A sensitive method for the determination of Sulfonamides in seawater samples by Solid Phase Extraction and UV–Visible spectrophotometry

Article abstract of DOI:10.1016/j.saa.2017.03.061

The authors have developed a sensitive spectrophotometric method for determination of sulfonamide derivatives such as sulfanilamide (SAA), sulfadiazine (SDZ), sulfacetamide (SCT) sulfamethoxazole (SMX), sulfamerazine (SMR), sulfadimethoxine (SDX), sulfamethiazole (SMT) and Sulfathiazole (STZ). This method is based on the Bratton-Marshall reaction, which involves the diazotization of sulfonamides with sodium nitrite under acidic conditions, followed by coupling with N-(1-naphtyl) ethylenediamine dihydrochloride (NED) to form a pink colored compound. Therefore, the Bratton-Marshall method was modified by optimizing the reaction conditions, which allows us to determine a low concentration range of sulfonamides compared to the reported methods. The limits of detection and quantification obtained were 0.019–0.05 and 0.06–0.16?μg?mL^??1, respectively. In comparison with other reported methods using different coupling agents, the proposed method was found to be the most simple and sensitive for sulfonamides determination. In this paper, the modified method was successfully employed for the determination of sulfonamides in drinking water, seawater and pharmaceutical and veterinary formulations. The purpose of this work is to optimize and develop a simple method for extraction and concentration of sulfonamides present as residues in seawater and their quantification with the recommended spectrophotometric method. Solid phase extraction (SPE) of sulfonamides from seawater samples was evaluated using Oasis HLB cartridges (3?mL, 540?mg). The recovery efficiency was investigated in the sulfonamides concentration range comprised between 0.19 and 126?ng?mL^??1. The ease of use of this extraction method makes it very useful for routine laboratory work.

Full text of DOI:10.1016/j.saa.2017.03.061

Accepted Manuscript
A sensitive method for the determination of Sulfonamides in
seawater samples by Solid Phase Extraction and UV–Visible
spectrophotometry
Sophia Ait Errayess, Abdellatif Ait Lahcen, Laila Idrissi, Caterina
Marcoaldi, Salvatore Chiavarini, Aziz Amine
PII:
DOI:
Reference:
S1386-1425(17)30247-0
doi: 10.1016/j.saa.2017.03.061
SAA 15040
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Received date:
Revised date:
Accepted date:
31 December 2016
15 March 2017
26 March 2017
Please cite this article as: Sophia Ait Errayess, Abdellatif Ait Lahcen, Laila Idrissi,
Caterina Marcoaldi, Salvatore Chiavarini, Aziz Amine , A sensitive method for the
determination of Sulfonamides in seawater samples by Solid Phase Extraction and
UV–Visible spectrophotometry. The address for the corresponding author was captured
as affiliation for all authors. Please check if appropriate. Saa(2017), doi: 10.1016/
j.saa.2017.03.061
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ACCEPTED MANUSCRIPT
A sensitive method for the determination of Sulfonamides in seawater
samples by Solid Phase Extraction and UV-Visible spectrophotometry
Sophia Ait Errayessa, b, Abdellatif Ait Lahcena, Laila Idrissia, Caterina Marcoaldib, Salvatore Chiavarinib
a
,
and Aziz Amine *
a Laboratoire Génie des Procédés et Environnement, Faculté des Sciences et Techniques de Mohammedia, Hassan II
University of Casablanca, B.P. 146, 20650 Mohammedia, Morocco
b ENEA, C.R. Casaccia, via Anguillarese 301, 00123 Rome, Italy
*Corresponding author. Tel.: +212 523314705; Fax: +212523315353
E-mail addresses: azizamine@yahoo.fr ; a.amine@univh2m.ac.ma (A. Amine)

