Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Introduction
16
Vitamin B12 (cobalamin) is an organic complex that contains a cobalt ion in its structure.[1] This vitamin is an important coenzyme
for cell development and growth, and its deficiency leads to weakness, fatigue, nausea, constipation, weight loss, pernicious anemia
and nerve degeneration.[2] Peculiarly, vitamin B12 is essential for
the human diet, but cannot be synthesized by the body. This vitamin has to be obtained from natural sources such as fish, dairy
products, egg, meat and poultry. However, compared to other vitamins, the daily requirement of vitamin B12 is relatively low.[3] The
determination of vitamin B12 in human serum at picogram level becomes necessary for the detection of its deficiency.[4] Hence, sensitive analytical detection devices are required. To counter the
deficiency of vitamin B12, many supplements are available in the form
of tablets, capsules and injections. Methods for determination of vitamin B12 remain limited due to their low sensitivity and poor selectivity.
Several methods have been so far applied for determination of
vitamin B12 including microbiological assay that uses Lactobacillus
leichmannii (ATCC 7830) as the test organism,[5] radioisotopic dilution assay,[6,7] high-performance liquid chromatography (HPLC)
with different detection methods including UV detection,[8,9] electrochemical detection,[8] flame atomic absorption spectrometry
(FAAS),[10] fluorometry,[11,12] inductively coupled plasma mass spectrometry (ICP-MS)[1315] and biosensor assay based on surface plasmon resonance (SPR).[16] These conventional methods have their
own advantages, but at the same time, they have certain drawbacks such as being laborious, time consuming, tedious, less safe,
too expensive and showing low sensitivity and selectivity. Other
disadvantages of these methods have been mentioned in the
literature.[17] But there is no report about determination of vitamin
B12 by using X-ray fluorescence (XRF).
XRF analysis is a powerful analytical tool for the spectrochemical
determination of almost all the elements present in a sample. XRF
radiation is induced when photons of sufficiently high energy, emitted from an X-ray source, impinge on a material. These primary
X-rays undergo interaction processes with the analyte atoms. This
allows the identification of the elements present in the analyte
and the determination of their mass or concentration. Measurement of the spectrum of the emitted characteristic fluorescence radiation is performed using wavelength dispersive (WD) and energy
dispersive (ED) spectrometers. In principle, XRF analysis is a multielement analytical technique and in particular, the simultaneous determination of all the detectable elements present in the sample is
inherently possible with EDXRF. In WDXRF both the sequential and
the simultaneous detection modes are possible.[18]
New idea for analysis of organic compounds by XRF methods extends the applicability of this methodology in further studies. So far,
only a few paper presented an indirect method for the determination XRF determination of organic compound. The method is based
on the precipitation of an ion-associate complex formed between
terazosin (as target analyte) and [Zn(SCN)4]2 and the formation
of a thin film on a membrane filter.[19] To the best of our knowledge,
this is the first report about the application of the XRF for the determination of vitamin B12 which have one cobalt atom in its structure.
XRF possesses a number of limitations when dealing with aqueous samples, such as short linear range, the requirement of closely
matching standards to overcome matrix effects, bubbles released
from solutions due to inadequate sample holder filling and heating
of the solution.[20] The extraction of analytes from a liquid onto a
solid (SPE) is an important field in sample handling of elements
Sample preparation
for their detection by XRF. The basic principle of such procedure is
based on transfer of analytes from the aqueous phase to the active
sites of the adjacent solid phase. One of the clear trends in science
and technology of the future is nanotechnology. Several sorbents
have been reported in the literature to preconcentrate metals prior
to their determination by XRF techniques, such as ion-exchange
materials,[21] polyurethane foam (PUF),[22] filter discs,[23] activated
carbon[24] and membrane filters.[25] Yamini et al.[26] reported determining the nanogram per milliliter level of V, Cr, Mn, Fe, Co, Ni, Cu
and Zn in water by XRF after using a silica gel powder. In another
work, the preconcentration of Co, Ni, Cu, Zn and Pb using graphene
oxide (GO) as solid sorbent without using chelating agent is proposed by Zawisza et al..[27] The proposed procedure is based on dispersive solid-phase microextraction (DSPME). Some new efficient
variations of these methods and new techniques extending the
possibilities of XRF for liquid solutions analysis have been proposed
in recent years. In 2010, Margu et al. reviewed practical aspects related to the use of extractive and non-extractive sample preparation
of elements before their detection by XRF.[28] However, the interest in
preconcentration methods for XRF has increasingly turned towards
the refinement of its modes for use in practical sample preparations.
In this research, GO are dispersed in aqueous samples, which promotes the immediate interaction between the vitamin B12 and GO
and shortens the time of sample preparation in comparison with a
classical SPE. After the adsorption process, the GO with adsorbed vitamin were collected onto a filter and measured directly using XRF
spectrometry without the necessity of analyte elution.
