ANALYTICAL CHEMISTRY ARTICLE SUBJECT STUDYING THE ANALYTICAL CONDITIONS OF SULFONAMIDES BY CHROMATOGRAPHIC METHOD.

Socialist Republic of Vietnam
Independence-Freedom-Happiness

SCHOOL OF MEDICINE VIET NAM NATIONAL UNIVERSITY
HO CHI MINH CITY

ANALYTICAL CHEMISTRY ARTICLE
SUBJECT
STUDYING THE ANALYTICAL CONDITIONS OF SULFONAMIDES BY
CHROMATOGRAPHIC METHOD.
CLASS: D3-2020
GROUP MEMBERS
Mai Nguyễn Đăng

207720201006

Nguyễn Xuân Hiệp

207720201014

Phạm Nhật Hoàng

207720201015

Nguyễn Thanh Huyền

207720201017

Nguyễn Tấn Tài

207720201033

Binh Duong, September 29, 2


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Abstract

HPLC optimization includes choosing the detector's wavelength, the stationary phase, and the
mobile phase's pH, composition, and speed. The separation conditions are also optimized.
Linear interval survey; detection and quantification limits; Analyze the measurement's
accuracy and repeatability. fecal sample handling conditions optimization Choose a
representative treatment approach, then assess the effectiveness of recovery. To examine
various shrimp samples, develop analytical processes and use research processes.

Keyword

Chromatographic method; 
Analytical chemistry; 
Sulphonamides; 
Antibiotics; 
Shrimp;

1.

Identify problem:

Shrimp farming is now the most significant industry in Vietnam, and it is also the aquaculture
development plan's primary goal. The fast growth of shrimp aquaculture contributes to a rise
in medication and chemical usage. MTD, a member of the broad range antibacterial
medication family SAs, is frequently used in shrimp farming to prevent and cure a variety of
bacterial illnesses in animals. If the animals are not given time to clean themselves after using
these chemicals, there will be leftover antibacterial compounds that might be harmful to
health.
Metronidazole sulfaguanidin (SGU), sulfamethoxypyridazine (SMP), sulfadoxine (SDO), and
sulfamethoxazole (SMX), all of which are members of the SAs antibacterial family and are
often employed in blankets, are the analytical topics that we decided to research. Based on
this need, we chose the HPLC reverse phase adsorption method as the research method, with a
UV-Vis detector as the linked detector.

1.1 Introduction to Sulphonamides (SAs) and Metronidazole (MTD)
1.1.1 Molecular structure:

Figure 1: General molecular structure of SAs
When we replace groups R1, R2 with different radicals, we have different SAs.

Figure 2: Structure of Metronidazole (MTD)


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It is a nitroimidazole-family antibiotic used mostly to treat anaerobic bacteria and protozoa.
MTD is a component of animal feed, also an antibiotic used in aquaculture (with the trade
name Enro DC).

1.1.2 Physical

and

chemical

properties

of

the

Sulphonamides,

Metronidazole
1.1.2.1

Physical properties:

SAs are white or pale yellow crystals, odorless, sparingly soluble in water, soluble in acids
and alkaline solutions (except sulfaguanidine). 
MTD is a crystalline or crystalline powder that is odorless, yellowish, stable in the air, and
gradually darkens when exposed to light. The melting point ranges between 159 and 163℃.
Metronidazole hardly dissolves in acetone and water.

1.1.2.2

Chemical properties: 

SAs are amphoteric.
SAs form complex salts that precipitate with Ag+ ions, and precipitate colored complexes
with Cu2+, Co2+ ions, ...
In the primary amine group of SAs, there are free electron pairs, helping SAs to carry out

charge transfer complexation with phenosafranine (PSF) giving a purple complex with a
maximum absorption wavelength at 270-273 nm.

1.1.3 Pharmacological properties and spectrum of action of Sulphonamides,
Metronidazole:
With therapeutic doses, SAs do not kill bacteria, only make bacteria weaker, unable to grow
and reproduce, easily destroyed by white blood cells.
SAs is a broad-spectrum antibiotic: they are effective against many anthrax bacteria, cholera
bacteria, Shigella, E.coli, and bacilli.
Along with protozoa and anaerobes, Metronidazole also has a wide range of action against
Eubacterium, Peptococcus, Peptostreptococcus, Fusobacterium Veillonella, Clostridium,
including C.difficile and C.perfringens. It works well against isolates of B. fragilis that are
clindamycin resistant.

1.1.4 Mechanism of antibacterial action of Sulphonamides, Metronidazole
1.1.4.1 Antibacterial mechanism of SAs:
Inhibits folic acid metabolism.
Inhibits the synthesis of folic acid by bacteria.

