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Vitamin B12 is a water-soluble vitamin. It is one of the eight vitamins of vitamin B complex, needed for blood and cell maturation. It helps maintain healthy nerve cells and red blood cells, and it is needed in DNA replication. Its deficiency may cause megaloblastic anemia (amidst others health issues). For these and many similar reasons, it sometimes becomes necessary to measure its concentration. This article has carefully reviewed the different methods used for measuring vitamin B12 concentration, and the unique principles involved. The principles, basically, depend on the molecular structure of Vitamin B12 and its reactions with other substances. The methods include microbiological assay and spectrophotometric methods – these are old methods: they were the first available methods, but they are still in use for reference purposes. Another method is electroluminescent (ECL) which involves highly reactive materials. However, inductive-coupled plasma-mass spectrometry (ICP-MS) is a very important method, which is used routinely, even in many research. On the other hand, atomic absorption spectroscopy depends on measuring the amount of energy involved in the reaction; while radioimmunoassay (RIA) is a highly sensitive immunoassay technique. In addition, there are different techniques for separating and preparing samples to be used in the various measurement methods. High-performance liquid chromatography (HPLC) is used for non-validate analyst, while capillary-electrophoresis (CE) that have high resolving power than traditional electrophoresis, which when they are coupled with certain detectors they afford us another principle for measuring this vitamin. Choosing the best method for measuring vitamin B12 concentration depends on many factors – including the type of sample, purpose of the test, necessity of pre-processing, time limitations, cost, sensitivity, specificity.
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The IIOAB Journal
ISSN: 0976-3104
©IIOAB-India OPEN ACCESS Vol. 2; Issue 2; 2011: 23-32
Ola Karmi*, Ashraf Zayed, Suheir Baraghethi, Muhammad Qadi, Rasha Ghanem
Department of Medical Laboratory Sciences, Faculty of Health Professions, Al-Quds University, (Master Program in
Hematology and Microbiology), Jerusalem, PALESTINE
Received on: 9th-July-2010; Revised on: 22nd-Nov-2010; Accepted on: 30th-Nov-2010; Published on: 10th-Jan-2011
*Corresponding author: Email: Tel: +972-598-242829
Vitamin B12 is a water-soluble vitamin. It is one of the eight vitamins of vitamin B complex, needed for
blood and cell maturation. It helps maintain healthy nerve cells and red blood cells, and it is needed in
DNA replication. Its deficiency may cause megaloblastic anemia (amidst others health issues). For
these and many similar reasons, it sometimes becomes necessary to measure its concentration. This
article has carefully reviewed the different methods used for measuring vitamin B12 concentration, and
the unique principles involved. The principles, basically, depend on the molecular structure of Vitamin
B12 and its reactions with other substances. The methods include microbiological assay and
spectrophotometric methods these are old methods: they were the first available methods, but they
are still in use for reference purposes. Another method is electroluminescent (ECL) which involves
highly reactive materials. However, inductive-coupled plasma-mass spectrometry (ICP-MS) is a very
important method, which is used routinely, even in many research. On the other hand, atomic
absorption spectroscopy depends on measuring the amount of energy involved in the reaction; while
radioimmunoassay (RIA) is a highly sensitive immunoassay technique. In addition, there are different
techniques for separating and preparing samples to be used in the various measurement methods.
High-performance liquid chromatography (HPLC) is used for non-validate analyst, while capillary-
electrophoresis (CE) that have high resolving power than traditional electrophoresis, which when they
are coupled with certain detectors they afford us another principle for measuring this vitamin.
Choosing the best method for measuring vitamin B12 concentration depends on many factors
including the type of sample, purpose of the test, necessity of pre-processing, time limitations, cost,
sensitivity, specificity.
Keywords: Electroluminescence; Inductive-Coupled Plasma-Mass Spectrometry; microbiological assay; radioimmunoassay;
capillary-electrophoresis; vitamin B12 concentration
Vitamin B12 is a water-soluble vitamin. It is one of the “B
complex vitamins,” which play roles in red blood cell formation,
nerve cell maintenance, and methyl donation in DNA synthesis.
Deficiency of vitamin B12 affects immunologic and hematologic
parameter in the body [1].
Human’s source of vitamin B12 is of animal origin. It was in
1948 that vitamin B12 was first isolated from liver juice, and it
was used in treating pernicious anemia [2]. Vitamin B12 consists
of corrin ring (synthesized by bacteria) and cobalt ion; and this
cobalt-corrin ring complex gives vitamin B12 its red colouration.
Different forms of vitamin B12 are similar in the cobalt central
ion, the four parts of the corrin ring and a dimethylbenzimidazole
group, but differ in the sixth site which may contain cyano group
(CN), hydroxyl group (OH), methyl group (CH3) and/or 5'-
deoxyadenosyl group (C-CO) [3,4].
There are several methods to assay and calculate vitamin B12.
Some of these methods are used in medical field, and some
others in pharmacological studies/investigations. This review
The IIOAB Journal
ISSN: 0976-3104
©IIOAB-India OPEN ACCESS Vol. 2; Issue 2; 2011: 23-32
focuses on some of the weaknesses and strengths of these
methods, and aims to identify the best method for measuring the
concentration of vitamin B12.
2.1. Microbiological and spectrophotometric
Microbiological method is one of the oldest methods for
measuring the concentration of vitamin B12. Information
regarding this method has been extensively documented [5].
Ross (1950) was the first scientist to describe microbiological
method using Euglena gracilis var-bacillaris as test organism.
Thereafter there was introduction of Z strain of Euglena gracilis
so as to shorten the growth period required for the test to as low
a five days [6]. Further experiments on measurement of vitamin
B12 focused on either changing microorganism test or
developing test techniques, such as adding heating step or some
substances to the test procedures for converting vitamin B12 to
the active free form [6]. Also, several microorganisms were
proposed for the microbiological assay. These methods include
Euglena gracilis tube method, filter paper disc method (FPD),
Escherichia coli tube method, plate method, Lactobacillus
leishmanii tube method, bioautographic method, and
Ochromonas malhamensis tube method [7]. However, Euglena
gracilis and Lactobacillus leishmanii are the most commonly
used methods [5,7,8].
Davis et al [6] described a fully automated method for the
microbiological measurement of vitamin B12 using
chloramphenicol-resistance strain of Lactobacillus leishmannii
as test organism. Chloramphenicol eliminates the need for
sterilization. Using this method, results could be available within
24 hours. This automated method solved the challenge of how to
dissociate vitamin B12 from its protein carrier. This was possible
by treating the sample with a solution containing glutamic/malic
acid [6].
