HP Lcms Ms Clinical Lab

Available online at www.sciencedirect.
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Clinical Biochemistry 41 (2008) 649 662
Review
A decade of HPLCMS/MS in the routine

clinical laboratory Goals for further developments
Michael Vogeser a,, Christoph Seger b,
a
Institute of Clinical Chemistry, Hospital of the University of Munich, Marchioninistrae 15, D-81377, Munich, Germany
Division of Mass Spectrometry and Chromatography, Institute of Medical and Chemical Laboratory Diagnostics (ZIMCL), University Hospital Innsbruck,
Anichstrae 35, A-6020, Innsbruck, Austria
Received 24 October 2007; received in revised form 17 February 2008; accepted 20 February 2008
Available online 14 March 2008
Abstract
During the past decade, tandem mass spectrometry hyphenated to liquid chromatography separation systems (HPLCMS/MS) has developed
to an important technology in clinical chemistry not only for research purposes but also for routine use. At present, most important application
fields are target analyses in therapeutic drug monitoring (TDM) and metabolic disorders diagnosis. The essential strengths of HPLCMS/MS
include potentially high analytical specificity, wide range of applicability to small and large molecules, capability of multi- and mega-parametric
tests, and the opportunity to develop powerful assays with a high degree of flexibility within a short time frame. The technique has overcome
important limitations of GCMS and is characterized by short analytical runtimes, applicability to thermo labile, polar and large molecules, and
straightforward sample preparation. However, implementation of HPLCMS/MS assays still requires substantial expertise and know-how. At the
present, its application is limited to a rather small number of clinical routine laboratories. Nonetheless, HPLCMS/MS has the potential to be
further developed to a commonly applied high-throughput technique in clinical chemistry, complementary to present standard techniques as
photometry and ligand binding methods. This review intends to characterize working characteristics of present day HPLCMS/MS instrumentations used in clinical routine laboratories. Limitations of currently available systems and applications will be critically discussed.
Required instrument improvements supporting the successful spreading of HPLCMS/MS in laboratory medicine within the next decade will be
outlined.
2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Keywords: High performance liquid chromatographytandem mass spectrometry (HPLCMS/MS); Routine application; Therapeutic drug monitoring; Clinical
toxicology; Metabolomics
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Characteristics of current HPLCMS/MS applications in clinical laboratories
Areas of application . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instrumentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Costs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Handling and robustness . . . . . . . . . . . . . . . . . . . . . . . . .
Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Analytical limitations . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Corresponding authors. M. Vogeser is to be contacted at fax: +49 89 7095 6220. C. Seger, fax: +43 512 504 24088.
E-mail addresses: Michael.Vogeser@med.uni-muenchen.de (M. Vogeser), christoph.seger@uki.at (C. Seger).
0009-9120/$ - see front matter 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.clinbiochem.2008.02.017
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M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662
Goals for further developments in clinical HPLCMS/MS applications

System configurations . . . . . . . . . . . . . . . . . . . . . . . .
Instrument control software . . . . . . . . . . . . . . . . . . . . .
Ion sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fluid system . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample throughput. . . . . . . . . . . . . . . . . . . . . . . . . .
Instrument costs . . . . . . . . . . . . . . . . . . . . . . . . . . .
Application specific reagents and support . . . . . . . . . . . . . .
Potential future role of HPLCMS/MS in the clinical laboratory . . . .
Clinical pharmacology and xenobiotic compounds . . . . . . . . .
Target and multi-target analyses of endogenous compounds . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction
Beginning in the 1970s, gas chromatographymass spectrometry (GCMS) instrumentation has evolved to an essential
tool for both biomedical research and some important routine
clinical chemistry applications. However, it was the routine
introduction of non-disintegrating soft ionization techniques as
atmospheric pressure ionization (API) and sophisticated ion
analysis methods in the 1990s that finally made the majority of
biologically relevant analytes (endogenous metabolites as well
as xenobiotics) amenable to highly specific mass spectrometric
analyses.
From our current perspective, the hyphenation of high
performance liquid chromatography to tandem mass spectrometry [1] via an API interface (HPLCMS/MS) is the only
liquid chromatographysoft ionizationmass spectrometry
technique which has been introduced into routine clinical
laboratories so far [2,3]. This technology seems to be of similar
novelty for clinical chemistry as immunoassays in the 1970s
and PCR in the 1980s. The use of HPLCMS/MS systems in
bio-analytical research started already in the late 1980s [4,5].
Tandem mass spectrometry found its way into clinical laboratories in the early 1990s when first flow injection analysis
(FIA)MS/MS methods were introduced for neonatal screening
assays [6,7]. A significant methodological progress was the
introduction of HPLCMS/MS systems featuring API interfaces, which were broadly acknowledged in bio-analysis in
the mid-1990s [8]. First implementations in clinical routine
laboratories started about a decade ago with realizing the first
therapeutic drug monitoring (TDM) assay [9,10]. Consequently,
this technology allowed a number of important novel assay
applications to be realized in such laboratories, improving their
overall quality. Nevertheless, at present the availability of this
powerful technology is still restricted to a rather small number
of clinical laboratories.
The aim of this article is to comprehensively characterize
the utilization of HPLCMS/MS technologies in today's clinical chemistry, to critically discuss the limitations of currently
available systems and applications, and to outline perspectives and goals of further developments of this emerging
technology.
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Characteristics of current HPLCMS/MS applications in

clinical laboratories
Essential strengths of the HPLCMS/MS technology for
laboratory medicine include:
(i) Specificity. The potentially very high analytical specificity of tandem mass spectrometry as HPLC detector
results from using the molecular mass of the analyte
and its specific disintegration behaviour as detection
principle.
(ii) Wide range of applicability with good practicability. In
contrast to GCMS as the classical mass spectrometry
technique, the application of HPLCMS/MS is not limited
to volatile molecules (usually with molecular weights
below 500 Da). Furthermore, aside from highly polar
analytes (i.e. amino acids), sample preparation is usually
simple and does not include derivatization techniques.
Mass spectrometry detected HPLC assays are generally
optimized to shorter runtimes. Hence, compared to GC
MS, far higher sample throughput can be realized.
(iii) Flexibility. New assays can typically be developed inhouse with a high degree of flexibility and within a short
time.
(iv) Information rich detection. A large number of quantitative
or qualitative results can be obtained from a single analytical HPLCMS/MS run, since due to the fast ion
selection electronics, multi-parametric, quasi parallel analyses can be performed with a mass spectrometer.
Areas of application
In many industrialized countries FIAMS/MS is now used in
the routine neonatal screening for inherited diseases of metabolism. Typically, these analyses are performed by a few specialized
laboratories per country and a specified set of metabolites is traced
in a semi-quantitative manner [11]. Besides these applications, at
least one HPLCMS/MS system is available in most university
hospital laboratories in these states (e.g., in clinical chemistry
institutes, pharmacological and forensic institutes, institutes of
occupational health, mass spectrometric core facilities). Here,
they are often used for both research and routine analytical
purposes, serving application fields like therapeutic drug
monitoring (TDM; in particular quantification of immunosuppressants and new generation anticonvulsive and antipsychotic
drugs), pharmacology, endocrinology, and toxicology. For more
detailed information, the readership is referred to the literature
[1219]. Beyond university hospital laboratory centres only few
tertiary care hospitals in Europe are equipped with a HPLCMS/
MS instrument at present. In addition, the majority of larger
laboratory trusts are nowadays equipped with HPLCMS/MS
instruments, mostly in centralized core facilities. These instruments are predominantly used for TDM. HPLCMS/MS
methods replace more and more of the numerous conventional
HPLCUV and HPLCFLD methods in such laboratories since
they offer superior specificity, shorter runtimes, and less laborious
method development and sample preparation.
651
Fig. 2. Online-SPEHPLCMS/MS system in one of our laboratories. Besides