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Abstract
The authors have developed a sensitive spectrophotometric method for determination of sulfonamide
derivatives such as sulfanilamide (SAA), sulfadiazine (SDZ), sulfacetamide (SCT)
sulfamethoxazole (SMX), sulfamerazine (SMR), sulfadimethoxine (SDX), sulfamethiazole (SMT)
and Sulfathiazole (STZ). This method is based on the Bratton-Marshall reaction, which involves the
diazotization of sulfonamides with sodium nitrite under acidic conditions, followed by coupling with
N-(1-naphtyl) ethylenediamine dihydrochloride (NED) to form a pink colored compound. Therefore,
the Bratton-Marshall method was modified by optimizing the reaction conditions, which allows us to
determine a low concentration range of sulfonamides compared to the reported methods. The limits of
detection and quantification obtained were 0.019-0.05 and 0.06-0.16 µg mL-1, respectively. In
comparison with other reported methods using different coupling agents, the proposed method was
found to be the most simple and sensitive for sulfonamides determination. In this paper, the modified
method was successfully employed for the determination of sulfonamides in drinking water, seawater
and pharmaceutical and veterinary formulations.
The purpose of this work is to optimize and develop a simple method for extraction and concentration
of sulfonamides present as residues in seawater and their quantification with the recommended
spectrophotometric method. Solid phase extraction (SPE) of sulfonamides from seawater samples was
evaluated using Oasis HLB cartridges (3 mL, 540 mg). The recovery efficiency was investigated in the
sulfonamides concentration range comprised between 0.19 and 126 ng mL-1. The ease of use of this
extraction method makes it very useful for routine laboratory work.
Keywords: Sulfonamides; spectrophotometric method; drug analysis; seawater; solid phase
extraction; pre-concentration

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1. Introduction
Sulfonamides (SNs) are a group of compounds having the general structure reported in Fig.1. The
members of this group, also known as sulfa drugs are synthetic derivatives of sulfanilamide (R=H),
which competitively block the synthesis of folic acid in microorganisms [1]. Because of the low cost,
low toxicity and relevant efficiency against many common bacterial infections, sulfonamides are
among the most common antibacterial agents used in both veterinary and human medicine [2]. They
are effective against Gram-positive and Gram-negative bacteria [3]. Recently, this drug class attracted
attention for their high consumption and pollution potential in the natural environments. An extensive
range of pharmaceuticals has been detected in fresh and marine waters; some of these substances have
the capacity to damage the aquatic middle. Scientific research has been carried to study their effect,
transport to natural aqueous systems and their environmental impact [4].
Regrettably, the extensive discharge of sulfa-drugs contaminated sewage in the environment both
results in a selective pressure on microorganisms which develop antibiotic resistance and causes an
ecological impact on fish and other water life, with consequent threat to animal and human health, due
to the admission of these substances in the food chain. The highest contamination levels of
sulfonamides were found nearby to animal and hospital wastewater outlets. Among them,
sulfamethoxazole and sulfamethazine, respectively used for the cure of humans and animals, were
detected in natural waters with a maximum of few ng mL-1 levels [5]. These emerging pollutants
require a simple, rapid and accurate method for routine analysis. It is in this context that we aimed to
develop a spectrophotometric method, for the determination of sulfonamides present as residues in
seawater, which may offer many advantages such as a simple implementation, low cost and widely
available instrumentation and low consumption of reagents, coupled with a high sensitivity and low
detection limits.
Several analytical methods have been reported in the literature for the determination of sulfonamides
including high-performance liquid chromatography (HPLC) [6-8], capillary electrophoresis (CE) [9-
11], gas chromatography (GC) [12, 13], liquid chromatography (LC) [14-16], UV/Visible
Spectrophotometry [17-23], enzymatic methods [24-25] and electrochemical methods [26-28]. The

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most extensively used assay procedure for sulfonamides and their dosage forms is the titration with
sodium nitrite solution to determine the aromatic amine function; this method is cited in the official
methods of British and United State pharmacopoeias [29, 30].
Herein, we present a developed UV-Visible spectrophotometric method for the quantitative estimation
of eight sulfonamides in water samples. This proposed method is based on the diazotization of the
aromatic amino group present in sulfonamides with nitrite in acidic medium (NaNO2/HCl) at a room
temperature to form a diazonium salt. The excess of nitrite was removed by the treatment with
sulfamic acid (H3NSO3), and then the diazonium salt was coupled with N-(1-naphthyl)
ethylenediamine dihydrochloride (NED), to form a colored product with maximum absorption λmax at
536 nm. A comparative study of different spectrophotometric methods reported in the literature was
investigated, the modified Bratton-Marshall method is found to be simpler and more sensitive than the
other methods used for sulfonamides determination.
Due to the low environmental concentrations in seawater, a pre-concentration step is necessary for the
determination of these compounds. Solid phase extraction is considered as the most popular sample
preparation method and commonly used for a broad range of contaminants [31-33], and it was adopted
in this research work for sulfonamides extraction in seawater samples. Therefore, the aim of this work
is the development and the optimization of a simple method for pre-concentration of sulfonamides,
present as residues in seawater, using the Solid Phase Extraction on Oasis HLB columns, followed by
a UV-Visible spectrophotometric detection.
Fig.1.
2. Experimental
2.1.Chemicals and Reagents
All the reagents and solvents used were of analytical grade: acetone, hydrochloric acid and sulfamic
acid were purchased from Solvachim (Morocco), acetonitrile and ethylene diamine tetra acetic acid
(Na2-EDTA) from Sigma-Aldrich (USA), sodium nitrite from Riedel-deHaën (Germany), N-(1-