Experimental
Sample preparation
Extraction procedure
2.3 ml of 1.0 mg ml-1 suspension of GO was added to 77.7 ml of
aqueous sample. The pH of the solution was adjusted to 2. Next,
the mixture was stirred by a magnetic stirrer for 10.5 min. After that,
the sample was collected onto the Millipore filter (cellulose esters,
0.2-mm pore size) using a vacuum filtration assembly. Loaded filters
were placed between two 6.0-mm-thick Mylar X-ray foils (supplied
by Chemplex Industries, Inc., New Cork, USA) mounted in special
liquid sample holders which incorporate a snap-on ring at the
end of the cell for attachment of thin-film supports. Afterwards,
samples were sealed in the sample holder of the equipment for
WDXRF analysis. The blank sample was prepared using the described
procedure and high purity water instead of the analyzed solution.
Optimization strategy
There are several factors like pH, sample volume, sorbent amount
and extraction time that affect the extraction process. In order to
obtain the optimum conditions for extraction of vitamin B12, effects
wileyonlinelibrary.com/journal/xrs
17
M. Moradi et al.
of sample pH on the extraction of analyte by the proposed method
were optimized using one-variable-at-a-time (OVAT) process. In
OVAT strategy we can only control one variable at a time and other
factors are fixed. Optimization of the three other parameters on the
extraction efficiency was performed using a multivariate optimization. The software package Design-Expert 8.0.6 trial version
(Stat-Ease Inc., MN, USA) was used for experimental design, data
analysis and response surfaces.
The extraction recovery for solid phase extraction was expressed
as the percentage of total analyte amount, ns, initial (number of
moles of the analyte amoun toriginally present in the sample) transferred into the extraction phase at the end of the extraction, na, final
(number of moles finally collected in the extraction phase) was calculated by the following equation:
R% 100 na;final =na;initial
(1)
18
Figure 1. Characterization of graphene oxide nanosheets: (a) XRD pattern; (b) Raman spectrum and (c) TEM image.
wileyonlinelibrary.com/journal/xrs
Sample preparation
Figure 2. Ioniziation plot of vitamin B12. Purple circle is positive charge and green circle is negative.
wileyonlinelibrary.com/journal/xrs
19
(2)
M. Moradi et al.
Table 1. BoxBehnken response surface design and responses value
Run
X1
X2
X3
cps
1
2
3(C)
4
5(C)
6
7
8
9(C)
10
11
12
13
14(C)
15
16
1
1
0
1
0
0
1
0
0
1
1
0
1
0
0
1
1
1
0
0
0
1
0
1
0
0
0
1
1
0
1
1
0
0
0
1
0
1
1
1
0
1
1
1
0
0
1
0
1.0
3.0
2.0
3.0
2.0
2.0
3.0
2.0
2.0
1.0
1.0
2.0
3.0
2.0
2.0
1.0
50.0
50.0
75.0
75.0
75.0
100.0
75.0
50.0
75.0
75.0
75.0
50.0
100.0
75.0
100.0
100.0
10.0
10.0
10.0
15.0
10.0
15.0
5.0
15.0
10.0
15.0
5.0
5.0
10.0
10.0
5.0
10.0
1.84
3.32
4.02
3.50
3.95
3.21
2.88
2.03
3.91
1.96
2.07
2.11
3.02
4.10
2.55
2.51
(C)Center point
20
than 0.0500 indicate model terms are significant. It was also shown
that GO amount (p = 0.0002), sample volume (p = 0.0117) and the
interactions between GO amount and sample volume (p = 0.0485)
have a significant effect upon the extraction.
As can be seen, all of the quadratic components of have the significant effect (p < 0.0001) on the extraction efficiency and the related RSM behavior agrees with the literature.[35] Quadratic
component is a non-linear relation between factors and response.
The effect of quadratic components is very complicated, and up
to now the reasons of their effect are not clear.
The response equation fitted the experimental data with R2 of
0.9765, indicating 97.65% of the variability in the response. The
goodness-of-fit of the model to the experimental data shown in
Fig. 4 has an adjusted R2-value of 0.9412. According to Joglekar
and May,[36] R2 should be at least 0.80 for a good fit of a model.
As is observed, the coefficient of determination, R2, was more than
0.80 which means that the obtained equation is adequate for correlating the experimental results. In this figure, red color is related to
higher cps and blue is related to lower cps. Plotting predicted
wileyonlinelibrary.com/journal/xrs
Sample preparation
Figure 5. Response surfaces for: (A) sample volume-sorbent amount, (B) extraction time-sorbent amount and (C) extraction time-sample volume.
time even for larger volumes of samples. The results are presented
in Fig. 5B, C. It is found that the extraction of elements is maximized
when the stirring time is more than 11 min. So stirring of the solution was done for 11 min at subsequent analysis.
Quantitative analysis
According to the overall results of the optimization study, the following experimental conditions were chosen: sorbent amount,
2.3 mg; sample volume, 80 ml and extraction time, 11 min. In order
to proceed with the current evaluation of the proposed extraction
technique, linearity, LOD, enhancement factor (EF) and repeatability
were investigated under optimized conditions with the standard
solutions of the analyte. The performance of the developed procedure is summarized in Table 2. Calibration curve was plotted using
10 spiking levels of vitamin B12 over a range between 10 and
1000 g l1 and analyzing each level in triplicates. Good linearity
of response (LDR) was observed in the range of 251000 g l1 with
a correlation of determination more than 0.998. Figure 6 shows
these calibration curves with their equation. It can be seen that it
is linear. The LOD was obtained from the formula CLOD = 3Sb/m,
where Sb is the standard deviation of ten replicates of blank measurements and m is the slope of the calibration curve. Also, EF, calculated as the ratio of slopes of the calibration curve after
preconcentration of the analyte and direct calibration equation,
was obtained 46. Precision of the method (RSD%) was found to
be less than 8.1% based on four-replicate analysis during 1 day at
a concentration of 100 g l1.
