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Flow-through amperometric determination of ampicillin using a copper electrode in a batch injection analysis system
Journal Pre-proofs Flow-through amperometric determination of ampicillin using a copper electrode in a batch injection analysis system William Barros Veloso, Geyse Adriana Corrêa Ribeiro, Cláudia Quintino da Rocha, Auro Atsushi Tanaka, Iranaldo Santos da Silva, Luiza Maria Ferreira Dantas PII: DOI: Reference:
S0263-2241(20)30053-1 https://doi.org/10.1016/j.measurement.2020.107516 MEASUR 107516
To appear in:
Measurement
Received Date: Revised Date: Accepted Date:
12 November 2019 10 January 2020 15 January 2020
Please cite this article as: W.B. Veloso, G.A. Corrêa Ribeiro, C.Q. da Rocha, A.A. Tanaka, I. Santos da Silva, L.M.F. Dantas, Flow-through amperometric determination of ampicillin using a copper electrode in a batch injection analysis system, Measurement (2020), doi: https://doi.org/10.1016/j.measurement.2020.107516
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Original Article
1 2 3
Flow-through amperometric determination of ampicillin using a
4
copper electrode in a batch injection analysis system
5 6 7
William Barros Veloso1, Geyse Adriana Corrêa Ribeiro1, Cláudia Quintino da Rocha1, Auro
8
Atsushi Tanaka1,3, Iranaldo Santos da Silva2*, Luiza Maria Ferreira Dantas2*
9 10 11 12 13 14 15 16 17 18
1
Departamento de Química, Centro de Ciências Exatas e Tecnologia, Universidade Federal do Maranhão, CEP 65080-805, São Luís, MA, Brasil 2
3
Departamento de Tecnologia Química, Centro de Ciências Exatas e Tecnologia, Universidade Federal do Maranhão, CEP 65080-805, São Luís, MA, Brasil
Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Caixa Postal 6154, CEP 13083970, Campinas, SP, Brasil.
19 20 21 22 23 24 25 26 27 28 29 30 31
*Corresponding authors: [email protected], [email protected]
32
Phone: +55 98 3272 8244, +55 98 3272-8252
1
33
Abstract
34 35
This paper presents, for the first time, an amperometric electroanalytical method for ampicillin
36
determination using a copper electrode in a batch injection analysis (BIA) system. Initial
37
voltammetric measurements were performed to establish the working potential in the
38
amperometric assays and to optimize parameters such as the supporting electrolyte and the pH
39
of the medium. The method was shown to be precise (RSD = 3.5%, n = 18), reliable (confirmed
40
by an application using a drug sample), sensitive (LOD = 7.11 μmol L−1; LOQ = 23.7 μmol
41
L−1), and fast (86 injections h−1). The proposed method was successfully applied for the
42
determination of ampicillin in commercial drug samples. The results were compared with those
43
obtained using HPLC-UV/Vis and UV-Vis spectrophotometry. There were no significant
44
differences, indicating that the new technique could be used in routine analyses.
45 46
Keywords: ampicillin; batch injection analysis; copper electrode; rapid determination.
2
47
1. Introduction
48
Ampicillin, classified as a semi-synthetic amino-penicillin (Fig. 1) [1], has been used as
49
an antimicrobial agent for more than 80 years [2]. It is a member of the β-lactam class of
50
compounds, which have a basic structure consisting of a β-lactam ring linked to another five-
51
membered heterocyclic thiazolidine ring [3]. The β-lactam ring present in the structure of
52
ampicillin inhibits the synthesis of the layer of peptidoglycan monomers important for the
53
structural integrity of bacterial cell walls [4], so the compound is frequently used for the
54
treatment of infectious diseases in both humans and animals. The quality control in drug
55
production since the World Health Organization guidelines emphasize the need to ensure that
56
all drugs produced to meet specific standards of quality, effectiveness, and safety, to avoid
57
compromising the work of health services [5]. Also, the uncontrolled use of antibiotics can lead
58
to bacterial resistance and increased concentrations of antibiotics in foods (such as meat and
59
milk), wastewater, and other media [6]. For these reasons, it is essential to develop sensitive,
60
selective, and low-cost methods capable of precise and accurate quantification of these
61
compounds in diverse matrices.
62
INSERT FIG. 1
63
Various analytical techniques have been used for the determination of ampicillin in
64
drugs and biological fluids, including chromatography [7,8], spectrophotometry [9,10], and
65
amperometry [11]. The most widely used methods employ high-performance liquid
66
chromatography (HPLC) combined with mass spectrometric or UV-Vis detection [3].
67
Electroanalytical techniques offer several advantages for the determination of bioactive
68
compounds and the active agents in pharmaceutical formulations, compared to the
69
aforementioned conventional techniques, highlighting their operational simplicity, fast
70
response, relatively low cost, and high sensitivity [11-14]. Electroanalytical methods reported
71
in the literature for the determination of ampicillin in different samples have used modified 3
72
electrodes such as aptamer sensors [15,16], a carbon paste electrode modified with
73
ferrocendicarboxylic acid [11], and Pt electrodes coated with a molecularly imprinted polymer
74
(MIP), gold nanoparticles, and multi-walled carbon nanotubes [17]. However, to the best of our
75
knowledge, there are no previously reported methods for the determination of ampicillin using
76
a bare copper electrode in combination with batch injection analysis (BIA).
77
The BIA technique has grown in popularity in recent years and, when used in association
78
with an amperometric detection system, has proved to be a powerful tool for the analysis of
79
pharmaceutical, environmental, and food samples [18-20]. As proposed by Wang and Taha,
80
small volumes of the standard solution or sample to be analyzed are injected directly onto the
81
surface of the working electrode, using a micropipette [21]. The working electrode remains
82
immersed in a large volume of supporting electrolyte so that when the sample is injected, it is
83
immediately diluted in the electrolyte after the transient signal has been acquired. Although this
84
technique has some similarities with flow injection analysis (FIA), an advantage is that there is
85
no requirement for a complex system of injectors and pumps, while it maintains other attractive
86
characteristics including fast analysis, simplicity, high sensitivity, good repeatability, and the
87
use of low volumes of reagents and samples [18,21].
88
In this work, a BIA technique with amperometric detection was developed for the
89
determination of ampicillin, with an evaluation of analytical parameters including linearity,
90
repeatability, and the limits of detection and quantification. The proposed procedure was
91
validated by applying it to determine the ampicillin contents of commercial pharmaceutical
92
samples, comparing the results with those obtained using HPLC-UV/Vis and UV-Vis
93
spectrophotometry techniques.
94 95
4
96
2. Experimental
97
2.1. Reagents, solutions and drug samples
98
All solvents and reagents were analytical grade and were used without further
99
purification. Ultrapure water obtained from a Milli-Q Direct 8 water purification system
100
(Millipore, USA) was used to prepare all the solutions of standards and drug samples. Glacial
101
acetic acid and boric acid were acquired from Merck S.A. (Cotia, Brazil), phosphoric acid (85%
102
v/v) was from Isofar (Duque de Caxias, Brazil), and sodium hydroxide was from Dinâmica
103
(Diadema, Brazil).