1.1.4.2

Antibacterial mechanism of Metronidazole:

Metronidazole's action against obligate anaerobes occurs through a four-step process:
Attack on microorganisms
Decreased activation by intracellular protein transport
Decreased cell-mediated subunit interactions - toxic intermediates interact with host DNA,
leading to DNA strand breakage and DNA strand destruction.
Breakdown of cytotoxic intermediates - toxic intermediates break down into inactive end
products.

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2.

Design the experimental procedure 

2.1 Research method

In the article, samples of prawn are collected and examined, with highly complicated
components. Therefore, high performance liquid chromatography (HPLC) was chosen to
survey the conditions of extraction and quantitative analysis.

2.1.1 General principles and equipment required for HPLC.
High performance liquid chromatography (HPLC) is a segregation of compounds based on
the movement of solutes through a separation column that has either pellicular and porous
material as the inner coating. Solute will move through the mobile phase at different
velocities, depending on its distribution constant.

2.1.2 Quantitative analysis using HPLC
In the chosen conditions, every substance has a unique quantity t Ri, indicating the amount of
time that a certain substance is moving through the column. We can use this time to conduct
qualitative analysis onto the substance based on a standard sample. Then, quantify the
substance based on analytical properties of the result. Normally, the concentration of
substance is usually depending on the height (H) or area (S) of the peek.
    H = k.Cb   or   S = k.Cb

 

 

In which
H: height of the peek
S: area of the peek
k: experimental constant (depends on the conditions of the experiment)
b: nature constant, satisfied 0 < b ≤ 1

2.2 Solid phase extraction
When the amount of analyte is small, analyte enrichment through solid phase extraction is
essential. Moreover, food samples have a complicated composition of substance, with many
types of fat beside the analytes. Therefore, solid phase extraction is necessary to isolate the
analyte quantitatively, remove and evict any impurities.

3.

Conduct an experiment 

3.1 Investigation of chromatographic conditions
3.1.1 Determination the wavelength of the detector
Based on the similarity of the maximum absorption capacity, we decided to choose a common
wavelength of 270 nm for 5 substances. MTD is more sensitive (320 nm); therefore, the
detector has two channels of 320 nm and 270 nm for efficient quantification.


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Fig. 3. The absorption capacity of wavelength of 5 substances

3.1.2 Exploring the separation ability of sulfonamides on RP-C18 column
Retention time is calculated when the sample is loaded into the chromatographic separation

column until the solute is eluted from the column at the point of maximum concentration.
Retention time can determine the order of a substance's appearance. 
The retention time of 5 standard solution 1,0ppm on chromatographic mobile phase (20%
ACN – 80% buffer solution acetate, buffer solution concentration 10mM, pH=4,5). Temp:
30oC. Speed of mobile: 1ml/min. Detector UV-Vis: 2 channels 270 nm and 320 nm.
No
Retention time (min)
Analytes
1
3,30
SGU
2
4,48
MTD
3
8,49
SMP
4
13,23
SDO
5
15,08
SMX
Table 1: Retention time and peak order of analytes
Purpose: realizing the specific retention time of each substance as a basis for qualitative and
quantitative determination of sulphonamides, metronidazole in research subjects.

3.2 Determination stationary phase.
To research separation ability and determine SAs, MTD which are polarizers. Therefore, most
published works use separation columns containing reversed phase stuffing such as RP- C4,

RP - C12 .... RP - C18 column is chosen to separate above substances with packed particle
size of 5 µm. 

3.3 Mobile phase optimization
3.3.1 Acetate buffer concentration of mobile phase
The selected buffer concentration values are in the range of 2 - 20mM.
As a result, the capacity factor is almost unchanged. Moreover, the buffer concentration did
not have a significant effect on the separation ability, peek appearance time and peek
chromatographic area.
The capacity factor, the separation ability, peek appearance time and peek chromatographic
area are almost not changed show that the reflecting separation ineffectively.
At a certain buffer concentration in the mobile phase, the capacity factors of the analytes are
significantly different, so it reflects effective separation between substances in the
chromatographic process. With the buffer concentration range, they show chromatographic


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peek that are very sharp and distinct. Based on these observations, the appropriate buffer
concentration in the mobile phase was selected as 10mM.

Fig. 4. The capacity factors of analytes depend on the acetate buffer concentration

3.3.2 pH of buffer acetate
Analytes are amphoteric with pKa values between 5.4 and 7.4 so the use of acidic buffer often
gives sharp chromatographic peaks. For that reason, we investigated the composition of buffer
solution CH3COOH/CH3COONa with pKa=4.75 in the concentration range of 10mM with
pH values: 3.5; 4.0; 4.5; 5.0; 5.5. 
The higher the pH, the weaker the separation between SMX and SDO (pH=5.5), the lower
pH, the long elution time (pH=3.5). At pH = 4.5, the chromatographic peaks have more
distinct separation, more balanced peaks and less time for chromatographic peaks to appear.

