Renuka, et al.

Int J Pharm 2015; 5(3): 911-917 ISSN 2249-1848

International Journal of Pharmacy

Journal Homepage: http://www.pharmascholars.com

Review Article CODEN: IJPNL6

LIQUID CHROMATOGRAPHY - MASS SPECTROMETRY AND ITS APPLICATIONS

Renuka Jajikore*, G. Ushasree, A. Ajitha, V. Umamaheswararao

Department of Pharmaceutical Analysis and Quality Assurance, CMR College of Pharmacy,

Secunderabad, India

*Corresponding author e-mail: [email protected]

ABSTRACT

Liquid chromatography is a fundamental separation technique in the life sciences and related fields of chemistry. An
LC-MS is an HPLC system with a mass spec detector. Liquid chromatography-mass spectrometry is now a routine
technique with the development of electrospray ionisation providing a simple and robust interface. It can be applied
to a wide range of biological molecules and the use of tandem MS and stable isotope internal standards allows
highly sensitive and accurate assays to be developed although some method optimisation is required to minimise ion
suppression effects.

Keywords: Liquid chromatography, mass spectrometers, electrospray ionisation

INTRODUCTION For most compounds, a mass spectrometer is more

sensitive and far more specific than all other LC
Liquid chromatography is a fundamental separation detectors. It can analyze compounds that lack a
technique in the life sciences and related fields of suitable chromospheres. It can also identify
chemistry. Unlike gas chromatography, which is components in unresolved chromatographic peaks,
unsuitable for non-volatile and thermally fragile reducing the need for perfect chromatography. Mass
molecules, liquid chromatography can safely separate spectral data complements data from other LC
a very wide range of organic compounds, from small- detectors. While two compounds may have similar
molecule drug metabolites to peptides and proteins. UV spectra or similar mass spectra, it is uncommon
Traditional detectors for liquid chromatography for them to have both. The two orthogonal sets of
include refractive index, electrochemical, data can be used to confidently identify, confirm, and
fluorescence, and ultraviolet-visible (UV-Vis) quantify compounds.
detectors. Some of these generate two-dimensional Some mass spectrometers have the ability to perform
data; that is, data representing signal strength as a multiple steps of mass spectrometry on a single
function of time. Others, including fluorescence and sample. They can generate a mass spectrum, select a
diode array UV-Vis detectors, generate three- specific ion from that spectrum, fragment the ion, and
dimensional data. Three-dimensional data include not generate another mass spectrum; repeating the entire
only signal strength but spectral data for each point in cycle many times. Such mass spectrometers can
time. Mass spectrometers also generate three- literally deconstruct a complex molecule piece by
dimensional data. In addition to signal strength, they piece until its structure is determined.
generate mass spectral data that can provide valuable
information about the molecular weight, structure, INSTRUMENTATION
identity, quantity, and purity of a sample. Mass
spectral data add specificity that increases confidence Mass spectrometers work by ionizing molecules and
in the results of both qualitative and quantitative then sorting and identifying the ions according to
analyses. their mass-to-charge (m/z) ratios. Two key
components in this process are the ion source, which

