Antalgin 2
Antalgin 2
3, 134-144
Available online at http://pubs.sciepub.com/wjce/6/3/6
©Science and Education Publishing
DOI:10.12691/wjce-6-3-6
Abstract A drastic increase in the consumption of pharmaceuticals has resulted in a high load of pharmaceuticals
in wastewater. Many pharmaceuticals are non-biodegradable and are resistant to conventional wastewater treatments.
For this reason there is an obvious need to first detect these substances and, second, to detoxify them. Metamizole is
a typical representative of an analgesic non-steroidal. In this first part, a rapid, sensitive, and inexpensive detection
method with different commercial screen-printed electrodes (SPEs) were used to quantitatively detect metamizole.
(Spectro-)Electrochemical methods such as cyclic voltammetry (CV), electrogenerated chemiluminescence (ECL),
and amperometry (AM) are discussed in detail and the sensitivities of the electrochemical methods are compared to
the sensitivity of conventional gas chromatography-mass spectrometry detection (GC-MSD). The limit of detection
(LOD) is 1 mol/L for GCMS and 5 to 50 mol/L for electrochemical detection depending on the method used.
Keywords: metamizole (dipyrone), 4-aminoantipyrine, 4-methylaminoantipyrine, electrochemical detection, mass
spectrometric detection
Cite This Article: Achim Habekost, “The Analgesic Metamizole (Dipyrone) and Its Related Products
Antipyrine, 4-Aminoantipyrine and 4-Methylaminoantipyrine. Part 1: Mass Spectrometric and Electrochemical
Detection.” World Journal of Chemical Education, vol. 6, no. 3 (2018): 134-144. doi: 10.12691/wjce-6-3-6.
Metamizole is a prodrug, which hydrolyses to toxicity of the harmful substances, are also important
4-methylaminoantipyrine and then into many other metabolites. topics in this declaration. Therefore, chemical education
These metabolites are known to be non-biodegradable plays a pivotal role in revealing how water security is
[7]. Gyenge-Szabo et al. [8] determined the main important for the survival of humanity.
metabolites to be 4-aminoantipyrine, 4-acetylaminoantipyrine, It is generally agreed that analytical chemistry courses
4-formylaminoantipyrine and 4-methylaminoantipyrine. are an important component of college and school curricula.
These were found in communal wastewater that was In first-year courses, typical preparatory chemistry combined
collected; after reversed-phase solid phase extraction, the with qualitative and quantitative courses dominate the
probes were analysed with a Micromass Q-TOF instrument. laboratory work. In advanced courses, electrochemical and
The limit of detection (LOD) was in the microgram per litre photometric investigations are conducted. Final analytical
range. Zhang et al. [9] investigated 4-methylaminoantipyrine, courses intend to encourage interest in analytical
4-formylaminoantipyrine and 4-acetylaminoantipyrine in chemistry. Some topics handle complex apparatus, such as
goat tissues using liquid chromatography-tandem mass mass spectrometers and NMR spectrometers, AAS, HPLC,
spectrometry and found values ranging from 0.4 to 6 g/kg. potentiostats, etc. This is done by solving chemical
In environmental chemistry, there is an obvious need to problems, e.g., determining the LOD of the different
first detect these substances and then to detoxify them. In analytical methods.
this paper, several analytical procedures will be discussed In a chemical sense, the analytical chemistry of harmful
and compared regarding the speed of the tests and the and threatening substances and their detoxification is a
LOD. The elaborate and expensive gas chromatography-mass great challenge for chemical education both in university
spectrometry (GC-MS) technique, operating in different and in (high) school. However, the elaborate apparatus
modes (scan and selected ion monitoring SIM mode), is and the analytical methods used suggest that the topic is
compared to a rapid, sensitive and inexpensive electrochemical suitable for university-level lessons [13].