the bench top MS machine and a HPLC block additional instruments as prevacuum rotary pumps, additional HPLC related equipment, sufficient gas supply
and well vented exhaust lines are needed to operate the HPLCMS/MS
platform.
Instrumentations
Fig. 1. Three different HPLCMS experiments of a plasma sample illustrating

the power of selected reaction monitoring (SRM) and multiple reaction
monitoring (MRM) experiments. Whereas the top trace (A) is acquired in full
scan mode covering a m/z range of 5001800 and illustrates the complexity of
the sample, the middle trace (B) is a selected ion chromatogram recorded at m/z
of the precursor ion of pep2, a IFG-1 reporter peptide. Only in the tandem MS
experiment the specificity of combining two mass transitions allows to gain the
sensitivity needed for successful quantification [41].
HPLCMS/MS instruments capable of selected reaction

monitoring (SRM, also known as multiple reaction monitoring
MRM) and related experiments (Fig. 1), are currently manufactured by six different companies: Agilent (http://www.agilent.
com), MDS Sciex (http://www.sciex.com), GSG (http://www.
gsg-analytical.com), Thermo-Fischer (http://www.thermo.com),
Varian (http://www.varianinc.com), and Waters (http://www.
waters.com). Among these, MDS Sciex, Thermo-Fisher, and
Waters have produced tandem mass spectrometer instruments for
more than 10 years, whereas Agilent and Varian have included
HPLCMS/MS systems into their portfolio substantially later.
GSG is a rather new competitor, manufacturing HPLCMS/MS
instruments technically very similar to widely used instruments
but at a lower price level. Most manufacturers offer several
tandem mass spectrometry systems with different ion sources and
technical specifications.
While the footprint of HPLCMS/MS instruments itself is
rather small (beginning with about 2 m2 for compact instruments),
the installation of these machines requires significantly more
overall space. Besides a MS/MS analyzer and a HPLC, naturally
being the core pieces of a HPLCMS/MS system, one or two prevacuum rotary pumps (typically placed below the instrument's
bench), a nitrogen generator (run with pressurized air) if no
nitrogen supply is available, an uninterrupted power supply
(UPS), and for some older instruments a water cooling system
serving as heat exchanger for the vacuum pump system (Fig. 2)
have to be situated, installed, and maintained.
Due to the technical complexity of HPLCMS/MS systems,
particular know-how for the adaptation of laboratory environment is needed: Mandatory site requirements communicated
by the vendors in detail include an efficient air-conditioning
system and a well designed ventilation system, since waste heat
(typically several kWh 1) and exhaust air (containing toxic
organic solvent residuals from the MS and oil mist from the
652
rotary pumps) produced by the system has to be dissipated and

vented, respectively. Accidental blockage of the waste air flow
(e.g. by fire protecting systems) or a failing air-conditioning
system can cause system down-times due to overheating or even
may cause damage to the instruments. Furthermore, the mass
spectrometer has to be protected from substantial vibrations.
Pressurized oil free (zero) air or very clean nitrogen gas with a
continuous pressure usually exceeding conventional installation
limits (e.g. 5 bar for pressurized air) has to be delivered at high
flow rates. If no nitrogen gas supply system (e.g. from liquid
nitrogen tanks) is available, pressurized nitrogen can be produced
from ambient air by nitrogen gas generators. These are operating
with pressurized air, which might require to run compressor
systems at the laboratory site. Care must be taken, that such
devices do not produce any impurities negatively impacting the
mass spectrometer performance. Usually, a specifically approved
generator type is recommended by the producer of the mass
spectrometer. Whereas a nitrogen generator is a silent device, the
noise pollution of a compressor system prohibits its installation
within the laboratory; a separate sound insulated room has to be
provided. HPLCMS/MS instrument noise (mainly arising from
the vacuum pumps) typically makes the room not longer suitable
as regular working place. Either sound insulating housing systems
have to be constructed for the systems, or work places for sample
preparation and post-run data exploration have to be located in a
separate room.
During the first decade of clinical routine application of
HPLCMS/MS the sensitivity of high end instruments has been
improved by an approximate factor of 1020. This advancement
has been achieved by optimizing the process of atmospheric
pressure ion generation and ion transfer into and throughout the
high vacuum area of the analyzer. One major innovation was the
introduction of ion source housings directing the HPLC effluent
and ion spray orthogonal to the main axis of the mass spectrometer. Hereby mass spectrometer contamination and background noise from co-elution matrix constituents were
significantly reduced leading to improved instrument robustness
and sensitivity.
An important trend to be observed in liquid chromatography
over the past years was the improvement of stationary phases.
Besides the successful application of micro- and nano-HPLC
MS/MS equipment in sample limited research applications (e.g.
proteomics), the use of sub-2 m HPLC column packing meant
a methodological revolution in chromatography. With such
stationary phases the chromatographic separation is independent from the applied flow rate [20]. As a consequence, narrow
chromatographic peaks are realized within a fraction of time
needed for conventional column hardware. Resulting shorter
runtimes translate into increased sample throughput (= productivity) of the expensive HPLCMS/MS equipment. As a drawback of small particle diameters, a significant increase of the
systems' working pressure (up to 600 bar) has to be accepted,
which cannot be handled with conventional HPLC instrumentation. Furthermore, higher sampling rates of the detecting devices are necessary to allow for a sufficient number of
data points to characterize the narrow chromatographic peaks
obtained.
Costs
HPLCMS/MS systems are expensive analytical tools.
Investments ranging from 300,000 to 500,000 have to be
expected to purchase systems suitable for abovementioned
routine applications in clinical laboratories. This roughly corresponds to twenty ultrasound instruments, ten intensive care
respirators, or half a modern CT scanner and exceeds the costs
for immunoanalyzer systems widespread in routine laboratories.
If instrument leasing is preferred, expenses N 10,000 /month
have to be taken into account. Maintenance costs of HPLCMS/
MS systems are substantial as well, yearly expenses of up to 10%
of the primary instrument costs can be expected for a contract
suiting the needs of a routine operation. Only recently more
economic manufacturer-independent services have become
available. HPLCMS ion-trap instruments, allowing MS/MS
data acquisition too, are 30% less expensive than triple stage
tandem mass spectrometers. However, these instruments are
clearly inferior to triple stage methods if used for quantitative
applications [21,22]; their strengths lie in more qualitative
screening applications as the identification and structural characterization of unknown metabolites.
It is optimistic but not unrealistic to expect that the typical
lifetime of a HPLCMS/MS is about 10 years. Given extremely
good utilization of an instrument (e.g. 350 days per year, 22 h per
day, 2 min per sample) about 2 million samples could be analyzed
during this time. Given a degree of utilization which is rather
typical today for most laboratories (e.g. 250 days per year, 15 h
per day, 5 min per sample), about 500,000 samples could be
analyzed during these 10 years. Assuming costs of 350,000 for
the instrument and cumulative maintenance costs of 150,000 ,
the instrument related expenses per sample are about 0.25 in the
highest throughput scenario and about 1 for the standard
throughput calculation. Additional per sample expenses for consumables (vials, columns), chemicals (mobile phases, standards,
internal standards), and gas/electricity supply are rather low for
HPLCMS/MS assays. Even costs for isotope labelled internal
standard materials are negligible in most cases. However, if no
suitable labelled internal standard material is available and a de
novo synthesis has to be considered, additional (once only) costs
of several thousand may have to be taken into account. Current
HPLCMS/MS systems require substantial hands-on time of
skilled technicians typically one instrument occupies one
person fully. Hence, over the theoretical 10 year life-span of an
instrument at least 500,000 seem to be realistic cumulative
expenses for human resources. Taken together, in the highest
throughput scenario, overall expenses of about 2 would result
for one LCMS/MS analysis, while about 6 per sample are
required in a standard throughput scenario.
Summarizing, present overall costs for HPLCMS/MS analyses
are substantially higher compared to automated clinical chemistry
tests based on photometry; are about equal to high volume immunoassay tests (like TSH), and are substantially lower compared to the costs of most expensive low volume immunoassays
(e.g. immunosuppressant TDM assays). This calculation applies if
HPLCMS/MS is used for single analyte quantification; if multi- or
mega-parametric analyses are performed with this technique (as for
example several hundred analytes in toxicology and food analyses)

