Hyphenated Techniques Breathing New Life into the Analytical

Instrumentation Industry

Hyphenated Techniques combine chromatographic and spectral methods to exploit

the advantages of both.

Chromatography - Produces pure or nearly pure fractions of chemical

components in a mixture.

Spectroscopy – Produces selective information for identification using

standards or library spectra.

Being a part of a mature market, with marginal and steady growth at best,
analytical instrumentation companies have been looking for a facelift. These
veterans are looking to be able to cash in on new applications, new marketplaces
and new prices for their products. On a macro level, technological advancements in
pharmaceutical and bio-technology industries and therefore increased investment
in R&D on these advancements has tremendously improved demand for
application-specific analytical instruments in the past five years.

Extensive research in biochemistry, drug discovery, environmental testing, and

even space research, has increased the need for high-performance analytical
equipment. Each new improvement offers much more than its predecessors.
Greater emphasis on quality control in manufacturing processes and prospects of a
vibrant recovering economy are also creating a positive impact.

Improvements in instrument design such as miniaturization, incorporation of

statistical methods, and user-friendly software are aiding customers in easy use and
maintenance of equipment. Vendors are focusing on compatibility of instruments
with research facilities such as Lab-on-a-chip and Laboratory Information
Management Systems (LIMS) to enable complete interconnectivity. Application-
specific equipment such as surface analyzers for semiconductor chip, and infrared
spectrometers are increasing the demand for these instruments as a whole.

Integrated Equipment Edging Out the Stand Alone?

Traditional analytical approaches including HPLC (High-Performance Liquid

Chromatograph), GC (Gas Chromatograph), UV (Ultraviolet) detection, etc., have
become insufficient to effectively address the growing number of challenges in
analyses of species- specificity and sensitivity. Modern speciation analyses, whose
developments are referred to as hyphenated techniques, originate from the
traditional use of molecule or element specific detection in electrophoresis or
chromatography.

Hyphenated techniques and instruments have been around for over two decades
now. The GC-MS (Mass Spectrophotometer), the ICP-MS (Inductive Coupled Plasma –
Mass Spectrophotometer) have been finding applications in several research efforts
across end-user segments. Apart from routine research, these techniques are
finding increasing application in commercial uses as well.

Elemental speciation analysis in biological, environmental and clinical sample

matrices are important applications of several new hyphenated techniques
including the GC-ICP-MS. Stand alone instruments could analyze various physical
properties such as boiling point, toxicity, solubility and metabolic pathways in
different living organisms. With integrated equipment such the HPLC-ICP-MS or the
GC-ICP-MS, sample preparation before analysis is minimized and ‘on-line’ species
separation is much faster. These integrated techniques are becoming increasingly
popular in applications where analyses of complex matrices with low detection
limits and high specificity are expected.

New Techniques on the Block

One of the most important industry trade shows, Pittcon 2004 is expecting to see
several such innovations and integration of erstwhile stand alone instrumentation.
One of the leading participants in this market, Thermo Electron is planning to
announce the launch of new innovations in its X-series ICP-MS line of
instrumentation. The company is planning to include new HPLC and GC coupling
packs such as the Finnigan™ Surveyor™, Finnigan™ Focus and Finnigan™ Trace
into its ICP-MS series. These new hyphenated techniques are expected to have the
benefits of an open architecture sample introduction system that enables quick and
easy interchangeability between ICP-MS, HPLC-ICP-MS and GC-ICP-MS
configurations. These configurations are expected to offer optimum
chromatographic separations in both aqueous and organic mobile phases.

The Thermo Electron initiative is merely a glimpse of what is expected in the future.
Increasing number of hyphens in the technology used in analytical instrumentation
is a reflection of more complex requirements in several end user segments and
their insatiable need for high-performance instrumentation.

In addition, a portable GC-IMS (Ion Mobility Spectrometry) instrument was

demonstrated by FemtoScan to be used for detection of toxic chemicals in military
and defense applications. Apart from being a highly powerful hyphenated analytical
instrument, its portability offers an exciting range of applications from an in-situ
manufacturing process monitor to on-site environmental testing.
The biggest advantage of hyphenated speciation techniques is the ability to detect
species other than the pre-conceived compounds. This has been found to be
especially true in the analysis of drinking and wastewater, drug discovery,
biochemistry and biotechnology, where focus on research is maximum the world
over.