detection method employing different commercial We would like to summarise the didactic reasons
screen-printed electrodes (SPEs) (i.e. single-walled carbon for presenting the unusual method electrogenerated
nanotubes SWCNT, graphite and Au SPE). The chemiluminescence (ECL) to an audience of chemistry
electrochemical detection technique can operate on students. ECL is not only an aesthetic technique but also a
site and can identify contaminated probes very relatively new and sound electrochemical method. The
quickly. Spectroelectrochemical methods such as cyclic goals of using ECL are the following:
voltammetry (CV), electrogenerated chemiluminescence - to demonstrate that ECL correlates to CV predictions,
(ECL) and amperometry (AM) are discussed in detail and - to investigate electrochemical instruments (potentiostat,
the sensitivities of these methods are compared to the light detection), and
sensitivity of the different modes of GC-MS detection. - to use ECL in analytical techniques—in particular,
to investigate pharmaceutical substances such as
metamizole. We refer the reader to a review on this
2. Pedagogical Objectives subject [14].
Figure 1. UV spectra of antipyrine (top left), 4-aminoantipyrine (top right), methylaminoantipyrine (bottom left) and metamizole (bottom right). See
also Table 1
Figure 3. GC-MS of a mixture of aminoantipyrine, methylaminoantipyrine and metamizole. The substance at about 14 min could not be identified
Figure 5. Concentration dependence of dimethylaminoantipyrine, the thermal decomposition product of metamizole, in the range of 55 mol/L–
500 mol/L. Insert: 55 mol/L–100 mol/L
In the SIM mode the LOD is about fifty times lower than in the scan mode (selected mass: 231 amu).
Figure 6. Concentration dependence of dimethylaminoantipyrine. The curve shows a linear dependence between 55 and 500 mol/L
The calculated LOD is 50 mol/L * 1 L = 50 pmol or 15.6 ng (with molecular weight of metamizole = 313 g/mol).
140 World Journal of Chemical Education
Table 2. Masses and structures of the main fragments of the four 4.3. Electrochemical Characterisation via
antipyrine derivatives [18]
Cyclic Voltammetry
Substance Mass / atomic mass unit amu
Antipyrine (188 amu) 188, 96, 77, 56 Several electrochemical investigations of metamizole have
Aminoantipyrine (203 amu) 203, 147, 121, 106, 84, 56 been carried out with different types of electrodes, including
Methylaminoantipyrine (215 amu) 217, 203, 123, 98, 83, 56
glassy carbon electrodes [20] and carbon paste electrodes
[21]. Metal electrodes as platinum were used by Basaez et
Metamizole (311 amu) 231, 216, 123, 98, 83, 56
al. [22] while Munoz [23] used gold electrodes. Dadamos
Mass / amu Structure et al. [19] modified a carbon paste electrode with nickel-
doped potassium strontium niobate. This electrode exhibits
reversible behaviour, high conductivity and durability. In
231 addition, the electrode promotes the oxidation of dipyrone
by first oxidising niobium(IV) to niobium(V) at the electrode
surface. The niobium(V) then undergoes a reduction by
dipyrone back to niobium(IV). Baranowska et al. [24]
used a hanging mercury drop and a graphite electrode for
216 / 215 differential pulse voltammetry for the simultaneous detection
of different drugs (paracetamol, furosemide, dipyrone,
dexamethasone, and cefazolin) in spiked urine samples.
We used different SPEs to determine the best current
response and electrochemical resolution. The scanning
203 electron microscope image (SEM) of the Au SPE used is
shown in Figure 7. Figure 8 shows the best result with an
Au SPE.
At about 0.4 V and 0.6 V, the enamine moiety
(–C=C-N-, see the circle in the structure)
147
123 unknown
96
oxidizes and forms an iminium radical cation. In the
82 C4H6NO reversed scan, a small cathodic peak can be observed at
0.25 V. The voltage difference between the anodic and
cathodic peak is about 150 mV. Therefore, the redox
reaction is quasi-reversible.
77 After Bacic et al. [15], the oxidized metamizole can
subsequently react (dimerize) at 0.4 V (EC mechanism). This
was demonstrated when the reverse scan was started at different
voltages. When it was started at 0.5 V, the oxidation peak
56 CH2=N=CH-CH3 is more pronounced (see dotted line in Figure 9).
Figure 8. Cyclic voltammogram (CV) of metamizole with gold as the working electrode
The oxidation process at 0.8 V is common to all antipyrines. Therefore, the most plausible oxidation possibility is the
nitrogen adjacent to the phenyl ring [15].
