SOLUTION: Trinity College Anti Metabolite Chemistrylab Report

SOLUTION: Trinity College Anti Metabolite Chemistrylab Report.

01 Quantitation by Extraction (Hilary Term)
________________________________________________________________
Title: Anti-metabolite Chemistry – Quantitation by Extraction
ANALYSIS 01
Scope:
Assay of SEPTRIN® Oral Suspension
Reference:
British Pharmacopoeia Vol. III – Co-trimoxazole Oral Suspension
Trimethoprim
Sulfamethoxazole
Action and use Dihydrofolate reductase inhibitor + sulfonamide antibacterial.
Definition Co-trimoxazole Oral Suspension is a suspension containing 1.6% w/v of
Trimethoprim and 8.0% w/v of Sulfamethoxazole in a suitable flavoured vehicle.
The oral suspension complies with the requirements stated under Oral Liquids and
with the following requirements.
Content of trimethoprim, C14H18N4O3
1.44 to 1.76% w/v.
Content of sulfamethoxazole, C10H11N3O3S
7.40 to 8.60% w/v.
ASSAY
For sulfamethoxazole
To 4 g of the oral suspension add 30 ml of 0.1M sodium hydroxide, shake and extract
with four 50 ml quantities of chloroform, washing each extract with the same two 10
ml quantities of 0.1M sodium hydroxide. Reserve the combined chloroform extracts
for the Assay for trimethoprim. Dilute the combined aqueous solution and washings to
250 ml with water, filter and dilute 5 ml of the filtrate to 200 ml with water (solution A).
Carry out the following procedure protected from light using 2 ml of solution A. Add
0.5 ml of 4M hydrochloric acid and 1 ml of a 0.1% w/v solution of sodium nitrite and
allow to stand for 2 minutes. Add 1 ml of a 0.5% w/v solution of ammonium sulfamate
and allow to stand for 3 minutes. Add 1 ml of a 0.1% w/v solution of N-(1-naphthyl)
ethylenediamine dihydrochloride and allow to stand for 10 minutes. Dilute the resulting
solution to 25 ml with water and measure the absorbance at 538 nm, Appendix II B,
using in the reference cell a solution prepared in the same manner but using 2 ml of
water in place of solution A. Dissolve 0.25 g of sulfamethoxazole BPCRS in 50 ml of
0.1M sodium hydroxide and dilute to 250 ml with water. Dilute 5 ml of the resulting
solution to 200 ml with water (solution B). Repeat the procedure using 2 ml of solution
B and beginning at the words ‘Add 0.5 ml of …’. Calculate the content of C10H11N3O3S
from the values of the absorbances obtained using the declared content of
C10H11N3O3S in sulfamethoxazole BPCRS. Determine the weight per ml of the oral
suspension, Appendix V G, and calculate the content of C10H11N3O3S, weight in
volume.
For trimethoprim
Extract the chloroform solution reserved in the Assay for sulfamethoxazole with four
50 ml quantities of 1M acetic acid. Wash the combined extracts with 5 ml of chloroform
and dilute the aqueous extracts to 250 ml with 1M acetic acid. To 10 ml of this solution
01 Quantitation by Extraction (Hilary Term)
________________________________________________________________
add 10 ml of 1M acetic acid and sufficient water to produce 100 ml and measure the
absorbance of the resulting solution at the maximum at 271 nm, Appendix II B.
Calculate the content of C14H18N4O3 taking 204 as the value of A(1%, 1 cm) at the
maximum at 271 nm. Using the weight per ml of the oral suspension, calculate the
content of C14H18N4O3, weight in volume.
Storage
Co-trimoxazole Oral Suspension should be protected from light and stored at a
temperature not exceeding 30°.
Co-trimoxazole Oral Suspension contains, in 5 ml, 80 mg of Trimethoprim and 400 mg
of Sulfamethoxazole.
The extraction procedure has been changed slightly for use in our practicals.
Please refer to the flow-chart on the next page.
Chloroform is toxic!
Warning
Hazard statements:
H315 Causes skin irritation.
H373 May cause damage to organs through prolonged or repeated exposure.
H302 Harmful if swallowed.
H351 Suspected of causing cancer.
Precautionary statement:
P281 Use personal protective equipment as required.
All procedures involving chloroform must be carried out in a fume hood.
Chloroform waste is disposed of
in the Chlorinated Organic Waste container!
➢
Shake the Oral Suspension well.01
Transfer
2.5 ml of oral
(use
spoon Term)
provided with formulation) into a 250 ml separating funnel
Quantitation
by suspension
Extraction
(Hilary
(labelled A) ________________________________________________________________
containing 30 ml 0.1 M NaOH.
➢
Pour 50 ml of chloroform into the same separating funnel (A). Shake the separating funnel for at least 1 min, venting the funnel several times.
➢
Allow the layers to settle and dispense the bottom layer into a second separating funnel (labelled B).
➢
A contains aqueous NaOH and Sulfamethoxazole. B contains Trimethoprim in Chloroform.
➢
Wash B with 10 ml of 0.1 M NaOH. Dispense the bottom layer into a beaker (labelled C).
➢
Add another 50 ml of chloroform to separating funnel A, shake and dispense the bottom layer into separation funnel B (still containing the
10 ml 0.1 M NaOH from the previous washing) and wash with the same 10 ml of 0.1 M NaOH. Dispense bottom layer into beaker C, combining
the organic extracts. Repeat this this step twice more (total of 200 ml Chloroform in beaker C).
➢
The combined chloroform extracts in beaker C contain trimethoprim (continue on left).
➢
Dispense the top layers of separation funnels A + B (aqueous, containing sulfamethoxazole) into a 250 ml volumetric flask and make up to
250 ml with water. (continue on right).
Sulfamethoxazole – Top Layer ( Aqueous NaOH ):
Filter approximately 10 ml of the solution and transfer 5.0 ml of
the filtrate to a 250 ml volumetric flask and make up to volume.