Future of the Hyphen in Analytical World

Currently the most common techniques for trace element speciation include a
combination of separation technique coupled with a detection technique that is
more sensitive. Earlier such hyphenated techniques were the coupling of separation
of a special sample preparation off-line and later adding a detection technique.
Presently, online coupling of the two techniques have become increasingly
common. As will be witnessed in Pittcon 2004, benefits from using integrated
analytical instrumentation are plenty. These hyphenated techniques offer

• Shorter analysis time

• Higher degree of automation

• Higher sample throughput

• Better reproducibility

• Reduction of contamination because it is a closed system

• Enhanced combined selectivity and therefore higher degree of information

All these factors are expected to improve the commercial availability of these
hyphenated techniques in the next couple of years. Environmental and biological
research users are already using these type of instruments in varied applications.
Introduction of commercial versions of this equipment is expected to find demand in
industrial applications as well. Chemical and petrochemical, food and beverage,
plant and animal biochemistry, clinical chemistry, etc., are all areas where research
and manufacturing will be greatly benefited by these hyphenated techniques.

The advancements in technological compatibility and integration are signs of

serious movement in the analytical instrumentation industry worldwide. Saturation
of demand and market maturity of several conventional, stand-alone instruments
are driving industry participants such as Thermo Electron, PerkinElmer and several
others to investigate and offer highly advanced hyphenated techniques that meet
the needs of high-technology oriented applications. These market leaders are acting
on foresight that indicates the need to offer innovative instruments that proactively
cater to the changing requirements of their customers.
Liquid chromatography-mass
spectrometry

Ion trap LCMS system

Acronym LCMS
Classificatio Chromatography
n Mass spectrometry
Analytes organic molecules
biomolecules
Agilent
Bruker
Manufactur MDS
ers Shimadzu Scientific
Thermo Fisher Scientific
Varian, Inc.
Waters Corporation

Liquid chromatography-mass spectrometry (LC-MS, or alternatively HPLC-

MS) is an analytical chemistry technique that combines the physical separation
capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities
of mass spectrometry. LC-MS is a powerful technique used for many applications
which has very high sensitivity and specificity. Generally its application is oriented
towards the specific detection and potential identification of chemicals in the
presence of other chemicals (in a complex mixture).

Contents

• 1 Liquid chromatography

○ 1.1 Scale

○ 1.2 Flow splitting

• 2 Mass spectrometry

○ 2.1 Mass analyzer

○ 2.2 Interface

• 3 Applications

○ 3.1 Pharmacokinetics

○ 3.2 Proteomics

○ 3.3 Drug development

• 4 See also

• 5 References

• 6 Bibliography

Liquid chromatography

Scale

A major difference between traditional HPLC and the chromatography used in LC-MS
is that in the latter case the scale is usually much smaller, both with respect to the
internal diameter of the column and even more so with respect to flow rate since it
scales as the square of the diameter. For a long time, 1 mm columns were typical
for LC-MS work (as opposed to 4.6 mm for HPLC). More recently 300 µm and even
75 µm capillary columns have become more prevalent. At the low end of these
column diameters the flow rates approach 100 nL/min and are generally used with
nanospray sources.[1]

Flow splitting
When standard bore (4.6 mm) columns are used the flow is often split ~10:1. This
can be beneficial by allowing the use of other techniques in tandem such as MS and
UV. However splitting the flow to UV will decrease the sensitivity of
spectrophotometric detectors. The Mass Spec on the other hand will give improved
sensitivity at flow rates of 200 μL/min or less. This is because the analyte ions must
be vaporised (nebulized) in order to become charged.

Mass spectrometry

Mass analyzer

There are a lot of mass analyzers that can be used in LC/MS. Single Quadrupole,
Triple Quadrupole, Ion Trap, TOF (time of Flight) and Quadrupole-time of flight (Q-
TOF).