In addition, there is a significant difference in volume coefficient. After that, we chose a
buffer solution having suitable pH.

Fig. 5. The capacity factors of analytes depend on the pH of buffer acetate

3.3.3 Ratio of mobile phase components
The ratio of solvent components to the mobile phase affects the elution of the samples from
the separation column. As the mobile phase composition ratio changes, the elution force of
the mobile phase changes. As a result, it makes the change of the retention time and the
volume coefficient of the analyte.
Buffer solution acetate has pH=4,5 and organic solution Acetonitrile (ACN) that change ratio
of mobile phase components following below conditions:
If we follow the way that ratio of mobile phase components is higher, the elution time from
the column is faster. Moreover, the volume coefficients are closer together and the


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chromatographic peaks are not clearly separated that affect the area of the chromatographic
peaks (like 30% ACN – 70% buffer). If we reduce the ACN rate, it takes a long time to elute
the analyte.
In conclusion, experiment need an effective ratio of elution time (not too fast and not too
slow), clear peek and chosen ratio is 20% ACN – 80% buffer.

Fig. 6. The capacity factors of analytes depend on ratio of mobile phase components

3.3.4 Speed of mobile phase
The speed of the mobile phase is a decisive factor in the elution of substances in the
chromatographic column, because it affects the process of establishing a solute equilibrium
between the stationary and mobile phases. The mobile phase speed is too small will cause the
phenomenon of peak dispersion and increase the elution time but too large mobile phase

speed lead to overlapping peaks. Therefore, it is necessary to choose an appropriate mobile
phase rate.
For a definite split column which has an optimal rate. In this case, we survey mobile phase
rate in the range from 0.6 to 1.4 ml/min with optimal conditions and conduct the above survey
(acetate buffer with pH=4.5; buffer concentration 10mM; composition mobile phase
consisting of 20% ACN-80% buffer), the results are obtained: 
At the speed of 1ml/min, the separation is quite good and does not take much time to separate
the substance from the column.
According to the figure, we choose a mobile phase rate of 1ml/min for the next experiments.

Fig. 7. The capacity factors of analytes depend on speed of mobile phase


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4.

Analyze the experimental data

4.1 Survey to establish a standard curve in the concentration range of 0.05 --1,000ppm
From the conditions that have been optimized above, proceed to observe the linear
interval of the measurement with the following condition
Stationary phase

RP C18 (25cmì4.6cm; 5àm

Mobile phase

20% ACN-80% acetate buffer 10mM (pH=4.5)

Mobile phase rate

1 ml/min

Separation column temperature 30oC
Detector

UV: 270 nm, 320nm

Concentration range

0.05ppm – 1.00 ppm

Quantitative method

Charge of peak

Table 2. Optimal conditions for chromatography


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Fig.8. Standard curve of analyte in the concentration range 0.05 – 1.00ppm
a. Sulfaguanidine Standard Curve (SGU)
b. Metronidazole (MTD) Standard Curve
c. Standard curve of Sulfamethoxypyridazine (SMP)
d. Standard curve of Sulfadoxine (SDO)
e. Standard curve of Sulfamethoxazole (SMX)

4.2 Limit of Detection (LOD); Limit of Quantification (LOQ)
Standard deviation:
s=

√

∑ ( Si−Stb )2
n−1

In there:



Si - Value of the analyzed signal at the i-time measurement. (mAU.s)
Stb - Average signal value of i measurements. (mAU.s)

LOD (ppm):   LOD =
LOQ (ppm): LOQ =

3 Sy
B

10 Sy
B

In there:



Sy - The standard deviation of the blank, also determined by the regression equation.
B - The slope in the regression equation.
Substance Coefficient of angle b Standard deviation LOD (ppm) LOQ (ppm)
SGU

117814,44

772,12

0,020

0,066

MTD

124010,14

1028,82

0,025

0,083

SMP

129146,83

1017,35

0,024

0,079

SDO

139792,32

1336,46

0,029

0,096

SMX

126666,70

502,18

0,012

0,040


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Table 3. LOD, LOQ calculated by regression equation

4.3 Measurement accuracy, repeatability:
To evaluate measurement error, select analytical samples whose concentrations are in
the linear range at the beginning, middle, and end of the linear range investigated. We
proceed to prepare three standard samples with a concentration of 0.08; 0.4 and 0.8ppm with
the same condition as the linear interval survey condition.