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generates the ions, and the mass analyzer, which sorts concentration in the droplets increases. Eventually,
the ions. Several different types of ion sources are the repulsive force between ions with like charges
commonly used for LC/MS. Each is suitable for exceeds the cohesive forces and ions are ejected
different classes of compounds. Several different (desorbed) into the gas phase. These ions are
types of mass analyzers are also used. Each has attracted to and pass through a capillary sampling
advantages and disadvantages depending on the type orifice into the mass analyzer.
of information needed. Some gas-phase reactions, mostly proton transfer and
charge exchange, can also occur between the time
ions are ejected from the droplets and the time they
reach the mass analyzer. Electrospray is especially
useful for analyzing large biomolecules such as
proteins, peptides, and oligonucleotides, but can also
analyze smaller molecules like benzodiazepines and
sulfated conjugates. Large molecules often acquire
more than one charge. Thanks to this multiple
charging, electrospray can be used to analyze
molecules as large as 150,000 u even though the mass
range (or more accurately mass-to-charge range) for a
typical LC/MS instruments is around 3000 m/z. For
example: 100,000 u / 10 z = 1,000 m/z When a large
Fig 1 : Instrumentation molecule acquires many charges, a mathematical
process called deconvolution is often used to
Ion Sources determine the actual molecular weight of the analyte.
Much of the advancement in LC/MS over the last ten
years has been in the development of ion sources and Atmospheric pressure chemical ionization
techniques that ionize the analyte molecules and In APCI, the LC eluent is sprayed through a heated
separate the resulting ions from the mobile phase. (typically 250°C – 400°C) vaporizer at atmospheric
Earlier LC/MS systems used interfaces that either did pressure. The heat vaporizes the liquid. The resulting
not separate the mobile phase gas-phase solvent molecules are ionized by electrons
molecules from the analyte molecules (direct liquid discharged from a corona needle. The solvent ions
inlet, thermo spray) or did so before ionization then transfer charge to the analyte molecules through
(particle beam). The analyte molecules were then chemical reactions (chemical ionization). The analyte
ionized in the mass spectrometer under vacuum, often ions pass through a capillary sampling orifice into the
by traditional electron ionization. These approaches mass analyzer. APCI is applicable to a wide range of
were successful only for a very limited number of polar and nonpolar molecules. It rarely results in
compounds. The introduction of atmospheric pressure multiple charging so it is typically used for molecules
ionization (API) techniques greatly expanded the less than 1,500 u. Due to this, and because it involves
number of compounds that can be successfully high temperatures, APCI is less well-suited than
analyzed by LC/MS. In atmospheric pressure electrospray for analysis of large biomolecules that
ionization, the analyte molecules are ionized first, at may be thermally unstable. APCI is used with
atmospheric pressure. The analyte ions are then normal-phase chromatography more often than
mechanically and electro statically separated from electrospray is because the analytes are usually
neutral molecules. Common atmospheric pressure nonpolar.
ionization techniques are:
• Electro spray ionization (ESI) Atmospheric pressure photoionization
• Atmospheric pressure chemical ionization (APCI) Atmospheric pressure photoionization (APPI) for
• Atmospheric pressure photo ionization (APPI) LC/MS is a relatively new technique. As in APCI, a
Electro spray relies in part on chemistry to generate vaporizer converts the LC eluent to the gas phase. A
analyte ions in solution before the analyte reaches the discharge lamp generates photons in a narrow range
mass spectrometer. The LC eluent is sprayed of ionization energies. The range of energies is
(nebulized) into a chamber at atmospheric pressure in carefully chosen to ionize as many analyte molecules
the presence of a strong electrostatic field and heated as possible while minimizing the ionization of
drying gas. The electrostatic field causes further solvent molecules. The resulting ions pass through a
dissociation of the analyte molecules. The heated capillary sampling orifice into the mass analyzer.
drying gas causes the solvent in the droplets to APPI is applicable to many of the same compounds
evaporate. As the droplets shrink, the charge that are typically analyzed by APCI. It shows

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particular promise in two applications, highly mass analyzers have a wide mass range and can be
nonpolar compounds and low flow rates very accurate in their mass measurements.

