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96 | P a g e International Standard Serial Number (ISSN): 2319-8141
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International Journal of Universal Pharmacy and Bio Sciences 4(5): September-October 2015
INTERNATIONAL JOURNAL OF UNIVERSAL
PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.093***
ICV 5.13***
Pharmaceutical Sciences REVIEW ARTICLE …………!!!
A REVIEW ON CAPILLARY ELECTROPHORESIS MASS SPECTROMETRY
Ch. Aparna* and D. Gowrisankar
University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra
Pradesh, Pin code - 530003.
KEYWORDS:
CEMS, modes of
separation interfaces
applications.
For Correspondence:
Ch. Aparna*
Address:
University College of
Pharmaceutical Sciences,
Andhra University,
Visakhapatnam, Andhra
Pradesh, Pin code -
530003.
ABSTRACT
Capillary electrophoresis (CE) has wide range of applicability for the
separation of the compounds depending on their charge and mass. It is
the choice of separation technique for large and small molecules
especially biological samples. Coupling of CE with mass spectrometry
increases the applicability of the technique by providing
characterization of the samples separated by CE. Capillary
electrophoresis mass spectrometry (CEMS) became popular analytical
technique due to efficient and selective separation in combination with
powerful detection allowing identification and detailed
characterization. Here, detailed information about interfaces used in
CEMS, modes of separations and applications are reviewed and
discussed.
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INTRODUCTION:
CEMS (Capillary Electrophoresis-Mass Spectrometry) is an analytical technique combined the uses of
liquid separation process of capillary electrophoresis with mass spectrometry. CEMS provide high
separation efficiency and molecular mass information in a single analysis. Since its introduction in
1987, new developments and application has made CEMS powerful separation and identification
technique. Use of CEMS has increased for protein and peptides analysis and other biomolecules. The
original interface between capillary zone electrophoresis and mass spectrometry was developed in
1987 by Richard D. Smith and coworkers at Pacific Northwest National Laboratory, and who also later
were involved in development of interfaces with other CE variants, including capillary
isotachophoresis and capillary isoelectric focusing [1]
.
Fig.1. Block diagram of Capillary Electrophoresis mass spectrometry
Principle:
CEMS work by the both the principles of capillary electrophoresis and mass spectrometry. The
separated molecules are converted into ions by the interface present between the capillary
electrophoresis and mass spectrometer. Capillary electrophoresis is a separation technique which
uses high electric field to produce electro osmotic flow for separation of ions. Analytes migrate
from one end of capillary to other based on their charge, viscosity and size. Higher the electric
field, greater is the mobility. Mass spectrometry is an analytical technique that identifies chemical
species depending on their mass-to-charge ratio. During the process, an ion source will convert
molecules coming from CE to ions that can then be manipulated using electric and magnetic field.
The separated ions are then measured using a detector [1]
.
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Capillary:
The capillary is the most important part of the system. The usual range of inner diameters is from
20–100 μm. Typically the capillaries that are used in CE are circular in cross-section. However,
capillaries with square cross-sections have been produced but to date they have not been widely
used. Capillaries were introduced into electrophoresis as an anti-convective and heat controlling
innovation. In wide tubes thermal gradients cause band mixing and loss of resolution.
Many materials have been suggested and tested for the construction of capillaries. These include
fused silica, borosilicate glass, and polytetrafluoroethylene (Teflon). Fused silica is now the
preferred material for the construction of capillaries. The silica used is of very high purity, similar
to that used to produce silicon chips for electronic components. Tubes of silica are heated to over
1000°C, and then stretched to produce the final dimensions. Fused silica capillaries are extremely
brittle. To facilitate handling they are coated with a layer of polyimide 10–25 μm thick, rendering
the capillary very flexible. The thickness of the wall of the capillary and the polyimide coating is
generally much greater than the internal diameter of the capillary. The thickness of the wall is
critical, because the heat that is produced when an electrical current passes through the electrolyte
must be removed through this wall. Reducing the wall thickness increases the fragility of the
capillary at the same time as it increases the heat transfer capacity [2]
.