104
The supporting electrolytes used were 0.10 mol L−1 Britton-Robinson (BR) buffers at
105
different pH values, prepared by dilution of 0.40 mol L−1 BR stock solution. The pH values of
106
the final solutions were adjusted by the addition of volumes of 2.00 mol L−1 NaOH solution,
107
employing a pH meter 827 pH lab (Metrohm, Switzerland). All the experiments were carried
108
out at room temperature.
109
A stock solution of ampicillin (Sigma-Aldrich, St. Louis, MO) at 10 mmol L−1 was
110
freshly prepared daily by dissolving the solid compound in the supporting electrolyte. Working
111
solutions containing ampicillin at different concentrations were prepared by appropriate
112
dilution of the stock solution in the supporting electrolyte. Commercial ampicillin samples were
113
obtained from a local pharmacy. The samples used for the measurements were prepared by
114
grinding 10 tablets in a porcelain mortar and dissolving an accurately weighed portion of the
115
powder in 100 mL of water, using an ultrasonic bath. The resulting solution was diluted in 0.10
116
mol L−1 BR buffer (pH 7.0) in a 50 mL volumetric flask. The solution was filtered, and further
117
dilutions were made to obtain the ampicillin working solutions used in the analyses.
118
5
119
2.2. Apparatus
120
The electrochemical measurements were performed using an Ivium-n-Stat potentiostat
121
controlled with IviumSoftTM Electrochemistry Software (Ivium Technologies, Eindhoven,
122
Netherlands). The voltammetric measurements employed a conventional three-electrode
123
system consisting of a copper wire (ϕ = 2 mm), a platinum wire, and Ag/AgCl (KClsat) as the
124
working, auxiliary, and reference electrodes, respectively. For the BIA measurements, the
125
working electrode was a copper plate obtained by cutting out a small piece (2 cm × 6 cm) of a
126
printed circuit board containing high purity copper. In the BIA system, the injections of standard
127
or sample solutions were performed using a motorized electronic micropipette EDP1-Plus
128
(Rainin Instrument, MA, USA). All the amperometric measurements were performed using a
129
home-made 3D-printed BIA cell, described previously [22]. The cell body was a cylindrical
130
vessel with a maximum capacity of 100 mL, which had a small round hole to allow contact of
131
the working electrode with the internal solution. The top cover was securely attached using
132
three screw fittings located equidistantly around the cell body. The cover contained two orifices
133
for inserting the auxiliary and reference electrodes, a hole for liquid manipulation (or the
134
introduction of a mechanical stirrer), and another small hole for insertion of the micropipette
135
adapter.
136 137
2.3. Cleaning and activation of the copper electrode
138
The copper wire electrode was activated by cleaning it mechanically using felt and a
139
suspension of alumina (0.30 μm). The copper printed circuit boards were treated by immersing
140
them in HNO3 solution (10%, v/v) before use. After the cleaning processes, the electrodes were
141
ultrasonicated for 2 min and rinsed with copious amounts of deionized water.
142
6
143
2.4. Measurements by HPLC-UV/Vis
144
The BIA method was validated by comparing the results with those obtained by HPLC
145
analysis according to a method described in the literature for the determination of ampicillin in
146
drugs [23]. The analysis was performed using a Shimadzu HPLC system (Shimadzu
147
Corporation, Kyoto, Japan) consisting of a solvent delivery module with a double-plunger
148
reciprocating pump, a SPA-10A UV/Vis detector (λ = 280 nm), and an AQUA RP C18 column
149
(150 mm × 4.6 mm, 5 µm). The mobile phase components were 0.01% formic acid in water
150
(A) and 0.01% formic acid in acetonitrile (B), in gradient elution mode, with the concentration
151
of B ranging from 2 to 98% between 0 and 20 min. The solutions were filtered and degassed
152
before use. The flow rate used was 1.00 mL min-1. The stock standard solution and the samples
153
to be analyzed were diluted with the mixture used for the mobile phase. The separation was
154
performed at room temperature, and the sample injection volume was 10.0 µL. The data were
155
collected and processed using Shimadzu LC Solution v. 1.25 software (Shimadzu Corporation,
156
Kyoto, Japan).
157 158
2.5. UV-Vis measurements
159
The results obtained with the proposed method were also compared to those obtained
160
by UV-Vis spectrophotometry employing a Shimadzu UV-1800 spectrophotometer controlled
161
by UVProbe 2.52 software (Shimadzu Corporation, Kyoto, Japan). The wavelength to be used
162
was selected by scanning the absorbance of a 1.00 mmol L−1 ampicillin standard solution in the
163
range of 200-800 nm.
164
Quantification of ampicillin in the drug samples by UV-Vis spectrophotometry was
165
achieved using a calibration curve constructed by measuring the absorbance of ampicillin
166
standard solutions at concentrations between 0.13 and 0.80 mmol L−1, followed by reading the
167
absorbance of the samples.
7
168
3. Results and Discussion
169
3.1. Voltammetric behavior of ampicillin
170
Firstly, the cyclic voltammetry (CV) technique was used to investigate the voltammetric
171
behavior of the antibiotic on the copper wire electrode (ϕ = 2 mm) in 0.10 mol L−1 BR solution
172
(pH 7.0). Fig. 2 shows typical results obtained for potential scanning from −0.800 to +0.500 V
173
vs. Ag/AgCl (KClsat) at a scan rate of 50 mV s−1. The electrochemical response of the copper
174
electrode, before the addition of ampicillin, showed two peaks (I and II, at +0.025 V and −0.215
175
V, respectively). According to the literature, the first potential indicated an oxidation process
176
associated with the formation of a surface anodic layer composed mainly of copper(I) and
177
copper(II) oxides (Cu2O and CuO) [24], while the second potential was related to the reduction
178
of the oxides formed in the previous sweep. However, following the addition of 1.00 mmol L−1
179
ampicillin, a substantial current enhancement was observed, at a potential range corresponding
180
to the Cu(II) formation, and attributed to the surface anodic layer dissolution, followed by a
181
Cu-Ampicillin complex formation. The consumption of the Cu(II) species by ampicillin leads
182
to a decrease in the reduction current, which was similar to the behavior associated with the
183
formation of complexes by reactions between Cu(II) and analytes, as previously reported by
184
Coutinho et al. [25]. Hence, a strategy of complex formation between ampicillin and copper
185
ions from the oxide surface layer was used to obtain the analytical signal of interest.
186 187
INSERT FIG. 2
188 189
For further enhancement of the current obtained for the detection of ampicillin on the
190
copper electrode, an evaluation was made of different 0.10 mol L−1 supporting electrolyte
191
solutions, including phosphate buffer (pH 7.0), BR buffer (pH 7.0), acetate buffer (pH 4.5),
192
KCl, and NaOH. For this, cyclic voltammograms were acquired using an electrochemical cell 8
193
containing 1.00 mmol L−1 of ampicillin. Comparison of the background voltammograms
194
(supporting electrolyte only) with those recorded after the addition of 1.00 mmol L−1 of
195
ampicillin showed that the use of the 0.10 mol L−1 BR buffer (pH 7.0) resulted in the highest
196
peak current and the best definition of the oxidation peak (Fig. 1S). Therefore, the BR buffer
197
was selected for use in the subsequent experiments.