Figure 10 shows the CVs of antipyrine and the three derivatives with a single-walled carbon nanotube working
electrode (note the slight voltage shift compared to the working electrode used above).
142 World Journal of Chemical Education
Figure 10. CVs of the four antipyrine derivates at pH 10. Thick line: metamizole; thin line: antipyrine; dotted line: 4-aminoantipyrine; dashed line:
methylaminoantipyrine
4.4. Electrogenerated Chemiluminescence given by Miao [25], Kapturwicz [26], Hercules [27],
(ECL) Richter [28], Parveen [29] and Bard [30].
The ECL system used is a combination of [Ru(bpy)3]2+
Since the 1960s, the ECL technique has become and proline (as co-reactant), which produces one of
increasingly attractive in analytical chemistry. ECL the most intense ECLs [14]. In addition, this system
involves the generation of an excited state of the has excellent water solubility and is less toxic than
commonly used and intensively investigated tris(2,2’- tripropylamine, one of the most common co-reactants in
bipyridyl)ruthenium (II) [Ru(bpy)3]2+ on an electrode ECL studies. The ECL is quenched by adding metamizole.
surface. First, the [Ru(bpy)3]2+ is oxidized; then, it Quenching can be used to quantitatively determine
undergoes an electron-transfer reaction with a co-reactant. metamizole. Figure 11 shows the ECL quenching at three
During the latter process, an excited state is created that different metamizole concentrations (1 mmol/L, 0.5 mmol/L
subsequently decays and emits light. Reviews of ECL are and 0.1 mmol/L).
Figure 11. Cyclic voltammograms and electrogenerates curves. Solid lines: cyclic voltammograms; dotted lines: ECL of [Ru(bpy)3]2+ / proline with
(concentrations from 1 mmol, 0.5 mmol and 0.1 mmol (13)) and without (4) metamizole. Working electrode: Au 220 AT
World Journal of Chemical Education 143
Figure 12. Amperometric detection of metamizole at pH 10. Injection volume: 10 L. Three different concentrations—1 mmol/L, 90 mol/L and
8 mol/L—injected three times. LOD = 8*10-11 mol = 25 ng
4.5. Amperometric Detection inside the electrochemical HPLC cell and the injection of
the aqueous metamizole solution (pH 10) was done via a
A simple, rapid and low-cost method to quantify Rheodyne valve (injection volume 10 L). The pumping
metamizole is amperometric detection (AM). In association speed was 2 mL/min. The current was recorded as 0.25 V
with flow injection analysis (FIA), AM provides a fast at three different metamizole concentrations (1 mmol/L,
analysis [23]. Perez-Ruiz [31] used a glassy carbon 90 mol/L and 8 mol/L). All solutions were injected
electrode for amperometric detection and measured the three times. Figure 12 shows the amperometric curves.
LOD as a function of flow rate and coil length between The LOD is about 5 mol/L (signal to noise ratio 3:1).
1 and 10 g/mL.
Burcinova et al. [32] determined antipyrine and
4-aminoantipyrine in urine with HPLC and amperometry 5. Discussion
detection (carbon fibre array as working electrode). The
authors concluded that photometric detection is more This paper has described mass spectrometry and three
sensitive than amperometric detection. However, photometric (spectro)electrochemical methods for identifying metamizole:
detection requires pre-concentration of urine samples by CV, ECL and AM. We have shown that these methods
extraction. have different LODs and that the ECL signal has the
In our own AM measurements, we tested two electrodes: disadvantage of having poor reproducibility. The
SWCNT SPE and Au SPE. No difference was observed electrodes used had to be cleaned before each
between these electrodes but practicability was better for measurement to assure that the electrode had no ageing
the latter. This was ascribed to the better cleaning procedure: effect through passivation.
the gold electrode is rinsed with concentrated nitric acid Table 3 compares the LODs and gives a final appraisal
and then with distilled water. The electrode was installed of the methods used.