Surface modifications:
The inner surface of a capillary is an extremely important factor in CEMS because the sample of
interest is in direct contact with the inner wall of the capillary. The fused silica inner wall is
naturally negative due to ionization of the free silanol groups at pH above 3.0, which then exist in
deprotonated anionic form. The ionized silica wall thus shows a tendency to interact strongly with
positively charged analytes. Such adsorption leads to fluctuation in the EOF and subsequently
irreproducible migration times, severe band broadening, low recovery, decreased sensitivity and
reduced separation efficiency. Therefore, modification of the inner wall of fused silica capillaries is
highly beneficial for reducing analyte wall interaction. Moreover, capillary wall modifications are
advantageous for alteration of the EOF to achieve rapid separation, to increase resolution,
reproducibility and to improve selectivity. In some instances, the adjustments of the pH of the
running buffer to extreme pH values that give either a highly negatively or positively charged
capillary wall were used. For instance, the electrostatic interaction of proteins with the silica
surfaces is affected by the pH. However, even if the net charge of proteins is the same as the net
charge of the capillary surface, hydrophobic domains can still interact and cause problems. In
addition, the use of pH to control such adsorption effects has in many cases some limitations, such
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as protein aggregation. Other alternatives used to minimize analyte adsorption on the silica
capillaries are high-ionic strength buffers or buffer additives such as amines, low conductive
zwitterions or alkali metal salts. However, for CE-MS, high amount of salts in BGE (background
electrolyte) are not desirable as this causes loss of MS sensitivity. Additionally, high amount of
salts causes Joule heating, due to the increase in current, leading to peak distortion.
Covalent Cationic Coatings:
Covalent coupling to the ionized silanol group can be achieved either by using neutral or charged
coatings. The most commonly used are polyacrylamide (PAA) and polyvinlyalcohol (PVA). The
non-ionic nature of the neutral coating eliminates the EOF. The migration of the analytes therefore
depends only on the electrophoretic mobility, which in turn prevents detection of both acidic and
basic proteins in the same run. Other limitations include instability of the siloxane bond (Si-O-C) at
neutral pH and the coating process (silanization) is often time-consuming, involving multi-step
processes which may introduce problems with irreproducibility. The major concern is the difficulty
to regenerate the coatings, especially when dealing with complex biological samples. Such
problems can be circumvented by using charged coatings (positive or negative) covalently attached
to the silica wall, which generally give high EOF. For cationic coatings, the pH of the BGE should
generally be low, whereas for anionic coatings high pH is recommended. With many covalent
cationic coatings, the capillary can rapidly be regenerated within a few minutes by rinsing it with
the coating solution.
Electrostatic Cationic Coatings:
Non-covalent modifications are usually prepared by flushing the capillary with the adsorbed
coating reagent or by adding a certain amount to the BGE to continuously coat the capillary wall.
Different types of amines, surfactants and polymers have been applied, which are reversibly bound
to the negatively charged silica wall. Based on the type of coating used, the negative charge of a
silica surface is reduced, neutralized or even reversed. Wide variety of noncovalent coatings have
been reported that have been used successfully for CE separations including for instance;
polybrene, polyethylenimine poly (methoxyethoxyethyl) ethylenimine, poly
(diallymethylammonium chloride), Cetyltrimethylammonium bromide and polyargenine. However,
from a CE-MS perspective, the presence of additives in the BGE, or bleeding of some adsorbed
polymers, contaminate the ion source and cause ion suppression which in turn decreases the
sensitivity [3]
.
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Sample Introduction Techniques:
Samples are introduced into the capillary for separation by two different methods. They are Electro
kinetic injection and Hydrodynamic (Vacuum or Pressure) Injection.
Electro kinetic injection works when the capillary is placed into the catholyte on one end and into
the analyte (containing the sample to be analyzed) on the other end. When a voltage is applied, the
EOF moves from the tip of the capillary to the end of the capillary. A siphoning effect occurs,
dragging a representative sample into the capillary. Also, ions begin moving into the capillary from
the buffer solution due to electrophoretic mobility as part of the sample loading. This can be an
advantage when trying to analyze small concentrations of these ions. These injections usually last
for 1-5 seconds.
Fig.2 Electro kinetic injection
Hydrodynamic injection works when a pressure is applied at one end of the capillary or a vacuum
is applied. The pressure differential between the two opposite sides of the capillary will make liquid
move into the capillary.
Fig.3 Hydrodynamic injection
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Modes of Separation
Fig. 4 modes of separations in CEMS
Capillary Zone Electrophoresis (CZE)
CZE is characterized by the use of open capillaries and relatively low viscosity buffer systems.