Automated microbiological method was designed to use
Mecolab M which is a multi-instrument that provides facilities
for sample dilution, reagent addition and mixing, as well as
measurement and digital estimation of bacterial growth. It
consists of sample preparation unit, autocolorimeter, A/D
converter and calculator [6].
Years after, scientists have tried to develop a microbiological
method by using microtiter plates. They used chloramphenicol
resistance strain of lactobacillus casei on serum and red blood
cell folates. Then they compared the results with traditional
microbiological method. They obtained better results with better
intra-assay precision for both serum and red cells (CV% of <5).
However, the previous method was more compact, less time
consuming, has a lower cost, need smaller amount of sample,
and easy to perform in medical laboratories [9].
Microbiological methods are facing difficulties in the assay of
vitamin B12, mainly because they are tedious, and time
consuming; they have poor precision, and relatively low
specificity [10]. Other disadvantage of this method is that
whenever the patient serum contains antibiotics, the growth of
some assay organisms will be inhibited and false low/negative
results would be obtained [10]. Also, L. leichmannii assay may
give falsely low results in the presence of some antibiotics and
antimetabolites. In addition, the E. gracilis assays produce
falsely low results with sulfonamide and chlorpromazine [11].
The reference normal range of vitamin B12 concentration for
which using Euglena gracilis method would be good is 200-900
g/cc [12].
In 1972, scientists started working on photochemistry of vitamin
B12 by Pratt [13]. The conversion of cyanocobalamin to
hydroxycobalamin takes place readily in the pH range between
“3.5 6.5” under the action of light. The quantum of the
photoaquation reaction of cyanocobalamin is 10^-4 [14]. The
photo degradation of cyanocobalamin plays an important role in
the stability of vitamin B12 solutions. If the primary
photochemical change leading to the formation of hydroxyl
cobalamin could be minimized, the photo stability of
cyanocobalamin could be enhanced. Spectrophotomery for
vitamin B12 measuring was very diverse according to the use of
many light spectra like gamma-ray counter spectrophotometer
[15]. Ultraviolet (UV) -vis spectrophotometer different types [16,
17] or some reagents were added as 6,7-dimethoxy-1-methyl-
2(1H)-quinoxaline-3-prpionyl carboxylic acid hydrazine
(DMEQ) to produce a highly fluorescence vitamin B12
derivative [14], and 4,4’-diazobenzenediazoaminoazobenzene
(BBDAB) [18]. In UV and visible spectrophotometry, aqueous
solutions of cyanocobalamin exhibit maximum UV and visible
region at 278nm, 361nm, and 550 nm [19]. However, several
factors such as changes in solvent, temperature, and pH can
affect the spectrum [20].
Several many colorimetric methods had been reported for the
determination of cyanocobalamin. These methods are based on
the determination in the content of cobalt which forms
complexes with many compounds at different wavelengths. A
colorimetric catalytic kinetic method has been developed for the
determination of trace amounts of cobalt in vitamin B12
preparation. In acetate buffer (pH.4), cobalt (II) catalyses the
reduction of colorless ferric-dipyridyl complex to pink ferrous-
dipridyl complex in the dark. The linear determination range is
0-10 mg/10ml cobalt (III) [15].
Finally, application to injections containing vitamin B12 gave
results closer to the results obtained by capillary electrophoresis
[20]. Spectrophotometric method has low cost and acceptable
specificity in comparison with radio ligand assay [20]. However,
it is not suitable for complex samples, and the sensitivity is
relatively low in such cases so it is not used routinely [10].
The IIOAB Journal
ISSN: 0976-3104
©IIOAB-India OPEN ACCESS Vol. 2; Issue 2; 2011: 23-32
3.1. Electroluminescence (ECL)
Electroluminescence (ECL) is a process in which reaction of
highly reactive molecules are generated from stable state
electrochemically by an electron flow cell forming highly reacted
species on a surface of a platenium electrode producing light
[21]. This method uses ruthenium (II)-tris (bipyridyl) [Ru (bpy)3
]2+ complex and triproplamine (TPA) and react them with each
other to emit light. The applied voltage creates an electrical field
that causes the reaction of all materials. Tripropylamine (TPA)
oxidized at the surface of the electrode, releases an electron and
forms an intermediate which may further react by releasing a
proton. In turn the ruthenium complex releases an electron at the
surface of the electrode forming an oxidized form of Ru(bpy)33+
cation, which is the second reaction component for the
chemiluminescent reaction. Then this cation will reduce and
form Ru(bpy)3 2+ and an exited state via energy transfer which is
unstable and decays with emission of photon at 620 nm to its
original state [21, 22].
The florescence emitted by Ru(bpy)32+ is detected by standard
photomultiplier, and the results are expressed as ECL intensity,
which is the measurement of the whole luminescence emitted
from the sample [23]. This method employs various test
principles (such as competitive principle, sandwich and bridging)
for the measurement [22]. The most important one in measuring
vitamin B12 concentration is the competitive principle. The
competitive principle is applied to low molecular weight
molecules. It uses antibodies (intrinsic factor) for vitamin B12
labeled with ruthenium complex. These antibodies are incubated
with the sample, then biotinylated vitamin B12 and streptavidin
which is coated with paramagnetic miroparticles are added to the
mixture. The free binding sites of the labeled antibody become
occupied with the formation of an antigen-hapten complex. Then
the entire complex is bonded to biotin and streptavidin. After
incubation the reaction mixture is transported into the measuring
cell where the immune complexes are magnetically entrapped on
the working electrode and the excess unbound reagent and
sample are washed away. Then the reaction is stimulated
electrically to produce light which is indirectly proportional to
the amount of vitamin B12 that is measured [Figure1] [22].
Fig: 1. Electroluminescence method, competitive principle for measuring vitamin B12. Step 1: Vitamin B12 from serum sample
enters to the flow channel. Step 2: Vitamin B12 particles from the reagent are bonded to streptavidin-biotin to help in their attachment
to the magnet part. Step 3: Intrinsic factor (which acts as antibodies) are bounded to ECL particles to enhance the reaction. Step 4:
Vitamin B12 from the sample with the particles from the reagent bind to the intrinsic factor. Step 5: Only intrinsic factor that is bonded
to the vitamin B12 labeled with Streptavidin-biotin particles is attached to the working electrode (by magnetic action) were the ECL
reaction will take place and the signal will be measured. The free intrinsic factor with the ones that binds to the vitamin B12 from the
sample will be washed away.