Table 3. Summary of the methods used
[15] Bacil, R.P., Buoro, R.M., Campos, O.S., Ramos, M.A., Sanz, C.G.,
Acknowledgements Serrano, S.H.P., Electrochemical behavior of dipyrone (metamizol)
and others pyrazolones, Electrochim. Act. 273, 358-366, 2018.
A. H. thanks the Vector foundation, the Fonds der [16] Isaacs, R.C.A., Harper, M.M., Miller, E.C., Analytical challenge
Chemischen Industrie and the University of Ludwigsburg in the confirmative identification of dipyrone as an adulterant in
illicit drug sample, Forensic Sci. Int. 270, 185-192, 2017.
for financial support.
[17] Rogosch, T., Sinning, C., Podlewski, A., Watzer, B., Schlosburg,
J., Lichtman, A.H., Cascio, M.G., Bisogno, T., Di Marzo, V.,
Nüsing, R., Imming, P., Novel bioactive metabolites of dipyrone
References (metamizol), Bioorg. & Med. Chem., 20, 101-107, 2012.
[18] Zitz, E., Spiteller, G., Location of Functional Groups in Antipyrine
Metabolites by Mass Spectrometry, Biomed. Mass. Spectro., 4,
[1] Kümmerer, K., Drugs in the environment: emission of drugs,
155-158, 1977.
diagnostic aids and disinfectants into wastewater and surface
water by hospitals in relation to other sources - a review, [19] Dadamos, T.R.L., Freitas, B.H., Genova, D.H.M., Espirito-Santo,
Chemosphere, 45, 957-969, 2001. R.D., Gonzales, E.R.P., Lanfredi, S., Teixeira, M.F.S.,
Electrochemical characterization of the paste carbon modified
[2] Feldmann, D.F., Zuehlke, S., Heberer, T., Occurrence, fate and
assessment of polar metamizole (dipyrone) residues in hospital electrode with KSr2Ni0.75O15- solid in catalytic oxidation of the
and municipal wastewater, Chemosphere, 71, 1754-1764, 2008. dipyrone, Sens. and Act. B, 169, 267-273, 2012.
[3] Thomas, K.V., Dye, C., Schlabach, M., Langford, K.H., Source to [20] Bacil, R.P., Buoro, R.M., da Silva, R.P., Medinas, D.B., Lima,
sink tracking of selected human pharmaceuticals from two Oslo A.W.G., Serrano, S.H.P., Mechanism of electro-oxidation of
city hospitals and a wastewater treatment works, J. Environ. metamizol using cyclic voltammetry at a glassy carbon electrode,
Monitor. 9, 1410-1418, 2007. ECS Trans. Electrochem. Soc. 43, 251-258, 2012.
[4] Gomez, M.J., Petrovic, M., Alba, A.R.F., Barceló, D., [21] Marcolino-Junior, L.H., Bergamini, M.F., Teixeira,, M.F.S.,
Determination of pharmaceuticals of various therapeutica classes Cavalheiro, E.T.G., Fatibello-Filho, O., Flow injection
by solid-phase extraction and liquid-chromatography-tandem mass amperometric determination of dipyrone in pharmaceutical
spectrometry analysis in hospital effluent wastewaters, J. formulations using a carbon paste electrode, Farmaco, 58, 999-
Chromatogr. A 1114, 224-233, 2006. 1004, 2003.
[5] Verlicchi, P., Al Aukidy, A., Galletti, A., Petrovic, M., Barceló, D., [22] Basaez, L., Peric, I.M., Jara, P.A., Soto, C.A., Contreras, D.R.,
Hospital effluent: Investigation of the concentration and Aguirre, C., Vanysek, P., Electrochemical and electrophoretic
distribution of pharmaceuticals and environmental risk assessment, study of sodium metamizol, J. Chil. Chem Soc. 53, 1572-1575,
Sci. of Total Environ., 430, 109-118, 2012. 2008.