Analyte molecules move from one end of the capillary to the other according to the vector sum of
electrophoresis and electroosmotic mobility. CZE is the most widespread mode of CE. It has been
used for analytes as diverse as sodium ions, drugs, and protein molecules. Analyte species can be
separated by CZE if they migrate at different velocities in the electrical field [4]
.
Fig: 5 Capillary zone electrophoresis
Capillary Gel Electrophoresis (CGE)
Capillary gel electrophoresis (CGE) is separation based on viscous drag. In this mode the capillary
is filled with a gel or viscous solution. EOF is often suppressed so that the migration of the analytes
is solely by electrophoresis. Larger molecules tend to be retarded more by the viscous separation
medium than are smaller molecules, so that the separation is effectively based on the molecular
size. This is the method of choice for molecules that differ in size. For example, DNA molecules
can vary greatly in length, but the charge per unit length is quite constant. In a pure CZE separation,
all the molecules move at very nearly the same velocity and no separation results. In a viscous
medium, the longer molecules are retarded more than shorter molecules.
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Micellar Electro kinetic chromatography (MEKC)
Electrophoresis is not possible for analytes that are not charged. In order to analyze such analytes, it
is necessary to employ some agent in the separation buffer that will transport them through the
capillary. The most commonly used mode of CE for these analytes is MEKC. In this technique a
suitable charged detergent, such as SDS (sodium dodecyl sulphate), is added to the separation
buffer in a concentration sufficiently high to allow the formation of micelles. These micelles are
arrangements of detergent molecules that have a hydrophobic inner core and a hydrophilic outer
surface. Micelles are dynamic and constantly form and break apart. For any given analyte, there is a
probability that the molecules of that analyte will associate within the micelle at any given time.
This probability is the same as the partition coefficient in classical chromatography. When
associated with the micelle, the analyte molecule will migrate at the velocity of the micelle. When
not in the micelle, the analyte molecule will migrate with the EOF. Differences in the time that
analytes spend in the micellar phase will determine the separation. MEKC is useful for a wide range
of small molecules such as drugs, pesticides, and food additives that are not charged and are
sufficiently hydrophobic to associate with the micelle. While SDS is probably the most widely used
detergent for this purpose, cationic detergents such as TTAB can also be employed. Nonionic
detergents by themselves do not provide mobility to uncharged analytes, but in combination with
charged detergents they will modify the separation. Some detergents are useful in specific
applications. For example, sodium cholate is useful in the separation and analysis of a variety of
steroids. The micelles formed in this case are not the classical spherical shape but are probably
sodium cholate molecules arranged on each other like a stack of coins. Different uncharged steroids
differ in their tendencies to participate in these stacks [4]
.
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Fig.6 Micellar electrokinetic chromatography
Capillary Electrochromatography
Capillary electrochromatography (CEC) is a hybrid technique between liquid chromatography and
electrophoresis. It is a partitioning technique in which molecules distribute between a stationary and
a moving phase. As described for MEKC, different analytes will tend to associate to a greater or
lesser extent with the stationary phase, effecting a separation.
CEC capillaries are packed with particles like those used in HPLC columns. Unlike conventional
LC techniques, CEC uses electroosmotic flow to drive the mobile phase down the column. The
resulting plug flow improves the separation efficiency over that of the laminar flow of pressure
driven systems. As of this writing, most of the work done on CEC has used model systems, such as
polyaromatic hydrocarbons. CEC is very useful when coupled to mass spectroscopy (CEC-MS).
Chiral Electrophoresis
Chiral molecules are molecules that can exist in two stereo-specific forms. These chiral forms or
enantiomers are identical in molecular weight and chemical formula but differ in the arrangement
of the atoms in space. Separation of these enatiomeric forms depends on the tendency to associate
differentially with other chiral molecules known as chiral selectors. By incorporating a chiral
selector into the buffer, it is often possible to separate enantiomers of a chiral molecule. The
complex of the analyte and the selector will migrate at a different rate than will the analyte alone.
Because one of the two enantiomers associates more strongly with the selector than does the other
form a separation can be achieved. The most commonly employed chiral selector is cyclodextrin,
ring shaped carbohydrates made up of 6, 7, or 8 D-glucose subunits. Cyclodextrins may be
chemically modified to alter their hydrophobicity or charge. Uncharged cyclodextrins are not
suitable for the analysis of uncharged analytes since the complex will move with the EOF.