the costs per single analyte result decrease dramatically.
Handling and robustness
Although routine handling of HPLC-MS/MS systems is easier
compared to GCMS instruments, it is much more complex than
operating modern day clinical chemistry analyzers. Incorrect use
can cause substantial machine damage and a training for several
weeks is usually required for technicians to run an instrument.
While everyday handling and basic maintenance procedures can
doubtlessly be performed by skilled technicians, the main
responsibility for HPLCMS/MS installations is typically in the
hands of an academic, in most cases holding a PhD in chemistry.
Such an expert is particularly necessary for the development and
validation of new HPLCMS/MS methods, which are aside
some exceptions individualized (home brewed) assay setups
tailored to the equipment available. A comprehensive assay
validation including a detailed risk assessment has to be carried
out. National or international guidelines (e.g. as published by
CLSI, ICH, IFCC, or FDA) are usually the basis of such an
undertaking, especially in a clinical environment where special
legal regulations have to be met in some countries (e.g. as the
IVDD directive 98/79/EG in the European Community).
It is a key feature of HPLCMS/MS using API techniques as
ESI (electrospray ionization) or APCI (or atmospheric pressure
chemical ionization), that ideally only a clean beam of ions is
transferred into the high vacuum area of an instrument while
unionized molecules (HPLC solvent and sample matrix residuals)
do not enter the mass spectrometer. Solid contaminants typically
precipitate in the ion source housing around the mass spectrometer's vacuum area entrance orifice. In most cases, contaminated
hardware components can be cleaned without venting the mass
spectrometer. This is in contrast to GCMS where essentially the
entire effluent of the chromatographic procedure enters the high
vacuum area, cleaning of which is very difficult and laborious.
Hence, state of the art HPLCMS/MS instruments are by far more
robust than GCMS instruments and allow the continuous
analysis of large sample batches. Daily measurement series up to
24 h duration with short, simple maintenance interventions (e.g.
the exchange of the HPLC stationary phases) after several days to
weeks can be achieved. Taking into account the high analyte
specificity of tandem mass spectrometry, chromatographic
analyte separation prior to MS/MS can be minimized, if ion
suppression effects (see below) are managed. State of the art
HPLCMS/MS instruments with one- or two-dimensional
chromatography setups allow to run up to 20 analyses per hour.
Consequently, within 24 h several hundred quantitative analyses
can be performed with one HPLCMS/MS system in a
continuous work mode. In most clinical laboratories far smaller
series are run in daily routine, especially if switching from one
assay to another requires hardware changes (e.g. of HPLC
columns) causing significant down times.
Typically HPLCMS/MS instruments do work for months
with minimal maintenance but can cause unexpected substantial
problems without prior warning. In general, more down-times are
related to the HPLC modules with its large number of mechanical
653
parts compared to the mass spectrometer. Although the

chromatographic methods in routine applications are kept as
simple as possible (no complex gradients, no salt-buffered mobile
phases), typical HPLC problems (e.g. clogging of capillaries, gas
bubbles in the fluid system, crystallization of mobile phase
additives, microbial growth, abrasion, erosion, and leakage
problems) also occur in HPLCMS/MS. Mass spectrometry
related problems most frequently arise from the API spray
capillary (erosion, blockage) and problems within the ion source
housing like matrix accumulation or salt precipitation. Hence,
preventive interventions as changing the spray capillary and
cleaning the vacuum entrance area have to be done regularly and
require special training. More severe problems result from
substantial contamination within the vacuum area, problems in
the vacuum system or electronic faults. Such events typically
require intervention of a service engineer but may be prevented by
regular planned maintenance visits.
Troubleshooting, e.g. for decreasing sensitivity or insufficient
precision of an individual method, has to include complex considerations. They have to encompass all aspects of the assay as
the sample preparation (e.g. fluctuating quality of extraction
cartridges lots, prevention of handling errors), the chromatography (e.g. injected volume check, early blocking/leakage detection, errors in the composition of mobile phases), the MS/MSbased analyte detection (e.g. hampered by decreasing quality of
nitrogen supply causing high background signals, an instable ion
spray, source contaminations, electrical noise due to a failure of
the detector, or decreasing vacuum quality), and used chemicals
(e.g. contamination of mobile phases causing ion suppression
effects, the instability of internal standards or analytes [23,24]).
It might happen, that after some maintenance actions or with
increased contaminations of some hardware components, the reoptimization of about fifteen MS instrument settings (if running
SRM experiments) is necessary. Several of these tuning operations have to be performed in a more the less intuitive trial and
error manner. Ion spray adjustments (e.g. re-optimization of the
capillary position) are typically done manually without the use
of a servo drive. Consequently, no software read-back is offered,
and the documentation of these settings is hardly possible.
Since almost all HPLCMS/MS systems are customized by
the individual user, not two installations are identical. This
makes professional troubleshooting via hotline or service
engineers on site additionally difficult; especially if HPLC and
MS are purchased from different manufacturers. With few
exceptions, there is no comprehensive support available from the
MS industry which covers all components of an installation or
specific applications.
Summarizing, it has to be stated, that present day routine
HPLCMS/MS assays are no turn-key applications. They
require continuous technical control and optimization by an inhouse expert. It has to be emphasized that currently a gapping
difference in the quality of technical support has to be observed if
major diagnostic companies are compared to mass spectrometry
vendors. Both costs and response delays are significantly higher
from MS industries. Even the most expensive premium
service contracts guarantee only a 48 h response time until the
beginning of a repair visit. Under these optimized conditions, a
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total HPLCMS/MS platform downtime of one week can easily