198
Optimization of the pH, to maximize the electrochemical signal for ampicillin detection,
199
was performed by recording cyclic voltammograms using the electrode in 0.10 mol L−1 BR
200
buffer containing 1.00 mmol L−1 ampicillin, at pH values from 5 to 9. As shown in Fig. 3, the
201
peak current intensity (Ip) increased between pH 5.0 and pH 7.0, followed by a marked decrease
202
in pH values above 7.0. Since some β-lactam antibiotics undergo acid degradation [23], pH 7.0
203
was established as the ideal pH, since it avoided possible degradation of the analyte, while
204
obtaining a high peak current and satisfactory definition of the analytical response. The working
205
potential for the amperometric experiments (+0.025 V) was defined as the potential at which
206
the highest current value was obtained in the voltammetric tests, after optimization of the
207
previous parameters.
208
It can be seen from Fig. 3 that as the pH was increased, there was a linear shift of the
209
anodic peak potential (Epa) to less positive values. The curve formed by the peak potential
210
presented a slope of 0.056 V pH−1, which was close to the Nernstian value (0.059 V pH−1),
211
indicating that the number of electrons transferred in the reactions associated with formation of
212
the electrode layer (complexation reactions between Cu(II) and ampicillin) was equal to the
213
number of protons [26].
214 215
INSERT FIG. 3
216
9
217 218 219
3.2. Optimization of BIA parameters The previous voltammetric experiments established some of the parameters to be used in the BIA system, including the supporting electrolyte, pH, and working potential.
220
The next step was to investigate the effects of the injection volume and dispensing rate
221
to obtain the highest signal for ampicillin determination. Fig. 2S shows the influences of the
222
injection volume (10-190 μL) and dispensing rate (22.7-76.9 μL s−1) on the ampicillin current
223
signal. The analytical signal increased proportionally with the injection volume up to 160 μL,
224
followed by a small change in the range between 160 and 190 μL (employing a dispensing rate
225
of 76.9 μL s−1). However, volumes above 100 μL resulted in higher relative standard deviation
226
(RSD) values, as well as higher reagent consumption and, consequently, higher generation of
227
waste. Therefore, to obtain a satisfactory response, with low RSD and low waste generation, an
228
injection volume of 100 μL was established in the subsequent experiments. An increase in the
229
programmable micropipette dispensing rate within the entire range studied (22.7 to 76.9 μL s−1)
230
caused no change in the current signal when a constant injection volume of 100 μL was used
231
(Fig. 2S). Therefore, to obtain a favorable compromise between the analytical response and the
232
RSD value, a dispensing rate of 76.9 μL s−1 was selected in the subsequent amperometric
233
measurements.
234
The influence of stirring during the BIA assays was investigated using an injection of
235
100 μL aliquots of ampicillin standard solution (at 120 μmol L−1) and measuring the time
236
required for the analytical signal to return to the baseline. It was found that in the presence of
237
stirring (Fig. 3S), the current rapidly returned to the baseline after the formation of the transient
238
signal, hence increasing the analytical frequency and providing faster analysis.
239
10
240
3.3. Analytical performance
241
A repeatability experiment was performed to evaluate the precision of the method. Fig.
242
4S shows the signals recorded for 18 successive injections of 100 μmol L−1 ampicillin into the
243
BIA cell. A low RSD of 3.1% was obtained, indicating excellent performance and stability of
244
the copper electrode used for ampicillin determination.
245
The linear range of the proposed method was determined by injecting a series of
246
ampicillin standard solutions of different concentrations, in ascending and descending order,
247
into the BIA cell. Fig. 4 presents the series of amperometric signals obtained for the sequential
248
injection (in triplicate) of the seven standard solutions. A satisfactory linear response was
249
obtained in the concentration range used (30-250 μmol L−1), with a correlation coefficient better
250
than 0.999. The limits of detection (3Sb/slope) and quantification (10Sb/slope) were 7.11 and
251
23.7 μmol L−1, respectively. Furthermore, considering the time required for the analytical signal
252
to return to the baseline, the analytical frequency was 86 injections h−1.
253 254
INSERT FIG. 4
255
INSERT TABLE 1
256 257
Table 1 compares the proposed method with other methods described in the literature
258
using electrochemical techniques for the determination of ampicillin. The BIA method presents
259
a linear range and LOD suitable for the quantification of ampicillin in commercial samples of
260
pharmaceutical products. It should be pointed out that in Table 1, the linear range varies
261
according to each type of sample to be analyzed, and either they depend on stages of preparation
262
of the electrochemical sensor and some of them require several complicated steps to prepare
263
the sensor, such as the molecularly printed polymers (MIPs) and DNA [16,27,30]. Thus, the
11
264
results obtained here demonstrated the use of the BIA method for rapid, direct, and low-cost
265
ampicillin determination in drug samples with this analyte as the active ingredient.
266
The proposed BIA method was applied for the quantification of ampicillin in
267
pharmaceutical samples, under the optimized conditions, using 100 μL injections in triplicate
268
(Fig. 5). The linear working range was determined using injections of ampicillin standard
269
solutions at increasing concentrations between 30 and 200 μmol L−1 (a-e), alternating with
270
triplicate injections of two drug samples (s1 and s2), as well as sample s1 enriched with two
271
different concentrations of standard ampicillin solution (r1 and r2). The results obtained by the
272
proposed method showed that the ampicillin contents in the samples were close to the values
273
stated on the labels, with a difference of less than 5%. The method presented satisfactory results
274
for the determination of ampicillin in the drug samples, with recovery values of over 95% for
275
both fortification levels (Table 2).
276 277
INSERT FIG. 5
278 279
INSERT TABLE 2
280 281
The accuracy of the results obtained with the proposed method was evaluated by
282
comparison with the values obtained using the HPLC-UV/Vis and spectrophotometric methods
283
(Table 3). Fig. 5S shows the results for the determination of ampicillin in the same
284
pharmaceutical samples using the two latter methods. The peak asymmetry observed for the
285
HPLC method was probably caused by the manual injection onto the column.
286 287
INSERT TABLE 3
288
12
289
According to the Student’s t-test, at the 95% confidence level, there were no significant
290
differences between the results obtained using the two literature methods, and it could also be
291
concluded that the values obtained using the proposed BIA method were statistically equal
292
(tcalculated < ttabulated) to those obtained using the literature methods. The values obtained using
293
the different methodologies were in excellent agreement and were very close to the labeled
294
values. The advantages of the new method include speed, low consumption of reagents, minor
295
waste generation, and ease of operation. The results for the analysis of the samples, without any
296
pretreatment, also showed that the presence of solid particles from the excipients did not
297
interfere in the electrochemical measurements. This achieves highlighted another advantage of
298
this technique since sample clean-up is an indispensable and time-consuming step in optical
299
and chromatographic methods.
13
300
4. Conclusions
301
This work presents a method for the amperometric determination of ampicillin in drugs
302
using an unmodified copper electrode in a BIA system and exploring the complex formation
303
between copper and ampicillin. Evaluation of the proposed method in real samples showed no
304
significant differences with the results obtained with the other two well-established methods in
305
the literature, leading to the conclusion that the proposed method could be used in routine
306
analyses with low-cost equipment and avoiding time-consuming sample preparation steps.