Trimethoprim – Bottom Layer ( Chloroform ):
Extract the chloroform solution with four 50 ml
quantities of 1 M acetic acid. Keep and combine the
upper layers (acetic acid containing Trimethoprim,
total of 200 ml). Wash the combined extracts with 5 ml
of chloroform. Dispense the top layer (aqueous acetic
acid) into a 250 ml volumetric flask.
Standard: Instead of above solution, in a
250 ml volumetric flask dissolve 0.200 g of
sulfamethoxazole BPCRS in 50 ml of 0.1 M
sodium hydroxide, adding sufficient water to
produce 250 ml. Dilute 5.0 ml of the resulting
solution to 250 ml with water in a volumetric
flask.
Blank: Instead of above
solution, take 2.5 ml of
water and add reagents as
outlined below. Use this as
your blank to auto-zero the
spectrophotometer.
Add 2.5 ml of each of the above solutions to a separate 25 ml
volumetric flask. With each of the three flasks do the following:
• Add sufficient 1 M acetic acid to the volumetric flask to
produce 250 ml.
• Transfer 10 ml of this solution to a 100 ml volumetric flask
• Add 10 ml of 1 M acetic acid and dilute with water to
volume.
• Blank the spectrophotometer at 271 nm with 0.2 M acetic
acid and measure the absorbance of the above solution.
• Add 1 ml of 2 M hydrochloric acid and 1 ml of a 0.1% w/v
solution of sodium nitrite and allow to stand for 2 minutes.
• Add 1 ml of a 0.5% w/v solution of ammonium sulfamate and
allow to stand for 3 minutes.
• Add 1 ml of a 0.1% w/v solution of N-(1-naphthyl) ethylene
diamine (NED) dihydrochloride, allow to stand for 10 minutes.
• Add sufficient water to produce 25 ml
Use the ‘Blank’ to autozero and then measure the absorbance of
the standard and sample solutions at 538 nm.
01 Quantitation by Extraction (Hilary Term)
________________________________________________________________
General Procedure for single wavelength readings (Shimadzu UVMini-1240)
0.0 In the spectrophotometers main menu select option 1. Press the go to
wavelength key, and set the measuring wavelength to the specified wavelength
(consult demonstrator).
0.1 Blank – Fill a 1 cm cuvette with solvent or solution to be used to determine
background reading.
0.2 Make sure sides of cuvette are dry and clean. Place in instrument with the clear
side facing the light path.
0.3 Set Abs zero by pressing the auto zero key.
0.4 Sample/Standard – transfer 2.5 ml of your sample solution to a 1 cm cuvette
and place in the compartment of the instrument.
Record absorbance readings in your Report Sheet.
Final Report:
1.
Calculate the amount (weight) per 5 ml in SEPTRIN® for Sulfamethoxazole and
Trimethoprim. Remember dilutions! Show your calculation strategy.
2.
Sulfamethoxazole: Calculate the content of C10H11N3O3S from the values of the
absorbances obtained using the declared content of C 10H11N3O3S in
sulfamethoxazole BPCRS. Calculate the content of C10H11N3O3S in SEPTRIN®,
weight in volume, and decide whether it complies with the specifications set by
the Pharmacopoeia.
Tips for the calculation:
➢ Work out the concentration (in %) of the standard that is prepared.
➢ Calculate the specific absorbance of the reaction product by taking the
absorbance reading of the standard and the concentration in % (determined
above) using Beer’s Law.
➢ Then, using the calculated specific absorbance, calculate the concentration of
the sample by taking its absorbance reading and applying Beer’s Law.
➢ From the calculated sample concentration, by working backwards through the
dilutions, determine how much Sulfamethoxazole was present in the original
2.5 ml that you measured out for the assay.
➢ Compare this to the label claim / the specifications of the pharmacopoeia.
01 Quantitation by Extraction (Hilary Term)
________________________________________________________________
3.
Trimethoprim: Calculate the content of C14H18N4O3 taking 204 as the value of
A(1%, 1 cm) at the maximum at 271 nm. Calculate the content of C14H18N4O3,
in SEPTRIN®, weight in volume, and decide whether it complies with the
specifications set by the Pharmacopoeia.
Tips for the calculation:
➢ Calculate the concentration of the sample solution by using the measured
absorbance and the given specific absorbance value using Beer’s Law.
➢ Work out the dilution factor.
➢ Adjust the calculated concentration for dilutions.
➢ Compare this to the label claim / the specifications of the pharmacopoeia.
Questions:
1.
Illustrate and describe the extraction procedure.
2.
Explain, with illustration, why sulfamethoxazole is soluble in the alkaline
aqueous solution and trimethoprim is not.
3.
Write out the chemical reaction which leads to the coloured product (BrattonMarshall Reaction).
4.
Why is ammonium sulfamate added? What would happen if this was forgotten?
Tip: Review the reaction of amines (primary and secondary, aliphatic and
aromatic amines (such as NED) with nitrous acid).
5.
Draw the structure of the protonated form of Trimethoprim.
6.
Explain why the extraction procedure is carried out in this particular sequence
(1st NaOH, 2nd HOAc).
7.
Calculate the theoretical absorbance that you would expect to be measured in
this experiment if the Trimethoprim content was exactly as stated on the label.
Reflect on differences to the value that you obtained in the experiment.
Include a general reflection – what worked, what didn’t? What did you learn when
preparing for this practical? What did you learn while doing it? During the write-up?
Is there any way the procedure or the instructions could be improved?
01 Quantitation by Extraction (Hilary Term)
________________________________________________________________
01 – Quantitation by Extraction
Sulfamethoxazole
Absorbance
Standard
Absorbance
Sample
Trimethoprim
Absorbance
Sample
Please note any deviations to the procedure:
A NEW
COUPLING
AMIDE
BY A. CALVIN
COMPONENT
DETERMINATION
BRATTON
FOR
AND E. K. MARSHALL,
BABBITT
the Department
of Pharmacology
and Experimental
The Johns Hopkins
University,
Baltimore)
(Received
for publication,
March
JR.