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naphthyl) ethylenediamine dihydrochloride from ACROS Organics (Belgium), sulphuric acid from
LOBA Chemie (India) and methanol, HPLC grade, from ProLab (Canada). Diuron-d6 (3-(3, 4-
Dychlorophenyl)-1, 1-dimethylurea-d6), diuron (3-(3, 4-dichlophenyl)-1, 1-dimethylurea),
trimethoprim, l-Histidine and N-Acetyl-Sulfamethoxazole were kindly provided by Dr. S. Chiavarini
(ENEA, Italy). The sulfonamide derivatives selected for this study were purchased from Sigma-
Aldrich (USA), and their chemical structures are given in Table 1S: sulfanilamide (SAA), sulfadiazine
(SDZ) ≥ 99.0 %, sulfamerazine (SMR) ≥ 99.0 %, sulfathiazole (STZ) ≥ 98.0 %, sulfadimethoxine
(SDX) ≥ 98.5 %, sulfacetamide (SCT) ≥ 98.0 %, sulfamethoxazole (SMX) ≥ 99.0 %, sulfamethiazole
(SMT) ≥ 99.0 %.
2.2.Instrumentation and Apparatus
A JENWAY Model 6850 UV-Visible Double beam Spectrophotometer (Bibby Scientific Brand-UK)
with 1 cm matched cells was used for all spectral and absorbance measurements.
A vacuum filtration apparatus, including glass funnel; frit support; clamp; adapter; stopper; filtration
flask; and vacuum tubing, was used for filtration of the seawater. The filters used were Whatman glass
microfiber filters; type GF/B, 1 μm pore size, 90 mm and ALBET cellulose acetate filter, 0.45 μm
pore size, 47 mm. Sulfonamide compounds were extracted using an automated system of extraction,
SPE Vacuum manifold (Supelco). The samples are automatically pumped through the conditioned
SPE material by using a pump KnF LABOPORT. The SPE cartridge adopted during this work was
Oasis® HLB (Waters, Milford, USA) Sorbent weight: 540 mg, 3 mL reservoir.
The pH meter used in this work for the determination of the pH values of the solutions was a HANNA
instrument (HI 8521) pH meter.
Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis, purchased from
Waters, was carried out by an Acquity IClass UPLC coupled to a Xevo G2-XS QTof high resolution
mass spectrometer, managed by through Masslynx 4 software.
2.3. Preparation of standard solutions