307 308
Conflicts of Interest
309
The authors declare that there are no conflicts of interest
310 311
Acknowledgments
312
The authors are grateful for the financial support provided by the Brazilian agencies
313
FAPEMA (grant number #UNIVERSAL-00863/16, #IECT-03/2016, #INFRA-03170/18, and
314
#UNIVERSAL-01372/17), CNPq (grant number #465389/2014-7, #205220/2018-5) and
315
CAPES (Finance Code 001). The authors thank prof. Kagan Kerman from the University of
316
Toronto (ON, Canadá), for kindly revising and improving this manuscript. We are also very
317
grateful to Professor Rodrigo Alejandro Abarza Munõz from the Federal University of
318
Uberlândia (MG, Brazil), for providing us with the 3D-printed BIA cell.
14
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404 [26]. Hrioua A, Farahi A, Lahrich S, Bakasse M, Saqrane S, Mhammedi MAE. 405
Chronoamperometric Detection of Amoxicillin at Graphite Electrode using Chelate Effect
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408 [27]. Liu X, Hu M, Wang M, Song Y, Zhou N, He L, Zhang Z. Novel nanoarchitecture of Co409
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18
415 [29]. Yu Z, Sutlief AL, Lai RY. Towards the development of a sensitive and selective 416
electrochemical aptamer-based ampicillin sensor. Sens. Actuators B: Chem. 2018; 258: 722-
417
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418 [30]. Wang J, Ma K, Yin H, Zhou Y, Ai S. Aptamer based voltammetric determination of 419
ampicillin using a single-stranted DNA binding protein and DNA functionalized gold
420
nanoparticles. Microchim. Acta 2017; 185: 1-7. https://doi.org/10.1007/s00604-017-2566-8
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423
1053-1065. https://doi-org.ez14.periodicos.capes.gov.br/10.1007/s00216-018-1533-5
424
19
425
Figure captions
426
Fig. 1 – Chemical structure of ampicillin.
427
Fig. 2 – Cyclic voltammogram obtained using the copper electrode (ϕ = 2.0 mm) in BR buffer (pH 7.0), in the
428
absence and presence of 1.0 mmol L−1 of ampicillin, at v = 50 mV s−1.
429
Fig. 3 – Effect of pH on the anodic peak current (Ipa) and the anodic peak potential (Epa).
430
Fig. 4 – Amperometric responses obtained for triplicate injections of solutions containing different concentrations
431
of ampicillin (μmol L−1): a = 30; b = 60; c = 110; d = 150; e = 200; f = 250; g = 300. Insert: response obtained for
432
injection of the supporting electrolyte. Electrolyte: BR buffer (pH 7.0); injection volume: 100 μL; dispensing rate:
433
76.9 μL s−1; potential: +0.025 V vs. Ag/AgCl (KClsat.)
434
Fig. 5 – Amperometric responses obtained for triplicate injections of solutions containing different concentrations
435
of ampicillin (μmol L−1: a = 30; b = 60; c = 110; d = 150; e = 200), the drug samples (s1 and s2), and drug sample
436
s1 enriched with ampicillin at two levels (r1 and r2). Electrolyte: BR buffer (pH 7.0); injection volume: 100 μL;
437
dispensing rate: 76.9 μL s−1; potential: +0.025 V vs. Ag/AgCl (KClsat.)
438 439 440
20
441 442
Fig. 1
443 444
21
445 446
Fig. 2
40
III I
I / µA
20 0 -20
IV
-40
II
-0.8 447
electrolyte -1 [AMP] 1.0 mmol L
0.4 0.0 -0.4 E/V vs Ag/AgCl/KClsat
448
22
449 450
Fig. 3
0,2
Ip / µA
24
0,1
16
Ep / V
32
0,0
8 0 5 451
6
7 pH
8
9
-0,1
452
23
453
Fig. 4
8
I / µA
6
A
0.5 A
e
Electrolyte
c
4
a
g
f
f
e d
d
c
b
b
a
2 0
1000
1500
2000
time / s
454
4
Ascending
B
I / µA
2
R = 0.999 Ip (µA) = 0.017 + 0.17 x [AMP]
0 4
Descennding
2
R = 0.998 Ip (µA) = -0.021 + 0.17 x [AMP]
0 50
455
2500
100
150
200
[AMP] / µmol L
-1
250
300
456
24
457
Fig. 5
0.8 A
5
d
I / µA
electrolyte
4 3
a
b
c
e
s2 s1
r2 r1
2 1 400 458
800
1200 time / s
1600
459 460 461 462
25
464
465 466 467 468 469 470
Table 1. Performance comparison of the proposed technique and methods reported in the literature. Electrode type
Analysis method
Linear range (µmol L−1)
LOD (µmol L−1)
Reference
E – AB
ACV
5 - 5000
1.0
[28]
E – AB
ACV
0.2 - 15000
0.03
[29]
DNA-AuNPs/GCE
DPV
1.0x10-6 – 0.005
3.8x10-7
[30]
FDCMCPE
DPV
2.34 - 30
0.67
[10]
MIP/MWCNTs/AuNPs/Pt
DPV
0.01 – 5.0
0.001
[16]
Co-MOF@TPN-COF
EIS
2.86x10-9 – 5,72x10-3
6.21x10-10
[27]
APT-modified gold chip
AMP
2.5 - 1000
1.0
[32]
Copper
BIA-AMP
30.0 – 250.0
7.11
This work
E – AB: electrochemical aptamer-based sensor. ACV: Alternating Current Voltammetry. DNA-AuNPs: DNA functionalized gold nanoparticles. GCE: glassy carbon electrode. FDCMCPE: carbon-paste electrode spiked with ferrocenedicarboxylic acid. DPV: Differential Pulse Voltammetric. MIP/MWCNTs/AuNPs/Pt: platinum electrode modified with multiwalled carbon nanotubes, gold nanoparticles and a thin film of molecularly imprinted polymers. CoMOF@TPN-COF: nanoarchitecture of Co-based metal-organic frameworks and terephthalonitrile-based covalent organic framework. EIS: electrochemical impedance spectroscopy. AMP: amperometry.
471 472
27
473
Table 2. Ampicillin concentrations per tablet, obtained by the proposed BIA method, and recovery values for the
474
analysis of drug samples.
475 Ampicillin content (g/tablet)
476
*S
Recovery test [ampicillin] (µmol L−1)
Labeled
Founded
Added
Founded
Recovery (%)
S1*
0.50
0.54 ± 0.060
46.9
49.7 ± 3
105.9
S2*
0.50
0.52 ± 0.062
115.6
124.6 ± 3
107.7
1
= sample 1; S2 = sample 2.
477
28
478
Table 3. Ampicillin concentrations per tablet, obtained by the proposed BIA method, HPLC-UV, and UV-Vis
479
spectrophotometry.
Ampicillin (mg/tablet)
480
Labeled value
BIA
HPLC-UV
UV-Vis
500
540 ± 62
570 ± 93
550 ± 62
n = 3, 95% confidence level.
481 482
HIGHLIGHTS
483 484
A rapid and sensitive method for the determination of the ampicillin antibiotic;
485
The method uses a batch injection analysis system with bare copper electrodes;
486
Development of a simple, low cost, and portable instrumentation system
487
This system presents competitive performance for ampicillin routine analyses.
488 489 490
William Barros Veloso: Formal analysis, Writing- Original draft preparation.: Geyse Adriana
491
Corrêa Ribeiro: Formal analysis, Visualization.: Cláudia Quintino da Rocha: Visualization,
492
Formal analysis.: Auro Atsushi Tanaka: Writing - Review & Editing.: Iranaldo Santos da
493
Silva: Conceptualization, Writing - Review & Editing.: Luiza Maria Ferreira Dantas:
494
Conceptualization, Writing - Review & Editing.