AND ALMA
Therapeutics,
4, 1939)
The method proposed for the determination
of sulfanilamide
(l-3) has been widely used in estimating the drug in blood and
urine both in experimental
work and in controlling
the dosage of
the drug for patients.
During the 2 years since the method has
been in use, certain disadvantages
have become apparent.
The
use of N, N-dimethyl-1-naphthylamine
(dimethyl-cu-naphthylamine) as the coupling component
for the diazotized sulfanilamide is not entirely satisfactory
on account of the necessity of a
catalyst for rapid development
of color in dilute solutions, the
need of a large excess of the reagent, and the necessity of a certain
amount of alcohol to keep the resultant azo dye in solution.
A
coupling component which can be obtained in the form of a crystalline salt of reproducible
composition and which gives a soluble
azo dye in acid solution appeared desirable.
The other defect
which was discovered in the method was that certain samples
of dimethyl-a-naphthylamine
did not give complete recovery of
sulfanilamide
added to normal blood.
This was found to be due
to the salts (mainly chloride) present in the blood filtrate catalyzing the destruction of the azo dye by the excess nitrite.
Modifications
of our method by various authors offer no real
advantages.
In the main, these procedures have consisted in
altering the amount of blood and reagents used (4), a purification
of the coupling component (5), or a restatement of slight modifica* This investigation
R. Markle Foundation.
has been aided by a grant
537
from
the John and Mary
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WITHTHETECHNICALASSISTANCE
OF DOROTHEA
R. HENDRICKSON
(From
SULFANIL*
538
Sulfanilamide
Determination
1Coupling in alkaline
been cited
(3).
solution
has certain
disadvantages
which
have
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tions already described by the author (6, 7). Two important
improvements
in the method were described about a year ago
(8); namely, the destruction
of excess nitrite by ammonium
sulfamate and the buffering of the diazotized solution before coupling
with
dimethyl-oc-naphthylamine.
The destruction
of excess
nitrite, by preventing
the formation of nitroso compounds, allows
the use of a much wider variety of coupling components than is
otherwise possible.
Previously,
but unknown to us at the time,
Hccht (9) detcrmincd N4-sulfanilyl-Nr,
N1-dimethylsulfanilamide
in urine and blood by coupling the diazotized compound with
N-ethyl-1-naphthylamine
after destruction
of the excess nitrite
with sulfamic acid or urea.
We decided that the ideal coupling agent for determination
of
sulfanilamide
should exhibit rapidity
of coupling,
sensitivity,
purity, and reproducibility,
be unaffected in rapidity of coupling
by changes of pH from 1 to 2, and that the azo dye formed should
be acid-soluble and not affected in color by pH changes from 1
to 2. A number of compounds which appeared to be promising
in these respects and which couple in acid solution’ have been
examined.
The rapidity of coupling (speed) was noted for diazotized 0.1
and 0.01 mg. per cent solutions of sulfanilamide
buffered to pH
1.3; also the influence of pH on the rapidity of coupling with a
1 mg. per cent solution, the sensitivity with a 0.01 mg. per cent
solution, the solubility of the azo dye with a 10 mg. per cent solution, and the effect of pH on the color of the dye in the same
solutions used for determining
the effect of pH on speed. Trichloroacetic
acid was used for acidification,
excess nitrite was
destroyed with ammonium
sulfamate,
and pH was varied by
adding excess acid or sodium dihydrogen
phosphate.
In Table
I are summarized these preliminary
tests on seventeen compounds.
Two aqueous solutions of each coupling component were used,
one of such a strength that 10 moles per mole of diazonium salt
were used for the 10 mg. per cent solution of sulfanilamide
and the
other one-tenth as strong for use with the other dilutions of the
A minimal
quantity
of hydrochloric
acid was used to
drug.
dissolve coupling Compounds
1 to 3, 6 to 9, and 13; sodium
A. C. Bratton
and E. K. Marshall,
Jr.
539
Preparation
and Purification
of N-(I-Naphthyl)Ethylenediamine
Dihydrochloride
No adequate chemical description was given by Newman (10,
11) who first prepared this compound through the Gabriel synthesis. Therefore,
the following
modifications
of Newman’s
synthesis and a rather detailed characterization
of the compound
seem justified.
/3-(I-Naphthylamino)Ethylphthalimide-This
was prepared
by
essentially the procedure of Newman, except that 2.5 moles of
l-naphthylamine
to 1 mole of /?-bromoethylphthalimide
were
used, and the product was recrystallized
from glacial acetic acid.
2 This sample
had been aerated
of dimethyl-oc-naphthylamine
was
30 minutes at 267” (3), and distilled
a pure sample which
at reduced pressure.
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hydroxide
was employed
on Compounds
4, 10 to 12, 15, and
16; alcohol was necessary for Compound
17. Coupling Compounds 2 to 4, 7, and 10 to 17 were obtained
from E. I.
du Pont de Nemours and Company, Inc., through the courtesy of
Dr. H. A. Lubs and Dr. D. E. Kvalnes.
Compounds 1,2 2,4,7,8,
15, and 17 were prepared or purified from the commercial base or
salt before use, Compounds 1, 6, 8, and 9 were from the Eastman
Kodak Company, and Compound 5 was synthesized by us.
On the basis of rapidity
and sensitivity
only five compounds
(Nos. 2, 3, 5, 7, and 8) offer improvement
over N,N-dimethyl1-naphthylamine
(No. 1). Of these, Compounds
7 and 8 are
eliminated because they yield precipitates with diazotized 1 mg.
per cent sulfanilamide.
Compound
3 is eliminated
because of
the considerable influence of pH on rapidity of coupling, leaving
only Compounds 2 and 5 for consideration.
Compound 2 is very
difficult to purify, and possesses no definite physical properties
to aid in its characterization.
It is to be expected that different
batches of the compound would show considerable variation
in
purity, and the lack of reproducibility
would render it unsuitable
for use in the method.
