.

[19] Y. Rao, M. McCooeye, and Z. Mester, “Mapping of sulfur metabolic pathway by LC Orbitrap mass spectrometry,” Anal. Chim. Acta, vol. 721, pp. 129–136, 2012.

[20] Q. Hu, R. J. Noll, H. Li, A. Makarov, M. Hardman, and R. G. Cooks, “The Orbitrap:

A new mass spectrometer,” J. Mass Spectrom., vol. 40, no. 4, pp. 430–443, 2005.

[21] O. Fiehn, “Metabolomics - The link between genotypes and phenotypes,” Plant Mol. Biol., vol. 48, no. 1–2, pp. 155–171, 2002.

[22] U. Engelke, L. Kluijtmans, M. Huigen, A. Goudswaard, M. vd Graaf, A. Heerschap,

E. Morava-Kozicz, S. Wortmann-Hagemann, and R. Wevers, Handbook of 1H-NMR spectroscopy in inborn errors of metabolism, 3rd ed. Nijmegen, 2014.

[23] N. I. Wolf, M. a a P. Willemsen, U. F. Engelke, M. S. van der Knaap, P. J. W.

Pouwels, I. Harting, J. Zschocke, E. a Sistermans, D. Rating, and R. a Wevers,

“Severe hypomyelination associated with increased levels of N-acetylaspartylglutamate in CSF.,” Neurology, vol. 62, no. 9, pp. 1503–1508, 2004.

[24] A. Smolinska, L. Blanchet, L. M. C. Buydens, and S. S. Wijmenga, “NMR and

pattern recognition methods in metabolomics: From data acquisition to biomarker

discovery: A review,” Anal. Chim. Acta, vol. 750, pp. 82–97, 2012.

[25] J. Dénes, E. Szabo, S. Robinette, I. Szatmari, J. G. Kreuder, E. W. Rauterberg, and

Z. Takats, “Metabonomics of newborn screening dried blood spot samples – a

novel approach in the screening and diagnostics of inborn errors of metabolism,” Anal. Chem., vol. 84, no. 22, pp. 10113–10120, 2012.

[26] C. Lam and C. Law, “Untargeted Mass Spectrometry-Based Metabolomic Profiling

of Pleural Effusions: Fatty Acids as Novel Cancer Biomarkers for Malignant Pleural Effusions,” J. Proteome Res., vol. 13, pp. 4040–4046, 2014.

34

[27] A. Kaufmann, P. Butcher, K. Maden, S. Walker, and M. Widmer, “Quantitative and

confirmative performance of liquid chromatography coupled to high-resolution

mass spectrometry compared to tandem mass spectrometry,” Rapid Commun. Mass Spectrom., vol. 25, no. 7, pp. 979–992, 2011.

[28] C. Junot, G. Madalinski, J.-C. Tabet, and E. Ezan, “Fourier transform mass

spectrometry for metabolome analysis.,” Analyst, vol. 135, no. 9, pp. 2203–2219,

2010.

[29] E. D. Virus, T. G. Sobolevsky, and G. M. Rodchenkov, “Introduction of

HPLC/orbitrap mass spectrometry as screening method for doping control,” J. Mass Spectrom., vol. 43, no. 7, pp. 949–957, 2008.

[30] M. Hardman and A. a. Makarov, “Interfacing the orbitrap mass analyzer to an electrospray ion source,” Anal. Chem., vol. 75, no. 7, pp. 1699–1705, 2003.

[31] G. Madalinski, E. Godat, S. Alves, D. Lesage, E. Genin, P. Levi, J. Labarre, J. C.

Tabet, E. Ezan, and C. Junot, “Direct introduction of biological samples into a LTQ-

orbitrap hybrid mass spectrometer as a tool for fast metabolome analysis,” Anal. Chem., vol. 80, no. 9, pp. 3291–3303, 2008.

[32] J. J. J. van der Hooft, L. Ridder, M. P. Barrett, and K. E. V. Burgess, “Enhanced

Acylcarnitine Annotation in High-Resolution Mass Spectrometry Data:

Fragmentation Analysis for the Classification and Annotation of Acylcarnitines,” Front. Bioeng. Biotechnol., vol. 3, no. March, pp. 1–15, 2015.

[33] E. de Hoffmann and V. Stroobant, Mass Spectrometry - Principles and Applications. Chichester: John Wiley & Sons, Ltd, 2007.

[34] A. Makarov, “Electrostatic axially harmonic orbital trapping: A high-performance technique of mass analysis,” Anal. Chem., vol. 72, no. 6, pp. 1156–1162, 2000.

[35] A. Makarov, E. Denisov, A. Kholomeev, W. Balschun, O. Lange, K. Strupat, and S.

