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Express analysis of amoxicillin via colorimetric testing

Article in Chemical Papers · February 2020

DOI: 10.1007/s11696-020-01085-6

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Chemical Papers
https://doi.org/10.1007/s11696-020-01085-6

ORIGINAL PAPER

Express analysis of amoxicillin via colorimetric testing

Anastasia V. Marakaeva1 · Irina V. Kosyreva1

Received: 25 September 2019 / Accepted: 29 January 2020

© Institute of Chemistry, Slovak Academy of Sciences 2020

Abstract
Falsified drugs are a serious problem of modern society. Therefore, it is necessary to develop a fast and simple method of
drug screening for checking of their falsification. In this article, we present paper-based test methods that can be used to
identify amoxicillin, a β-lactam series antibiotic agent, used the mobile phone camera. The detection time is 10 min. Ninhy-
drin, p-dimethylaminobenzaldehyde, and Fehling’s reagent are used as reagents for the test. We have found out the optimal
conditions of express analysis, including temperature and duration of heating. We have obtained the color scales for visual
and colorimetric detection of the antibiotic agent. Optimal color parameters for RGB, CMYK, and HSV color models have
been analyzed. We have demonstrated linear correlations between optimal colorimetric parameters and antibiotic agent
concentration. Profiles of radar diagrams using color parameters as coordinates are obtained. Linear correlation between
area and perimeter of radar diagrams and amoxicillin concentration was found.

Keywords Express analysis · Indicator papers · Antibiotic agents · Amoxicillin · Phone camera-based detection ·
Colorimetry

Introduction Falsified drugs are a serious problem of modern society.

Antibiotics include the by-products of living cells (or their
synthetic counterparts) that selectively inhibit the functions
of various microbial agents, tumors, etc. (Lorian 2005; Arza-
mastsev 2004). Currently, this category of drugs is one of
the most common in medicine. Primary classes of antibiotics
contain: β-lactams, tetracyclines, quinolones, aminoglyco-
sides, etc. (Egorov 2004).
Amoxicillin is a broad-spectrum antibiotic that belongs to
the class of β-lactams. It is included into the list of vital and
essential drugs for medical use (WHO 2017). Amoxicillin
is produced in many forms includes pills, capsules, suspen-
sions, powders, etc. In recent years, the number of antibiotic-
resistant strains of microorganisms has increased due to the
widespread availability and often unreasonable application
of antibacterial drugs (Neu 1992). To preserve the antibac-
terial activity and make amoxicillin more effective against
microorganisms that are usually resistant to it, a combination
of β-lactamase inhibitors with this antibiotic is used.
* Anastasia V. Marakaeva
marakaeva_anastasiya@mail.ru
1
Chemistry Institute, Saratov State University,
Astrakhanskaya 83, 410012 Saratov, Russia
Often, patients receive substandard or counterfeit medicines
(Cockburn et al. 2005; Attaran et al. 2012; Arzamastsev et
al. 2004). Antibiotic detection in various objects (medi-
cal drugs, food products, and biological fluids) is mostly
performed via spectrophotometric, chromatographic, and
immunoassay analysis techniques (Cháfer-Pericás et al.
2010; Kahsay et al. 2014; Taranova et al. 2015). Screening
procedures are used as a first stage of antibiotic identifica-
tion. These procedures are designed for fast removal samples
without antibiotics, thus allowing to analyze a large number
of samples with minimal time and low cost. Then, positive
results of screening are clearly identified and subjected to
quantitative analysis. Decision of the European Union no.
2002/657/EC provides general definitions, requirements, and
criteria for evaluation the efficiency of screening methods in
accordance with Directive no. 96/23/EC (EC 2002).
To determine amoxicillin, spectrophotometric, chroma-
tographic, and electrochemical methods are mainly used
(Table 1).
There are also express methods for the detection of
amoxicillin, such as immuno- and receptor tests. To deter-
mine amoxicillin and other antibiotics in milk and meat,
commercial tests “Beta S ­ tar®”, “Beta S­ tar® Combo” have
been developed (Kalnitskaya 2008). Analysis time up to 10