Compound 5, N-(l-naphthyl)ethylenediamine dihydrochloride
may be readily prepared in a state of high
purity.
Its coupling is very rapid and uninfluenced
by pH in the
range of 1 to 2. This range of pH has no effect on the color of the
dye, and the dye is more soluble in this range than that from any
other coupler we have examined.
1. N,N-Dimethyl-l-naphthylamine
2. N-(1-Naphthyl)glucamine
3. N,N-Di-(hydroxyethyl)-lnaphthylamine
4. Sulfonated
N-ethyl-l-naphthylamine
5. N-(l-Naphthyl)ethylenediamine dihydrochloride
6. 2-Naphthylamine
7. N-Ethyl-1-naphthylamine
hydrochloride
8. N-Methyl-1-naphthylamine
acid sulfate
9. l-Naphthylamine
10. l-Hydroxyethylamino-5-naphthol
11. I-Amino-5-naphthol
12. Phenyl
J acid (N-phenyl-2amino-5-naphthol-7-sulfonic
acid)
Compound
Suitability
Color of dye,
IO mg. per cenl
solution
Violet
Orange-red
+
+
–
–
–
Violet-red
Purple-red
–
–
–
Violet
++
+
+
–
–
–
Orange
Purple
i -SfS
I+++
0
++
+
+
t-++-I
t -++-t
-I-SfS
:+++
-I-+++
++
pH 1.6
Purple-red
-i
–I
++
–
-+-t
t++i
+
+
pH 1.3
+
‘I
_-
pH 1.0
Influence
on coupling
speed
1 mg. per cent solution
for Sulfanilamide
Violet-red
Violet
Purple-red
I
Components
I
+
-fS
++
+
‘:;,“t”’
soluions a
IH 1.3
Speed
or 0.01
.nd 0.1
of Some Coupling
TABLE
_-
0
0
+
0
+
+
+
+
+
+
+
+
ensivity
0.01
mz.
Per
, cent
0lW
1tion
t+-t
++
t+-t
t+-t
t+-t
t+-t
t+-t
0
t+-t
-I-+
+
t++
Ppt.,
10 mg.
,er cm
olutiol
Determination
12
t+++
+
t++-t
t+++
++
pH 1.0
4
-I
4
i
t+++
t+++
+++
t+++
++
pH 1.3
pH 1.6
Effect on dye color,
1 mg. per cent solution
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0 = color no greater
than blank;
13. N,iY-Diethyl-l-naphthylamine
14. H acid (I-amino-8-naphthol3,6-disulfonic
acid)
15. Phenyl
peri acid (phenyl-lnaphthylamine-8-sulfonic
acid)
16. Tolyl
peri acid
(p-tolyl-lnaphthylamine-8-sulfonic
acid)
17. N-Phenyl-l-naphthylamine
‘I
–
= precipitate
Purple’
+
–
Violet
‘I
Red
++
+
0
formation;
–
–
–
i
I-
+ = degree of color, speed, etc.
–
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542
Sulfanilamide
Determination
C18H1~N50T.
Calculated.
Found.
C 52.02, H 4.13, N 16.87
“ 52.21, “ 4.12, “ 16.82
Smaller still charges (8 to 10 gm.) gave higher yields, probably
due to the lower heat gradient in the mass. Larger batches (60
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The yield was 68 to 78 per cent of theory.
If desired, half the
1-naphthylamine
in the synthesis may be replaced by an equivalent
amount of sodium bicarbonate,
resulting in a yield of 55 to 60
per cent.
The product is extensively
soluble in hot glacial acetic acid,
benzene, and ethyl acetate, moderately in alcohol.
It is moderately soluble in cold ethyl acetate and benzene, fairly soluble in
alcohol, and slightly soluble in ether.
It crystallizes from acetic
acid in greenish yellow, thin, irregular plates, m. p. 165.3-165.7”.
(A second recrystallization
from this solvent with the addition of
activated charcoal yields a golden yellow product.)
Hydrolysis of p-(I-Naphthylamino)Ethylphthalimide-The
use
of ordinary hydrolytic
agents, even fuming hydrochloric
acid as
employed by Newman, results in poor yields.
Either of the following two methods may be employed.
(a) By Fused Sodium Hydroxide-/3-(l-Naphthylamino)ethylphthalimide
was ground intimately
with an equal weight of
solid sodium hydroxide, and the mixture was distilled rapidly at
10 to 20 mm. pressure.
The mass melted at 160”, an oily yellow
distillate passed over at 200-300”, and the distillation
was discontinued when the residue began to char at 340”. The distillate
was extracted with the calculated amount of 0.05 N hydrochloric
acid (stronger acid yielded a turbid extract), and after the material
was decolorized with charcoal, the base was liberated with excess
sodium hydroxide,
taken up in benzene, and dried over solid
sodium hydroxide.
Upon distilling off the solvent and taking up
the base in a little alcohol, the calculated amount of hot alcoholic
picric acid was added and the solution was cooled.
The redbrown picrate was recrystallized
from 6 N acetic acid (or 95 per
cent alcohol), and precipitated
as tiny red octahedra in 40 to 70
per cent yield.
Its melting point is not sharp, and depends upon
the rate of heating: placed at 222” into a fairly rapidly rising bath,
m.p. 227-228” with decomposition;
in a more slowly heated bath,
m.p. 225-226” with decomposition.
A. C. Bratton
and E. K. Marshall,
Jr.
543
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to 70 gm.) gave lower yields because of a side reaction involving
scission of the side chain to yield 1-naphthylamine,
whose picrate
may be obtained from the alcoholic picric acid mother liquor by
addition of 5 volumes of water.
In one experiment with 0.0994
mole of fl-(1-naphthylamino)ethylphthalimide,
0.0398 mole of
N-(1-naphthyl)ethylenediamine
picrate
and 0.0124 mole of
1-naphthylamine
picrate were obtained.
Liberation of Base-The
picrate was suspended in warm water,
treated with sodium hydroxide in slight excess, and the liberated
N-(1-naphthyl)ethylenediamine
was taken up in benzene and
dried over solid sodium hydroxide.