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Distilled water was used to prepare all solutions. Standard solutions of sulfonamides (1000 μg mL-1)
were prepared by dissolving 100 mg of each sulfonamide in 10-40 mL of 1 M HCL or Acetone and
then diluting with distilled water to the final volume of 100 mL in volumetric flasks.
2.4. Preparation of working standard solutions
A working standard solution of each sulfonamide containing 10 µg mL-1 was prepared by further
dilution.
2.5. Reagent Solutions
1 % solution of sodium nitrite (NaNO2) in water, hydrochloric acid 1 M, 2 % aqueous sulfamic acid, 1
% aqueous solution of N-(1-napthyl) ethylenediamine dihydrochloride (NED), were used for the
experiments. 5 % diluted Na2-EDTA, 25 % sulphuric acid solution and 5 % acetonitrile solution were
used for the pre-concentration steps.
2.6. Procedure for the determination of sulfonamide derivatives
Aliquots of sulfonamide derivative solutions (sulfanilamide, sulfamethoxazole, sulfamethiazole,
sulfathiazole, sulfacetamide, Ssulfamerazine, sulfadiazine, sulfadimethoxine) ranging from 0.1-1.0 mL
(10 μg mL-1) were transferred into each of the series of 10 mL flasks, 1 mL of sodium (1 % w/v) and 1
mL of 1 M hydrochloric acid were added with swirling at room temperature. After 5 min, 1 mL of
sulfamic acid (2 % w/v) and 1 mL of N-(1-naphthyl) ethylenediamine dihydrochloride (1 % w/v) were
added with swirling. The volumes were adjusted to the mark with distilled water, and then the
solutions were mixed thoroughly. The absorbance of the light pink colored solutions were measured at
536 nm against the reagent blank. Fig.2 shows a picture of a gradient of concentrations of the pink
colored solutions tested.
Fig.2.

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2.7. Analytical application to real samples
Three samples of commercialized mineral water, purchased from a local market (Mohammedia-
Morocco), and tap water were analyzed using the optimized method to determine their content of
sulfonamide before and after their fortification with a known amount of SMX.
Two brands of commercial drug samples were purchased from a local drugstore in Mohammedia city
(Morocco) and analyzed using the proposed method. COTRIM® containing 800 mg of SMX per tablet
and TRIMETO-SULFA® containing 8.35 g of SDZ per 100 mL. The sample solutions were prepared
as follows:
Tablets - Twenty tablets were weighed and the average weight was calculated, tablets were cursed
thoroughly in a mortar and mixed. An accurate weight of the powder equivalent to one tablet was
dissolved in 60 mL of 1 M HCl in a 100 mL volumetric flask, and stirred for about 10 min. The
solution was filtered to separate any insoluble matter. The filtrate was made up to the mark with
distilled water in 100 mL volumetric flask and, after dilution, an appropriate aliquot of the drug
solution was treated as described above for the determination of sulfonamides.
Oral solution (veterinary formulation) – 2 mL of drug (equivalent to 0,164 g of SDZ) was diluted
with 60 mL of 1 M HCl in 100 mL volumetric flask and the volume was made up to the mark with
water. After dilution, the recommended procedure was then followed.
2.8. Pre-concentration of sulfonamides in seawater samples
2.8.1. Sample preparation
Seawater samples were filtered through glass fiber filters (1 μm) type GF/B to eliminate the suspended
matter and then filtrated through 0.45 μm cellulose acetate filters. The samples were acidified to pH
4.0 with diluted sulphuric acid solution (25 %). In order to minimize the degradation, 5 mL of 5 %
diluted Na2-EDTA were added.
2.8.2. Extraction and concentration procedure

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Figure 1S shows the Solid Phase Extraction (SPE) procedure adopted and used during this work.
Sulfonamides were extracted using an automated SPE (vacuum manifold), for this extraction step a
(540 mg, 3 mL) Oasis® HLB cartridges was tested. Sorbent of SPE have been previously conditioned
using 5 mL of acetonitrile (100 %) and 5 mL of water at pH 4.0. The samples have been loaded into
Oasis HLB cartridge at a flow rate 2 mL min-1. After loading, the cartridge was rinsed with 5 mL of
acetonitrile (5 %) and dried under the Vacuum Manifold for 20-30 min. The sulfonamides (selected
compounds) were eluted using acetonitrile (100 %). After elution, we obtained the extract which is
named the concentrate of sulfonamides.
2.9.Procedure of spectrophotometric analysis
The analysis of the obtained concentrate was performed as following:
6 mL of standard solution or seawater sample (after elution from SPE) were transferred into standard
flasks, and then 1 mL of sodium nitrite (1 % w/v) and 1 mL of 1 M of hydrochloric were added with
swirling at room temperature. After 5 min, 1 mL of sulfamic acid (2 % w/v) and 1 mL of N-(1-
naphthyl) ethylenediamine dihydrochloride (1 % w/v) were added with swirling. The absorbance of
the solution was measured at 536 nm against the reagent blank. In order to use a small volume of the
eluted concentrate (1.2 mL), the volume of each reagent used in the above procedure was reduced to
0.5 mL.
2.10. Procedure of UPLC MS ES+ analysis
25 µL of the internal standard solution (Diuron-d6) were added to 500 µL of the extract sample, and
then 200 µL of the sample were taken and added to 300 µL of water. The sample was filtered before
analysis. Sulfonamides were analyzed in positive electrospray ionization mode (+ESI). For
chromatography, solvent A and solvent B consisted of water and acetonitrile respectively, with
addition of 0.01 % of formic acid. The mobile phase flow rate was 0.3 mL min-1. Gradient conditions
were initiated with 0 % B (hold for 1 min) followed by a linear increase to 50 % A in 6 min, and then
to 100 % in 0.4 min (hold for 0.5 min).