Interface

Understandably the interface between a liquid phase technique which continuously

flows liquid, and a gas phase technique carried out in a vacuum was difficult for a
long time. The advent of electrospray ionization changed this. The interface is most
often an electrospray ion source or variant such as a nanospray source; however
fast atom bombardment, thermospray and atmospheric pressure chemical
ionization interfaces are also used.[2] Various deposition and drying techniques have
also been used such as using moving belts; however the most common of these is
off-line MALDI deposition.[3][4]

Applications
Pharmacokinetics

LC-MS is very commonly used in pharmacokinetic studies of pharmaceuticals. These

studies give information about how quickly a drug will be cleared from the hepatic
blood flow, and organs of the body. MS is used for this due to high sensitivity and
exceptional specificity compared to UV (as long as the analyte can be suitably
ionised), and short analysis time.

The major advantage MS has is the use of tandem MS-MS. The detector may be
programmed to select certain ions to fragment. The process is essentially a
selection technique, but is in fact more complex. The measured quantity is the sum
of molecule fragments chosen by the operator. As long as there are no
interferences or ion suppression, the LC separation can be quite quick. It is common
now to have analysis times of 1 minute or less by MS-MS detection, compared to
over 10 mins with UV detection.

Proteomics
LC-MS is also used in the study of proteomics where again components of a
complex mixture must be detected and identified in some manner. The bottom-up
proteomics LC-MS approach to proteomics generally involves protease digestion
and denaturation (usually trypsin as a protease, urea to denature tertiary structure
and iodoacetamide to cap cysteine residues) followed by LC-MS with peptide mass
fingerprinting or LC-MS/MS (tandem MS) to derive sequence of individual peptides.[8]
LC-MS/MS is most commonly used for proteomic analysis of complex samples where
peptide masses may overlap even with a high-resolution mass spectrometer.
Samples of complex biological fluids like human serum may be run in a modern LC-
MS/MS system and result in over 1000 proteins being identified, provided that the
sample was first separated on an SDS-PAGE gel or HPLC-SCX.

Drug development

LC-MS is frequently used in drug development at many different stages including

Peptide Mapping, Glycoprotein Mapping, Natural Products Dereplication, Bioaffinity
Screening, In Vivo Drug Screening, Metabolic Stability Screening, Metabolite
Identification, Impurity Identification, Degradant Identification, Quantitative
Bioanalysis, and Quality Control.

Gas chromatography-mass spectrometry

Example of a GC-MS

Gas chromatography-mass spectrometry (GC-MS) is a method that combines

the features of gas-liquid chromatography and mass spectrometry to identify
different substances within a test sample. Applications of GC-MS include drug
detection, fire investigation, environmental analysis, explosives investigation, and
identification of unknown samples. GC/MS can also be used in airport security to
detect substances in luggage or on human beings. Additionally, it can identify trace
elements in materials that were previously thought to have disintegrated beyond
identification.

The GC-MS has been widely heralded as a "gold standard" for forensic substance
identification because it is used to perform a specific test. A specific test positively
identifies the actual presence of a particular substance in a given sample. A non-
specific test merely indicates that a substance falls into a category of substances.
Although a non-specific test could statistically suggest the identity of the substance,
this could lead to false positive identification.

Contents

• 1 History

• 2 Instrumentation

○ 2.1 Split/Splitless GC-MS inlets

○ 2.2 Purge and Trap GC-MS

○ 2.3 Types of Mass Spectrometer Detectors

• 3 Analysis

○ 3.1 Full scan MS

○ 3.2 Selected ion monitoring

○ 3.3 Types of Ionization

 3.3.1 Electron Ionization

 3.3.2 Chemical Ionization

○ 3.4 GC-MS/MS

• 4 Applications

○ 4.1 Environmental Monitoring and Cleanup

○ 4.2 Criminal Forensics

○ 4.3 Law Enforcement

○ 4.4 Security

○ 4.5 Food, Beverage and Perfume Analysis

○ 4.6 Astrochemistry
 ○ 4.7 Medicine

• 5 See also

• 6 References

• 7 Bibliography

• 8 External links

History

The use of a mass spectrometer as the detector in gas chromatography was

developed during the 1950s by Roland Gohlke and Fred McLafferty.[1][2] These
sensitive devices were bulky, fragile, and originally limited to laboratory settings.
The development of affordable and miniaturized computers has helped in the
simplification of the use of this instrument, as well as allowed great improvements
in the amount of time it takes to analyze a sample. In 1996 the top-of-the-line high-
speed GC-MS units completed analysis of fire accelerants in less than 90 seconds,
whereas first-generation GC/MS would have required at least 16 minutes.[citation needed]
This has led to their widespread adoption in a number of fields.