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Table 1  Methods for the determination of amoxicillin in drugs
References Method Reagent
Mohamed (2001) Spectrophotometry Molybdenum and thiocyanate
Aly and Amin (2007) Spectrophotometry Sudan III
Shirole et al. (2018) HPLC Potassium dihydrogen phos-
phate, methanol
Pawar et al. (2017) Fluorimetry Quantum dots
Bergamini et al. (2006) Voltammetry [N,N-ethylenebis(salicylidenea
minato)]oxovanadium(IV)
Hatamie et al. (2015) Voltammetry ZnO NRs/gold/glass electrode
min and detection sensitivity from 2 to 20 μg/kg. However,
the disadvantage of such methods is the relatively high
cost. Chemical selective field-effect transistors designed
for detection antibiotics have been developed (Kellner
et al. 2004). However, these sensors are highly sensitive
to environmental changes, such as humidity, temperature,
and irradiation.
Recently, the registration of the analytical signal with
the help of various digital devices has become widespread.
Said devices include: photo and video cameras (Lapresta-
Fernández and Capitán-Vallvey 2011; Pena-Pereira et al.
2016) and smartphone cameras (Li et al. 2012; Martinkova
et al. 2018; García et al. 2011). This study shows the pos-
sibility of express identification of amoxicillin with help
of indicator papers and phone camera.
Experimental
Materials
Chemicals
The reagents were purchased from commercial sources and
used without further purification. Distilled water was used
to prepare the solutions.
Amoxicillin (in the form of amoxicillin sodium) is a
powder used to prepare an intravenous solution («Bio-
chemist», Russia). The initial antibiotic solution with a
concentration of 32 mg/mL was prepared by dissolving
the exact sample in a solution of 0.01 M hydrochloric acid
or distilled water. Working solutions with a concentration
of 1–32 mg/mL (in 0.01 M HCl or distilled water) were
prepared on the day of the experiment by successive dilu-
tion of the initial solution.
Ninhydrin, p-dimethylaminobenzaldehyde (DMAB), and
Fehling’s reagent («Reachem», Russia) prepared in accord-
ance with technique provided by (Feigl 1960) were used as
test reagents.
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Analytical ranges LOD Comments
7.5–75 μg/mL 0.75 μg/mL Simple, rapid, accurate sensitive
0.2–22 μg/mL 0.06 μg/mL Simple, rapid, accurate sensitive
0–60 µg/mL 0.28 μg/mL Expensive instrumentation
5–30 μg/mL 5.19 μg/mL Expensive instrumentation
28.5–82.6 μM 24.8 μM Time consuming synthesis,
expensive instrumentations for
19 μM 50–250 μM characterization and detection
Equipment
5.0-Megapixel camera of Lenovo A319 smartphone with
Android 4.4.2 operating system.
Photo box sized 22 × 23 × 24 cm with two strips of led
lighting (5 V, 1 A).
Dual-beam scanning spectrophotometer Shimadzu
UV-1800 (Japan), measuring range: 200–800 nm.
Data processing
The images obtained with the phone’s camera were pro-
cessed using Adobe P ­ hotoshop® software. First, we used the
“Average” filter, and then, we determined the values of the
color channels provided by RGB, CMYK, and HSV color
models.
Indicator paper preparation
Cheap and affordable cellulose paper was used as a sub-
strate. To obtain the test papers, 5 × 5 cm cellulose paper
sheets were immersed for 4–5 min in freshly prepared etha-
nolic solution of ninhydrin (DMAB or solution of Fehling’s
reagent) and dried at room temperature.
Linear correlations for colorimetric identification
of amoxicillin
We used the following procedure of colorimetric identifi-
cation: a drop of analyzed solution (within 1–32 mg/mL
concentration range) was applied to ninhydrin-covered
paper (colorimetric test 1), DMAB-covered paper (col-