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3. Results and Discussion
3.1.Principle of the color reaction
The color reaction is based on diazotization of the aromatic amine in the presence of a nitrite solution
in acidic medium to form a diazonium salt and the addition of sulfamic acid to the reaction to remove
the excess of nitrous acid. Then coupling the resulting diazonium salt with another aromatic amine as
a coupling agent to form a colored compound. This reaction has been used for determination of
sulfonamides by researchers. In the present study, we have tested the coupling agent N-(1-naphthyl)
ethylenediamine dihydrochloride in the modified Bratton-Marshall method. The chemical reactions are
shown in Fig.3.
Fig.3.
3.2.Absorption spectrum
The light pink colored products formed with NED have a λmax of 536 nm; the absorption spectrum is
shown in Fig.2S.
3.3.Optimization of reaction conditions
1 - 2.5 mL of 1 M HCl, 0.6 - 1.6 mL of 1% NaNO2 solution, 1 - 2.5 mL of 2 % sulfamic acid solution
and 0.8-2 mL of 1 % NED solution were necessary to achieve a maximum color intensity in the
modified Bratton-Marshall method. The obtained results of the optimization of reaction conditions are
shown in Fig.4.
Fig.4.

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3.4.Stability of the resulting product
Stability study of the pink colored product was carried out by measuring the absorbance values at
different time intervals. The obtained results showed that the resulting colored product was stable for
more than 60 h in the modified Bratton-Marshall method.
3.5. Analytical parameters
The optical characteristics such as Beer’s law limits, molar absorptivity, percent relative standard
deviation RSD % (calculated from three independent measurements), correlation coefficient,
regression equation, limits of detection (LOD) and quantification (LOQ), standard deviation of slope
and standard deviation of intercept were determined for all the sulfonamides tested. The internal
validation was achieved in agreement with ICH guidelines [34]. All the obtained values of optical and
analytical parameters for the spectrophotometric determination of eight sulfonamides derivatives by
using NED are given in Table 1.
Under the optimal conditions, the measurements of the absorbance at λmax were performed in the
concentration range of sulfonamides of 0.1-1.0 µg mL-1 for the modified Bratton-Marshall method.
The linear range obtained for each sulfonamides was presented in Table 1. Each point of the
calibration curve was the mean value of three independent measurements. As an example, the linear
regression equation for sulfamethoxazole determination is Y=0.213 X + 0.066 with a correlation
coefficient (R²) of 0.999.
The sensitivity in practical terms, is the slope of the calibration curve that is obtained by plotting the
response against the concentration of analyte [35], the obtained results of sensitivity were comprised
between 0.170 and 0.307 Abs μg-1 mL as indicated in Table 1. The precision of the method expressed
in term of repeatability from relative standard deviation (% RSD) values of intraday analyses. This
showed a satisfactory precision (1.2-6.4 %) of the method, as showed in Table 1.
3×푆
푆
10×푆
푆
푏
푏
The LOD and LOQ were calculated for eight sulfonamides according to
and
respectively,
where Sb is the standard error of the intercept and S is the slope of the linear calibration curve. LOD
and LOQ were found to be in the range of 0.019 to 0.05 µg mL-1 and in the range of 0.06 to 0.16 µg