The dihydrochloride
may be
prepared by bubbling
dry hydrogen chloride into the benzene
solution, or the solvent may be evaporated
and the base distilled
under reduced pressure.
(b) By Hydraxine-Refluxing
p-( l-naphthylamino)ethyIphthalimide in alcoholic suspension for 2 hours with hydrazine hydrate,
followed by addition of excess hydrochloric
acid and continuation
of refluxing for 1 hour, results in an 85 to 90 per cent yield of fairly
pure N-(1-naphthyl)ethylenediamine
dihydrochloride.
The procedure was essentially that of the general method of Ing and
Manske (12). Substitution
of the hydrazine
hydrate
by an
equivalent
amount of hydrazine acid sulfate, sodium carbonate,
and a minimal amount of water gave a more nearly pure product
in slightly lower yield (70 to 80 per cent).
Properties of N-(i-Naphthyl)Ethylenediamine-The
base is a
straw-yellow,
viscous liquid with an odor resembling that of the
alkyl naphthylamines;
the boiling point is 204” at 9 mm., about
320” with decomposition
at 760 mm.; nt5 = 1.6648; di5 = 1.114.
The solubility in water is about 0.2 gm. in 100 cc. at 25”, more
soluble in hot than in cold water; the pH of a saturated aqueous
solution is 10.5. The base is readily soluble in the common organic solvents, except petroleum
ether.
It distils very poorly
with steam, even from concentrated alkali.
Salts of Base-The
dihydrochloride
is prepared by introducing
dry hydrogen chloride into a solution of the base in benzene or
ether, or by dissolving the base in excess hot 6 N hydrochloric
acid. Recrystallized
from 6 N hydrochloric
acid, it precipitates
in long colorless hexagonal prisms.
Use of activated
charcoal
is of advantage in obtaining a perfectly white preparation.
We
Sulfanilamide
544
Determination
C,,H,~NElz.
Calculated.
Found.
C 55.58, H 6.23, N 10.82, Cl 27.37
“ 55.52, “ 6.20, “ 10.41, “ 26.81
Prolonged drying of the analytical sample at 110” raised the determined nitrogen to 11.60 per cent and lowered the halogen to
18.76 per cent.
The zinc chloride and mercuric chloride salts and the acid sulfate
were prepared but the first is too soluble, and the melting points
of the latter two too high, to be of use in purification
or identification of the base.
Determination
of Xulfanilamide
in Blood and Urine
Reagenk1. A solution of trichloroacetic
acid containing 15 gm. dissolved
in water and diluted to 100 cc.
2. A 0.1 per cent solution of sodium nitrite.
3. An aqueous solution
of N-(1-naphthyl)ethylenediamine
dihydrochloride
containing
100 mg. per 100 cc. This solution
should be kept in a dark colored bottle.
4. A solution of saponin containing 0.5 gm. per liter.
3 The reagents
can
Company,
Baltimore.
be obtained
from
LaMotte
Chemical
Products
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were unable to dry the dihydrochloride
without loss of a little
hydrogen chloride, which resulted in a poor analysis.
A mixture
of the mono- and dihydrochlorides
is perfectly satisfactory
for
use in the method, but it is, of course, advantageous
to strive for
a pure dihydrochloride
in order that the melting point may serve
in identification
and control of purity.
For this reason, the excess
mother liquor should be removed by pressing between filter paper
and the bulk of the remaining water should be removed in vacua
or by air drying.
The last traces of water are removed by heating
briefly at 110” and transferring
while still warm to a vacuum
desiccator.
M.p. (placed at 184’ in a fairly rapidly rising bath)
188-190”.
If the dihydrochloride
is distilled at reduced pressure,
a product is obtained which melts at 231-232” with slight decomposition,
and is probably
the monohydrochloride.
The
dihydrochloride
is easily soluble in 95 per cent alcohol, dilute
hydrochloric
acid, and hot water; it is rather difficultly
soluble in
cold water, acetone, and absolute alcohol.
A. C. Bratton
and E. K. Marshall,
Jr.
545
4 Sample
the minimal
and reagent volumes
can be proportionately
amount of filtrate necessary
for an accurate
reduced to give
color comparison.
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5. 4 N hydrochloric
acid.
6. A solution of ammonium
sulfamate, containing
0.5 gm. per
100 cc.
7. A stock solution of sulfanilamide
in water containing
200
mg. per liter.
This solution can be kept for several months in the
ice box. The most convenient
standards to prepare from the
stock solution are 1, 0.5, and 0.2 mg. per cent. To prepare these
5, 2.5, and 1 cc. of the stock solution plus 18 cc. of the 15 per cent
solution of trichloroacetic
acid are diluted to 100 cc.
Procedure for Blood4-2
cc. of oxalated blood are measured into
a flask and diluted with 30 cc. of saponin solution, and after 1 or
2 minutes precipitated
with 8 cc. of the solution of trichloroacetic
acid. The free sulfanilamide
is determined in the filtrate as follows: 1 cc. of the sodium nitrite solution is added to 10 cc. of the
filtrate.
After 3 minutes standing, 1 cc. of the sulfamate solution
is added, and after 2 minutes standing, 1 cc. of the solution of
N-(1-naphthyl)ethylenediamine
dihydrochloride
is added.
The
unknown is compared with an appropriate
standard which has
been treated as above.
This comparison can be made immediately
and no change in color is observed for 1 hour or more.
To determine the total sulfanilamide,
10 cc. of the filtrate are treated with
0.5 cc. of 4 N hydrochloric
acid, heated in a boiling water bath
for 1 hour, cooled, and the volume adjusted to 10 cc. The subsequent procedure is as stated above for determining
free sulf anilamide.
Procedure for Urine-Protein-free
urine is diluted to contain
about 1 to 2 mg. per cent of sulfanilamide
and 50 cc. of the diluted
urine plus 5 cc. of the 4 N hydrochloric
acid are diluted to 100 cc.