Instrumentation

Main articles: gas chromatograph and mass spectrometer

The insides of the GC-MS, with the column of the gas chromatograph in the oven on
the right.

The GC-MS is composed of two major building blocks: the gas chromatograph and
the mass spectrometer. The gas chromatograph utilizes a capillary column which
depends on the column's dimensions (length, diameter, film thickness) as well as
the phase properties (e.g. 5% phenyl polysiloxane). The difference in the chemical
properties between different molecules in a mixture will separate the molecules as
the sample travels the length of the column. The molecules take different amounts
of time (called the retention time) to come out of (elute from) the gas
chromatograph, and this allows the mass spectrometer downstream to capture,
ionize, accelerate, deflect, and detect the ionized molecules separately. The mass
spectrometer does this by breaking each molecule into ionized fragments and
detecting these fragments using their mass to charge ratio.

GC-MS schematic

These two components, used together, allow a much finer degree of substance
identification than either unit used separately. It is not possible to make an accurate
identification of a particular molecule by gas chromatography or mass spectrometry
alone. The mass spectrometry process normally requires a very pure sample while
gas chromatography using a traditional detector (e.g. Flame Ionization Detector)
detects multiple molecules that happen to take the same amount of time to travel
through the column (i.e. have the same retention time) which results in two or more
molecules to co-elute. Sometimes two different molecules can also have a similar
pattern of ionized fragments in a mass spectrometer (mass spectrum). Combining
the two processes makes it extremely unlikely that two different molecules will
behave in the same way in both a gas chromatograph and a mass spectrometer.
Therefore when an identifying mass spectrum appears at a characteristic retention
time in a GC-MS analysis, it typically lends to increased certainty that the analyte of
interest is in the sample.

Split/Splitless GC-MS inlets

Samples are introduced to the column via an inlet. This inlet is typically injection
through a septum. Once in the inlet, the heated chamber acts to volatilize the
sample. In a split system, a constant flow of carrier gas moves through the inlet. A
portion of the carrier gas flow acts to transport the sample into the column. Another
portion of the carrier gas flow gets directed to purge the inlet of any sample
following injection (septum purge). Yet another portion of the flow is directed
through the split vent in a set ratio known as the split ratio. In a splitless system,
the advantage is that a larger amount of sample is introduced to the column.
However, a split system is preferred when the detector is sensitive to trace amounts
of analyte and there is concern about overloading the column.
Purge and Trap GC-MS

For the analysis of volatile compounds a Purge and Trap (P&T) concentrator system
may be used to introduce samples. The target analytes are extracted and mixed
with water and introduced into an airtight chamber. An inert gas such as Nitrogen
(N2) is bubbled through the water; this is known as purging. The volatile compounds
move into the headspace above the water and are drawn along a pressure gradient
(caused by the introduction of the purge gas) out of the chamber. The volatile
compounds are drawn along a heated line onto a 'trap'. the trap is a column of
adsorbent material at ambient temperature that holds the compounds by returning
them to the liquid phase. The trap is then heated and the sample compounds are
introduced to the GC-MS column via a volatiles interface, which is a split inlet
system. P&T GC-MS is particularly suited to volatile organic compounds (VOCs) and
BTEX compounds (aromatic compounds associated with petroleum).[3]

Types of Mass Spectrometer Detectors

The most common type of mass spectrometer (MS) associated with a gas
chromatograph (GC) is the quadrupole mass spectrometer, sometimes referred to
by the Hewlett-Packard (now Agilent) trade name "Mass Selective Detector" (MSD).
Another relatively common detector is the ion trap mass spectrometer. Additionally
one may find a magnetic sector mass spectrometer, however these particular
instruments are expensive and bulky and not typically found in high-throughput
service laboratories. Other detectors may be encountered such as time of flight
(TOF), tandem quadrupoles (MS-MS) (see below), or in the case of an ion trap MSn
where n indicates the number mass spectrometry stages.