The IIOAB Journal
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Test sample needed is serum, and the sample duration time is 27
minutes, this test is very sensitive. It can even detect 22 pmol/L
(30 pg/ml). It is also very precise (CV% is >10%), and very
specific and cross reactivity rarely occurs. This test has high
reproducibility, and can be processed easily. Machines used in
this technique have extremely long life span with no
maintenance costs. An example of such machines is Elecsys
2010 and Cobas e 411. This technique is often used in
pharmacological, industrial, clinical and chemical research [24].
3.2. Inductive-coupled plasma (ICP) - mass
spectrometry (MS) (ICP-MS)
One of the best methods for the determination of vitamin B12
concentration is mass spectrometry (MS). This is because of its
speed, sensitivity, easy (fully automated) and its vast possible
application. It is one of the most important instruments for both
routine and research applications. In contrast to what its name
implies, MS actually measures mass to charge ratio and not just
the mass. However, when the charge of all particles (ions) is the
same, the mass spectrum plot is simplified to have only mass on
the X- axis and the relative abundance on the Y-axis [25].
There are deferent types of MS but they all have three main
components in common: an ionization source; mass analyzer;
and detector. Ionization process occur in deferent ways in the
different types of MS and that is what actually explains the
differences between the deferent MS types. Ionization is an
important step, and ensures the conversion of the the analyte of
interest into gaseous phase ions. The first described ionization
source is the electron ionization where the sample must be of low
molecular weight, vaporizable and thermally stable. The
analytes has to be vaporized and then ionized, and these limited
the availability of such method for many biological samples and
analytes so there was great need for developmental ionization
sources [25, 26]. This lead to the development of electrospray
ionization, atmospheric pressure chemical ionization and matrix
assisted laser desorption ionization [25].
The first type of ionization source is Electrospray ionization
(ESI). It depends on generation of electrons at atmospheric
pressure by exposing the sample to different voltage depending
on the boiling temperature of the liquid phase sample and the
diameter of the inner capillary tube. Most machines with EIS
also have additional or optional ionization technique which is
Atmospheric Pressure Chemical Ionization or Inductively
Coupled Plasma where ionization can also occurs at atmospheric
pressure. But these differ from ESI in that sample travels through
the different heating zones: when plasma torches it, it becomes
dried, vaporized, atomized, and ionized. During this time, the
sample is transformed from a liquid aerosol to solid particles,
then into a gas, so that they are excited and they gain enough
energy to release electrons from their orbits and generate ions.
Like ESI and ICP, matrix assisted laser desorption ionization
occurs in vacuum where laser irradiation pulsed is the source of
ion generation [25, 26, 27].
As Since the ionization sources can differ, there are also several
types of mass analyzers. One of the simplest types is “Time of
flight mass analyzer” where the velocity of the ions (which
depends on the mass to charge ratio) leads to the separation of
the ions in different speeds. When fixed potential force them
toward the detector, the speed (time) of an ion in reaching the
detectors is proportional to its mass to charge ratio lower ratio
is associated with higher velocity. The other type of the mass
analyzer is the sector analyzer (magnetic or electric sectors are
available). Here the ions are focused toward the detector after
they have left the sector, through a split by applying a fixed
accelerating potential.
The widely used mass analyzer is the quadruple, especially with
gas and liquid chromatography, since it is much smaller, easier,
and cheaper than other analyzers. By placing a direct current
(DC) field on one pair of rods and a radio frequency (RF) field
on the opposite pair, ions of a selected mass are allowed to pass
through the rods to the detector, while the others are ejected from
the quadrupole. The other ions of different mass to-charge ratios
will pass through the spaces between the rods and be ejected
[Figure2] [25, 27, 28].
To perform MS one needs to start with pure analyte to be able to
use different MS types. So it is important to combined MS with
other separation techniques such as capillary electrophoresis,
HPLC, gas chromatography, and liquid chromatography, where
Cobalamin in human urine and multivitamin tablet solutions can
be converted into free cobalt ions in acid medium. The linearity
of MS is over the cobalamin concentration range of 1.0 × 10−10
g /mL− to 8.0 × 10−5 g /mL and the limit of detection is 0.05 ng/
mL for both ICP-MS and HPLC-MS. MS is often used in
Pharmacology, industries, and in basic research, but not used in
clinical field due to its high cost [29].
3.3. Atomic absorption spectroscopy
Atomic absorption spectroscopy is an analytical chemistry
technique used for determining concentration of particular metal
element in a sample, and it is widely used in pharmaceutics.
This technique can be used to analyze the concentration of over
70 different metals in a solution [30]. The discovery of the
Fraunhofer lines in the sun's spectrum in 1802 marked the
beginning of the main phenomenon behind this technique.
However, it was not until 1953 that Sir Alan W (Australian
physicist) demonstrated the possibility of using atomic
absorption for quantitative analysis [31]. Simply put, atomic
absorption spectroscopy has to do with the measurement of the
absorption of light by vaporized ground state atoms and then
estimating the desired concentration from the absorption.
Basically, the incident beam (of light) is attenuated by the
absorption by atomic vapor according to Beer’s law [32].
A detector measures the wavelengths of light transmitted by the
sample (called the “after wavelengths”), and compares them to
the wavelengths, which was passed through the sample (the
The IIOAB Journal
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“before wavelengths”). Moreover, a signal processing unit then
processes the changes in wavelength, and gives the output for
discrete wavelengths as peaks of energy absorption. Since, an
atom is unique in its absorption pattern of energy at various
wavelengths due to the unique configuration of electrons in its
outer shell, the qualitative analysis of a pure sample can be
achieved [32]. This, in fact, makes it reasonable for this method
to measure the quantity of energy (in the form of photons of
light) absorbed by the sample. Using this technique, various
metals in organic samples can be analyzed. The basic structure of
the machine consists of 4 basic structural elements; a light source
(hollow cathode lamp), an atomizer section for atomizing the
sample (burner for flame, graphite furnace for electro thermal
atomization), a monochromatic for selecting the analysis
wavelength of the target element, and a detector for converting
the light into an electrical signal [Figure3] [33].