10 cc. of the product of this second dilution are treated as a blood
filtrate for free sulfanilamide,
and 10 cc. heated without further
addition of acid for total sulfanilamide.
If the urine contains
protein, it is diluted and treated by the procedure for blood.
Photoelectric Calorimeter-When
a photoelectric
calorimeter
is
available, dilutions of blood of 1: 50 or 1: 100 can be used. The
blood is diluted with water (saponin is unnecessary), allowed to
stand a few minutes, and precipitated
with trichloroacetic
acid
solution, with a volume which is one-fifth that of the final mixture.
546
Sulfanilamide
Determination
6For these dilutions
(1 : 200 or 1 : 400, a photoelectric
calorimeter
high sensitivity,
designed and constructed
by Dr. Morris
Rosenfeld
this department,
was used.
A description
of this instrument
will
published.
of
of
be
Downloaded from http://www.jbc.org/ at IReL (Trinity College Dublin) on October 8, 2015
This allows the use of 0.1 or 0.2 cc. samples of blood which are
measured with washout pipettes.
Determinations
on urine or
other body fluids are easily made after appropriate
dilution.
The
reagent blank on distilled water is quite low, but increases with
time if the solution is left in the light.
For this reason, solutions
to be read in the photoelectric
calorimeter
should be protected
from light unless the reading is made immediately.
Some reaction occurs between the trichloroacetic
acid and the N-(lnaphthyl)ethylenediamine,
since solutions acidified with hydrochloric acid do not show an increased color on exposure to light.
The blood blank is extremely low and negligible for most purposes.
With a 1:50 dilution of human blood the correction due to the
blood blank varies from 0 to 0.03 mg. per cent. This blank can
be easily determined
by performing
an analysis as usual except
that water is substituted
for the sodium nitrite solution.
When
small concentrations
of sulfanilamide
are to be determined
or
when a foreign dye such as prontosil or neoprontosil
is present,
The color of the normal urinary
this procedure is quite useful.
pigments can be conveniently
corrected for by the same procedure.
When only a very small amount of blood is available, as in the
case of small animals such as mice, a determination
can be made
with considerable accuracy on 0.02 cc. The adaptation
is essentially that described by Marshall and Cutting (13), the dilution
of blood being 1: 200 or 1: 400, depending on the concentration
of
sulfanilamide
present.
The proportion
of reagents used is the
same as in the other adaptations
of the method.
Centrifugation
before filtration
of the protein precipitate is useful in securing the
maximum amount of filtrate.5
In using a photoelectric
calorimeter
a filter is essential.
With
dimethyl-ol-naphthylamine
the peak of the absorption
of the
azo dye formed occurs at 530 rnp (14). When N-(l-naphthyl)ethylenediamine
is used, the peak of absorption
is shifted to
545 rnp, and the dyes from sulfapyridine
and N*-ethanolsulfanilamide show the same absorption peak. In Fig. 1 is reproduced an
absorption
curve of the azo dye from sulfanilamide.
We wish
A. C. Bratton
and E. K. Marshall,
Jr.
547
s
i= JO
9
$
42.5
:I
500
5/o
520
530
540
WAVELENG)TH
with
FIG. 1. Absorption
curve of dye from
N-(1-naphthyl)ethylenediamine.
550
Imp
560
570
580
I
coupling
diazotized
sulfanilamide
Average percentage
recoveries were for the 1~4 dilution
90.0,
for the 1: 10 dilution 94.3, for the 1: 20 dilution 96.9, and for the
1:50 dilution 99.5. Determinations
on other samples of blood
with added sulfanilamide
gave similar recoveries for the 1:20
and 1:50 dilutions.
With acetylsulfanilamide
recovery is not complete in a 1:20
dilution of blood (about 90 per cent) but is complete in a 1:50
dilution.
A series of eight bloods to which varying amounts of
acetylsulfanilamide
were added gave recovery of 97.6 per cent
Downloaded from http://www.jbc.org/ at IReL (Trinity College Dublin) on October 8, 2015
to thank Dr. Elizabeth E. Painter of the Department
of Physiology, Columbia University,
New York, for these absorption data.
Blood Dilution and Recovery-When
a 1: 4 dilution of blood is
used as suggested in Fuller’s method (15) and in Proom’s (6)
adaptation
of our method, a result 10 per cent too low may be
obtained.
Sulfanilamide
was added to three samples of mixed
human blood to give about 10 mg. per cent, and determinations
made with various dilutions for precipitation.
The filtrates were
diluted when necessary to read with the photoelectric
calorimeter.
548
Sulfanilamide
Determination
Downloaded from http://www.jbc.org/ at IReL (Trinity College Dublin) on October 8, 2015
(96.2 to 99.2) with the dimethyl-ar-naphthylamine
reagent in a
1:50 dilution.
Since experiments
show that solutions of pure
acetylsulfanilamide
give theoretical
results when hydrolyzed
with hydrochloric
acid and estimated by use of the new coupling
component, no recoveries of the acetyl compound with the new
reagent were made.
The accidental errors of the method can be best illustrated
by
some determinations
made with the photoelectric
calorimeter.
Sulfanilamide
was added to a sample of mixed human blood and
fifteen determinations
were made on the sample, 0.5 cc. of blood
(measured with a syringe pipette) being used each time in a 1: 50
dilution.
The mean value in mg. per cent was 4.888 =t 0.039
with a maximum deviation of 0.113.
Protein Precipitation-Precipitation
of the blood proteins with
trichloroacetic
acid has been adopted in place of p-toluenesulfonic
acid for the following
reasons: trichloroacetic
acid of constant
quality and purity can be obtained much more readily than can
toluenesulfonic
acid, no difficulty
is experienced in determining
total sulfanilamide
in a trichloroacetic
acid blood filtrate when the
excess nitrite is destroyed (8), and the blood blank given with
trichloroacetic
acid is much less than that obtained when toluenesulfonic acid is used. The last mentioned advantage is of great
importance when a photoelectric
calorimeter is used, as the blank
obtained on human blood with trichloroacetic
acid can usually be
neglected but must be taken into account when a toluenesulfonic
acid filtrate is used.