However, cyclodextrins modified to carry a charge by addition of sulfate groups, can serve both as
chiral selectors and as carrier molecules. Other molecules, such as the antibiotic vancomycin, have
also been employed as chiral selectors.
Chiral CE can be used to separate the enantiomeric forms of pharmaceuticals as well as natural
substances, such as amino acids. Impurities as small as 0.1% is easily detected by this method.
Capillary Isoelectric Focusing (cIEF)
Molecules that carry both positively and negatively charged groups exhibit, at a specific pH, an
equal number of positive and negative charges. At this pH, known as the isoelectric pH or pI, the
molecule, although charged, behaves as if it is neutral because its positive and negative charges
cancel each other. The molecule, therefore, has no tendency to migrate in an electrical field. In
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isoelectric focusing, special reagents called ampholytes are used to create a pH gradient within the
capillary. These ampholytes are mixtures of buffers with a range of pKa values. In an electrical
field, ampholytes will arrange themselves in order of pKa; this gradient is trapped between a strong
acid and a strong base. Analytes introduced into this gradient will migrate to the point where the pH
of the gradient equals their pI. At this point the analyte, having no net charge, ceases to migrate. It
will remain at that position so long as the pH gradient is stable, typically as long as the voltage is
applied. Capillary isoelectric focusing (cIEF) is used almost exclusively for the separation of
closely related protein species. Hemoglobin can be separated into several bands by this technique,
whereas separation by SDS-CGE usually results in a single form being identified. In this
application, the protein sample is mixed with the ampholyte solution and the mixture is pumped
into the capillary. When voltage is applied, the proteins and the ampholytes migrate to their
appropriate positions in the gradient.When focusing is complete, the proteins in the mixture are
distributed throughout the length of the capillary. In order to detect the proteins it is necessary to
mobilize them so that they pass by the detector in turn. There are two ways to accomplish this
mobilization. Pressure mobilization utilizes positive pressure applied to one end of the capillary to
drive the entire fluid column through the window. In order to prevent band distortion, this pressure
must be applied carefully, preferably while the voltage is still being applied. Chemical mobilization
requires that one end of the capillary be transferred to a salt solution after focusing has taken place.
Upon application of voltage, salt will migrate into the capillary, disrupting the pH gradient and
allowing the proteins to migrate past the detector by electrophoresis.
cIEF is also widely used for examining the distribution of carbohydrate isoforms of glycoproteins
[4].
Fig. 7 Capillary isoelectric focusing
Capillary Isotachophoresis (cITP)
In this technique the sample plug is introduced between two different buffers. One of these, the
leading electrolyte, has the highest mobility in the separation. The second, trailing electrolyte, has a
mobility lower than anything else does. The sign of the charge on the analytes and the buffers must
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be the same. When the voltage is applied the ions in the sample form discrete zones that are not
separated into peaks; one zone is adjacent to the next. The concentration of the analyte within a
zone is constant within that zone and the length of the zone is proportional to the concentration
within the zone.Capillary isotachophoresis (cITP) of peptides and proteins has been used with MS
detection.
Fig. 8 Capillary isotachophoresis
Interfacing CE with MS
The major problem faced when coupling CE to MS arises due to insufficient understanding of
fundamental processes when two techniques are interfaced. The separation and detection of
analytes can be improved with better interface. CE has been coupled to MS using various ionization
techniques like FAB (fast atom bombardment), ESI (electron spray ionization), MALDI (matrix
assisted laser desorption/ionization) and APCI (atmospheric pressure chemical ionization). The
most used ionization technique is ESI.
Fig. 9 Interfacing CE with Electrospray ionization-MS
Electrospray ionization interface
The first CE-MS interface had cathode end of CE capillary terminated within a stainless steel
capillary. An electrical contact was made at that point completing the circuit and initiating the
electrospray. This interface system had few drawbacks like mismatch in the flow rates of two
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systems. Since then, interface system has been improved to have continuous flow rate and good
electrical contact. At present, three types of interface system exist for CE/ESI-MS which are
discussed briefly [5]
.
Sheath less interface
CE capillary is coupled directly to ionization source in sheathless interface system. The capillary is
coated with conducting metal or gold or platinum wire is inserted into CE capillary to establish
suitable electrical connection. Since no sheath liquid is used, the system has high sensitivity, low
flow rates and minimum background. However, right choice of buffer solution has to be made
which is suitable for both CE separation and ESI operation.