accumulate. Therefore a detailed in-house training of the routine
staff as well as a long-term troubleshooting experience is of
outmost importance to minimize down-times of the HPLCMS/
MS instrumentation. If urgent HPLCMS/MS analyses require
result reporting within the same day (as applies for immunosuppressant monitoring), a back-up solution is obligatory (at
least immediate usage of an equivalent nearby back-up instrument or sample shipment to a cooperating laboratory).
Sample preparation
Although less laborious than GCMS sample work-up,
typical HPLCMS/MS protocols include several purification
steps. Insufficient sample preparation affects HPLCMS/MS
analyses in two major ways. Macromolecular and cellular
sample contaminations may lead to HPLC column clogging
resulting in instrument down-times. Insufficient removal of
substances co-eluting with an analyte into the mass spectrometer can lead to impaired analyte detection, resulting in sensitivity loss or reduced assay specificity and accuracy. Retaining
investigated analytes in the chromatographic separation system
is a major cornerstone in sample preparation. It allows to separate undesired sample matrix constituents (e.g. salts, phospholipids etc.) and other metabolites from the analyte elution
band.
Depending on the analyte concentration range to be addressed, sample concentration or dilution has to be achieved in the
course of sample preparation. The most widely applied simple
sample preparation technique is protein precipitation performed
by the addition of organic solvents and/or chaotropic substances
(which does not substantially modify the ionic/salt matrix of a
sample) [25]. It is often combined with on-line solid phase
extraction [26] (also termed two-dimensional HPLC) to remove
polar matrix contaminants prior to HPLC separation. Attempts
to miniaturize this approach onto a chip format have been
initiated by one of the vendors. Alternatively, protein filtration,
off-line solid phase extraction, or solvent extraction procedures are utilized, with the two latter approaches allowing to
concentrate analytes. If pipetting automates are used, a near
complete sample preparation is possible for many analytes [27
30]. However, such solutions require further expensive instrumentation and substantial qualified man power, since the
development of instrument protocols is demanding. Consequently, such systems are used in only few clinical laboratories at
present and most LCMS/MS applications are still characterized
by a substantial manual workload during sample preparation.
Current sample preparation and chromatography protocols
produce a high load of toxic organic (e.g. methanol, acetonitrile)
and inorganic (e.g. zinc sulphate) liquid waste which has to be
disposed off at high costs.
Analytical limitations
Even though the principle of tandem mass spectrometry can
allow very specific analyte detection, the quality of an individual
method has to be critically and carefully assessed. The choice of
the optimal internal standard (IS) is one of the most critical steps
in designing a HPLCMS/MS assay. Whenever a stable isotope
labelled derivative of a target analyte is available as IS, very
reliable isotope dilution mass spectrometry (IDMS) methods can
be developed. If homologue molecules (i.e. a molecule
structurally closely related to analyte) have to be used as IS
as is the case for most drug assays (e.g. immunosuppressants)
assay reproducibility and linearity may become a substantial
problem. Only IDMS assays can assure co-elution of the targeted
analyte with its IS. This is the only possibility to fully
compensate sudden signal fluctuations caused by individual
matrix induced reduced or increased ion yields (ion suppression and ion enhancement, e.g. caused by co-administered
drugs) and other unpredictable impairments of the ionization
efficacy. It should not be overlooked, that a constant drop in ion
yields, e.g. due to instrument contamination, will even hamper
the performance of an IDMS-based assay due to sensitivity loss.
A careful assessment of ion suppression effects has to be
included in the assay validation protocols. Efficient sample
clean-up and sufficient chromatographic pre-fractionation of the
extract prior to MS/MS analysis are the main approaches to
minimize such effects. Different experimental procedures as
post-extraction analyte addition (spiking experiments) or postHPLC analyte infusion (T-piece experiments) have been
recommended to get an overall impression of the ion yield in real
life samples [31,32]. However, it must be realized that the
presence or absence of ion suppression can be dependent on the
actual condition of an individual instrument since the contamination of the ion source after prolonged measurement series can
increase the ion suppression level. Furthermore, ion suppression
can occur in a sample specific manner due to unidentified
interfering compounds in an individual specimen. Measures
have to be taken that such effects are easily and reliably detected
in daily routine work.
Additional pitfalls in the accuracy of HPLCMS/MS methods
can arise from in-source fragmentation of conjugate derivatives of
a target analyte; especially if glycosidic bonds are involved [33] or
from isobaric mass transitions of unrelated or related compounds
such as metabolites and isomers (Fig. 3) [34]. Interference from
isomers of the target analyte can make even IDMS methods
invalid [35]. Indeed, most clinical HPLCMS/MS methods
published for the quantification of serum testosterone (TE) have
the essential drawback, that the specificity towards the inactive
isobaric isomer epi-testosterone (ET) has neither been addressed
nor tested (e.g. see Kushnir et al. [36] for a recent example)
although TE/ET rations are known to be fluctuating [37]. This is
quite astonishing, since measuring TE/ET ratios is feasible and is
considered as the backbone of testosterone doping testing [38].
Manufacturers specify the sensitivity of HPLCMS/MS
instruments by applying solutions of different reference compounds. Reserpine is widely used for this purpose. It is relatively
stable and yields positive and negative ions in both electrospray
ionization (ESI) and atmospheric pressure chemical ionization
(APCI). However, due to different protocols used (i.e. flow rates,
analyte concentration, ion source tuning, ion transmission
conditions) direct comparison of the sensitivity of different
instruments is hardly possible and rather approximate; notably,
sensitivity of instruments may differ in a compound specific

manner. Therefore it is advisable to define analyte related
specifications if an instrument is to be implemented for a certain
assay. Moreover, it is useful to supply the vendor's application
laboratories with test samples (i.e. extracts from spiked samples)
in order to test the feasibility of a specific method intended to be
implemented by the customer. Nominal sensitivity documented
on installation of an instrument may differ significantly from the
level of sensitivity found in long-term routine application. Long
term sensitivity critically depends on the robustness of a
respective instrument system. Furthermore, substantial differences in sensitivity can be observed between identical individual
instruments even with similar serial numbers.
Protein and peptide quantification is by principle amenable
by HPLCMS/MS methods [3941]. However, currently such
assays are hardly used for routine purposes but are employed as
655
reference methods to evaluate secondary assay performance