495 496 497
Declaration of interests
498 499 500
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
501 502 503
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
29
504
505 506 507 508 509 510 511 512 513
Flow-through amperometric determination of ampicillin using a copper electrode in a batch injection analysis system William Barros Veloso, Geyse Adriana Corrêa Ribeiro, Cláudia Quintino da Rocha, Auro Atsushi Tanaka, Iranaldo Santos da Silva, Luiza Maria Ferreira Dantas
514 515 516
GRAPHICAL ABSTRACTS
517
518 519 520 30
S0263-2241(20)30053-1 https://doi.org/10.1016/j.measurement.2020.107516 MEASUR 107516
To appear in:
Measurement
Received Date: Revised Date: Accepted Date:
12 November 2019 10 January 2020 15 January 2020
Please cite this article as: W.B. Veloso, G.A. Corrêa Ribeiro, C.Q. da Rocha, A.A. Tanaka, I. Santos da Silva, L.M.F. Dantas, Flow-through amperometric determination of ampicillin using a copper electrode in a batch injection analysis system, Measurement (2020), doi: https://doi.org/10.1016/j.measurement.2020.107516
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© 2020 Elsevier Ltd. All rights reserved.
Original Article
1 2 3
Flow-through amperometric determination of ampicillin using a
4
copper electrode in a batch injection analysis system
5 6 7
William Barros Veloso1, Geyse Adriana Corrêa Ribeiro1, Cláudia Quintino da Rocha1, Auro
8
Atsushi Tanaka1,3, Iranaldo Santos da Silva2*, Luiza Maria Ferreira Dantas2*
9 10 11 12 13 14 15 16 17 18
1
Departamento de Química, Centro de Ciências Exatas e Tecnologia, Universidade Federal do Maranhão, CEP 65080-805, São Luís, MA, Brasil 2
3
Departamento de Tecnologia Química, Centro de Ciências Exatas e Tecnologia, Universidade Federal do Maranhão, CEP 65080-805, São Luís, MA, Brasil
Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Caixa Postal 6154, CEP 13083970, Campinas, SP, Brasil.
19 20 21 22 23 24 25 26 27 28 29 30 31
*Corresponding authors: [email protected], [email protected]
32
Phone: +55 98 3272 8244, +55 98 3272-8252
1
33
Abstract
34 35
This paper presents, for the first time, an amperometric electroanalytical method for ampicillin
36
determination using a copper electrode in a batch injection analysis (BIA) system. Initial
37
voltammetric measurements were performed to establish the working potential in the
38
amperometric assays and to optimize parameters such as the supporting electrolyte and the pH
39
of the medium. The method was shown to be precise (RSD = 3.5%, n = 18), reliable (confirmed
40
by an application using a drug sample), sensitive (LOD = 7.11 μmol L−1; LOQ = 23.7 μmol
41
L−1), and fast (86 injections h−1). The proposed method was successfully applied for the
42
determination of ampicillin in commercial drug samples. The results were compared with those
43
obtained using HPLC-UV/Vis and UV-Vis spectrophotometry. There were no significant
44
differences, indicating that the new technique could be used in routine analyses.
45 46
Keywords: ampicillin; batch injection analysis; copper electrode; rapid determination.
2
47
1. Introduction
48
Ampicillin, classified as a semi-synthetic amino-penicillin (Fig. 1) [1], has been used as
49
an antimicrobial agent for more than 80 years [2]. It is a member of the β-lactam class of
50
compounds, which have a basic structure consisting of a β-lactam ring linked to another five-
51
membered heterocyclic thiazolidine ring [3]. The β-lactam ring present in the structure of
52
ampicillin inhibits the synthesis of the layer of peptidoglycan monomers important for the
53
structural integrity of bacterial cell walls [4], so the compound is frequently used for the
54
treatment of infectious diseases in both humans and animals. The quality control in drug
55
production since the World Health Organization guidelines emphasize the need to ensure that
56
all drugs produced to meet specific standards of quality, effectiveness, and safety, to avoid
57
compromising the work of health services [5]. Also, the uncontrolled use of antibiotics can lead
58
to bacterial resistance and increased concentrations of antibiotics in foods (such as meat and
59
milk), wastewater, and other media [6]. For these reasons, it is essential to develop sensitive,
60
selective, and low-cost methods capable of precise and accurate quantification of these
61
compounds in diverse matrices.
62
INSERT FIG. 1
63
Various analytical techniques have been used for the determination of ampicillin in
64
drugs and biological fluids, including chromatography [7,8], spectrophotometry [9,10], and
65
amperometry [11]. The most widely used methods employ high-performance liquid
66
chromatography (HPLC) combined with mass spectrometric or UV-Vis detection [3].
67
Electroanalytical techniques offer several advantages for the determination of bioactive
68
compounds and the active agents in pharmaceutical formulations, compared to the
69
aforementioned conventional techniques, highlighting their operational simplicity, fast
70
response, relatively low cost, and high sensitivity [11-14]. Electroanalytical methods reported
71
in the literature for the determination of ampicillin in different samples have used modified 3
72
electrodes such as aptamer sensors [15,16], a carbon paste electrode modified with
73
ferrocendicarboxylic acid [11], and Pt electrodes coated with a molecularly imprinted polymer
74
(MIP), gold nanoparticles, and multi-walled carbon nanotubes [17]. However, to the best of our
75
knowledge, there are no previously reported methods for the determination of ampicillin using
76
a bare copper electrode in combination with batch injection analysis (BIA).
77
The BIA technique has grown in popularity in recent years and, when used in association
78
with an amperometric detection system, has proved to be a powerful tool for the analysis of
79
pharmaceutical, environmental, and food samples [18-20]. As proposed by Wang and Taha,
80
small volumes of the standard solution or sample to be analyzed are injected directly onto the
81
surface of the working electrode, using a micropipette [21]. The working electrode remains
82
immersed in a large volume of supporting electrolyte so that when the sample is injected, it is
83
immediately diluted in the electrolyte after the transient signal has been acquired. Although this
84
technique has some similarities with flow injection analysis (FIA), an advantage is that there is
85
no requirement for a complex system of injectors and pumps, while it maintains other attractive
86
characteristics including fast analysis, simplicity, high sensitivity, good repeatability, and the
87
use of low volumes of reagents and samples [18,21].
88
In this work, a BIA technique with amperometric detection was developed for the
89
determination of ampicillin, with an evaluation of analytical parameters including linearity,
90
repeatability, and the limits of detection and quantification. The proposed procedure was
91
validated by applying it to determine the ampicillin contents of commercial pharmaceutical
92
samples, comparing the results with those obtained using HPLC-UV/Vis and UV-Vis
93
spectrophotometry techniques.
94 95
4
96
2. Experimental
97
2.1. Reagents, solutions and drug samples
98
All solvents and reagents were analytical grade and were used without further
99
purification. Ultrapure water obtained from a Milli-Q Direct 8 water purification system
100
(Millipore, USA) was used to prepare all the solutions of standards and drug samples. Glacial
101
acetic acid and boric acid were acquired from Merck S.A. (Cotia, Brazil), phosphoric acid (85%
102
v/v) was from Isofar (Duque de Caxias, Brazil), and sodium hydroxide was from Dinâmica
103
(Diadema, Brazil).