Mass Analyzers Ion trap

Although in theory any type of mass analyzer could An ion trap mass analyzer consists of a circular ring
be used for LC/MS, four types: electrode plus two end caps that together form a
• Quadrupole chamber. Ions entering the chamber are “trapped”
• Time-of-flight there by electromagnetic fields. Another field can be
• Ion trap applied to selectively eject ions from the trap. Ion
• Fourier transform-ion cyclotron resonance (FT-ICR traps have the advantage of being able to perform
or FT-MS) multiple stages of mass spectrometry without
are used most often. Each has advantages and additional mass analyzers.
disadvantages depending on the requirements of a
particular analysis. Fourier transform-ion cyclotron resonance (FT-
ICR)
An FT-ICR mass analyzer (also called FT-MS) is
another type of trapping analyzer. Ions entering a
chamber are trapped in circular orbits by powerful
electrical and magnetic fields. When excited by a
radio-frequency (RF) electrical field, the ions
generate a timedependent current. This current is
converted by Fourier transform into orbital
frequencies of the ions which correspond to their
mass-tocharge ratios. Like ion traps, FT-ICR mass
analyzers can perform multiple stages of mass
Fig 2: Quadrupole spectrometry without additional mass analyzers. They
also have a wide mass range and excellent mass
Quadrupole resolution. They are, however, the most expensive of
A quadrupole mass analyzer consists of four parallel the mass analyzers.
rods arranged in a square. The analyte ions are
directed down the center of the square. Voltages Collision-Induced Dissociation and Multiple-Stage
applied to the rods generate electromagnetic fields. MS
These fields determine which mass-to-charge ratio of The atmospheric pressure ionization techniques
ions can pass through the filter at a given time. discussed are all relatively “soft” techniques. They
Quadrupoles tend to be the simplest and least generate primarily:
expensive mass analyzers. Quadrupole mass • Molecular ions M+ or M–
analyzers can operate in two modes: • Protonated molecules [M + H]+
• Scanning (scan) mode • Simple adduct ions [M + Na]+
• Selected ion monitoring (SIM) mode • Ions representing simple losses such as the loss of a
In scan mode, the mass analyzer monitors a range of water [M + H – H2O]+
mass-to-charge ratios. In SIM mode, the mass The resulting molecular weight information is very
analyzer monitors only a few massto-charge ratios. valuable, but complementary structural information is
SIM mode is significantly more sensitive than scan often needed. To obtain structural information,
mode but provides information about fewer ions. analyte ions are fragmented by colliding them with
Scan mode is typically used for qualitative analyses neutral molecules in a process known as
or for quantitation when all analyte masses are not collisioninduced dissociation (CID) or collisionally
known in advance. SIM mode is used for quantitation activated dissociation (CAD). Voltages are applied to
and monitoring of target compounds. the analyte ions to add energy to the collisions and
create more fragmentation.
Time-of-flight
In a time-of-flight (TOF) mass analyzer, a uniform CID in single-stage MS
electromagnetic force is applied to all ions at the CID is most often associated with multistage mass
same time, causing them to accelerate down a flight spectrometers where it takes place between each
tube. Lighter ions. travel faster and arrive at the stage of MS filtering, but CID can also be
detector first, so the mass-to-charge ratios of the ions accomplished in single-stage quadrupole or time-of-
are determined by their arrival times. Time-offlight flight mass spectrometers. In single-stage mass

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spectrometers, CID takes place in the ion source and Adapting LC Methods
is thus sometimes called source CID or in-source Early LC/MS systems were limited by fundamental
CID. Analyte (precursor) ions are accelerated and issues like the amount of LC eluent the mass
collide with residual neutral molecules to yield spectrometer could accept. Significant changes to LC
fragments called product ions. The advantage of methods were often required to adapt them to MS
performing CID in singlestage instruments is their detectors. Modern LC/MS systems are more versatile.
simplicity and relatively low cost. The disadvantage Many mass spectrometers can accept flow rates of up
is that ALL ions present are fragmented. There is no to 2 ml/min. With minor modifications, the same
way to select a specific precursor ion so there is no instruments can also generate good results at
sure way to determine which product ions came from microliter and nanoliter flow rates. Ion sources with
which precursor ion. The resulting spectra may orthogonal (off-axis) nebulizers are more tolerant of
include mass peaks from background ions or nonvolatile buffers and require little or no
coeluting compounds as well as those from the adjustment, even with differing solvent compositions
analyte of interest. This tradeoff may be acceptable and flow rates. Changes to LC methods required for
when analyzing relatively pure samples, but does not modern LC/MS systems generally involve changes in
give good results if chromatographic peaks are not sample preparation and solution chemistry to:
well resolved or background levels are high. • Ensure adequate analyte concentration
• Maximize ionization through careful selection of
CID and multiple-stage solvents and buffers
MS Multiple-stage MS (also called tandem MS or • Minimize the presence of compounds that compete
MS/MS or MSn ) is a powerful way to obtain for ionization or suppress signal through gas-phase
structural information. In triple-quadrupole or reactions
quadrupole/quadrupole/time-of-flight instruments Sample preparation
(see Figure 16), the first quadrupole is used to select Sample preparation generally consists of
the precursor ion. CID takes place in the second stage concentrating the analyte and removing compounds
(quadrupole or octopole), which is called the collision that can cause background ions or suppress
cell. ionization. Examples of sample preparation include:
The third stage (quadrupole or TOF) then generates a • On-column concentration to increase analyte
spectrum of the resulting product ions. It can also concentration
perform selected ion monitoring of only a few • Desalting to reduce the sodium and potassium
product ions when quantitating target compounds. In adduct formation that commonly occurs in
ion trap and FT-ICR mass spectrometers, all ions electrospray
except the desired precursor ion are ejected from the • Filtration to separate a lowmolecular-weight drug
trap. The precursor ion is then energized and collided from proteins in plasma, milk, or tissue
to generate product ions. The product ions can be Ionization chemistry
ejected to generate a mass spectrum, or a particular Because formation of analyte ions in solution is
product ion can be retained and collided to obtain essential to achieving good electrospray results,
another set of product ions. This process can be careful attention must be paid to proper solution
sequentially automated so that the most abundant chemistry. For electrospray:
ion(s) from each stage of MS are retained and • Select more volatile buffers to reduce the buildup of
collided. This is a very powerful technique for salts in the ion source
determining the structure of molecules. It is also a • Adjust solvent pH according to the polarity of ions
powerful technique for obtaining peptide mass desired and the pH of the sample
information that relates to the sequence of amino • Use solvents that have low heats of vaporization
acids in a peptide. A major advantage of multiple- and low surface tensions to enhance ion desorption
stage MS is its ability to use the first stage of MS to • Make sure that gas-phase reactions do not neutralize
discard nonanalyte ions. Sample cleanup and ions through proton transfer or ion pair reactions
chromatographic separation become much less If pH adjustments interfere with proper
critical. With relatively pure samples, it is quite chromatography, postcolumn modification of the
common to do away with chromatographic separation solvent may be a good solution. This can improve
altogether and infuse samples directly into the mass MS response without compromising chromatography.
spectrometer to obtain product mass spectra for Solution chemistry is less critical for APCI operation
characterization or confirmation. because ionization occurs in the gas phase, not the
liquid phase, but solvent selection can still have a
significant effect on APCI analyte signal response.
• Select more volatile solvents