[6] Bacil, R.P., Buoro, R.M., da Silva, R.P., Medinas, D.B., Lima, [23] Munoz, R.A.A., Matos, R.C., Angnes, L, Amperometric Detection
A.W.O., Serrano, S.H.P., Mechanism of Electro-Oxidation of of Dipyrone in Pharmaceutical Formulations with a Flow Cell
Metamizole using Cyclic Voltammetry at a Glassy Carbon Containing Gold Electrodes from Recordable Compact Discs, J.
Electrode, ECS Transactions, 43(1), 251-258, 2012. Pharma. Sci., 90, 1972-1977, 2001.
[7] Moldovan, Z., Occurrences of pharmaceutical and personal care [24] Baranowska, I., Markowski, P., Gerle, A., Baranowski, J.,
products as micropollutants in rivers from Romania, Chemosphere, Determination of selected drugs in human urine by differential
64, 1808-1817, 2006. pulse voltammetry technique, Bioelectrochem. 73, 5-10, 2008.
[8] Gyenge-Szabo, Z., Szoboszlai, N., Frifyes, D., Zaray, G., Mihucz, [25] Miao, W., Choi, J.P., Bard, A. J., Electrogenerated
V.G., Monitoring of four dipyrone metabolites in communal Chemiluminescence 69: The Tris(2,2'-bipyridine)ruthenium(II),
wastewater by solid phase extraction liquid chromatography (Ru(bpy)32+)/Tri-n-propylamine (TPrA) System Revisited - A New
electrospray ionization quadrupole time-of-flight mass Route Involving TPrA•+ Cation Radicals, J. Am. Chem. Soc.,
spectrometry, J. Pharmaceut. and Biomed. Anal., 90, 58-63, 2014. 124(48), 14478-14485, 2002.
[9] Zhang, C., Zhang, L., Cao, S., Jiang, Z., Wu, H., Yan, M., Zhang, [26] Kapturkiewicz, A., Electrogenerated chemiluminescence from the
X., Jiang, S., Xue, F., Simultaneous determination of residues of tris(2,2'-bipyridine)ruthenium(II) complex, Chem. Phys. Lett. 236,
dipyrone metabolites in goat tissue by hydrophilic interaction 389-394, 1995.
liquid chromatography tandem mass spectrometry, Food Chem. [27] Hercules, D.M., Lytle, F.E., Chemiluminescence from reduction
196, 83-89, 2016. reactions, J. Am. Chem. Soc., 88, 4795-4796, 1966.
[10] Anatas, P.T., Warner, J.C., Green Chemistry: Theory and Practice, [28] Richter, M.M., Electrochemiluminescence (ECL), Chem. Rev. 104,
Oxford University Press, New York, p. 30, 1998. 3003-3036, 2004.
[11] Hjereresen, D. L., Schutt, D.L., Boese, J.M., Green Chemistry and [29] Parveen, S., Aslam, M.S., Hu, L., Xu, G., Electrogenerated
Education, J. Chem. Educ. 77 (12), 1543-1547, 2000. Chemiluminescence. Protocols and Applications, Springer,
[12] Hill, J., Kumar, D.D., Verma, R.K., Challenges for Chemical Heidelberg, 2013.
Education: Engaging with Green Chemistry and Environmental [30] Bard, A.J. (Ed.), Electrogenerated Chemiluminescence, Marcel
Sustainability, J. Chem. Educ. 86 (1), 24-31, 2013. Dekker, New York, 2004.
[13] Stearns, C., Environmental Chemistry in the High School [31] Perez-Ruiz, T., Lozano, C.M, Tomás V., Flow-injection
Curriculum, J. Chem. Educ. 65 (3), 232-234, 1988. determination of Novalgin using amperometric detection at
[14] Habekost, A. Investigations of some reliable a glassy carbon electrode, J. Pharm & Biomed. Anal., 12 (9),
electrochemiluminescence systems on the basis of 1109-1113, 1994.
tris(bipyridyl)ruthenium(II) for HPLC analysis, World J. Chem. [32] Burcinova, A., Tichy, M., Pacakova, V., Stulik, K., Application of
Educ. 4, 13–20, 2016. amperometric detechtion to the high-performance liquid
chromatographic determination of antipyrine and 4-aminoantipyrine in
urine, J. Chromatogr. 455, 420-424, 1988.