Fig: 2. Inductive-Coupled Plasma-Mass Spectrometry (ICP-MS). A. Mass Spectrometry: Shows the basic components of a typical
mass spectrometry. All mass spectrometry shares three main components; an ionization source, mass analyzer, and detector. B.
Inductive-Coupled Plasma: It serves as the ionization source in some particular types of MS called ICP-MS. The sample travels
through the different heating zones and are finally ionized.
Here, the atomizers used are pyrocoated tubes and tubes with
centre fixed platforms. In addition, a cobalt hallow cathode lamp
is used and a wavelength of 242.5nm could be used for assaying.
Argon serves as a protective gas and serum or urine could be
introduced into the graphite furnace (GF) directly with
equivalent volume of modifiers. H2O2 is used to prevent carbon
residue formation in graphite tube. The electro thermal atomic
absorption correctly and optimally measures Cobalt (and thus,
vitamin B12) in serum and urine. It has a detection bound of 0.02
μg/L Co in serum samples with a relative standard deviation of
10-18% [34].
The main advantages of this method is that it has a high sample
throughput, it is easy to use, and it has high precision. But the
main disadvantages involve its less sensitivity, its requirement of
large sample, and the problems with refraction [34]. Another
method that is used is the Flame atomic absorption spectrometry.
The lowest concentration for quantitative recovery is 4 ng/cm3 of
vitamin B12. The method is used for vitamin B12 determination in
pharmaceutical samples. It is used in pharmacology, industry,
clinical and chemical basic research. [35].
3.4. Radioimmunoassay (RIA)
Radioimmunoassay (RIA) is a highly sensitive laboratory
technique used to measure minute amount of substrate (such as,
hormones, antigen and drugs) in the body. RIA is a primer
immunoassay techniques developed for detecting extremely
small concentrations [36]. Berson and Yallow developed the first
radio-isotopic technique to study blood volume and iodine
metabolism and had used it for the determination of insulin
levels in human plasma. Later the technique was adapted for
studying how hormones (especially insulin) are being used in the
body [35]. This method is so sensitive that it can measure one
trillionth of grams of substance per milliliter of blood and only
The IIOAB Journal
ISSN: 0976-3104
©IIOAB-India OPEN ACCESS Vol. 2; Issue 2; 2011: 23-32
small samples are required. These (among other reasons) made
RIA to quickly become a standard laboratory tool [37].
RIA is based on the reaction of antigen and antibody in which
very small amounts of the radio-labeled antigen competes with
endogenous antigen for limited binding sites of the specific
antibody against the same antigen. The radio-labeled antigen
have been an analogous in biological activity and/or immuno-
reactivity to the native antigen. For vitamin B12 we use
Modified intrinsic factor (IF) fractions which have R-proteins
that bind many porphyrin-ring-containing compounds (i.e.,
cobinamides) by radio assay with [57Co] vitamin B12 [38].
Fig: 3. Schematic diagram of an atomic absorption spectrometer. The basic structure of the machine consists of 4 basic structural
elements; a light source (hollow cathode lamp), an atomizer section for atomizing the sample, a monochromatic for selecting the
analysis wavelength of the target element, and a detector for converting the light into an electrical signal, amplifier and readout.
Most commonly used radio-isotope in RIA is 125-I. Other
emitting isotope such as C14 and H3 have also been used. Some
other important aspects of RIA are the use of specific antibody
against particular antigen, and the use of pure antigen as the
standard or calibrator [37] is attached to tyrosine. These radio
labeled IFs are then mixed with a known amount of
cyancoblamine, and they become chemically bound to each
other. A serum from a patient which contains an unknown
quantity of IF is added, so the unlabeled (or "cold") IF from the
serum competes with the radio labeled (or "hot”) IF for
cyanocoblamine binding sites [39].
If the concentration of the “cold" IF increased, more of it binds
to cyanocoblamine and this will lead to displacement of the
radio-labeled variant, so the ratio of “cyanocoblamine bound to
radio labeled antigen” to “free radio labeled IF” is reduced.
After that, the bound IF is separated from the unbound ones and
the radioactivity of the free IF that remains in the supernatant are
measured [39]. The separation of radio-labeled IF bound to
cyanocoblamine from unbound radio-labeled IF occurs after
optimal incubation conditions (buffer, pH, time and
temperatures) [37].
Polyethylene glycol joined with double antibody method is
regularly used to separate bound and free radio-labeled IF. Some
other techniques in use are the double antigen, charcoal,
cellulose, chromatography and solid phase technique [37].
Calibrations or standard curves are formed from sets of known
concentrations of the unlabeled standards and from such curves
the quantity of IF in the unknown samples can be determined.
Improving the sensitivity of the assay is possible by decreasing
the amount of radio-labeled analyst and/or antibody, or by
disequilibrium incubation format in which radio-labeled IF is
added after initial incubation of IF and cyanocoblamine. This
technique (just like the others) is supposed to meet the criteria of
sensitivity, specificity, precision, recovery and linearity and
dilution [39]. For this technique, the precision has been said to be
7.9% for 200 ng/L as the concentration of vitamin B12, 6.6 % for
400 ng/L, and 6.7 % for 800 ng/L. The sensitivity of the assay
has also been documented as 37 ± 9 ng/L [39]. RIA is well used
in pharmacology, industry, clinical, and chemical research [38].
The principle of radioisotope dilution is based on using unknown
quantity of non-radioactive vitamin B12 released from serum to
dilute the specific activity of a known quantity of [57Co] vitamin
B12. A solution of intrinsic factor concentrate (IFC) with a
vitamin B12 binding capacity less than the quantity of added
[57Co] vitamin B12 is used to bind a portion of the mixture of
radioactive and no radioactive vitamin B12 i.e., to “biopsy” the
pool of vitamin B12. The vitamin B12 not bound to IFC is
removed by the addition of coated charcoal [40].
The IIOAB Journal
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3.5. High-performance liquid chromatography
It is a liquid chromatography used for non-volatile analyst in
which the elute do not flow under the force of the gravity but it is
derived under a hydrostatic pressure of 5000 to 10000
pounds/square inch through a stainless steel column [41, 42].
The HPLC system uses a mobile-phase pump, a reagent pump,
an auto-sampler, a detector and a data system for data processing
and system control [43].
The system is a chromatography, in which the eluent is filtered
and pumped through the column, then the sample is loaded and
injected onto the column and the effluent is monitored using a
detector, and the peaks are recorded. The pump of the system
must be able to generate high pressure, performing a pulse-free
output and deliver flow rates ranging from 0.1 to 10 ml/min [42].