104
The supporting electrolytes used were 0.10 mol L−1 Britton-Robinson (BR) buffers at
105
different pH values, prepared by dilution of 0.40 mol L−1 BR stock solution. The pH values of
106
the final solutions were adjusted by the addition of volumes of 2.00 mol L−1 NaOH solution,
107
employing a pH meter 827 pH lab (Metrohm, Switzerland). All the experiments were carried
108
out at room temperature.
109
A stock solution of ampicillin (Sigma-Aldrich, St. Louis, MO) at 10 mmol L−1 was
110
freshly prepared daily by dissolving the solid compound in the supporting electrolyte. Working
111
solutions containing ampicillin at different concentrations were prepared by appropriate
112
dilution of the stock solution in the supporting electrolyte. Commercial ampicillin samples were
113
obtained from a local pharmacy. The samples used for the measurements were prepared by
114
grinding 10 tablets in a porcelain mortar and dissolving an accurately weighed portion of the
115
powder in 100 mL of water, using an ultrasonic bath. The resulting solution was diluted in 0.10
116
mol L−1 BR buffer (pH 7.0) in a 50 mL volumetric flask. The solution was filtered, and further
117
dilutions were made to obtain the ampicillin working solutions used in the analyses.
118
5
119
2.2. Apparatus
120
The electrochemical measurements were performed using an Ivium-n-Stat potentiostat
121
controlled with IviumSoftTM Electrochemistry Software (Ivium Technologies, Eindhoven,
122
Netherlands). The voltammetric measurements employed a conventional three-electrode
123
system consisting of a copper wire (ϕ = 2 mm), a platinum wire, and Ag/AgCl (KClsat) as the
124
working, auxiliary, and reference electrodes, respectively. For the BIA measurements, the
125
working electrode was a copper plate obtained by cutting out a small piece (2 cm × 6 cm) of a
126
printed circuit board containing high purity copper. In the BIA system, the injections of standard
127
or sample solutions were performed using a motorized electronic micropipette EDP1-Plus
128
(Rainin Instrument, MA, USA). All the amperometric measurements were performed using a
129
home-made 3D-printed BIA cell, described previously [22]. The cell body was a cylindrical
130
vessel with a maximum capacity of 100 mL, which had a small round hole to allow contact of
131
the working electrode with the internal solution. The top cover was securely attached using
132
three screw fittings located equidistantly around the cell body. The cover contained two orifices
133
for inserting the auxiliary and reference electrodes, a hole for liquid manipulation (or the
134
introduction of a mechanical stirrer), and another small hole for insertion of the micropipette
135
adapter.
136 137
2.3. Cleaning and activation of the copper electrode
138
The copper wire electrode was activated by cleaning it mechanically using felt and a
139
suspension of alumina (0.30 μm). The copper printed circuit boards were treated by immersing
140
them in HNO3 solution (10%, v/v) before use. After the cleaning processes, the electrodes were
141
ultrasonicated for 2 min and rinsed with copious amounts of deionized water.
142
6
143
2.4. Measurements by HPLC-UV/Vis
144
The BIA method was validated by comparing the results with those obtained by HPLC
145
analysis according to a method described in the literature for the determination of ampicillin in
146
drugs [23]. The analysis was performed using a Shimadzu HPLC system (Shimadzu
147
Corporation, Kyoto, Japan) consisting of a solvent delivery module with a double-plunger
148
reciprocating pump, a SPA-10A UV/Vis detector (λ = 280 nm), and an AQUA RP C18 column
149
(150 mm × 4.6 mm, 5 µm). The mobile phase components were 0.01% formic acid in water
150
(A) and 0.01% formic acid in acetonitrile (B), in gradient elution mode, with the concentration
151
of B ranging from 2 to 98% between 0 and 20 min. The solutions were filtered and degassed
152
before use. The flow rate used was 1.00 mL min-1. The stock standard solution and the samples
153
to be analyzed were diluted with the mixture used for the mobile phase. The separation was
154
performed at room temperature, and the sample injection volume was 10.0 µL. The data were
155
collected and processed using Shimadzu LC Solution v. 1.25 software (Shimadzu Corporation,
156
Kyoto, Japan).
157 158
2.5. UV-Vis measurements
159
The results obtained with the proposed method were also compared to those obtained
160
by UV-Vis spectrophotometry employing a Shimadzu UV-1800 spectrophotometer controlled
161
by UVProbe 2.52 software (Shimadzu Corporation, Kyoto, Japan). The wavelength to be used
162
was selected by scanning the absorbance of a 1.00 mmol L−1 ampicillin standard solution in the
163
range of 200-800 nm.
164
Quantification of ampicillin in the drug samples by UV-Vis spectrophotometry was
165
achieved using a calibration curve constructed by measuring the absorbance of ampicillin
166
standard solutions at concentrations between 0.13 and 0.80 mmol L−1, followed by reading the
167
absorbance of the samples.
7
168
3. Results and Discussion
169
3.1. Voltammetric behavior of ampicillin
170
Firstly, the cyclic voltammetry (CV) technique was used to investigate the voltammetric
171
behavior of the antibiotic on the copper wire electrode (ϕ = 2 mm) in 0.10 mol L−1 BR solution
172
(pH 7.0). Fig. 2 shows typical results obtained for potential scanning from −0.800 to +0.500 V
173
vs. Ag/AgCl (KClsat) at a scan rate of 50 mV s−1. The electrochemical response of the copper
174
electrode, before the addition of ampicillin, showed two peaks (I and II, at +0.025 V and −0.215
175
V, respectively). According to the literature, the first potential indicated an oxidation process
176
associated with the formation of a surface anodic layer composed mainly of copper(I) and
177
copper(II) oxides (Cu2O and CuO) [24], while the second potential was related to the reduction
178
of the oxides formed in the previous sweep. However, following the addition of 1.00 mmol L−1
179
ampicillin, a substantial current enhancement was observed, at a potential range corresponding
180
to the Cu(II) formation, and attributed to the surface anodic layer dissolution, followed by a
181
Cu-Ampicillin complex formation. The consumption of the Cu(II) species by ampicillin leads
182
to a decrease in the reduction current, which was similar to the behavior associated with the
183
formation of complexes by reactions between Cu(II) and analytes, as previously reported by
184
Coutinho et al. [25]. Hence, a strategy of complex formation between ampicillin and copper
185
ions from the oxide surface layer was used to obtain the analytical signal of interest.
186 187
INSERT FIG. 2
188 189
For further enhancement of the current obtained for the detection of ampicillin on the
190
copper electrode, an evaluation was made of different 0.10 mol L−1 supporting electrolyte
191
solutions, including phosphate buffer (pH 7.0), BR buffer (pH 7.0), acetate buffer (pH 4.5),
192
KCl, and NaOH. For this, cyclic voltammograms were acquired using an electrochemical cell 8
193
containing 1.00 mmol L−1 of ampicillin. Comparison of the background voltammograms
194
(supporting electrolyte only) with those recorded after the addition of 1.00 mmol L−1 of
195
ampicillin showed that the use of the 0.10 mol L−1 BR buffer (pH 7.0) resulted in the highest
196
peak current and the best definition of the oxidation peak (Fig. 1S). Therefore, the BR buffer
197
was selected for use in the subsequent experiments.