Fig. 10 sheath less interface
Sheath Flow Interface
It is most widely used interface system. The electrical connection is established when the CE
separation liquid is mixed with sheath liquid flowing coaxially in a metal capillary tubing.
Commonly used sheath liquid is 1:1 mixture of water-methanol with 0.1% acetic acid or formic
acid. The system is more reliable and has wide selection range of separation electrolyte. There
might be some decrease in sensitivity due to sheath liquid.
Fig. 11 Sheath flow interface
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Liquid junction interface
This technique uses a stainless steel tee to mix separation electrolyte from CE capillary with
makeup liquid. The CE capillary and ESI needle are inserted through opposite sides of the tee and a
narrow gap is maintained. The electrical contact is established by makeup liquid surrounding the
junction between two capillaries. This system is easy to operate. However, the sensitivity is reduced
and the mixing of two liquids could degrade separation
Mass analyzers:
Most commonly used analyzers are quadrupole analyzer, time of flight analyzer, Fourier transform
ion cyclotron resonance (FTICR).
Quadrupole mass analyzer consists four rods, opposite rods connected electrically. Only ions of a
certain mass to charge ratio will reach the detector for a given ratio of voltages, other ions have
unstable trajectories and will collide with the rods.
Time of flight analyzer determined via a time measurement. Ions are accelerated by an electric field
of known strength. The time that it subsequently takes for the particle to reach a detector at a known
distance is measured.
FTICR determining the mass-to-charge ratio (m/z) of ions based on the cyclotron frequency of the
ions in a fixed magnetic field.
Factors effecting CEMS
Some of the factors which influence the separation process by CE there by effects the analysis by
mass are given below
1. Voltage: the separation time is universally proportional to applied voltage. However an
increase in the voltage can cause excessive heat production, giving rise to temperature and
viscosity gradients in the buffer inside the capillary which causes band broadening and
decreases resolution.
2. Temperature: the main effect of temperature is observed on buffer viscosity and electrical
conductivity, thus affecting migration velocity. In some cases an increase in capillary
temperature can cause conformational changes of some proteins modifying their migration
time and the efficiency of the separation. Temperature also effects the fragmentation pattern
of the separated molecules in MS.
3. Capillary: the length and internal diameter of the capillary affects the analysis time, the
efficiency of separations and the load capacity. Increasing both effective length and total
length can decrease the electric fields, at a constant voltage which will increase migration
time. The absorption of sample components on the capillary wall limits efficiency.
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Therefore methods to avoid these interactions should be considered in the development of
the separation method. This is critical in samples containing proteins. Strategies have been
devised to avoid adsorption of the proteins on the capillary wall. These strategies include
both the use of extreme pH and the absorption of positively charged buffer additives that
only require modification of the buffer composition. Other strategies include the coating of
the internal wall of the capillary with a polymer covalently bonded to the silica that prevents
interaction between the proteins and the negatively charged silica surface.
4. Buffer concentration: an increase in the buffer concentration at a given pH will decrease
electroosmotic flow and solute velocity.
5. Buffer pH: the pH of the buffer can affect separation by modifying the charge of the
analyte or other additives and by charging the EOF. For protein and peptide separation a
change in the pH of the buffer from above isoelectric point to below the pI changes the net
charge of the solute from negative to positive. An increase in the buffer pH generally
increases the EOF.
6. Organic solvents: organic modifiers such as Methanol, Acetonitrile and other are added to
the aqueous buffers to increase the solubility of the solute or other additives and or to affect
the ionization degree of the sample components. The addition of these organic modifiers to
the buffer generally causes a decrease in the EOF.
Applications:
Determination of proteins & peptides
CEMS has wide range of applications in protein analysis. Mass spectrometry combined with
capillary zone electrophoresis (CZE-MS) is widely employed for protein/peptides separation and
analysis. Some of the examples are analysis of N- linked glycopeptides, 8- aminopyrene-1,3,6
trisulfonate (APTS) labeled recombinant antibodies, apolipo proteins, bovain insulin, myoblobin,
and cytchrome C was separated by employing IT mass analyser [6]
. Hemoglobin analysed by using
CEMS was reported. Also used in determination of glycoproteins, phycobiliproteins from blue-
green algae Metal binding proteins can be analysed by using isotachophoresis as mode of
separation. Somatostatin and insulin were analysed by CZE-MALDI-MS was reported [7]
. The
analysis of carbonic anhydrase I and II in red blood cells was reported.