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• Select solvents with a lower charge affinity than the Determining the molecular weight of green
analyte fluorescent protein
• Protic solvents generally work better than nonprotic Green fluorescent protein (GFP) is a 27,000- Dalton
solvents for positive ion mode protein with 238 amino acids. It emits a green light
• For negative ionization, solvents that readily when excited by ultraviolet light. During electrospray
capture an electron must be used ionization, GFP acquires multiple charges. This
• Ammonium salts in the mobile phase can cause allows it to be analyzed by a mass spectrometer with
ammonium adduct formation a relatively limited mass (mass-to-charge) range.
Vaporizer temperature also affects APCI ionization Mass deconvolution is then used to determine the
results. The temperature must be hot enough to molecular weight of the protein. The upper part of the
vaporize the solvent but not so hot as to cause display in Figure 23 shows the full scan mass
thermal degradation of the analyte molecules spectrum of GFP. The pattern of mass spectral peaks
is characteristic of a multiply charged analyte. Each
Capillary LC/MS and CE/MS peak represents the molecule with a different number
Capillary LC and capillary electrophoresis (CE) are of charges. The lower display is a deconvoluted mass
commonly used alongside HPLC. Capillary LC at spectrum generated by the data system for the singly
microliter to nanoliter flow rates often provides better charged analyte.
sensitivity than conventional flow rates for extremely
small sample quantities. It is commonly used for Structural Determination
protein and peptide analysis but has also proven very Another fundamental application of LC/MS is the
useful for the analysis of small quantities of drugs. determination of information about molecular
Capillary electrophoresis (CE) is a technique with a structure. This can be in addition to molecular weight
high separation efficiency and the added benefit of information or instead of molecular weight
being able to handle very complex matrices. It has information if the identity of the analyte is already
proven useful for such diverse samples as: peptides, known.
drugs of abuse, drugs in natural products, flavanoids,
and aromatic amines. With specialized nebulizers and Structural determination of ginsenosides using
method adjustments, electrospray ionization can be MSn analysis
used for both capillary LC/MS and CE/MS. The mass Ginseng root, a traditional Chinese herbal remedy,
spectrometry benefits of selectivity and sensitivity are contains more than a dozen biologically active
available to both of these separation techniques. saponins called ginsenosides. Since most
ginsenosides contain multiple oligosaccharide chains
APPLICATIONS at different positions in the molecule, structural
elucidation of these compounds can be quite
LC/MS is suitable for many applications, from complicated. MSn analysis in an ion trap mass
pharmaceutical development to environmental spectrometer permits multiple stages of precursor ion
analysis. Its ability to detect a wide range of isolation and fragmentation. This stepwise
compounds with great sensitivity and specificity has fragmentation permits individual fragmentation
made it popular in a variety of fields. pathways to be followed and provides a great deal of
structural information. Figure 24 shows the full scan
Molecular Weight Determination mass spectrum from a direct infusion of the
One fundamental application of LC/MS is the ginsenoside Rb1. The most prominent feature is the
determination of molecular weights. This information sodium adduct ion [M + Na]+ at m/z 1131.7. MS/MS
is key to determining identity. of m/z 1131.7 yields a product ion at m/z 789.7
corresponding to cleavage of a single glycosidic bond
Differentiation of similar octapeptides (Figure 25). Subsequent isolation and fragmentation
The only difference in the sequence is at the C- of m/z 789.7 (Figure 26) yields two products: a more
terminus where one peptide has threonine and the abundant ion at m/z 365.1 corresponding to loss of
other has threonine amide. The smaller fragments are the oligosaccharide chain (–Glc 2 –Glc), and a less
identical in the two spectra, indicating that large abundant ion at m/z 627.5 representing the loss of a
portions of the two peptides are very similar. The deoxyhexose sugar.
larger fragments contain the differentiating peptides.
Pharmaceutical Applications
Rapid chromatography of benzodiazepines
The information available in a mass spectrum allows
some compounds to be separated even though they