Analysis

A mass spectrometer is typically utilized in one of two ways: Full Scan or Selective
Ion Monitoring (SIM). The typical GC/MS instrument is capable of performing both
functions either individually or concomitantly, depending on the setup of the
particular instrument.

Full scan MS

When collecting data in the full scan mode, a target range of mass fragments is
determined and put into the instrument's method. An example of a typical broad
range of mass fragments to monitor would be m/z 50 to m/z 400. The determination
of what range to use is largely dictated by what one anticipates being in the sample
while being cognizant of the solvent and other possible interferences. A MS should
not be set to look for mass fragments too low or else one may detect air (found as
m/z 28 due to nitrogen), carbon dioxide (m/z 44) or other possible interferences.
Additionally if one is to use a large scan range then sensitivity of the instrument is
decreased due to performing fewer scans per second since each scan will have to
detect a wide range of mass fragments.
Full scan is useful in determining unknown compounds in a sample. It provides more
information than SIM when it comes to confirming or resolving compounds in a
sample. During instrument method development it may be common to first analyze
test solutions in full scan mode to determine the retention time and the mass
fragment fingerprint before moving to a SIM instrument method.

Selected ion monitoring

In selected ion monitoring (SIM) certain ion fragments are entered into the
instrument method and only those mass fragments are detected by the mass
spectrometer. The advantages of SIM are that the detection limit is lower since the
instrument is only looking at a small number of fragments (e.g. three fragments)
during each scan. More scans can take place each second. Since only a few mass
fragments of interest are being monitored, matrix interferences are typically lower.
To additionally confirm the likelihood of a potentially positive result, it is relatively
important to be sure that the ion ratios of the various mass fragments are
comparable to a known reference standard.

Types of Ionization

After the molecules travel the length of the column, pass through the transfer line
and enter into the mass spectrometer they are ionized by various methods with
typically only one method being used at any given time. Once the sample is
fragmented it will then be detected, usually by an electron multiplier diode, which
essentially turns the ionized mass fragment into an electrical signal that is then
detected.

The ionization technique chosen is independent of using Full Scan or SIM.

Electron Ionization

By far the most common and perhaps standard form of ionization is electron
ionization (EI). The molecules enter into the MS (the source is a quadrupole or the
ion trap itself in an ion trap MS) where they are bombarded with free electrons
emitted from a filament, not much unlike the filament one would find in a standard
light bulb. The electrons bombard the molecules causing a hard ionization that
fragments the molecule, and the way in which a molecule fragment is usually
typical for all EI techniques.

Chemical Ionization

Main article: Chemical ionization

In chemical ionization a reagent gas, typically methane or ammonia is introduced

into the mass spectrometer. Depending on the technique (positive CI or negative CI)
chosen, this reagent gas will interact with the electrons and analyte and cause a
'soft' ionization of the molecule of interest. A softer ionization fragments the
molecule to a lower degree than the hard ionization of EI. One of the main benefits
of using chemical ionization is that a mass fragment closely corresponding to the
molecular weight of the analyte of interest is produced.

Positive Chemical Ionization

In Positive Chemical Ionization (PCI) the reagent gas interacts with the target
molecule, most often with a proton exchange. This produces the species in
relatively high amounts.

Negative Chemical Ionization

In Negative Chemical Ionization (NCI) the reagent gas decreases the impact of the
free electrons on the target analyte. This decreased energy typically leaves the
fragment in great supply.

This section requires expansion with:

Updating. The following information is in the process of being updated:.
The primary goal of instrument analysis is to quantify an amount of substance. This
is done by comparing the relative concentrations among the atomic masses in the
generated spectrum. Two kinds of analysis are possible, comparative and original.
Comparative analysis essentially compares the given spectrum to a spectrum
library to see if its characteristics are present for some sample in the library. This is
best performed by a computer because there are a myriad of visual distortions that
can take place due to variations in scale. Computers can also simultaneously
correlate more data (such as the retention times identified by GC), to more
accurately relate certain data.