[42]. Methodological advantages of HPLCMS/MS approaches
using SRM experiments for protein quantification over matrix
assisted laser desorption (MALDI) mass spectrometry instrumentations are well known [43,44]. It can be expected, that
MALDI based investigations will remain a major technology in
profiling approaches for biomarker discovery (e.g. proteomics),
whereas HPLCMS/MS assays will be the backbone of
quantitative biomarker verification in targeted assays.
In summary, it is evident that HPLCMS/MS holds great
potential as a complementary technology for laboratory
medicine, although at present there are still important limitations of its routine application which have to be dealt with.
These are in particular issues related to the ease of hardware and
software use, the instrument performance, the practicability of
HPLCMS/MS assays in a routine laboratory setup, and last
Fig. 3. Multiple reaction monitoring (MRM) chromatograms of cyclosporine A (mass transition 1220 N 1203) and its internal standard (IS) cyclosporine D (mass
transition 1234 N 1217) illustrating the need of sufficient chromatographic separation prior to tandem mass spectrometry based analyte detection. The top row
chromatograms (A,B) were recorded with a HPLC system successfully separating the IS peak (at 4.55 min) from isobaric cyclosporine A metabolite (AM1/AM9)
derived peaks co-eluting at 3.25 min. It can be clearly seen, that this peak is only recorded in patient samples (panel B,D) and is missing in calibrator samples (panel A,
C). Hence, insufficient chromatography with co-eluting IS and metabolite peaks (panel C,D) will lead to an underestimation of the analyte content doe to IS
overestimation. Such a method limitation, based on too fast chromatography and only occurring in patient samples, would have been overlooked, if only spiked
materials are included in method validation experiments. Reprinted with permission from [34].
656
but not least to instrument costs. Nonetheless, there is a

realistic perspective that these limitations will be overcome
within the next decade. In the following section we discuss
areas which require improvements in order to make the entire
potential of HPLCMS/MS accessible for medicine in the
future.
Goals for further developments in clinical
HPLCMS/MS applications
System configurations
For use in typical clinical laboratories it would be favourable
to have complete HPLCMS/MS systems available from the
manufacturers as stand-alone systems. Such system should
include all components which at present have to be procured
more or less separately (e.g. HPLC pumps, auto sampler,
column ovens, column switching devices, tubings (with/without
static splitter), rotary pump, nitrogen generator, uninterrupted
power supply). All of these should come assembled on a table as
one instrument. Furthermore, an appropriate noise protecting
cover is necessary in any case. Such systems should be more or
less mobile with only electrical power, pressurized air, and
exhaust line to be connected to the laboratory supply lines. The
footprint should not substantially exceed about 1.0 1.5 m. A
large number of different, fast and analyte specific assays
should be implementable on such an instrument. Short analysis
series can be expected. To be usefully applied in clinical
decision making (e.g. caffeine in neonatal care, methotrexate,
aminoglycosides), the generated results have to be available
within short time. Consequently, a generalized analytical
approach needs to be developed, which allows short runtimes
without changing the HPLC hardware.
Instrument control software
While in industry research facilities chemists, engineers, and
specialized technicians are typically available to operate the
instruments, the situation is quite different in typical hospital
laboratories. Here, technical personnel have to cope with a
number of distinctively different techniques, as manual ELISA
assays, microscopy or PCR methods, besides high-throughput
clinical chemistry analyzers. HPLCMS/MS systems which
still have the handling characteristics of research instruments
will have to mature to user friendly routine instruments in order
to achieve a breakthrough of this powerful technology in the
setting of clinical laboratories.
The software of currently available HPLCMS/MS systems
differs substantially with regard to its practicability. The main
issues of daily use as instrument start-up and shut-down,
generation of a sample list, and control of quantification with
inspection of each chromatogram is solved differently by each
vendor. The routine applicability of most HPLCMS/MS instrument configurations could be substantially improved by
introducing rather simple software solutions which should not
create too much additional cost for the manufacturer. Such
modifications seem rather trivial in relation to the complexity of
the mass spectrometric basic technology of ion sorting but can be

of substantial importance in daily routine application. Unfortunately at least to our experience even implementing simple
additional software tools to any of the software packages is
hardly possible and the customer contact policy of most if not all
MS vendors needs urgent improvement.
We would like to give the following example to stress what
type of problems we are thinking of: In some MS systems, the
desolvation gas flow has to be started first by the operator; only
then may the capillary voltage be applied and the source can be
heated to several hundred degrees. If the source heating is
accidentally started by the operator without starting the
desolvation gas flow before, severe damage of the source can
occur. If the HPLC flow is started before the desolvation gas
flow is opened, solvents can float into the analyzer and
substantial damage can arise as well. It should be easy for any
of the software developers to block heating of the source and
starting the HPLC flow unless the desolvation gas flow has been
started via the software.
In general, user friendly and simple function check tools in
the instrument software are desirable (e.g. solvent bottle filling
status, easy to use injection counting systems to monitor column
aging, pressure profile availability to name only a few). Software based trouble shooting or maintenance instruction tools
could as well be helpful but are hardly found. Standardized
performance check procedures could be introduced; e.g. simple
but reliable system suitability tests to be performed on a daily or
weekly basis could be proposed by the manufactures. Currently,
the development of such tests is more or less left to the choice of
the user. Improved tuning procedures (e.g. supporting the
current practice of syringe pump based manual tuning) with
respective hardware and software modules seem warranted to
improve practicability and performance of the systems.
As is realized with modern clinical chemistry analyzers, a
short-turnaround time (STAT) feature allowing to define priority
samples would be helpful in daily routine as well as a forecast of
the completion time of individual samples. Sample list generation could be greatly simplified if auto samplers would be able
to read bar codes from sample vials with a direct transfer of
identification data into a sample list. Since incorrect positioning
of samples within the auto sampler is one essential source of
errors in HPLCMS/MS analyses, such solutions would also
increase the reliability of results.
Ion sources
Adjustments of parts of the ion source (e.g. the utmost critical
electrode/capillary positioning) and all gas flows should be
wherever feasible controlled by the software. If this is not
possible by servo drives, at least a software based read-back of
positions and of all flows should be realized in order to improve
the standardization of instrument control. A stable electrospray
is the key to constant high sensitivity. The shape of this spray can
change during use; it may become wider in angle or asymmetric
since the electrically charged spray capillary is subject to
corrosion and depositions from the solvent flow; furthermore
pulsations of the spray can arise e.g. from dead volumes in the
tubings. If a post-column/pre-source split is used the spray