Through the analysis table and the above calculation results, we see that for samples with high

concentration, the error is small, for samples with small concentration, the error is large.
According to statistical theory, the allowable error is within 15%, so for the concentration
range investigated, the accuracy of this measurement is reliable.
5.

Propose a solution: 

CHƯƠNG 5.

5.1 The optimal conditions for chromatography have been selected:

RP-C18 column: 25 cm ì 4.6 mm; 5 àm
UV-VIS detector: two-channel = (1) 270 nm (2) 320 nm, Rise time = 0.1 s; Range = 0.01
AUFS
Recorder: paper speed = 1 mm/min; write potential = 10 mV
Mobile phase composition: acetate buffer (pH = 4.5) 10mM /aceto-nitrile: 80/20 (V/V)
Mobile phase speed: 1 ml/min.

5.2 The analytical method has been evaluated:
The linear range of sulfates: 0.05 – 1.00ppm
LOD: 0.012 – 0.029 ppm
LOQ: 0.040 - 0.096 ppm
Coefficient of variation: 0.2% - 5% in the concentration range 0.08 - 0.8ppm.

5.3 Survey of real samples:
Select the appropriate sample handling procedure, the recovery rate in shrimp samples

reached 71-90%.

Determination of SGU residue in white leg shrimp samples: 0.16 ± 0.01ppm, banana prawn:

0.21±0.03ppm, greasyback shrimp: 0.12±0.02ppm, black tiger shrimp :0.26±0.02ppm, SMP
residue in black tiger shrimp 0.09 ± 0.01 ppm.
No MTD, SDO, SMX, or SMP (except black tiger shrimp) were detected in the shrimp
samples.
From the obtained results, we see that the HPLC - UV-Vis Detector method has high
sensitivity, suitable for simultaneous analysis of metronidazole and antibacterial agents SGU,
SMP, SDO, and SMX in shrimp.

6.

Conclusion:

We anticipate that these studies will advance the use of HPLC methods in general and UVVis, in particular, to determine metronidazole and sulfonamide in food to efficiently improve
the science industry, particularly in the area of food safety and hygiene, helping to protect the
health of humans.


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7.

References

Vietnamese
1. Chu Đình Bính, Phạm Luận, Nguyễn Thị Ánh Nguyệt, Nguyễn Phương Thanh (2007),
“Xác định dư lượng các chất kháng khuẩn họ sulfamit trong thực phẩm bằng phương pháp sắc
ký lỏng hiệu nâng cao”, Tạp chí Khoa học và Công nghệ, 45(1B).
2. Vũ Cẩm Tú (2009), Xác định các sulfamit trong mẫu Dược phẩm và thực phẩm bằng
phương pháp sắc ký lỏng hiệu năng cao(HPLC), Khóa luận tốt nghiệp, Đại học Khoa học Tự
nhiên.
3. Tiêu chuẩn ngành (2004), Sulfonamit trong sản phẩm thuỷ sản-Phương pháp định lượng

bằng sắc ký lỏng hiệu năng cao, 28 TCN 196:2004
English
1.A. V. Pereira, Q. B. Cass(2005), “High- performance liquid chromatography method for the
simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk using an
on-line clean-up column”, Journal of chromatography B, 826.

2.Craig D.C. Salisbury, Jason C. Sweet, Roger Munro(2004), “ Determination of
sulfonamide residues in the Tissues of food animals using automated precolumn
derivatization and liquid chromatography with fluorescence detection”, Journal of
AOAC international, 87( 5).
3.Qiong-Hui Zou, Xiang-Feng Wang,Yuan Liu, Jin Wang, Jia Song, Hui Gao, Jie Han(2007),
“ Determination of sulphonamides in animal tissues by high performance liquid
chromatography with pre-column derivatization of 9- fluorenylmethyl chloroformate”,
Journal of Separation Science ,30(16).
4.Ticiano Gomes do Nascimento, Eduardo de Jesus Oliveira and Rui Oliveira
Macedo(2005),’’ Simultaneous determination of ranitidine and metronidazole in human
plasma using high performance liquid chromatography with diode array detection’’, Journal
of Pharmaceutical and Biomedical Analysis, 37(4).
5.Theresa A. Gehring, Bill Griffinb, Rod Williams , Charles Geiseker ,Larry G. Rushing ,
Paul H. Siitonen(2006), “ Multiresidue determination of sulfonamides in edible catfish,
shrimp and salmon tissues by high-performance liquid chromatography with postcolumn
derivatization and fluorescence detection”, Journal of Chromatography B, 840.
6.

PVinas,

C.Lopez

Erroz,N.Campillo,

M.Hernandez(1996),

“

Determination

of

sulphonamides in foods by liquid chromatography with postcolumn fluorescence
derivatization”, Journal of Chromatography A, 726(1-2).


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