Horning, “Performance Evaluation of a Hybrid Linear Ion Trap / Orbitrap Mass Spectrometer,” Anal. Chem., vol. 78, no. 7, pp. 2113–2120, 2006.

[36] J. V Olsen, B. Macek, O. Lange, A. Makarov, S. Horning, and M. Mann, “Higher-

energy C-trap dissociation for peptide modification analysis.,” Nat. Methods, vol. 4, no. 9, pp. 709–712, 2007.

[37] Thermo Scientific, “Schematic of the Q Exactive Plus,” 2015. [Online]. Available:

http://planetorbitrap.com/data/fe/image/QEPlus.png. [Accessed: 16-Jul-2015].

[38] H. Henry, H. R. Sobhi, O. Scheibner, M. Bromirski, S. B. Nimkar, and B. Rochat,

“Comparison between a high-resolution single-stage Orbitrap and a triple

quadrupole mass spectrometer for quantitative analyses of drugs,” Rapid Commun. Mass Spectrom., vol. 26, no. 5, pp. 499–509, 2012.

[39] Agilent Technologies, “Eseminar mass accuracy and mass resolution in TOF MS,”

2011. [Online]. Available:

https://www.chem.agilent.com/Library/eseminars/Public/Mass Accuracy and Mass Resolution - October 2011.pdf. [Accessed: 16-Jul-2015].

35

[40] J. C. L. Erve, W. Demaio, and R. E. Talaat, “Rapid metabolite identification with

sub parts-per-million mass accuracy from biological matrices by direct infusion

nanoelectrospray ionization after clean-up on a ZipTip and LTQ/Orbitrap mass spectrometry,” Rapid Commun. mass Spectrom., vol. 22, pp. 3015–3026, 2008.

[41] Thermo Scientific, “Figure Mass Resolution,” 2015. [Online]. Available:

http://www.thermoscientific.de/content/dam/tfs/ATG/CMD/cmd-support/exactive-

plus/brochures-and-catalogs/product-brochures/MassResolution-ResolvingPower.pdf. [Accessed: 16-Jul-2015].

[42] A. Makarov and M. Scigelova, “Coupling liquid chromatography to Orbitrap mass spectrometry,” J. Chromatogr. A, vol. 1217, no. 25, pp. 3938–3945, 2010.

[43] Chromacademy, “Figure Costs vs Resolution MS systems,” 2015. [Online].

Available: http://www.chromacademy.com/lms/sco156/Fundamental_LC-

MS_Orbitrap_Mass_Analyzers.pdf. [Accessed: 16-Jul-2015].

[44] a. C. Hogenboom, J. a. van Leerdam, and P. de Voogt, “Accurate mass screening

and identification of emerging contaminants in environmental samples by liquid

chromatography-hybrid linear ion trap Orbitrap mass spectrometry,” J.

Chromatogr. A, vol. 1216, no. 3, pp. 510–519, 2009.

[45] C. L. Austin, Z. S. Wilson, T. M. Ting, and J. Xiang-yu, “Complete profiling and

characterization of in vitro nefazodone metabolites using two different tandem

mass spectrometric platforms,” Rapid Commun. mass Spectrom., vol. 21, pp. 4001–4008, 2007.

[46] T. Nemkov, A. D’Alessandro, and K. C. Hansen, “Three-minute method for amino

acid analysis by UHPLC and high-resolution quadrupole orbitrap mass spectrometry,” Amino Acids, 2015.

[47] A. Michalski, E. Damoc, J.-P. Hauschild, O. Lange, A. Wieghaus, A. Makarov, N.

Nagaraj, J. Cox, M. Mann, and S. Horning, “Mass Spectrometry-based Proteomics

Using Q Exactive, a High-performance Benchtop Quadrupole Orbitrap Mass

Spectrometer.,” Mol. Cell. proteomics, vol. 10, no. 9, p. M111.011015, 2011.

[48] W. B. Dunn, D. Broadhurst, M. Brown, P. N. Baker, C. W. G. Redman, L. C. Kenny,

and D. B. Kell, “Metabolic profiling of serum using Ultra Performance Liquid

Chromatography and the LTQ-Orbitrap mass spectrometry system,” J.

Chromatogr. B Anal. Technol. Biomed. Life Sci., vol. 871, no. 2, pp. 288–298,

2008.

[49] W. Lu, E. Kimball, and J. D. Rabinowitz, “A high-performance liquid

chromatography-tandem mass spectrometry method for quantitation of nitrogen-

containing intracellular metabolites,” J. Am. Soc. Mass Spectrom., vol. 17, no. 1, pp. 37–50, 2006.