198
Optimization of the pH, to maximize the electrochemical signal for ampicillin detection,
199
was performed by recording cyclic voltammograms using the electrode in 0.10 mol L−1 BR
200
buffer containing 1.00 mmol L−1 ampicillin, at pH values from 5 to 9. As shown in Fig. 3, the
201
peak current intensity (Ip) increased between pH 5.0 and pH 7.0, followed by a marked decrease
202
in pH values above 7.0. Since some β-lactam antibiotics undergo acid degradation [23], pH 7.0
203
was established as the ideal pH, since it avoided possible degradation of the analyte, while
204
obtaining a high peak current and satisfactory definition of the analytical response. The working
205
potential for the amperometric experiments (+0.025 V) was defined as the potential at which
206
the highest current value was obtained in the voltammetric tests, after optimization of the
207
previous parameters.
208
It can be seen from Fig. 3 that as the pH was increased, there was a linear shift of the
209
anodic peak potential (Epa) to less positive values. The curve formed by the peak potential
210
presented a slope of 0.056 V pH−1, which was close to the Nernstian value (0.059 V pH−1),
211
indicating that the number of electrons transferred in the reactions associated with formation of
212
the electrode layer (complexation reactions between Cu(II) and ampicillin) was equal to the
213
number of protons [26].
214 215
INSERT FIG. 3
216
9
217 218 219
3.2. Optimization of BIA parameters The previous voltammetric experiments established some of the parameters to be used in the BIA system, including the supporting electrolyte, pH, and working potential.
220
The next step was to investigate the effects of the injection volume and dispensing rate
221
to obtain the highest signal for ampicillin determination. Fig. 2S shows the influences of the
222
injection volume (10-190 μL) and dispensing rate (22.7-76.9 μL s−1) on the ampicillin current
223
signal. The analytical signal increased proportionally with the injection volume up to 160 μL,
224
followed by a small change in the range between 160 and 190 μL (employing a dispensing rate
225
of 76.9 μL s−1). However, volumes above 100 μL resulted in higher relative standard deviation
226
(RSD) values, as well as higher reagent consumption and, consequently, higher generation of
227
waste. Therefore, to obtain a satisfactory response, with low RSD and low waste generation, an
228
injection volume of 100 μL was established in the subsequent experiments. An increase in the
229
programmable micropipette dispensing rate within the entire range studied (22.7 to 76.9 μL s−1)
230
caused no change in the current signal when a constant injection volume of 100 μL was used
231
(Fig. 2S). Therefore, to obtain a favorable compromise between the analytical response and the
232
RSD value, a dispensing rate of 76.9 μL s−1 was selected in the subsequent amperometric
233
measurements.
234
The influence of stirring during the BIA assays was investigated using an injection of
235
100 μL aliquots of ampicillin standard solution (at 120 μmol L−1) and measuring the time
236
required for the analytical signal to return to the baseline. It was found that in the presence of
237
stirring (Fig. 3S), the current rapidly returned to the baseline after the formation of the transient
238
signal, hence increasing the analytical frequency and providing faster analysis.
239
10
240
3.3. Analytical performance
241
A repeatability experiment was performed to evaluate the precision of the method. Fig.
242
4S shows the signals recorded for 18 successive injections of 100 μmol L−1 ampicillin into the
243
BIA cell. A low RSD of 3.1% was obtained, indicating excellent performance and stability of
244
the copper electrode used for ampicillin determination.
245
The linear range of the proposed method was determined by injecting a series of
246
ampicillin standard solutions of different concentrations, in ascending and descending order,
247
into the BIA cell. Fig. 4 presents the series of amperometric signals obtained for the sequential
248
injection (in triplicate) of the seven standard solutions. A satisfactory linear response was
249
obtained in the concentration range used (30-250 μmol L−1), with a correlation coefficient better
250
than 0.999. The limits of detection (3Sb/slope) and quantification (10Sb/slope) were 7.11 and
251
23.7 μmol L−1, respectively. Furthermore, considering the time required for the analytical signal
252
to return to the baseline, the analytical frequency was 86 injections h−1.
253 254
INSERT FIG. 4
255
INSERT TABLE 1
256 257
Table 1 compares the proposed method with other methods described in the literature
258
using electrochemical techniques for the determination of ampicillin. The BIA method presents
259
a linear range and LOD suitable for the quantification of ampicillin in commercial samples of
260
pharmaceutical products. It should be pointed out that in Table 1, the linear range varies
261
according to each type of sample to be analyzed, and either they depend on stages of preparation
262
of the electrochemical sensor and some of them require several complicated steps to prepare
263
the sensor, such as the molecularly printed polymers (MIPs) and DNA [16,27,30]. Thus, the
11
264
results obtained here demonstrated the use of the BIA method for rapid, direct, and low-cost
265
ampicillin determination in drug samples with this analyte as the active ingredient.
266
The proposed BIA method was applied for the quantification of ampicillin in
267
pharmaceutical samples, under the optimized conditions, using 100 μL injections in triplicate
268
(Fig. 5). The linear working range was determined using injections of ampicillin standard
269
solutions at increasing concentrations between 30 and 200 μmol L−1 (a-e), alternating with
270
triplicate injections of two drug samples (s1 and s2), as well as sample s1 enriched with two
271
different concentrations of standard ampicillin solution (r1 and r2). The results obtained by the
272
proposed method showed that the ampicillin contents in the samples were close to the values
273
stated on the labels, with a difference of less than 5%. The method presented satisfactory results
274
for the determination of ampicillin in the drug samples, with recovery values of over 95% for
275
both fortification levels (Table 2).
276 277
INSERT FIG. 5
278 279
INSERT TABLE 2
280 281
The accuracy of the results obtained with the proposed method was evaluated by
282
comparison with the values obtained using the HPLC-UV/Vis and spectrophotometric methods
283
(Table 3). Fig. 5S shows the results for the determination of ampicillin in the same
284
pharmaceutical samples using the two latter methods. The peak asymmetry observed for the
285
HPLC method was probably caused by the manual injection onto the column.
286 287
INSERT TABLE 3
288
12
289
According to the Student’s t-test, at the 95% confidence level, there were no significant
290
differences between the results obtained using the two literature methods, and it could also be
291
concluded that the values obtained using the proposed BIA method were statistically equal
292
(tcalculated < ttabulated) to those obtained using the literature methods. The values obtained using
293
the different methodologies were in excellent agreement and were very close to the labeled
294
values. The advantages of the new method include speed, low consumption of reagents, minor
295
waste generation, and ease of operation. The results for the analysis of the samples, without any
296
pretreatment, also showed that the presence of solid particles from the excipients did not
297
interfere in the electrochemical measurements. This achieves highlighted another advantage of
298
this technique since sample clean-up is an indispensable and time-consuming step in optical
299
and chromatographic methods.
13
300
4. Conclusions
301
This work presents a method for the amperometric determination of ampicillin in drugs
302
using an unmodified copper electrode in a BIA system and exploring the complex formation
303
between copper and ampicillin. Evaluation of the proposed method in real samples showed no
304
significant differences with the results obtained with the other two well-established methods in
305
the literature, leading to the conclusion that the proposed method could be used in routine
306
analyses with low-cost equipment and avoiding time-consuming sample preparation steps.