In this method, samples are treated very carefully and the
working pH, heating, agitation, centrifugation and filtration are
correctly adjusted in accordance with the source of the sample;
and the resulting solution is injected into the instrument that does
the measurement. he HPLC must be connected to a suitable
detector e.g. Micro-mass electrospray mass spectrometer. Its
results are often precise, and it is very sensitive with detection
limits of 50 nmol/L [43]. An example of this Instrument is
Kontron HPLC-system 400. This method is frequently used in
pharmacology, industry and basic research [43].
3.6. Capillary electrophoresis
Capillary electrophoresis (CE) was first documented in 1981. It
is used to separate peptides. CE have high resolving power than
traditional electrophoresis and do not require extremely great
skills as high-performance liquid chromatography (HPLC) [44].
CE is quantitative rather than semi quantitative or qualitative,
and very small samples (< 10 nL to 1 nL) can be used [43, 44].
The schematic structure for CE is composed of sample vial, two
buffer vials (source & destination), capillary, electrodes, high-
voltage power supply, detector, and data output. Electroosmotic
flow forms the main principle in CE [44]. Generally sample for
CE does not require preparations, but in low concentrations
biological sample such as serum or plasma, there could be a need
for pretreatment to prevent ionic strength and protein-rich matrix
from effecting the migration [44,45].
CE can be used for cobalamin separation and for differentiation
between different forms of the vitamin B12. The procedure of
cobalamin separation is done by using 70 cm capillary length
with 20 KV voltage supply, and 9.0 pH tris buffer 25 mM that
contain 15 mM sodium dodecyl sulfate as electrophoretic buffer
The main disadvantage of this technique is in its low ability to
detect the sample (i.e. low sensitivity) due to the wall of the
capillary, which is dissolved by coupling system of capillary
electrophoresis-inductively coupled plasma mass spectrometry
(CE-ICP-MS). It is mainly used in basic research. [46, 47]
Another source of error that is unique to CE but absent in CE-
ICP-MS is the electrokinetic sample injection [45]. On the other
hand, no problems are unique to the coupled system method
mentioned above. In general controlling the column over
loading, calibration to prevent sample aging and facilitate
analysis, and buffering according to the sample pH, are
important aspects that should always be taken care of while
separating vitamin B12 [45].
The microbiological method remains the routine method for the
determination of vitamin B12 concentration, despite the fact that
it is time consuming, and has relatively poor precision, and low
specificity. This might be because ECL and radioimmunoassay
which are simpler and faster are very expensive - since they
require pure intrinsic factor and some special reactants. Also,
ECL and atomic absorption spectrometry depends on indirect
measurement of the cobalt. On the other hand, Capillary
electrophoresis and HPLC methods include the use of UV or
visible photometry, atomic absorption and ICP-MS.
Determination of the best way of measuring vitamin B12
concentration would require critical consideration of the
required/desired sensitivity and specificity, the available time,
and the process of preparation of the sample, as well as cost.
Some of the important characteristics of the different methods
have been summarized in Table1.
Finally, we should say that cases of serious discrepancies
between results of vitamin B12 concentration determined by
different methods is highly common. We therefore think that it
would be important that every laboratory specifies on it reports
the method that had been used when reporting the results of
vitamin B12 concentration. This might present clear picture to
physician and patient. The reasons for deciding to measure
vitamin B12 concentration should also play a crucial role in
determining the most appropriate method. For example, the
investigator might want to use ECL, RIA or atomic absorption
spectroscopy if the results would be used for clinical/medical
purposes, while ICP-Ms might be preferred for industrial or
pharmaceutical needs or for basic research purposes. More
importantly, several advantages and disadvantages of each of
these methods govern the choosing of the suitable methods.
Table2 has clearly summarized some of these.
The IIOAB Journal
ISSN: 0976-3104
©IIOAB-India OPEN ACCESS Vol. 2; Issue 2; 2011: 23-32
Table: 1. Comparison of the Sensitivity of different methods used in measuring the concentration of vitamin B12
Table: 2. Advantages, disadvantages and applications of each of the methods used in measuring the concentration of vitamin
The IIOAB Journal
ISSN: 0976-3104
©IIOAB-India OPEN ACCESS Vol. 2; Issue 2; 2011: 23-32
Special thanks for Dr. Samira Barghouthi the dean of Scientific
Research at Al-Quds University, who supported and encouraged us all
the way, and lightened our path with her words and wisdom.
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©IIOAB-India OPEN ACCESS Vol. 2; Issue 2; 2011: 23-32
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Ola Talal Karmi, has a Bachelor degree in Medical Technology, class 2006, from Al-Quds University,
East Jerusalem, Palestinian Territories, since 2009 enrolled in the master degree of Hematology
graduated studies at Al-Quds University- Faculty of Health Professions- Medical Laboratory Science
Department. Have a cytogenetics training experience. Currently working at the medical diagnostic
laboratory of Augusta Victoria Hospital- Jerusalem.
Ashraf Zayed, Master student- Microbiology, Department of Medical Laboratory Sciences, Faculty of
Health Professions, Al-Quds University. Shared in the 6th Palestinian conference for clinical laboratories,
2010, Al-Quds University, in a research title “Effect of oral antibiotic on serum vitamin level” as main
author. Also, shared in 2nd Conference on Biotechnology Research and Application in Palestine, 2010
An-Najah National University, in a research title “Isolation of putative soil Streptomyces spp showing
antibacterial activity”. Currently doing a master thesis about Streptomyces spp.
Suhair F. Baraghithy, has a Bachelor degree in Medical Technology, from Al-Quds University East
Jerusalem Palestinian Territories, since 2009 enrolled in the master degree of Hematology graduated
studies at Al-Quds University- Faculty of Health Professions- Medical Laboratory Science Department. 3
years working in clinical laboratories, currently work in Ramallah Governmental hospital.
Mohammad Alqadi, a master degree student at Microbiology & Immunology program at Al-Quds
University, East Jerusalem, Palestinian Territories. Received a Bachelor degree in Medical Laboratory
Science from Arab American University, class 2009, Jenin, Palestine. Currently interested in working at
Molecular Typing for Bacteria.