orimetric test 2), and Fehling’s reagent-covered paper
(colorimetric test 3); the papers were dried at 95–100 °C
for 10 min; as a result, we observed formation of purple
(ninhydrin), yellow (DMAB), or brownish red (Fehling’s
reagent) coloration (see Fig. 2). Analytical signal was reg-
istered by a mobile phone (Lenovo A319) camera; the test
forms were put into a photo box and the photos were made.
Photos were processed using Adobe ­Photoshop® software.
Chemical Papers
We isolated a part of the photo and averaged it using the
“Average” filter, and then, we determined the values of the
color channels provided by RGB, CMYK, and HSV color
models. Next, we determined the intensity values of the
color parameters, and found linear correlations between
the intensities of the color parameters and the concentra-
tion of amoxicillin.
Results and discussion
Selection of optimal conditions for express analysis
To optimize the conditions of express analysis, we per-
formed some preliminary tests. As a result, the follow-
ing concentrations of reagents were selected as optimal:
ninhydrin (0.1 M), DMAB (0.1 M), and Fehling’s reagent
as prepared according to the technique described in (Feigl
1960).
We have found that the strongly acidic medium (pH
2–3) provides the best results for the amoxicillin reaction
with ninhydrin and DMAB on indicator papers; therefore,
standard antibiotic solutions were prepared in 0.01 M HCl.
We used the aqueous solution of amoxicillin in the case of
its reaction with Fehling’s reagent having basic pH.
We have determined the optimal heating time for
the indicator paper. To do this, a solution of amoxicil-
lin was applied to indicator papers covered with ninhy-
drin, DMAB and Fehling’s reagent; the paper was then
heated at 95–100 °C for 1–15 min, placed in a box, and
photographed.
One of the advantages of express methods of analysis
is their rapidness. As we can see from Fig. 1, the optimal
heating time is 10–15 min for colorimetric test 1, 2, and 3,
because we have the maximum color change as compared
Fig. 1  Correlation between intensity of a color channel B and con-
centration of amoxicillin (8 mg/mL) after 1–15 min of heating at
95–100 °C (colorimetric test 1, 2, 3)
to the control. Further express analysis using colorimetric
tests 1, 2, and 3 was carried out after 10 min.
Development of color scales for visual
and colorimetric identification of amoxicillin
Standard amoxicillin solutions with concentrations of 0, 1,
2, 4, 8, 16, and 32 mg/mL were used to obtain color scales.
A drop of the solution was applied to colorimetric test 1, 2,
or 3; the test paper was then heated, placed in a box, photo-
graphed, and processed via colorimetric technique. Figure 2
shows the resulting color scales after 10 min of heating indi-
cator papers at 95–100 °C.
Linear correlations for colorimetric identification
of amoxicillin
Photos of color scales were processed using Adobe
­Photoshop® software; the intensity of the color parameters
was determined via RGB model. We selected the best chan-
nels by comparing their sensitivity (tg α) and regression
coefficient values. For test papers with ninhydrin, the best
color channel is Blue (B) (47.6) with a regression coefficient
of 0.986 (Fig. 3a). The concentration curves of test papers
covered with DMAB showed that the largest tilt angle was
selected for a Blue color channel (B) (24.6) with regres-
sion coefficient equal to 0.987 (Fig. 3b). Similarly, for test
papers covered with Fehling’s reagent, we selected a Blue
(B) (− 59.2) channel with a regression coefficient of 0,989
(Fig. 3c). Limit of detection (LOD) is 1.5 mg/mL (colori-
metric test 1); 2,2 mg/mL (colorimetric test 2); 1,9 mg/mL
(colorimetric test 3).