307 308
Conflicts of Interest
309
The authors declare that there are no conflicts of interest
310 311
Acknowledgments
312
The authors are grateful for the financial support provided by the Brazilian agencies
313
FAPEMA (grant number #UNIVERSAL-00863/16, #IECT-03/2016, #INFRA-03170/18, and
314
#UNIVERSAL-01372/17), CNPq (grant number #465389/2014-7, #205220/2018-5) and
315
CAPES (Finance Code 001). The authors thank prof. Kagan Kerman from the University of
316
Toronto (ON, Canadá), for kindly revising and improving this manuscript. We are also very
317
grateful to Professor Rodrigo Alejandro Abarza Munõz from the Federal University of
318
Uberlândia (MG, Brazil), for providing us with the 3D-printed BIA cell.
14
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424
19
425
Figure captions
426
Fig. 1 – Chemical structure of ampicillin.
427
Fig. 2 – Cyclic voltammogram obtained using the copper electrode (ϕ = 2.0 mm) in BR buffer (pH 7.0), in the
428
absence and presence of 1.0 mmol L−1 of ampicillin, at v = 50 mV s−1.
429
Fig. 3 – Effect of pH on the anodic peak current (Ipa) and the anodic peak potential (Epa).
430
Fig. 4 – Amperometric responses obtained for triplicate injections of solutions containing different concentrations
431
of ampicillin (μmol L−1): a = 30; b = 60; c = 110; d = 150; e = 200; f = 250; g = 300. Insert: response obtained for
432
injection of the supporting electrolyte. Electrolyte: BR buffer (pH 7.0); injection volume: 100 μL; dispensing rate:
433
76.9 μL s−1; potential: +0.025 V vs. Ag/AgCl (KClsat.)
434
Fig. 5 – Amperometric responses obtained for triplicate injections of solutions containing different concentrations
435
of ampicillin (μmol L−1: a = 30; b = 60; c = 110; d = 150; e = 200), the drug samples (s1 and s2), and drug sample
436
s1 enriched with ampicillin at two levels (r1 and r2). Electrolyte: BR buffer (pH 7.0); injection volume: 100 μL;
437
dispensing rate: 76.9 μL s−1; potential: +0.025 V vs. Ag/AgCl (KClsat.)
438 439 440
20
441 442
Fig. 1
443 444
21
445 446
Fig. 2
40
III I
I / µA
20 0 -20
IV
-40
II
-0.8 447
electrolyte -1 [AMP] 1.0 mmol L
0.4 0.0 -0.4 E/V vs Ag/AgCl/KClsat
448
22
449 450
Fig. 3
0,2
Ip / µA
24
0,1
16
Ep / V
32
0,0
8 0 5 451
6
7 pH
8
9
-0,1
452
23
453
Fig. 4
8
I / µA
6
A
0.5 A
e
Electrolyte
c
4
a
g
f
f
e d
d
c
b
b
a
2 0
1000
1500
2000
time / s
454
4
Ascending
B
I / µA
2
R = 0.999 Ip (µA) = 0.017 + 0.17 x [AMP]
0 4
Descennding
2
R = 0.998 Ip (µA) = -0.021 + 0.17 x [AMP]
0 50
455
2500
100
150
200
[AMP] / µmol L
-1
250
300
456
24
457
Fig. 5
0.8 A
5
d
I / µA
electrolyte
4 3
a
b
c
e
s2 s1
r2 r1
2 1 400 458
800
1200 time / s
1600
459 460 461 462
25
464
465 466 467 468 469 470
Table 1. Performance comparison of the proposed technique and methods reported in the literature. Electrode type
Analysis method
Linear range (µmol L−1)
LOD (µmol L−1)
Reference
E – AB
ACV
5 - 5000
1.0
[28]
E – AB
ACV
0.2 - 15000
0.03
[29]
DNA-AuNPs/GCE
DPV
1.0x10-6 – 0.005
3.8x10-7
[30]
FDCMCPE
DPV
2.34 - 30
0.67
[10]
MIP/MWCNTs/AuNPs/Pt
DPV
0.01 – 5.0
0.001
[16]
Co-MOF@TPN-COF
EIS
2.86x10-9 – 5,72x10-3
6.21x10-10
[27]
APT-modified gold chip
AMP
2.5 - 1000
1.0
[32]
Copper
BIA-AMP
30.0 – 250.0
7.11
This work
E – AB: electrochemical aptamer-based sensor. ACV: Alternating Current Voltammetry. DNA-AuNPs: DNA functionalized gold nanoparticles. GCE: glassy carbon electrode. FDCMCPE: carbon-paste electrode spiked with ferrocenedicarboxylic acid. DPV: Differential Pulse Voltammetric. MIP/MWCNTs/AuNPs/Pt: platinum electrode modified with multiwalled carbon nanotubes, gold nanoparticles and a thin film of molecularly imprinted polymers. CoMOF@TPN-COF: nanoarchitecture of Co-based metal-organic frameworks and terephthalonitrile-based covalent organic framework. EIS: electrochemical impedance spectroscopy. AMP: amperometry.
471 472
27
473
Table 2. Ampicillin concentrations per tablet, obtained by the proposed BIA method, and recovery values for the
474
analysis of drug samples.
475 Ampicillin content (g/tablet)
476
*S
Recovery test [ampicillin] (µmol L−1)
Labeled
Founded
Added
Founded
Recovery (%)
S1*
0.50
0.54 ± 0.060
46.9
49.7 ± 3
105.9
S2*
0.50
0.52 ± 0.062
115.6
124.6 ± 3
107.7
1
= sample 1; S2 = sample 2.
477
28
478
Table 3. Ampicillin concentrations per tablet, obtained by the proposed BIA method, HPLC-UV, and UV-Vis
479
spectrophotometry.
Ampicillin (mg/tablet)
480
Labeled value
BIA
HPLC-UV
UV-Vis
500
540 ± 62
570 ± 93
550 ± 62
n = 3, 95% confidence level.
481 482
HIGHLIGHTS
483 484
A rapid and sensitive method for the determination of the ampicillin antibiotic;
485
The method uses a batch injection analysis system with bare copper electrodes;
486
Development of a simple, low cost, and portable instrumentation system
487
This system presents competitive performance for ampicillin routine analyses.
488 489 490
William Barros Veloso: Formal analysis, Writing- Original draft preparation.: Geyse Adriana
491
Corrêa Ribeiro: Formal analysis, Visualization.: Cláudia Quintino da Rocha: Visualization,
492
Formal analysis.: Auro Atsushi Tanaka: Writing - Review & Editing.: Iranaldo Santos da
493
Silva: Conceptualization, Writing - Review & Editing.: Luiza Maria Ferreira Dantas:
494
Conceptualization, Writing - Review & Editing.
495 496 497
Declaration of interests
498 499 500
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
501 502 503
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
29
504
505 506 507 508 509 510 511 512 513
Flow-through amperometric determination of ampicillin using a copper electrode in a batch injection analysis system William Barros Veloso, Geyse Adriana Corrêa Ribeiro, Cláudia Quintino da Rocha, Auro Atsushi Tanaka, Iranaldo Santos da Silva, Luiza Maria Ferreira Dantas
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GRAPHICAL ABSTRACTS
517
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