Rasha Emil Ghanem, has a Bachelor degree in Medical Laboratory Sciences from An-Najah National
University-Nablus- Palestinian Territories. since 2009 enrolled in the master degree of Hematology
graduated studies at Al-Quds University- Faculty of Health Professions- Medical Laboratory Science
Department. Currently working at Bethlehem Arab Society- Specialized Surgical Hospital Laboratory.
Have training programs in Gel Technique for blood banking and Quality Control in Laboratories.
Participated in several lectures about Thalassemia and Haemophilia.
... The microbiological tests generally involve the use of Lactobacillus delbrueckii [14]; these methods were originally developed for the analysis of vitamin preparations but are also applied to fortified foods and food matrices in general. Regarding chemical-physical methods, spectrophotometry is applicable, but with a complex matrix, it is difficult to achieve good sensitivity [15]. According to the literature, most of the studies on the chemical quantification of vitamin B12 in different matrices are based on radioisotopic assays [16] or chromatographic techniques combined with UV [17][18][19][20], fluorescence [21], or diode array detectors [22]. ...
... Another method is electroluminescent (ECL), which involves the use of highly reactive materials. Inductive-coupled plasmamass spectrometry (ICP-MS) is also routinely used in various research [15,23]. ICP-AES (inductively coupled plasma atomic emission spectroscopy) was also used by Bartosiak et al. (2018) [24] for the determination of cobalt species in dietary supplements. ...
Full-text available
The analysis of natural cobalamins in dairy products still represents an analytical challenge. The matrix’s complexity, low concentration level, light sensitivity, and binding to proteins are just some of the aspects that make their quantification a difficult goal to achieve. Vitamin B12 plays a fundamental role in human nutrition, and its intake is closely linked to a diet that includes the consumption of food of animal origin. In the current literature, few studies have been carried out on the quantitation of cobalamin in ripened cheeses. A sensitive, selective, and robust ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method was developed, validated, and applied on ripened cheeses from different species (cow, sheep, and goat) purchased from local Italian markets, highlighting species-dependent differences in vitamin B12 concentrations. The vitamin B12 extraction procedure was performed by converting all cobalamins to the cyanocobalamin form. Furthermore, solid-phase extraction was used for matrix clean-up and analyte preconcentration. The proposed method showed good performance in terms of linearity, sensitivity, reproducibility, and repeatability. The mean vitamin B12 content ranged from <LOQ to 38.9 ng/g. Sheep cheese showed the highest concentrations of vitamin B12, with a mean content of 29.0 ng/g.
... Transferrin saturation (%) was calculated by the percentage of serum iron levels divided by the TIBC value [33]. The serum levels of folate [34] and vitamin B 12 [35] were measured using SimulTRAC-SNB radioimmunoassay kits (MP Biomedicals, Santa Ana, CA, USA) with 125 I or 57 Co as the tracer, respectively. Serum 25(OH) vitamin D levels were determined by an electrochemiluminescence immunoassay using an Elecsys vitamin D total reagent kit with ruthenium-labeled vitamin-D-binding protein (Roche Diagnostics Ltd., Taipei, Taiwan) [36]. ...
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Vitamin D is involved in the pathophysiology of anemia. This cross-sectional study was conducted using the Nationwide Nutrition and Health Survey in Pregnant Women in Taiwan database. We investigated associations among dietary patterns (DPs), vitamin D, and iron-related biomarkers in pregnant women. The principal component analysis revealed four DPs. Linear and logistic regression analyses were performed to investigate the association of DPs with anemia-related biomarkers. Plant-based, carnivore, and dairy and nondairy alternatives DPs were positively associated with serum vitamin D levels. After adjusting covariates, the pregnant women consuming plant-based DPs at the mid-tertile (T2) were associated with reduced risks of low serum folate and vitamin D levels, and those consuming carnivore DPs at higher tertiles (T2 and/or T3) were correlated with an increased risk of low serum iron levels but decreased risks of low serum transferrin saturation, vitamin B 12 , and vitamin D levels. The pregnant women consuming dairy and nondairy alternatives DPs at the highest tertile (T3) were associated with reduced risks of low serum folate and vitamin B 12 levels. However, the processed food DP was not correlated with anemia-related biomarkers. Thus, plant-based, carnivore, and dairy and nondairy alternatives DPs were associated with the risk of low-serum-anemia-related variables.
... Some of these methods are used in the medical field and others in the pharmacological studies. 28 In this study, RBC folate was measured by the electrochemiluminescence binding assay method using the Elecsys folate RBC assay analyzer that employs a competitive test principle using natural folate-binding protein (FBP) specific for folate. Folate in the sample competes with the added folate (labeled with biotin) for the binding sites on FBP. 29 Whole blood treated with anticoagulant (EDTA) mixed with 1% ascorbic acid solution was incubated for liberation and stabilization of the intracellular folate. ...
Full-text available
Objective Folate is an essential vitamin for de novo DNA synthesis and cell proliferation. Folate insufficiency at the time of conception and during the first trimester of pregnancy is associated with unintended pregnancy and birth outcomes, particularly neural tube defects. Hence, this study aimed to assess folate status and associated factors of folate insufficiency among pregnant women attending antenatal care during their first trimester of pregnancy in Addis Ababa, Ethiopia. Materials and methods A cross-sectional study was conducted from 8 August 2017 to 3 January 2018 in Addis Ababa. In this study, 160 participants were enrolled via the convenience sampling method. Red blood cell folate was measured by the electrochemiluminescence binding assay method. Data were entered into Epi-Data version 3.1 and analyzed by SPSS version 22.0. Descriptive statistics were used to describe demographic characteristics and to determine the magnitude of folate deficiency. Logistic regression was used to identify the risk factors for folate deficiency. A p-value of less than 0.05 was considered statistically significant. Results In this study, 44/160 (27%) participants had red blood cell folate level <400 ng/mL, insufficient to prevent neural tube defect. Multivariate regression showed that regular vegetable consumption was an independent determinant factor for red blood cell folate level (adjusted odds ratio: 0.41, confidence interval: 0.18–0.93). Conclusion This study shows that a large magnitude of the first-trimester pregnant women had red blood cell folate concentrations below levels that are maximally protective against neural tube defects. Folic acid supplementation and supplemental nutrition containing green leafy vegetables should be promoted during the periconceptional period. In addition, the policymakers should set rules for mandatory folic acid fortification.