A comparison between the figures of merit of the proposed
method and some published methods for extraction and
wileyonlinelibrary.com/journal/xrs
21
M. Moradi et al.
Table 3. Comparison of the proposed method with other methods developed for extraction and determination of vitamin B12
Matrix
1
Method
LOD (g l )
Urine
Multivitamin tablet
Food products
LPME/HPLC-UV
SPE/AASb
Immunoaffinity
/HPLC-UV
HPLC-UV
SPE/XRF
Food products
Pharmaceutical
formulations
1
Linear range (g l )
RSD (%)
Ref
90.0
2.7
3.0
4008000
2.7300
10200
4.5
4.0
3.2
[37]
500
20.0
100010 000
25.01000
8.1
[39]
[38]
[17]
This work
Added
(g/l) a
Multivitamintablet
Effervescent
tablet
Injection
100
100
100
Determined
amount
6.6b
6.0b
4.5
4.9b
310c
5.0b
333c
106
93
98
6.3
7.5
6.9
5.0
3.8
for in sample handling of metal-organic compounds for their detection by XRF. The results further demonstrated that the proposed GO-based extraction method has good precision,
linearity, and accuracy over the investigated concentration
range. Also, the proposed method offers a simple, fast, sensitive
and selective method for extraction and determination of vitamin B12. Furthermore, there is a possibility of extraction of vitamin B12 from large volumes of sample and therefore sample
diluting and decrease of matrix effects are possible.
Acknowledgements
The authors are grateful for the financial support of this work from
the Iran National Science Foundation (INSF) (Tehran, Iran).
References
effervescent tablets with concentration of 6.6 and 4.9 g per each
tablet, respectively. Also, the method was tested for extraction of vitamin from an injection sample containing mixture of three vitamins of B1, B6 and B12 (containing 333 mg l1 of vitamin B12). For
this purpose, 200 l of the injection sample was diluted to 80 ml
with deionized water. According to Table 4, the results obtained
by the current method agreed well with the labeled values of vitamin B12 for multivitamin tablet, effervescent tablet and injection
sample. The RSD (%) were in the ranges of 3.87.5%. As we know,
recovery (R%) in extraction methods represents the amount of
extracted analyte from sample to extraction phase. To determination of R%, we can use add-found manner, so that a known concentration of analyte was added into sample and after extraction using
proposed technique, the amount of extracted analyte was
determined. If the recovery of added amount is in the range of
90110%, the proposed method is validated. As can be seen in
Table 4, recovery values of real samples are in the range of 93 to
106%; thus, proposed extraction method prior to XRF analysis is
suitable method for vitamin B12 determination.
Conclusions
22
wileyonlinelibrary.com/journal/xrs
Sample preparation
[25] E. Margu, C. Fonts, K. Van Meel, M. Hidalgo, I. Queralt. Spectroscopy
Europe 2008, 20, 11.
[26] Y. Yamini, N. Amiri, M. Karimi. X-Ray Spectrom. 2009, 38, 474.
[27] B. Zawisza, R. Sitko, E. Malicka, E. Talik. Anal. Methods 2013, 5, 6425.
[28] E. Margu, R. Van Grieken, C. Fonts, M. Hidalgo, I. Queralt. Appl. Spectro.
Rev. 2010, 45, 179.
[29] W. S. Hummers Jr., R. E. Offeman. J. Am. Chem. Soc. 1958, 80, 1339.
[30] M. J. Allen, V. C. Tung, R. B. Kaner. Chem. Rev. 2009, 110, 132.
[31] J. W. Lee, A. S. Hall, J. D. Kim, T. E. Mallouk. Chem. Mater. 2012, 24, 1158.
[32] Q. Tu, L. Pang, Y. Chen, Y. Zhang, R. Zhang, B. Lu, J. Wang. Analyst 2014,
139, 105.
[33] http://www.chemicalize.org/structure/. Retrieved 18 January 2014.
[34] D. Bingol, M. Kulcu. Analyst 2011, 136, 4036.
[35] J. Xia, B. Xiang, W. Zhang. Anal. Chim. Acta 2008, 625, 28.
[36] A. M. Joglekar, A. T. May. Cereal Food World 1987, 32, 857.
[37] P. Berton, R. P. Monasterio, R. G. Wuilloud. Talanta 2010, 97, 521.
[38] M. R. Hadjmohammadi, V. Sharifi. J. Food Drug Anal 2007, 15, 285.
[39] L. S. S. Kumar, M. S. Thakur. Anal. Biochem. 2011, 418, 238.
23
wileyonlinelibrary.com/journal/xrs