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are chromatographically unresolved. In this example, full scan product ion (MS/MS) spectrum was acquire
a series of benzodiazepines was analyzed using both from the most abundant relevant ion in each mass
UV and MS detectors. The UV trace could not be scan throughout the entire run. All MS and MS/MS
used for quantitation, but the extracted ion data were acquire from a single analysis.
chromatograms from the MS could be used. The Protein identification was accomplished using
mass spectral information provides additional MASCOT software that correlated the uninterpreted
confirmation of identity. Chlorine has a characteristic MS/MS data with sequences in a database. Figure 30
pattern because of the relative abundance of the two demonstrates the excellent match between the
most abundant isotopes. the triazolam spectrum observed MS/MS spectrum from the most abundant
shows that triazolam has two chlorines and the ion (m/z 807.2) in the chromatographic peak at 17.55
diazepam spectrum shows that diazepam has only minutes and the theoretical y-ion series predicted for
one. a tryptic peptide from human apolipoprotein, one of
the proteins in the sample mixture.
Identification of bile acid metabolites
The MSn capabilities of the ion trap mass Clinical Applications
spectrometer make it a powerful tool for the High-sensitivity detection of trimipramine and
structural analysis of complex mixtures. Intelligent, thioridazine
data-dependent acquisition techniques can increase For most compounds, MS is more sensitive than
ion trap effectiveness and productivity. They permit other LC detectors. Trimipramine is a tricyclic
the identification of minor metabolites at very low antidepressant with sedative properties. Thioridazine
abundances from a single analysis. One application is is a tranquilizer. Figure 31 shows these compounds in
the identification of metabolic products of drug a urine extract at a level that could not be detected by
candidates. UV. To get the maximum sensitivity from a single-
This example uses the in vitro incubation of the bile quadrupole mass spectrometer, the analysis was done
acid deoxycholic acid with rat liver microsomes to by selected ion monitoring.
simulate metabolism of a drug candidate. Intelligent,
data-dependent acquisition was used to select the two Food Applications
most abundant, relevant ions in each MS scan. These Identification of aflatoxins in food
precursor ions were automatically fragmented and Aflatoxins are toxic metabolites produced in foods by
full scan product ion spectra collected. Figure 28A certain fungi. Figure 32 shows the total ion
shows the base peak chromatogram. Figure 28B chromatogram from a mixture of four aflatoxins.
shows the extracted ion chromatogram of the [M-H]– Even though they are structurally very similar, each
ion at m/z 407 aflatoxin can be uniquely identified by its mass
corresponding to a predicted minor metabolite (cholic spectrum .
acid) that eluted at 9.41 minutes. The full scan Determination of vitamin D3 in poultry feed
MS/MS product spectrum (Figure 28C) from the ion supplements using MS3
at m/z 407 confirms the identity. Significant time was Vitamin D is an essential constituent in human and
saved because the confirming MS/MS product ion animal nutrition. Livestock diets deficient in vitamin
spectra were acquired automatically in the same run D can cause growth abnormalities. Traditional
as the full scan MS data. GC/MS analysis methods for vitamin D3 in feed
extracts require extensive and time-consuming
Biochemical Applications sample preparation and derivatization prior to
Rapid protein identification using capillary analysis. Atmospheric pressure chemical ionization
LC/MS/MS and database searching with ion trap detection provides a sensitive analytical
Traditional methods of protein identification method without the need for extensive sample
generally require the isolation of individual proteins preparation and derivatization. Further, the multiple-
by two-dimensional gel electrophoresis. The stage MS capability of the ion trap eliminates the
combination of capillary LC/MS/MS with intelligent, need for chromatographic separation, greatly
data-dependent acquisition and probability-based speeding analyses. Flow injection analysis of a
database searching makes it possible to rapidly poultry feed extract yields a peak at m/z 385
identify as many as 100 proteins in a single analysis. suggesting the presence of vitamin D3. Isolation and
In this example, a capillary LC and ion trap mass fragmentation of the precursor ion at m/z 385 is
spectrometer were used to acquire data from a inconclusive. The full scan product ion spectrum
mixture of five tryptically digested proteins at a shows a prominent peak at m/z 367 representing the
concentration of 1 pmol/µl each (Figure 29). Using loss of a single water molecule but little other
intelligent, automated datadependent acquisition, a fragmentation .