... The available data indicate that there has been an overall increase in the consumption of animal foods in the country (26.2 g/capita per d in 2010 v. 20.8 g/capita per d in 2005) [33], which might have contributed to a variation in the prevalence of vitamin B 12 deficiency. In addition, Lindström et al. used radioimmunoassay (RIA) for the assessment of vitamin B 12 status, while we used electrochemiluminescence immunoassay, which is a more sensitive, reliable, and advanced method than RIA [34]. Thus, the methodological differences in the assessment of vitamin B 12 status between the two studies may also explain the differences in the prevalence of vitamin B 12 deficiency. ...
Full-text available
Vitamin B12 deficiency is associated with an increased risk of pregnancy complications and adverse birth outcomes. However, data on vitamin B12 deficiency in pregnant Bangladeshi women are limited. This study examines vitamin B12 deficiency and marginal deficiency in rural Bangladeshi women during early and late pregnancies. Some 522 women whose gestational age was <20 weeks were recruited. Serum vitamin B12 concentrations were measured at baseline and after 14 weeks of iron-folate supplementation. Logistic regression analysis examined the association of various socio-demographic, dietary, and pregnancy-related factors with vitamin B12 deficiency and marginal deficiency. Overall, 19% of the women during early pregnancy had vitamin B12 deficiency (serum vitamin B12 concentration < 203 pg/mL) and nearly 40% had marginal deficiency (serum vitamin B12 concentration 203 to <300 pg/mL). Vitamin B12 deficiency doubled to 38% during late pregnancy, while marginal deficiency slightly increased to 41.7%. The pregnant women with a gestational age of ≥27 weeks had a higher risk of developing vitamin B12 deficiency (OR = 2.61; 95% CI = 1.096–6.214) than those of a gestational age of <27 weeks. Vitamin B12 deficiency was significantly higher in pregnant women in rented accommodation (OR = 13.32; 95% CI = 1.55–114.25) than in those living in their own house. Vitamin B12 deficiency was significantly higher among women who consumed red or organ meat <3 times a week than in those who consumed it more often (OR = 2.327, 95% CI = 1.194–4.536). None of these factors were significantly associated with marginal vitamin B12 deficiency. In conclusion, vitamin B12 deficiency and marginal deficiency among pregnant rural Bangladeshi women increased as their pregnancies progressed. Increasing gestational age, living in a rented house, and the consumption of red or organ meat <3 times a week were identified as the independent risk factors of vitamin B12 deficiency in this population. Further research with more in-depth assessments of dietary vitamin B12 intakes is needed to develop an intervention program preventing vitamin B12 deficiency in this population.
Full-text available
Vitamins are a unit of organic chemical substances that are essential for the adequate working of the human body. Water-soluble B vitamins are involved in the regulation of many metabolic and regulatory processes. Due to the inability to synthesize endogenously, they must be supplied to the body with edibles or in the form of supplementation as drugs or dietary supplements. Maintaining the correct level of vitamins is extremely important in the treatment of various diseases. In the presented work, the qualitative and quantitative procedure of the assay of vitamins B1, B2, B5, B6 and B12 in pharmaceutical products by chromatographic technique coupled with densitometric detection was developed, optimized and validated. During the optimization process, TLC Silica gel 60 F254 plates were chosen as a suitable stationary, and the mixture consisted of chloroform: ethanol: water: glacial acetic acid (2:8:2:0.5 v/v/v/v) as a mobile phase. Densitometric detection was conducted at a maximum absorbance λ = 254 nm for vitamins B1, B2, B6 and B12 and λ = 550 nm for vitamin B5 (after dyeing with ninhydrin). In the next step, the developed procedure was validated in accordance with the ICH guidelines. The recorded correlation coefficients obtained in all tested concentrations of B vitamins, ranging from 0.9947 to 0.9996, confirmed good linearity. The method is characterized by good precision, RSD data ranging from 0.62 to 1.52% for direct precision and from 0.84 to 1.4% for intermediate precision. Accuracy was proven by a recovery test at three concentration levels, with values close to 100% with RSD less than 1%. The calculated LOD and LOQ data for all tested vitamins B1, B2, B5, B6 and B12 were belove 1 μg/spot. The developed method was then used to quantitatively and qualitatively assess the content of B vitamins in medicinal products and dietary supplements with satisfactory results.
Full-text available
Background: The brain volume loss also known as brain atrophy is increasingly observed among children in the course of performing neuroimaging using CT scan and MRI brains. While severe forms of brain volume loss are frequently associated with neurocognitive changes due to effects on thought processing speed, reasoning and memory of children that eventually alter their general personality, most clinicians embark themselves in managing the neurological manifestations of brain atrophy in childhood and less is known regarding the offending factors responsible for developing pre-senile brain atrophy. It was therefore the goal of this study to explore the factors that drive the occurrence of childhood brain volume under the guidance of brain CT scan quantitative evaluation. Methods: This study was a case-control study involving 168 subjects with brain atrophy who were compared with 168 age and gender matched control subjects with normal brains on CT scan under the age of 18 years. All the children with brain CT scan were subjected to an intense review of their birth and medical history including laboratory investigation reports. Results: Results showed significant and influential risk factors for brain atrophy in varying trends among children including age between 14-17(OR = 1.1), male gender (OR = 1.9), birth outside facility (OR = 0.99), immaturity (OR = 1.04), malnutrition (OR = 0.97), head trauma (OR = 1.02), maternal alcoholism (OR = 1.0), antiepileptic drugs & convulsive disorders (OR = 1.0), radiation injury (OR = 1.06), space occupying lesions and ICP (OR = 1.01) and birth injury/asphyxia (OR = 1.02). Conclusions: Pathological reduction of brain volume in childhood exhibits a steady trend with the increase in pediatric age, with space occupying lesions & intracranial pressure being the most profound causes of brain atrophy.
Full-text available
Objective: In medical laboratories, it is important to verify the precision and trueness studies made by the manufacturer before giving results to the patient. The aim of this study was to evaluate the verification of TSH, ferritin and vitamin B12 tests according to CLSI EP15 A2 guideline. Methods: In this study verification studies were carried out according to CLSI EP15 A2 guidelines for TSH, vitamin B12, ferritin. PreciControl quality control material were used for precision study. For trueness studies, patient samples were simultaneously assayed with two different immunoassay analyzers (Beckman Coulter DXI 800 and Advia Centaur). Microsoft Excel programme was used for statistical evaluation. Results: The precision estimate of TSH, vitamin B12, and ferritin for both control levels were lower than the verification value. In the trueness verification study, the estimated percent bias of TSH and vitamin B12 were within the verification limits. The estimated percent bias of ferritin was found beyond the verification limits.