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mL-1, respectively. As indicated in Table 1, the obtained results showed that the modified method was
sensitive to detect and quantify sulfonamide derivatives at a low concentration levels.
The recovery technique was performed to study the accuracy and the reproducibility of the method.
The recovery of the modified method was carried from solutions containing different concentrations of
SMX and was found to be between 99.5 % and 111.3 %, indicating that the method is accurate and
reproducible.
Table 1
3.6.Interferences study
The matrix effect is a major problem whenever trace level pharmaceuticals in seawater are analyzed
using solid phase extraction (SPE) combined with any other determination method. Therefore, a study
of the interferences effect on the proposed colorimetric response towards sulfonamides was performed
in the presence of some potential interfering species in the environmental samples, such as diuron,
trimethoprim, N-Acetyl-Sulfamethoxazole and l-Histidine. In the presence of these compounds, no
pink coloration was observed, indeed no reaction was established. Some of these common species,
which can often be present in environmental samples, do not interfere in the present
spectrophotometric method. From these results, it may be concluded that the presence of interferences
in these samples has no matrix effect on the spectrophotometric response and which confirms the good
selectivity of the developed spectrophotometric method toward sulfonamides. Although its good
selectivity, this method is not specific for one type of sulfonamide but is specific for all sulfonamide
derivatives tested and this was confirmed by their equivalency of molar absorptivity values, which is
in the range of 5.4 ± 1.1 L mol-1 cm-1 and by their sensitivity values.
3.7.Stability study of sulfonamides (sulfadiazine, sulfamethiazole)

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A stability study was performed on standard stock solutions of sulfadiazine (SDZ) and sulfamethiazole
(SMZ), stored for one month at room temperature in darkness and exposed to the light. The results
obtained from this study, showed that the absorbance values decreased dramatically after 7 days for
SDZ and SMZ stored at room temperature and exposed to the light (Fig.5A and B).
However, in case of storage at room temperature and in darkness, the absorbance values decreased
slightly only after 15 days. The absorbance decreased in one month by 29 % and 20 % for SDZ and
SMZ respectively when storage conditions are room temperature and light exposition. Slight decrease
of absorbance was observed 2.8 % and 3.6 % for SDZ and SMZ respectively when storage conditions
are room temperature and darkness.
To conclude, the standard stock solutions of sulfonamides should be stored at room temperature and
kept in the dark.
Fig.5.
3.8.Comparison of the reported spectrophotometric methods and the proposed method for
sulfonamides determination
A comparison between analytical parameters derived from the published method using NED as a
coupling agent of the reaction [36] and the modified method, is given in Table 2. The results show that
the LOD and LOQ values of the modified method are lower compared to those of the reported method.
The optimized method has a higher slope than the published method, meaning that a higher sensitivity
for measuring sulfonamides was obtained. Literature spectrophotometric methods, based on Bratton-
Marshall reagent [35-41] and other coupling agents [17-18, 23, 36, 38, 41, 43-52], were compared to
the proposed method for the analysis of sulfonamide derivatives and are presented in Table 2. The
Beer’s Law was obeyed in the concentration range of 0.1-1.0 µg mL-1 in the present work. However, in
the range of 4.0-12 µg mL-1 in method of Sharma et al., [36] and in the range of 0.48-8.64 µg mL-1 in

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method of Salinas et al., [42] as indicated in Table 2. The optimized method gives a high sensitivity,
low detection and quantification limits.
Table 2
3.9.Analytical Applications
A spike recovery analysis was performed on three samples of mineral water, purchased from a local
supermarket (Mohammedia-Morocco), and Tap water taken from our laboratory to determine their
content of sulfonamides before and after their fortification with a known amount of SMX. The
recovery was calculated as the ratio of the amounts found/added. The recovery of the method was
between 99.2 - 111.2 % as presented in Table 3.
The modified spectrophotometric method was applied to the determination of SMX and SDZ in
commercial pharmaceutical samples (tablets and oral solution). The results of the analysis of these
commercial samples are given in Table 4, and show that the method gave satisfactory recovery results.
Table 3
Table 4
3.10. Sulfonamide analysis in seawater
3.10.1. Sample preparation and extraction
Sample preparation is a critical and important step in extraction and concentration of pharmaceutical
residues present in seawater. The matrix effect and the presence of interferences are a major problem
and can affect the efficiency, recovery and the reproducibility of the sample preparation techniques
and analytical methods. However, these problems are reduced by using the SPE, which become the