Another method of analysis measures the peaks in relation to one another. In this
method, the tallest peak is assigned 100% of the value, and the other peaks being
assigned proportionate values. All values above 3% are assigned. The total mass of
the unknown compound is normally indicated by the parent peak. The value of this
parent peak can be used to fit with a chemical formula containing the various
elements which are believed to be in the compound. The isotope pattern in the
spectrum, which is unique for elements that have many isotopes, can also be used
to identify the various elements present. Once a chemical formula has been
matched to the spectrum, the molecular structure and bonding can be identified,
and must be consistent with the characteristics recorded by GC/MS. Typically, this
identification done automatically by programs which come with the instrument,
given a list of the elements which could be present in the sample.

A “full spectrum” analysis considers all the “peaks” within a spectrum. Conversely,
selective ion monitoring (SIM) only monitors selected peaks associated with a
specific substance. This is done on the assumption that at a given retention time, a
set of ions is characteristic of a certain compound. This is a fast and efficient
analysis, especially if the analyst has previous information about a sample or is only
looking for a few specific substances. When the amount of information collected
about the ions in a given gas chromatographic peak decreases, the sensitivity of the
analysis increases. So, SIM analysis allows for a smaller quantity of a compound to
be detected and measured, but the degree of certainty about the identity of that
compound is reduced.

GC-MS/MS

When a second phase of mass fragmentation is added, for example using a second
quadrupole in a quadrupole instrument, it is called MS/MS or Tandem MS. Tandem
mass spectrometry (MS/MS) is a more powerful technique to quantitate low levels of
target compounds in the presence of a high sample matrix background.

The first quadrupole (Q1) is connected with a collision cell (q2) and another
quadrupole (Q3). Both quadrupoles can be used in scanning or static mode,
depending on the type of MS/MS analysis being performed. Types of analysis
include product ion scan, precursor ion scan, Single Reaction Monitoring (SRM) and
Multiple Reaction Monitoring (MRM) and Neutral Loss Scan. For example: When Q1
is in static mode (looking at one mass only as in SIM), and Q3 is in scanning mode,
one obtains a so-called product ion spectrum (also called "daughter spectrum").
From this spectrum, one can select a prominent product ion which can be the
product ion for the chosen precursor ion. The pair is called a "transition" and forms
the basis for SRM (MRM is sometimes used as term). SRM is highly specific and
virtually eliminates matrix background.

Applications
Environmental Monitoring and Cleanup

GC-MS is becoming the tool of choice for tracking organic pollutants in the
environment. The cost of GC-MS equipment has decreased significantly, and the
reliability has increased at the same time, which has contributed to its increased
adoption in environmental studies. There are some compounds for which GC-MS is
not sufficiently sensitive, including certain pesticides and herbicides, but for most
organic analysis of environmental samples, including many major classes of
pesticides, it is very sensitive and effective.

Criminal Forensics

GC-MS can analyze the particles from a human body in order to help link a criminal
to a crime. The analysis of fire debris using GC-MS is well established, and there is
even an established American Society for Testing Materials (ASTM) standard for fire
debris analysis. GCMS/MS is especially useful here as samples often contain very
complex matrices and results, used in court, need to be highly accurate.

Law Enforcement
GC-MS is increasingly used for detection of illegal narcotics, and may eventually
supplant drug-sniffing dogs.[1] It is also commonly used in forensic toxicology to find
drugs and/or poisons in biological specimens of suspects, victims, or the deceased.

Security

A post-September 11 development, explosive detection systems have become a

part of all US airports. These systems run on a host of technologies, many of them
based on GC-MS. There are only three manufacturers certified by the FAA to provide
these systems,[citation needed] one of which is Thermo Detection (formerly Thermedics),
which produces the EGIS, a GC-MS-based line of explosives detectors. The other two
manufacturers are Barringer Technologies, now owned by Smith's Detection
Systems and Ion Track Instruments, part of General Electric Infrastructure Security
Systems.

Food, Beverage and Perfume Analysis

Foods and beverages contain numerous aromatic compounds, some naturally

present in the raw materials and some forming during processing. GC-MS is
extensively used for the analysis of these compounds which include esters, fatty
acids, alcohols, aldehydes, terpenes etc. It is also used to detect and measure
contaminants from spoilage or adulteration which may be harmful and which is
often controlled by governmental agencies, for example pesticides.