capillary can even become completely blocked. Visual control of
the spray shape is often very difficult, in particular if flow
splitting is performed and flow rates below 100 L/min are
sprayed. For controlling the spray as the probably most
important system control measure implementation of a fixed
camera device in the source area with transfer of the spray
picture to the PC screen would be very helpful not only in the
case of nano- and micro-HPLCMS/MS couplings, where these
features are already realized. Automated programs to regularly
rinse the spray capillary (e.g., with formic acid and acetonitrile)
might improve the lifetime of this critical part of the system. It is
desirable that changing the spray capillary can be done very
easily which would help to avoid prolonged down-times. One
possible future trend is the use of disposable chip based HPLC
and capillary devices as recently realized by one manufacturer.
A major cause of deteriorated analysis results is the
deposition of involatile matrix constituents and mobile phase
components spoiling the inlet (sampling cone) to the vacuum
area of a mass spectrometer. A straightforward solution to
exchanging contaminated sampling cones against cleaned ones
should be offered for any routine instrument.
Fluid system
Increased efforts are necessary to reduce the extent of handling
large quantities of toxic solvents in HPLCMS/MS; this
particularly applies to with on-line solid phase extractions
associated with large fluid volumes. Using a low flow regimen
(e.g. b 100 L/min) will support the routine applicability of such
assays. Unfortunately, most micro- or nano-flow HPLCMS/MS
technologies are rather cumbersome and at least at present
hardly fit for routing use. Again, the disposable nano-flow chip
LC technology might be of interest for coming routine HPLC
MS/MS applications.
Mobile phases are typically prepared by the user, e.g. a 9:1
mixture of methanol and water spiked with 0.1% formic acid has
to be mixed. Preparation or refilling errors are typical problems
causing prolonged down-times of a HPLCMS/MS system.
Hence, the commercialized availability of mobile phase blends is
desirable, as it is already common practice for certified HPLC
UV and HPLCFLD assays. Of course this has to go along with
the design of generic HPLCMS/MS assays. Furthermore, user
friendly tube connection systems should be introduced to
minimize potential contact with toxic liquid or gaseous substances
during mobile phase bottle exchange or refill. Completely closed
tube lock solutions for liquid waste should also be included in an
optimized liquid management system for HPLCMS/MS instruments. Implementation of such solutions should be easy, since
closed HPLC systems are already on the market and widespread;
e.g. for the analysis of haemoglobin variants.
Sensitivity
Current standard HPLCMS/MS instruments have a level of
sensitivity which typically allows the quantification of compounds down to a concentration of 1 g/L (e.g. rapamycine,
657
testosterone) without laborious sample pre-concentration steps

(Fig. 4). With this performance which actually can be maintained in a daily routine setting standard HPLCMS/MS
systems offer sufficient sensitivity for most analyses in the field
of TDM. However, for many endogenous analytes, today's
standard instruments are far too insensitive to facilitate routine
analyses. Important and economically attractive analytes currently hardly amenable with pre-concentration free HPLCMS/
MS assays include steroidal hormones and 1,25-dihydroxyvitamin D3. Extremely expensive high end instruments may allow
the quantification of these compounds. Unfortunately, the
availability of such instruments is typically restricted to
university hospitals or research laboratories. Only reaching a
goal of 500 fg on-column sensitivity threshold (LOD) of standard HPLCMS/MS platforms under routine conditions would
enable a more or less universal application of HPLCMS/MS
for low molecular weight analytes in the clinical laboratory.
Ongoing design optimization of mass spectrometers, especially of the ion source, collision cell, and mass selectors might
lead to significant ion yield increases. Generally, combining an
efficient and selective ion transfer into a very pure vacuum with
high mass resolution can be considered a general approach to
increase signal-to-noise ratios. Novel ion source designs
including new ionization strategies such as APPI (atmospheric
Fig. 4. Selected reaction monitoring (SRM) based ion trace chromatograms of a

lower limit of quantification (LLOQ) standard of a routine on-line-SPEHPLC
MS/MS method for immunosuppressive drug quantification [13]. The LLOQ
(expressed in ng analyte/mL whole blood) was established well below the
anticipated therapeutic ranges. A sensitivity in the lower fmol (analyte oncolumn) range can be easily achieved with the used high end mass spectrometer
(API 4000QTrap operated in the SRM mode). Analyte co-elution is generally
feasible due to the high selectivity of SRM/MRM experiments, if co-elution of
isobaric analyte isomers or ion source fragmentation of analyte metabolites can
be ruled out.
658
pressure photo ionization) can show an additional potential in

certain cases. An alternative and probably important approach to
more sensitive MS/MS methods might be the development and
introduction of adduct forming compounds increasing the
efficacy of the ionization process. However, reviewing technical
developments of recent years, it became evident that increased
analytical sensitivity can potentially introduce new problems
caused by contaminations e.g. of solvents [23,24] or by carryover effects. Consequently, these issues have to be addressed
carefully in the process of technical development. Increasing the
sensitivity of HPLCMS/MS has not only the goal to make more
analytes amenable to analyses. An improved signal-to-noise ratio
of analyte signals results in a decrease of assay imprecision, since
the influence of stochastic background noise fluctuations on the
analyte response is minimized. Furthermore, sample preparation
protocols with higher dilution factors could be used minimizing
both matrix effects and mass spectrometer contamination.
Sample throughput
Today for most HPLCMS/MS applications a sample
throughput of 10 to 20 analyses per hour is typical. This stands
in contrast to high-throughput workflows of most analytical
systems in a clinical routine laboratory. Increasing the productivity of HPLCMS/MS assays can be achieved by optimizing the chromatographic dimension of the method. Using
faster auto-sampler modules and multiplexing two or more
chromatographic installations to one MS/MS detector allows
shorter runtimes per sample. These options are already partially
fulfilled by some hardware vendors, but will have to be exploited
in depth within the coming years.
Instrument costs
As noticed for GCMS technology within the past decades, a
continuous decline of the HPLCMS/MS instrument price level
can be expected, if the number of competitors and sold instruments
will rise. Currently, the pricing seems to be mainly oriented towards
industrial applications. With general health care systems having
more limited financial resources, a level of 150,000 for a
complete competitive triple quadrupol HPLCMS/MS systems is
desired. This has to be understood as prerequisite for widespread
application of HPLCMS/MS in diagnostic medicine beyond
specialized central laboratories. Further cost reductions can be
expected by replacing personnel intensive manual sample preparation schemes by automated workflows.
Application specific reagents and support
Extremely important in particular for a European routine
hospital laboratory is the commercial availability of IVD/CE
certified kits for the HPLCMS/MS analysis of a variety of
compounds. The flexibility to implement assays for a number of
drug analytes is a substantial advantage of HPLCMS/MS
technology over immunoassay technology. However, if a broad
panel of analytes has to be monitored, searching for appropriate
internal standard compounds as well as producing materials for
calibration and quality control purposes is hardly possible for a