Body Fluids Other Than Blood and Urine–No
difficulty
has
arisen in est.imating sulfanilamide
and its acetyl derivative
in
other body fluids by the same procedure as used for blood.
When
tissues are to be analyzed, it appears desirable to extract the
ground tissue in a Soxhlet apparatus with a limited amount of
alcohol, dilute an aliquot portion of the extract with water, and
proceed as in blood, with a photoelectric
calorimeter.
This is
a simpler and less laborious method than the one previously
used
for tissues (16).
Sulfanilamide
Derivatives-As
previously
indicated
(3), our
method can be used for determining
diazotizable primary
aryl
amines containing either a free amino group or a blocked amino
group which can be freed by hydrolysis.
In the limited experi-
A. C. Bratton
and E. K. Marshall,
Jr.
549
DISCUSSION
We have already discussed some of the so called modifications
of our method which have been proposed.
It remains to mention
briefly other methods which have been suggested for the determination of sulfanilamide
or allied compounds.
Ktihnau
(17) has
described a method for estimating N4-sulfanilyl-N’,
Nl-dimethylsulfanilamide
by the color produced
with dimethylaminobenzaldehyde, and Schmidt (18) one for sulfanilamide,
wit,h the color
produced
by sodium fi-naphthoquinone-4-sulfonate.
We have
had no experience with either of these. Scudi (19) has described
a diazotization
procedure,
followed
by neutral
coupling with
chromotropic
acid, while Doble and Geiger (20) used diphenylamine as an acid coupling agent.
Neither of these latter methods
appears to be as satisfactory
as the method we have described.
With a new compound, a few preliminary
trials should indicate
Downloaded from http://www.jbc.org/ at IReL (Trinity College Dublin) on October 8, 2015
ence which we have had in applying our method to the estimation
of compounds other than sulfanilamide,
three points of importance
can be mentioned.
With a very difficultly
soluble substance, the
recovery in the blood filtrate is generally not quantitative
unless
high dilutions (1: 100 or greater) are used, so that either one must
use such dilution or must resort to the original alcohol precipitation method (1). In the latter case, ammonium
sulfamate
is
used to destroy excess nitrite and N-(1-naphthyl)ethylenediamine
dihydrochloride
is used as a coupling component.
With certain
derivatives,
the azo dyes formed will not be acid-soluble
and a
certain amount of alcohol must be added with or just before the
coupling component.
With some substances buffering is necessary
to obtain sufficient speed of coupling (e.g., aniline).
In the determination
of sulfapyridine
(Z(sulfanilamido)pyridine)
with the present method, the following
may be mentioned.
To a sample of mixed human blood sulfapyridine
was
added to make about 10 mg. per cent. Precipitation
in a 1:4
dilution gave 80.8 per cent recovery, in a 1:20 dilution 93.7 per
cent, and in a 1:50 dilution 99.4 per cent. A number of other
experiments
indicate incomplete recovery (average 91 per cent)
in 1:20 dilution with 5 to 10 mg. per cent in blood, but essentially
complete with values below 5 mg. per cent. With a 1:50 or
greater dilution, recovery is quantitative.
550
Sulfanilamide
Determination
what slight modification
of the method, if any, is necessary to
determine it accurately.
However, we must caution investigators
against accepting results with a new compound
until control
recoveries from blood and urine have been made.
SUMMARY
BIBLIOGRAPHY
1. Marshall,
E. K., Jr., Emerson,
K., Jr., and Cutting,
W. C., J. Am.
Med. Assn., 108, 953 (1937).
2. Marshall,
E. K., Jr., Proc. Sot. Exp. Biol. and Med., 36, 422 (1937).
3. Marshall,
E. K., Jr., J. Biol. Chem., 122, 263 (1937-38).
4. MacLachlan,
E. A., Carey, B. W., and Butler, A. M., J. Lab. and Clin.
Med., 23, 1273 (1938).
5. Stevens, A. N., and Hughes,
E. J., J. Am. Pharm. Assn., 27, 36 (1938).
6. Proom, H., Lancet, 1, 260 (1938).
7. Kamlet,
J., J. Lab. and Clin. Med., 23, 1101 (1938).
8. Marshall,
E. K., Jr., and Litchfield,
J. T., Jr., Science, 88, 85 (1938).
9. Hecht, G., Dermat.
Woch., 106, 20 (1938).
10. Newman,
H. E., Ber. them. Ges., 24, 2199 (1891).
11. British
patent 247,717, June 29, 1925.
12. Ing, H. R., and Manske,
R. H. F., J. Chem. Sot., 2348 (1926).
13. Marshall,
E. K., Jr., and Cutting,
W. C., Bull. Johns Hopkins
Hosp.,
63, 328 (1938).
14. Gregersen,
M. I., and Painter,
E. E., Am. J. Physiol.,
123, 83 (1938).
15. Fuller, A. T., Lance& 1, 194 (1937).
16. Marshall,
E. K., Jr., Emerson,
K., Jr., and Cutting,
W. C., J. Pharmacol. and Exp. Therap., 61, 196 (1937).
17. Ktihnau,
W. W., Klin. Woch., 17, 116 (1938).
18. Schmidt,
E. G., J. Biol. Chem., 122, 757 (193738).
19. Scudi, J. V., J. Biol. Chem., 122, 539 (1937-38).
20. Doble, J., and Geiger, J. C., J. Lab. and Clin. Med., 23, 651 (1938).
Downloaded from http://www.jbc.org/ at IReL (Trinity College Dublin) on October 8, 2015
In the determination
of sulfanilamide
by diazotization
and
coupling in acid solution, the use of N-(l-naphthyl)ethylenediamine dihydrochloride
offers the following
advantages
over N ,Ndimethyl-1-naphthylamine
(dimethyl-a-naphthylamine)
: (1) reproducibility
and purity,
(2) greater rapidity
of coupling,
(3)
increased sensitivity,
(4) elimination
of buffer, and (5) increased
acid solubility
of the azo dye formed.