Color stability of the developed colorimetric tests
over time
After express identification was performed, the indicator
papers were stored at room temperature in a dark place. For
Fig. 2  Color scales for amoxicillin identification (mg/mL): a—colori-
metric test 1, b—colorimetric test 2, and c—colorimetric test 3
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Fig. 3  Correlation between the intensity of color channels and concentration of amoxicillin: a—colorimetric test 1, b—colorimetric test 2, and
c—colorimetric test 3 (n = 3)
Fig. 4  Variation in the intensity of the Blue color channel for colori-
metric tests 1, 2, and 3, obtained as a result of interaction with 4 mg/
mL amoxicillin solution for 30 days
30 days, test papers were photographed and resulting photos
were processed as described above. As we see in Fig. 4, the
intensity of the Blue channel of the color test 1 decreased
with time and stopped changing after 10–30 days. Colori-
metric tests 2 and 3 have not changed their colors within a
month.
Construction and analysis of radar charts
Data visualization plays an important role in the processing
of analysis results. Visualization possibilities allow you not
only to use various geometric objects, but also utilize their
special properties, such as: color, texture, and surface. A
radar chart represents the values of each data category along
the axes starting at the center and ending at the outer ring
of the chart.
Colors can be described by various color models (Fair-
child 2013; Cheng et al. 2001). In this paper we used three
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primary models: RGB (Red, Green, Blue), HSV (Hue,
Saturation, Value), and CMYK (Cyan, Magenta, Yellow,
Key).
We used Microsoft Office E ­ xcel® to construct profiles
of radar charts that use the coordinates of various color
parameters (R, G, B, C, M, Y, K, H, S, and V) for various
concentrations of amoxicillin. Figure 5 shows the resulting
diagrams with seven axes using R, G, B, C, M, Y, and K
color channels as coordinates and six axes using R, G, B,
H, S, V color channels.
As we see from Fig. 5a, formation of a radar chart pro-
file for the amoxicillin–ninhydrin system involves all color
channels; however, the biggest variations are observed for
intensities of G (from 137 to 183), B (90 to 170), and S
(4 to 11) channels. In the case of radar diagram profiles
for amoxicillin–DMAB system (Fig. 5b), characteris-
tic changes are observed only for B (from 70 to 110), Y
(from 74 to 58), and S (from 50 to 22) parameters. In the
case of colorimetric test 3, we observed changes of all
color parameters for various concentrations of amoxicil-
lin (Fig. 5c).
The profile of radar chart changed for various colori-
metric reactions (Fig. 6).
We used MO ­Excel® software to calculate the area (S)
and perimeter (P) of constructed radar chart.
As we see from Fig. 5 and Table 2, the greatest area of
radar chart profiles is observed for the reaction between
amoxicillin and Fehling’s reagent solution. The best
regression coefficients for this combination are obtained
in the RGBCMYK and RGBHSV coordinate systems
(0.994 and 0.997, respectively). The best regression coef-
ficient for the colorimetric test 1 (0.973) is observed for
the RGBCMYK coordinate and correlation between the
radar chart area and logC. In the case of reaction between
DMAB and amoxicillin, the best regression coefficient
(0.991) is observed for RGBHSV coordinates and cor-
relation between the radar chart area and amoxicillin
concentration.
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Fig. 5  Radar chart profiles: a—colorimetric test 1, b—colorimetric test 2, c—colorimetric test 3; Amoxicillin concentration, mg/mL