typical hospital laboratory. Hence, an integrated support of HPLC
MS/MS assays which covers both the instrument and method
specific problems is highly desirable.
Practicability issues addressed so far might well contribute to
a somewhat more widespread use of HPLCMS/MS in
medicine. However, as long as the implementation and operation
of an HPLCMS/MS system still requires a specialist with
academic background in each laboratory, this technology will
remain a wallflower of laboratory medicine. Integration and
automation of the entire sample preparation and analysis process
is inevitable in order to develop HPLCMS/MS to a powerful
new standard technology as it has been for immunoassays
during the recent decades.
Only industry driven developments towards a closed MSbased random-access walk-away analyzer to be operated by
regularly trained technicians can make the entire analytical
power of HPLCMS/MS amenable for medicine. Operation
layouts which will allow to introduce serum samples in standard
tubes into a comprehensive HPLCMS/MS analyzer which
reports final results to a LIMS system are technically demanding
but without doubt realizable in the near future.
Potential future role of HPLCMS/MS in the
clinical laboratory
In present day laboratories, the quantification of highabundance marker compounds is performed by highly economic
photometric and potentiometric high-throughput methods. Target
analysis of low abundance markers is facilitated by immunoassay
technologies and for a minority of analytes by chromatographic techniques. A major strength of immunoassay is the
potential of full automation and high throughput. These methods
are the realm of the diagnostic industry; assay development is
difficult, labour intensive, and expensive. Designing chromatographic assays is more straightforward, but practicability and
throughput of HPLC methods is generally poor. HPLCMS/MS
seems to have the potential to overcome these limitations.
However, after more than a decade of application in the routine
clinical laboratory, HPLCMS/MS has found only a limited
number of important applications in a limited number of
specialized tertiary care laboratories. Due to the limited
usability of currently available hard- and software but also due
to the lack of experts, the number of laboratories using HPLCMS/MS will probably not increase substantially in the near future.
But has HPLCMS/MS a true potential to further improve
the performance of clinical chemistry and laboratory medicine
in a significant way? What are the shortcomings of today's
powerful standard clinical chemistry technologies photometry,
potentiometry, and ligand binding assays which HPLCMS/MS
may potentially help to overcome?
Clinical pharmacology and xenobiotic compounds
HPLCMS/MS technology is of particular importance for
clinical pharmacology and hospital drug monitoring. For a
significant number of drugs as ribavirin, imantinib, ganciclovir,
valganciclovir, 5-FU and many more, current literature suggests

dose optimization based on a pharmacokinetic monitoring
[18,4548]. Despite the plethora of recent data also derived
from the pharmacogenomic approach pleading for a more
widespread therapeutic drug monitoring, individualization of
drug dosage is rarely performed in most institutions mainly
because of lacking analytical resources. Indeed during the past
decade almost no single TDM immunoassay has been
introduced by the diagnostic industry. In this field, widespread
application of HPLCMS/MS with its high flexibility and
versatility of assay implementation may substantially improve
treatment outcomes for many diseases in the near future.
Moreover, this technology has the potential to act as a driving
force of clinical pharmacology in the era of pharmacogenomics.
Most drugs have favourable pharmacokinetic properties if
administered to outpatients with more or less normal gastrointestinal, renal, and hepatic function. In this situation, drug
monitoring is hardly providing an additional benefit for most
medications. However, in hospitalized patients with organ
failures as well as in many chronically ill outpatients,
pharmacokinetic properties (absorption, distribution, metabolization, excretion) of many drugs may differ substantial from
normal conditions. Toxic drug accumulation or ineffectively low
blood concentrations of the drug may result during treatment.
This can in particular apply to anti-infective and anti-neoplastic
drugs, as well as in neonatal and paediatric care. HPLCMS/MS
has the potential after maturing to random-access, walk-away
multi-analyte analyzers to enable routine monitoring of
essentially all drugs which are used in an individual hospital.
A larger scale pharmacokinetic monitoring of medications
which under normal conditions do not necessarily require TDM
also has the potential to save expenses for drugs; e.g. if the
accumulation of expensive second line antibiotics in intensive
care patients due to altered pharmacokinetic conditions can be
avoided [49]. Furthermore, latest developments show that a more
or less general unknown screening based on HPLCMS/MS
seems feasible [50]. This is an important option for emergency
departments. However, in contrast to GCMS, instrument specific
data bases have to be applied for HPLCMS/MS. HPLCMS/
MS-based phenotyping as methodological independent alternative to genotyping approaches in pharmacogenetics [51,52] (e.g.
as frequently performed to monitor thiopurin-methyltransferase
(TMPT) polymorphism [53,54]) represents another future option
to contribute to clinical pharmacology, especially if certain CYP
enzymes can be addressed by test mixtures [55]. Taken together,
there are good reasons to assume that the availability of a versatile
high-throughput TDM HPLC-MS/MS facility in a hospital may
improve outcomes for many diseases by optimized dose
individualization of a number of drugs.
In the future, HPLCMS/MS methods might also be developed
for the detection of microbial compounds in blood such as galactomannans or endotoxins. A large number of synthetic and
potentially harmful chemicals (e.g. persistent organic pollutants
(POP), endocrine disruptors) can be found in serum and urine of
humans [5658]. A more widespread and routine biomonitoring of
human sample materials for such compounds could be enabled by
automated high-throughput HPLCMS/MS techniques and might
659
help to better characterize respective exposures in the future. This

would be an important prerequisite to finally reduce such
exposures. It will probably be recognized during the next decade
that monitoring of chemical (and also biological) immission to
humans is an essential part of the responsibility of laboratory
medicine [59,60].
Target and multi-target analyses of endogenous compounds
A number of endogenous small molecule compounds have
been recognized as potentially diagnostically useful analytes
during recent years, however, many of them are hardly amenable
to standard clinical chemistry techniques. Since up to now the
availability of HPLCMS/MS methods is very limited, most of
these markers have not been introduced into practical medicine
yet. Widespread implementation of HPLCMS/MS might thus
improve the power of laboratory medicine with such new
markers, including for example: Asymmetric dimethyl arginine
(ADMA) [61]; 8-hydroxy-deoxy guanosine (8-OHDG) as a
marker of nucleotid denaturation and oxidative stress [62];
methylmalonic acid (MMA) as marker of cobalamin deficiency
[63,64]; urinary lactulose and mannitol as markers of intestinal
permeability [65]; analytes related to the optimization of
nutrition such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [66]; or oxysterols [67], trans-fatty acids
[68,69], and advanced glycation end-products (AGEs) as novel
markers of glycaemic control [7074].
Furthermore, there are important and well established small
molecule markers which might potentially be quantified using
HPLCMS/MS on a reference method level in a routine laboratory setting in the future (e.g., creatinine, urea, different
forms of bilirubin, glucose, HbA1c [7577]). In multi-analyte
organ function panels such assays might have the advantage of
high accuracy and specificity as well as method independent
reference ranges in contrast to current standard methods.
Metabolomics the attempt to comprehensively describe the
low-molecular phenotype of sample fluids relies on a variety of
techniques (such as NMR, GCMS, FTICR MS) used to
describe the small molecule phenotype of human sample materials in an untargeted discovery approach. Such a comprehensive biochemical analysis strategy (although better named
metabolic profiling, metabolic fingerprinting or targeted metabolomics [78,79]) will probably disclose a number of potential
diagnostic markers in the near future [80]. It can be assumed
that apart from this discovery process, where HPLCMS/MS
is hampered by its low de novo structure elucidation power
this platform will be the most powerful and convenient
methodology to quantify large numbers of newly discovered
small molecule analytes simultaneously in metabolic profiling
approaches in the future.
For several panels of structurally related endogenous small
molecular analytes, HPLCMS/MS multi-methods already have
been developed (e.g., endocannabinoid profiles [81], differentiated description of phospholipids [82], amino acid profiles,
sugar profiles, characterization of compounds from the urea
cycle). Such multi-parametric analytical approaches using
HPLCMS/MS might also include bile acid profiles to monitor
660
hepatic injury, carnitine profiles to disclose mitochondrial