Astrochemistry

Several GC-MS have left earth. Two were brought to Mars by the Viking program.[4]
Venera 11 and 12 and Pioneer Venus analysed the atmosphere of Venus with GC-
MS.[5] The Huygens probe of the Cassini-Huygens mission landed one GC-MS on
Saturn's largest moon, Titan.[6] The material in the comet 67P/Churyumov-
Gerasimenko will be analysed by the Rosetta mission with a chiral GC-MS in 2014. [7]

Medicine

In combination with isotopic labeling of metabolic compounds, the GC-MS is used for
determining metabolic activity. Most applications are based on the use of 13C as the
labeling and the measurement of 13C/12C ratios with an isotope ratio mass
spectrometer (IRMS); an MS with a detector designed to measure a few select
ions and return values as ratios.

NMR

High resolution nuclear magnetic resonance (NMR) spectroscopy is a very powerful

tool for the structural identification of xenobiotic metabolites in complex biological
matrices such as plasma, urine and bile. However, these fluids are dominated by
thousands of signals resulting from endogenous metabolites and it is advantageous
when investigating drug metabolites in such matrices to simplify the spectra by
including a separation step in the experiment by directly-coupling HPLC and NMR.
Naproxen (6-methoxy-alpha-methyl-2-naphthyl acetic acid) is administered as the
S-enantiomer and is metabolised in vivo to form its demethylated metabolite which
is subsequently conjugated with beta-D-glucuronic acid as well as with sulfate.
Naproxen is also metabolised by phase II metabolism directly to form a glycine
conjugate as well as a glucuronic acid conjugate at the carboxyl group. In the
present investigation, the metabolism of naproxen was investigated in urine
samples with a very simple sample preparation using a combination of directly-
coupled HPLC-1H NMR spectroscopy and HPLC-mass spectrometry (MS). A buffer
system was developed which allows the same chromatographic method to be used
for the HPLC-NMR as well as the HPLC-MS analysis. The combination of these
methods is complementary in information content since the NMR spectra provide
evidence to distinguish isomers such as the type of glucuronides formed, and the
HPLC-MS data allow identification of molecules containing NMR-silent fragments
such as occur in the sulfate ester.

See also

• Liquid chromatography-mass spectrometry

• Ion mobility spectrometry-mass spectrometry

• Prolate trochoidal mass spectrometer

References

1. ^ Gohlke, R. S. (1959). "Time-of-Flight Mass Spectrometry and Gas-Liquid

Partition Chromatography". Analytical Chemistry 31: 535.
doi:10.1021/ac50164a024.

2. ^ Gohlke, R (1993). "Early gas chromatography/mass spectrometry". Journal

of the American Society for Mass Spectrometry 4: 367. doi:10.1016/1044-
0305(93)85001-E.

3. ^ "Optimizing the Analysis of Volatile Organic Compounds - Technical Guide"

Restek Corporation, Lit. Cat. 59887A

4. ^ The Development of the Viking GCMS

5. ^ V. A. Krasnopolsky, V. A. Parshev (1981). "Chemical composition of the

atmosphere of Venus". Nature 292: 610–613. doi:10.1038/292610a0.

6. ^ H. B. Niemann, S. K. Atreya, S. J. Bauer, G. R. Carignan, J. E. Demick, R. L.

Frost, D. Gautier, J. A. Haberman, D. N. Harpold, D. M. Hunten, G. Israel, J. I.
Lunine, W. T. Kasprzak, T. C. Owen, M. Paulkovich, F. Raulin, E. Raaen, S. H.
Way (2005). "The abundances of constituents of Titan’s atmosphere from the
 GCMS instrument on the Huygens probe". Nature 438: 77–9–784.
doi:10.1038/nature04122.

7. ^ Goesmann F, Rosenbauer H, Roll R, Bohnhardt H (2005). "COSAC onboard

Rosetta: A bioastronomy experiment for the short-period comet
67P/Churyumov-Gerasimenko". Astrobiology 5 (5): 622–631.
doi:10.1089/ast.2005.5.622.

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