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most commonly used sample preparation technique and offers many advantages over classical
techniques [53]. In order to enhance the extraction efficiency and to minimize the degradation of some
antibiotics, it is necessary to adjust the pH of the sample and to add chelating agents (EDTA or Na2-
EDTA) during the extraction step. Sulfonamides have an amphoteric character and can react both as
an acid as well as a base. They are positively charged at pH ≤ 2.0 and negatively charged at pH ≥ 5.0.
Therefore, to increase the hydrophobicity on reversed-phase SPE cartridge such as Oasis HLB
columns, sulfonamides require pH in the range 2.0 - 4.0. The highest recoveries for sulfonamides are
achieved in their uncharged forms at pH 4.0 [54, 55].
3.10.2. Optimization of SPE combined with spectrophotometric detection
The extraction and concentration of sulfonamides was performed using an automated system of
extraction following the developed procedure. To find the most appropriate organic solvent for
elution, with respect to the developed colorimetric method, three tests were conducted on a blank
(seawater sample without fortification of sulfonamides) as follows:
-
-
-
Test 1: Extraction using Oasis HLB cartridge (540 mg 3 mL), conditioning and eluting with
methanol.
Test 2: Extraction using STRATA-X cartridge (200 mg 3 mL), conditioning and eluting with
methanol.
Test 3: Extraction using Oasis HLB cartridge (540 mg 3 mL), conditioning and eluting with
acetonitrile.
These tests have allowed us to determine the appropriate solvent of elution for the method which is the
acetonitrile (ACN).
In the case of the use of methanol as elution solvent, either for Oasis HLB or STRATA-X cartridges, it
can be seen from the Fig.6 that the color developed of the blank concentrate is due to the probably
effect of methanol on the polymeric layer of both kinds of cartridges. However, when the elution is
carried out by ACN there is no coloration of the blank concentrate after the addition of reagents
necessary for the colorimetric analysis. Therefore, the use of ACN as solvent of elution was found to

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be the appropriate solvent towards extraction. Since then, it was chosen as the extraction solvent for
further experiments.
Fig.6.
3.10.3. Solid Phase Extraction using Oasis HLB columns

Analysis of seawater concentrate without fortification ( blank test)
In order to check the presence of sulfonamides in the seawater samples, 500 mL of seawater was
loaded into Oasis Hydrophilic–Lipophilic Balanced (HLB) cartridge and eluted using the extraction
procedure described above. The analysis of the obtained concentrate was performed by UV-Visible
spectrophotometry based on the above method described in procedure section. The results obtained
indicated the absence of sulfonamide in the seawater samples which lead us to spike the samples
before analysis.

Analysis of fortified seawater samples
Real samples of seawater were filtered as described in the general procedure for pre-concentration and
then spiked with SMX in the concentration range of 0.19-126 ng mL-1. The samples were concentrated
into the SPE columns and eluted with pure acetonitrile, and then the analysis was performed according
to the proposed spectrophotometric method. The concentration of the eluted solution was calculated
from the calibration equation of standard solution of SMX in ACN, and was A= 0.353 C (µg mL-1) +
0.049, with correlation coefficient (R²) of 0.9978. It should be noted that the presence of ACN
increases the sensitivity compared to that of the analysis of SMX in distilled water (Fig.3) which may
considered as an advantage. The results obtained of the pre-concentration of sulfonamide using Oasis
HLB cartridges are summarized in Table 5. It can be seen from these results that this method can pre-
concentrate sulfonamide from seawater samples more than 800 times, and shows a good recoveries

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values for seawater samples which were comprised between 99 % and 104 % for the range of
concentration tested 0.19 and 126 ng mL-1.
Table 5
3.11. External validation of the proposed method
UPLC-MS has been used as one of the most powerful analytical techniques for its selectivity,
sensitivity and reproducibility. The aim of this work is to develop and validate a simple, sensitive,
rapid spectrophotometric method for quantitative estimation of sulfonamides from seawater samples.
SPE coupled colorimetric detection was validated by checking the reproducibility and the efficiency of
the proposed method.
An evaluation of the performance of the proposed method was investigated through a validation
procedure with spiked seawater. For our recovery experiments, two real samples of seawater were
spiked with 2.4 and 0.19 ng mL-1 of standard sulfamethoxazole, filtrated as described above,
concentrated on HLB sorbents and then analyzed according to two techniques; UPLC tandem mass-
spectrometry and the developed spectrophotometry methods. Although, the presence of SMX was well
identified in the chromatogram as presented in Fig.7, the obtained results using both
spectrophotometric and UPLC-MS methods were statistically compared using t-test and F-test and
showed a difference at the 0.05 level as indicated in Table 6. However, the concentrations found using
the developed spectrophotometric method were more close to the nominal concentrations in spiked
seawater samples with low standard deviations than those obtained using UPLC-MS method. Thus,
the above results confirm the accuracy of the proposed spectrophotometric method.
Fig.7.