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Environmental Applications analysis caused by a coeluting compound. Based on

Detection of phenylurea herbicides the MS/MS spectrum of the coeluting compound, a
Many of the phenylurea herbicides are very similar possible structure was assigned
and difficult to distinguish with a UV detector
(Figure 36). Monuron and diuron have one benzene CE/MS Applications
ring and differ by a single chlorine. Chloroxuron has Analysis of peptides using CE/MS/MS
two chlorines and a second benzene ring attached to Capillary electrophoresis (CE) is a powerful
the first by an oxygen. The UV-Vis spectra are complement to liquid chromatography. Different
similar for diuron and monuron, but different for selectivity and higher chromatographic resolution are
chloroxuron. When analyzed using electrospray its biggest advantages when analyzing clean samples
ionization on an LC/MS system, each compound has such as synthetic peptides. When analyzing ppm-
a uniquely identifiable mass spectrum. levels of analytes in complex matrices, minimal
sample preparation and short analysis times enable
Detection of low levels of carbaryl in food high throughput.
Pesticides in foods and beverages can be a significant CE/MS with an ion trap mass spectrometer is
route to human exposure. Analysis of the carbamate demonstrated using a standard peptide mix. The data-
pesticide carbaryl in extracts of whole food by ion dependent acquisition capability of the ion trap
trap LC/MS/MS proved more specific than previous triggers MS/MS on the most intense ion in each MS
analyses by HPLC fluorescence and single- scan. Figure 39 shows total ion electropherogram
quadrupole mass spectrometry. The protonated (TIE) and the base peak electropherogram (BPE).
carbaryl molecule (m/z 202) was detected in full scan Drops in the TIE indicate where MS/MS spectra were
mode using positive ion electrospray. A product ion acquired automatically. The averaged mass spectrum
at m/z 145 (Figure 37) generated by collisioninduced of peak 4 (Figure 40) shows a singly charged
dissociation provided confirmation of carbaryl and molecule with m/z of 574.3. This is confirmed as
was used for subsequent quantitative analysis. Met-enkephalin (molecular weight 573.7) by the
Ion trap analysis was more sensitive than previous examination of the MS/MS mass spectrum. The
analysis using a single-quadrupole mass spectrometer MS/MS spectrum shows all of the b- and y-series
operating in scanning mode and more sensitive than fragments within the scan range as well as fragments
fluorescence detection. Ion trap LC/MS/MS also from other series.
confirmed false positives in the HPLC fluorescence

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