Determination of drugs in biological matrices
Quantitation of pharmaceutical agents in biological samples like plasma is very important in drug
discovery and development. Analysis of antiepileptic drug Lamotrigine in human plasma was
reported by Zheng et al using different BGE and sheath liquids [8]
. Anticancer drug Imatinib
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analysed by CE-ESI-TOFMS with high resolution and sensitivity was reorted by Elhamili et al [9]
.
Rivastigmin in human plasma obtained from alzhemers disease patients was repoeted by employing
CZE-ESI-MS. CEMS also plays an important role in eantiomeric separation and determination.
Chiral selectors are used in this technique. Mostly used chiral selectros are cyclodextrins,
polymeric surfactants, cyclofructans, macrocyclic antibiotics, crown ethers etc. lansoprazole
analysis by CZE separation technique using β cyclodextrin as chiral selector was reported. chiral
selector hydroxypropyl-γ-cyclodextrin in order to separate the antifungal drug iodiconazole and the
structurally related triadimenol analogues with good resolution [10]
.
Human disease biomarker discovering
CE in coupling with ESI-TOF-MS was extensively applied to the identification of protein and
peptide disease biomarkers in urine. CE-ESI-TOF-MS is a proteomic approach was applied to the
analysis of specific peptide and protein patterns with the aim to identify biomarkers of
immunoglobulin A nephropathy, focal-segmental glomerulosclerosis, membrane
glomerulonephritis, lupus nephritis, diabetic nephropathy and renal transplantation diseases [11]
.
CEESI-TOF-MS was applied to the proteomic analysis of urine also in relation to cancer diseases.
22 polypeptides were identified by CE–MS as a diagnostic pattern for urothelial cancer with high
sensitivity and selectivity among the patients and healthy volunteers [12]
. Also helpful in the
biomarker discovering of muscle invasive bladder cancer. Many of the biomarkers found for
diabetes by CE–MS analysis of urine were identified as fragments of collagen type I and II,
respectively [13]
.
CEMS was also applied to the proteomic analysis of the CSF in association with Alzheimer’s
disease and schizophrenia. CE MALDI-TOF MS was used as biomarkers of neurodegenerative
diseases [14]
.
Detection of drugs of abuse in biological samples
Urine is the most commonly used biological sample for the analysis of drugs of abuse (e.g.,
amphetamines and opiates) in forensic laboratories. Morphine and related opioids detected by CE-
APEI-MS was reported. Cocaine, benzoylecgonine, cocaethylene, anhydroecgonine,
anhydroecgonine methyl ester, ecgonine methyl ester using the BGM as Formic acid (1 M) and
sheath liquid as formic acid (0.05 M) in methanol:water was reported. Plasma and serum are
typically used for emergency testing in forensics and toxicology, and in therapeutic monitoring, as
they are suitable as samples to determine the short-term use of drugs. 6-monoacetylmorphine,
morphine, amphetamine, methamphetamine, 3, 4-methyelenedioxyamphetamine, 3,4-
methylenedioxymethamphetamine, benzoylecgonine, ephedrine, cocaine analysed by
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CEIT(iontrap)-MS was reported. Hair is being recognized as the third fundamental biological
specimen for drug testing. 3,4-methylenedioxyamphetamine, 3,4 methylene dioxyethyl
amphetamine, 3,4-methylenedioxymethaamphetamine methadone, cocaine, benzoylecgonine,
morphine, codeine and 6-acetylmorphine these are the few examples reported for hair analysis [15]
.
Analysis of metals, metalloids and non metals
Various forms of metals, non metals and metalloids naturally occurring in living organisms can
analysed by CEMS. Identification of manganese species in CSF was reported by Michalke et al [16]
.
Arsenic in Fish and oyster tissue, Cadmium in Rat liver, Manganese in Porcine liver, Iodine in
seaweed are some of the examples.
CEMS also useful in identification and quantification of dietary substances. For example
determination of free amino amino acids in royal jelly was reported earlier [17]
.
CONCLUSION:
CE in combination with MS has certainly emerged as an analytical tool that shows wide
applications in pharmaceutical, biological fields. It is a good alternative for widely used separation
and quantification techniques.
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