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Table 6
4. Conclusions
A sensitive spectrophotometric method based on the modified Bratton-Marshall reaction was proposed
for the detection and quantification of eight sulfonamides. The LOD for each sulfonamide studied
were calculated and found to be in the range of 0.019 to 0.05 µg mL-1. The proposed method is simple,
accurate, sensitive, precise, repeatable and economical, it does not require any radical experimental
conditions. This method can be successfully applied for the routine determination of sulfonamides in
drinking water, seawater and pharmaceutical & veterinary formulations. In order to achieve a low
detection range at the ng mL-1 level, typical environmental concentration of sulfonamides, an
optimized pre-concentration procedure based on Oasis HLB columns was coupled to the proposed
spectrophotometric method and was successfully tested for sulfonamides determination in seawater
samples. Furthermore, sulfonamides were measured using the procedure of pre-concentration coupled
UPLC-MS, the results obtained are promising compared to the spectrophotometric method developed.
Even if the detection method is not selective for each single sulfonamide, thanks to the substantial
equivalency of Molar absorptivity values for the tested sulfonamides (Table 2), it gives an accurate
enough evaluation in µM (nM after pre-concentration) of the total sulfonamide content of the sample.
This makes it an excellent screening method to be applied before the more powerful and costly
reference analytical method.
Acknowledgements
This work was supported by the European project ‘’ Sensing toxicants in Marine waters makes Sense’’
(SMS) GRANT AGREEMENT N° 613844.

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Figure Captions
Fig. 1 Chemical structure of sulfonamides.
Fig.2. Gradient of concentrations of the pink colored solutions tested. From left to right the
sulfonamide concentrations ranging from 0.1 to 1 µg mL-1.
Fig. 3 Chemical reaction scheme of the tested method for sulfonamides detection.
Fig. 4 Optimization of reagent volumes of : A) 1% Sodium nitrite with 1 mL of 1 M HCl, 1 mL of 2 %
Sulfamic acid and 1 mL of 1 % NED; B) 1 M of HCl with 1 mL of 1 % Sodium nitrite, 1 mL of 2 %
Sulfamic acid and 1 mL of 1 % NED; C) 2 % Sulfamic acid with 1 mL of 1 % Sodium nitrite, 1 mL of
1 M HCl and 1 mL of 1 % NED; D) 1 % of NED with 1 mL of 1 M HCl, 1 mL of 1 % Sodium nitrite
and 1 mL of 2 % Sulfamic acid. The final volume of 10 mL was adjusted to the mark with distilled
water. The absorbance was measured at λmax = 536 nm.
Fig. 5 Stability study of standard solution of A) Sulfadiazine, B) Sulfamethiazole stored at (a)
Room temperature in darkness (b) Room temperature and exposed to light.
Fig. 6 Analysis of the extracts using (Test 1) Oasis HLB cartridge, conditioning and eluting
with methanol, (Test 2) STRATA-X cartridge, conditioning and eluting with methanol, (Test
3) Oasis HLB cartridge conditioning and eluting with acetonitrile.
Fig. 7 UPLC-MS chromatogram of sulfamethoxazole.

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Table titles
Table. 1 Some Optical & Analytical parameters for the spectrophotometric determination of
Sulfonamide derivatives.
Table. 2 Comparison of reported UV–VIS spectrophotometric methods for the determination of
sulfonamides.
Table. 3 Recovery of sulfamethoxazole in spiked water samples.
Table. 4 Determination of sulfonamide derivatives in commercial drug samples.
Table. 5 Recovery studies of seawater samples.
Table. 6 Comparison between the results obtained using UPLC-MS and Spectrophotometry.

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Fig.1.

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Fig.2.

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Fig.3.