damage, amino acid profiles to monitor protein breakdown,
decrease of glucogenic amino acids along with hepatic insulin
resistance, alterations of fatty acid derivatives profiles during
inflammatory states.
Doubtlessly multi-parametric analyses are of utmost importance for the elucidation of disease mechanisms. However, at
present it is still difficult to speculate about the potential of
metabolomics-based diagnostic approaches to actually
improve patients' care in the future. When dedicated to
diagnostic medicine, metabolomic research might in particular
address areas where available diagnostic strategies have obvious
shortcomings. This applies for example to the characterization of
subclinical chronical inflammatory states (in particular with
respect to cardiovascular diseases); defence reactions of the
body against still localized malignant processes; neurodegenerative diseases; and also entities of autoimmunity. However, in
an individual diagnostic work-up, it must be systematically
questioned where pre-symptomatic diagnostic procedures might
be indeed helpful for the patient. As long as no treatment is
available for a specific illness as is for example the case for
Alzheimer's disease pre-symptomatic diagnosis might substantially compromise the quality of life.
Whereas HPLCMS/MS is traditionally linked to small
molecule analyses, there have recently been a growing number
of HPLCMS/MS methods described for the quantification of
peptides and therapeutic oligonucleotides [83]. HPLCMS/MS
protein and peptide assays still typically require laborious
sample preparation and are applied rather as reference methods
[42]. One of the currently still unsolved obstacles of SRM based
protein quantification is the limited availability of appropriate
internal standard materials. Since a tryptic digest is used as
central sample preparation step to obtain the peptides to be
monitored, labelled synthetically obtained proteins [84] or
peptides are needed if the digest step is assumed to be quantitative. However, current developments make it likely, that
HPLCMS/MS will gain significant importance in routine
protein and peptide quantification within the years to come.
Considering the potential of HPLCMS/MS for diagnostic
medicine demonstrated in this text, one essential question
arises: Why have fully automated random-access HPLCMS/
MS based analyzer applicable in a widespread routine laboratory setting not yet been developed by the industry?
For the manufacturers of HPLCMS/MS systems, clinical invitro diagnostics is an interesting but very limited market,
accounting for about 10% of all instrument sales. The majority of
HPLCMS/MS systems are presently sold to rather specialized
laboratories (e.g., pharmaceutical industry) where improvements in the practicability are far less urgently requested compared to clinical users. Hence, major investments for the
development of random-access comprehensive MS-based clinical analyzers are not very interesting for these companies at
present.
Key players of the in-vitro diagnostics industry on the other
hand still seem to look upon the up-coming HPLCMS/MS
technology rather as a threat, since this technology may enable customers to switch from some of their most expensive
immunoassays to HPLCMS/MS analyses with their lower

running costs. However, some companies seem to begin to
realize that commercialization of the HPLCMS/MS technology may in contrast to current standard technologies open
the perspective to include an important number of novel and
innovative analytes into their portfolio, potentially also in megaparametric diagnostic approaches. Fully automated HPLCMS/
MS analyzer systems might be included into a comprehensive
instrument architecture of a company as a specialized/
customized testing module.
The general situation of the in-vitro diagnostic industry is at
present characterized by a number of large companies, each
offering a rather comprehensive portfolio of high-throughput
analyzers and sample management solutions. With some minor
exceptions, the spectrum of analytes is almost identical. In this
situation, it might indeed be an interesting option for a company
to open new diagnostic horizons to the customers by commercializing complete MS solutions compatible with the workflow of modern high-throughput laboratories. Since commercial
decisions have to take into account not traditional target analytes with their known sales volumes but rather innovative, not
yet established analytes, forecasting the financial perspectives
of commercializing this technology are complex and require
substantial scientific and clinical expertise. It seems that the
assumed market volume of such innovative analytes and diagnostic approaches has not yet reached a critical size to definitely initiate the expensive development of fully automated
MS/MS analyzers by the diagnostic industry.
It is evident that standard small molecule analytes which are
currently quantified by use of well performing and not very
expensive automated immunoassays (e.g. thyroxine) are not a
relevant target for the implementation onto a future automated
HPLCMS/MS module, although potentially feasible. Such a
module of a comprehensive multi-analyzer is in particular an
option for (i) analytes currently addressed with immunometric methods which are of inferior analytical quality (e.g. some
steroid hormones, metanephrins, drugs of abuse screening,
immunosupressants); (ii) customized assays for analytes rather requested rarely in general but of high importance for
specific institutions (e.g. busulphane, 5-FU) since the development of immunoassays for such analytes is inappropriately
expensive and hence not pursued by the IVD industry; (iii)
targeted multi-analyte panels or whole profiles ranging from a
few specialized analytes (e.g. as realized for the neonatal
screening) to general unknown screening in clinical toxicology
or clinical metabolic profiling.
It can be summarized that within the past decade HPLCMS/
MS has matured besides the essential role in clinical chemistry
research to an important tool for some rather specialized
analytical applications in laboratory medicine. However, only a
minor proportion of the entire analytical potential of this
fascinating technology has been opened for laboratory medicine
so far. A breakthrough to widespread application of this technology requires substantial further development from the side
of the diagnostic industry. Given such efforts it seems realistic
that HPLCMS/MS will become a further main technology for
the clinical laboratory besides photometry, potentiometry, and
ligand binding assays opening novel diagnostic perspectives

within the decade to come.
[21]
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Clinical Biochemistry 41 (2008) 649 662

A decade of HPLCMS/MS in the routine

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

Goals for further developments in clinical HPLCMS/MS applications

Characteristics of current HPLCMS/MS applications in

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

Fig. 2. Online-SPEHPLCMS/MS system in one of our laboratories. Besides

Fig. 1. Three different HPLCMS experiments of a plasma sample illustrating

HPLCMS/MS instruments capable of selected reaction

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

rotary pumps) produced by the system has to be dissipated and

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

example several hundred analytes in toxicology and food analyses)

parts compared to the mass spectrometer. Although the

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

total HPLCMS/MS platform downtime of one week can easily

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

sensitivity of instruments may differ in a compound specific

reference methods to evaluate secondary assay performance

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

but not least to instrument costs. Nonetheless, there is a

the mass spectrometric basic technology of ion sorting but can be

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

tubings. If a post-column/pre-source split is used the spray

testosterone) without laborious sample pre-concentration steps

Fig. 4. Selected reaction monitoring (SRM) based ion trace chromatograms of a

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

pressure photo ionization) can show an additional potential in

calibration and quality control purposes is hardly possible for a

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

valganciclovir, 5-FU and many more, current literature suggests

help to better characterize respective exposures in the future. This

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

hepatic injury, carnitine profiles to disclose mitochondrial

immunoassays to HPLCMS/MS analyses with their lower

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

ligand binding assays opening novel diagnostic perspectives

and by ion trap mass spectrometry. Rapid Commun Mass Spectrom

M. Vogeser, C. Seger / Clinical Biochemistry 41 (2008) 649662

utility and cost-benefit analysis of methylmalonic acid determination in

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