Fig. 6  Profiles radar charts (A for RGBCMYK coordinates; B for RGBHSV coordinates) obtained for colorimetric tests 1, 2, and 3 interacting
with 4 mg/mL amoxicillin solution

Table 2  Linear correlations between the square (perimeter) of radar chart and the concentration of amoxicillin
Type Coordinate system Regression equation for correlation r2 Regression equation for correlation r2
between area and logC/C (mg/mL) between perimeter and logC/C (mg/mL)

Colorimetric test 1 RGBCMYK y = − 14,946x + 65,090 0.973 y = − 78.2 x + 833 0.950

RGBHSV y = − 16,936x + 66,934 0.961 y = − 123x + 921 0.962
Colorimetric test 2 RGBCMYK y = − 2679.7 x + 43,315 0.932 y = 9.07 x + 678 0.953
RGBHSV y = − 76.158 x + 42,279 0.991 y = − 7.21x + 713 0.978
colorimetric test 3 RGBCMYK y = − 11,738x + 38,010 0.994 y = − 140x + 661 0.967
RGBHSV y = − 16,852x + 34,534 0.997 y = − 202x + 689 0.956

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Validation of express identification method
The validity of express identification method based on the
developed colorimetric tests was tested (Table 3).
The values of relative errors of identification do not
exceed 9% for colorimetric test 1, 11% for colorimetric test
2, and 7% for colorimetric test 3.
Analysis of commercial amoxicillin drugs
We checked our tests to identify amoxicillin in commercial
medical drugs produced in the form of 250 mg pills and
500 mg capsules. The analyzed drugs contain amoxicil-
lin trihydrate as the active substance, and talc, magnesium
stearate, and potato starch as additives. Figure 7 shows the
intensity values of the Blue color channel in the presence
of auxiliary components of the drug and their mixture. The
concentrations of the components indicated on the package
of the drug were selected: talc 3.7 mg; magnesium stearate
3.7 mg; potato starch 75.6 mg per 1 tablet (tablet weight
370 mg). The intensity value of the Blue channel does not
change in the presence of auxiliary components, as well as
their mixture in all three studied systems (Fig. 7a–c). The
intensity of the Blue channel was constant in all cases. For
this reason, we concluded that talc, magnesium stearate,
and potato starch do not interfere with the detection of
amoxicillin.
The express identification was performed by dissolving a
sample of drug, filtering the resulting solution, and adding
a drop of the analyzed solution to the surface of paper of
colorimetric tests 1, 2 and 3. The results of the quantitative
identification of the antibiotic are presented in Table 4.

Table 3  Determination of Added, mg/mL Found, mg/mL

amoxicillin via express methods
(n = 3, P = 0.95) Colorimetric Sr Colorimetric Sr Colorimetric Sr
test 1 test 2 test 3

10 11 ± 1 0.07 9±1 0.11 10 ± 1 0.05

30 29 ± 2 0.09 32 ± 3 0.08 28 ± 2 0.07

Fig. 7  Intensity of the Blue color channel in the presence of talc (1% solution), magnesium stearate (1%), starch (20%), and their mixture with
concentration of amoxicillin 10 mg/mL: a—colorimetric tests 1, b—colorimetric tests 2, and c—colorimetric tests 3

Table 4  Results of amoxicillin identification in drug samples via spectrophotometric and express testing methods (n = 3, P = 0.95)

Drug Stated (mg) Found (mg)

Spectrophotometry Sr Colorimetric test 1 Sr Colorimetric test 2 Sr Colorimetric test 3 Sr

“AVVA RUS” pills, 250 250 ± 4 0.01 253 ± 23 0.09 260 ± 28 0.11 234 ± 14 0.02
Russia
“Hemofarm” capsules, 500 495 ± 10 0.02 489 ± 36 0.07 540 ± 38 0.07 503 ± 25 0.03
Serbia

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Conclusions
The obtained express methods allow analysis outside the
laboratory and have rapidness comparable to well-known
commercial tests. However, the advantage of the developed
express methods is low cost in comparison with immuno-
and microbiological tests; and stability under various envi-
ronmental changes unlike to field electrochemical sensors.
We have developed a paper-based test system that can
be used for express identification of amoxicillin using the
mobile phone camera. The analysis takes 10 min for different
colorimetric tests. However, the best results, i.e., the short-
est analysis time, the smallest value of the relative standard
deviation in drug analysis results (0.03), and the greatest
stability (30 days) are characteristics of the colorimetric test
3, based on Fehling’s reagent.
The validity of our test was verified and the values of
relative errors of identification do not exceed 9% for colori-
metric test 1, 11% for colorimetric test 2, and 7% for colori-
metric test 3. The test was used to analyze commercial drugs
(250 mg pill and 500 mg capsules) that contain amoxicillin.
The content of the antibiotic, found with the help of devel-
oped colorimetric tests, corresponds to the value declared
by drug manufacturers.
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