A novel extracorporeal circuit for hemodialysis is described, which enhances the removal of middle- and high-molecular-weight toxins. To enhance the removal of especially high-molecular toxins, a recirculation pathway for dialysate flow is added to conventional circuit. The influence of an increase in the ratio of recirculation to dialysate flow rate and the ultrafiltration rate (UFR) on the removal of toxins was investigated to evaluate the removal of different toxins in clinical conditions. Removal of toxins was also modeled by an analytical method and solved by the MATLAB software (The MathWorks, Inc., Natick, MA). A significant increase in removal of urea (up to 31%) and vitamin B12 (11%) was achieved when the UFR is low (≤50 ml/h) or zero. The model showed an excellent agreement with the experimental results, which indicates its applicability for the removal of different toxins in an extracorporeal circuit. Increase in recirculation flow, while adjusting the UFR near zero, improves the mass transfer coefficient and can lead to enhanced especially middle- and high-molecular-weight toxin removal.
In order to find out whether algae-based nutritional supplements contain both physiologically active vitamin B12 and its non-active pseudo-form, a simple and fast ultra-high performance liquid chromatography method with UV detection was developed to separate these two forms. A total of 57 commercially available microalgae products based on Chlorella and Spirulina were analyzed. Results obtained showed a broad variety of the content of total vitamin B12 ranging from not-detectable to 445.9 µg 100g⁻¹ dry matter for Chlorella samples, and between 92.8 and 164.1 µg 100g⁻¹ dry matter for Spirulina products. However, there was a big variation in the concentration of pseudo-vitamin B12 within all samples analyzed. Chlorella products contained mainly physiologically active cobalamin, while pseudo-vitamin B12 was the prevailing form in Spirulina-labeled nutritional supplements. The importance of being able to differentiate between cobalamin and its pseudo-form in algae is not only relevant for food analysts, but also regarding consumer protection since they have to be able to rely on the correct labeling of nutritional supplements.
Vitamin B12 is the major dietary supplementary for most animals and has widespread applications in healthcare. The present study deals with the isolation of vitamin B12 producing strains from food waste soil samples. Three strains (VS1, VS2 and VS3) were isolated from the soil sample and the extraction was done using potassium ferrocyanide along with PBS buffer. Vitamin B12 was exhibited at about 114.1g/L from VS1 strains than the VS2 (11.4g/L) and VS3 (22.88g/L).
Biochemical analysis is a rapidly expanding field and is a key component of modern drug discovery and research. Methods of Biochemical Analysis provides a periodic and authoritative review of the latest achievements in biochemical analysis. Founded in 1954 by Professor David Glick, Methods of Biochemical Analysis provides a timely review of the latest developments in the field.
Various sampling tools such as laser ablation, flow injection, desolvation systems are considered critical to enhance the practical capabilities of inductively coupled plasma-mass spectrometry (ICP-MS) for real-world samples. The limitation of ICP-MS to directly analyze solid materials or powders led to the development of high-powered laser systems to ablate the surface of a solid and sweep the sample aerosol into the ICP MS in the conventional way. Flow injection is a powerful front-end sampling accessory for ICP-MS that can be used for preparation, pretreatment and delivery of the sample. A research group at Canada's National Research Council has developed a very practical on-line approach, using a flow injection sampling system coupled to an ICP MS.
The distribution concentration of chemical species in a sample is dictated by the physical and chemical properties of the matrix. As such, when a sample is pre-treated, in any way, there is a potential for redistribution of homologous species. The extent of this analyte redistribution is determined by both thermodynamic properties of species (e.g., changes in concentrations of species according to their equilibrium expressions) and kinetic properties (e.g., the rate of the reactions compared with the duration of sample preparation and analysis). The redistributions of analyte species as a function of several experimental parameters (e.g., time, solution pH, injection methods and calibration methods) are illustrated in this paper. Whereas rabbit metallothionein protein showed a stability of more than a few days under certain storage conditions, coenzyme-B12 was rapidly degraded in less than 2 h. pH studies showed that the migration of free Cd2+ ions in rabbit metallothionein was not significantly affected unless the pH of the solution exceeds the solubility limit of the metal hydroxide. However, pH-sensitive compounds such as vitamin B12 showed significant changes in the migration time and analyte composition. The injection studies suggested that electrokinetic injection may produce biased results, in favor of species that have higher electrophoretic mobility. Hydrodynamic injection will produce a result that is more representative of the initial sample composition.
The advantages of capillary electrophoresis (CE) as a powerful separation technique can be combined with the atomic specificity, multi-elemental character and extremely high sensitivity of an inductively coupled plasma mass spectrometry (ICP-MS) detector for metal-speciation studies in different fields of interest. However, the coupling of both commercially available instruments deserves particular attention if separative resolution, high analyte transport efficiency and sensitive detection are to be achieved. In that technical vein, the interface itself may be considered as the “key to success”. We review the different ways of performing CE-ICP-MS coupling so far and discuss interfaces based on conventional, micronebulizers and the generation of volatile species of analytes. We pay particular attention to those contributions aimed at improving the sensitivity achieved in CE-ICP-MS couplings. Finally, we review the latest applications (as reported from 2002 to June 2004) of CE-ICP-MS to tackle trace-element-speciation problems. It appears that initial technical problems for coupling CE to ICP-MS have now been overcome, so we expect an increasing number of applications of the technique in the field of trace-element speciation in the near future.
Aim: Deficiency of cobalamin and folate during pregnancy is associated with megaloblastic anemia. Lower levels of folate and vitamin B12 were reported in mothers whose offspring had neural tube defects compared to unaffected controls. Increased methylmalonic acid (MMA) levels are a sensitive indicator of mild vitamin B12 deficiency and elevated homocysteine levels denote vitamin B12 or folate deficiency. We aimed to evaluate cobalamin state in pregnancy by using urine MMA levels determined by an economic spectrophotometric method. Materials and Methods: For this cross-sectional study, plasma total homocysteine (tHcy), serum folate and vitamin B12, and urine MMA levels were measured in 186 uncomplicated pregnant women (30 in first, 58 in second and 98 in third trimester). MMA measurements were made in first morning urine samples with normalizing by creatinine concentrations. Results: The tHcy concentrations were not elevated (