An improved synthesis
and a complete characterization
of the new coupling component
is presented.
Slight modification
of previous technique and application to other primary aryl amines are described.
A. Calvin Bratton, E. K. Marshall, Jr. and
With the technical assistance of Dorothea
Babbitt and Alma R. Hendrickson
J. Biol. Chem. 1939, 128:537-550.
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ARTICLE:
A NEW COUPLING COMPONENT FOR
SULFANILAMIDE DETERMINATION
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 181 (2017) 276–285
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A sensitive method for the determination of Sulfonamides in seawater
samples by Solid Phase Extraction and UV–Visible spectrophotometry
Sophia Ait Errayess a,b, Abdellatif Ait Lahcen a, Laila Idrissi a, Caterina Marcoaldi b,
Salvatore Chiavarini b, Aziz Amine a,⁎
a
b
Laboratoire Génie des Procédés et Environnement, Faculté des Sciences et Techniques de Mohammedia, Hassan II University of Casablanca, B.P. 146, 20650 Mohammedia, Morocco
ENEA, C.R. Casaccia, via Anguillarese 301, 00123 Rome, Italy
a r t i c l e
i n f o
Article history:
Received 31 December 2016
Received in revised form 15 March 2017
Accepted 26 March 2017
Available online 28 March 2017
Keywords:
Sulfonamides
Spectrophotometric method
Drug analysis
Seawater
Solid phase extraction
Pre-concentration
a b s t r a c t
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.
© 2017 Elsevier B.V. All rights reserved.
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,
⁎ Corresponding author.
E-mail addresses: azizamine@yahoo.fr, a.amine@univh2m.ac.ma (A. Amine).
http://dx.doi.org/10.1016/j.saa.2017.03.061
1386-1425/© 2017 Elsevier B.V. All rights reserved.
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.
S.A. Errayess et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 181 (2017) 276–285
277
≥ 98.5%, sulfacetamide (SCT) ≥ 98.0%, sulfamethoxazole (SMX)
≥ 99.0%, sulfamethiazole (SMT) ≥ 99.0%.
2.2. Instrumentation and apparatus
Fig. 1. Chemical structure of sulfonamides.
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 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-(1naphthyl) 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 preconcentration 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.
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
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
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 (Na 2 -EDTA) from Sigma-Aldrich (USA), sodium nitrite from
Riedel-deHaën (Germany), N-(1-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-dimethyluread 6), 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)
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 was measured at 536 nm
against the reagent blank. Fig. 2 shows a picture of a gradient of concentrations of the pink colored solutions tested.
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
278
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2.9. Procedure of spectrophotometric analysis
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.
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
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:
2.7.1. 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.
2.7.2. 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.
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).
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.
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.
2.8. Pre-concentration of sulfonamides in seawater samples
3.3. Optimization of reaction conditions
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.
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
3.4. Stability of the resulting product
2.8.2. Extraction and concentration procedure
Fig. 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.
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 N60 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
S.A. Errayess et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 181 (2017) 276–285
279
Fig. 3. Chemical reaction scheme of the tested method for sulfonamides detection.
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
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.
280
S.A. Errayess et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 181 (2017) 276–285
Table 1
Some optical & analytical parameters for the spectrophotometric determination of sulfonamide derivatives.
Parameter
SAA
SdZ
SrZ
ScT
Stz
SDX
SMX
SMZ
Color
λmax (nm)
Beer’s law range (Μg mL−1)
Limit of detection (Μg mL−1)
Limit of quantiﬁcation (Μg mL−1)
Molar absorptivity (L mol−1 cm−1)a
Regression equationb
Slope (a)
Intercept (b)
Correlation coefﬁcient (r)
Standard deviation of slope (Sb)
Standard deviation of intercept (Sa)
Relative standard deviation of the slope (%)
Pink
536
0.06–1.0
0.019
0.06
5.2 × 104
Pink
536
0.13–1.0
0.04
0.13
4.2 × 104
Pink
536
0.15–1.0
0.04
0.15
4.5 × 104
Pink
536
0.12–1.0
0.04
0.12
5.3 × 104
Pink
536
0.16–1.0
0.05
0.16
5.5 × 104
Pink
536
0.15–1.0
0.04
0.15
8 × 104
Pink
536
0.07–1.0
0.02
0.07
5.4 × 104
Pink
536
0.1–1.0
0.03
0.10
5.3 × 104
0.307
0.060
0.999
6.12 × 10−3
1.41 × 10−3
1.9
0.170
0.078
0.998
1.1 × 10−2
4.96 × 10−3
6.4
0.173
0.075
0.997
4.7 × 10−3
5.76 × 10−3
2.7
0.250
0.064
0.997
6 × 10−3
9.8 × 10−3
2.4
0.218
0.064
0.996
1 × 10−2
2.46 × 10−3
4
0.258
0.058
0.997
3.12 × 10−3
2.2 × 10−3
1.2
0.213
0.066
0.999
3.12 × 10−3
2.2 × 10−3
1.4
0.198
0.066
0.998
5.5 × 10−3
2.3 × 10−3
2.8
a
b
Molar absorptivity = Slope × Molecular weight × 103.
Y = aX + b Where X is the concentration of sulfonamides in Μg mL−1 and Y is the absorbance at λmax, for three measurements.
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.
The LOD and LOQ were calculated for eight sulfonamides according
10Sb
b
to 3S
S and S 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 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.
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%
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 towards sulfonamides.
Although it’s 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)
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
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.
S.A. Errayess et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 181 (2017) 276–285
281
Table 2
Comparison of reported UV–Visible spectrophotometric methods for the determination of sulfonamides.
Coupling agent
λmax
(nm)
Derivative studied
Beer’s law range Sensitivity = Slope LODa
(μg mL−1)
(Abs μg−1 mL)
3-Aminophenol
460
0.05–8.0
0.142–0.188
0.0232–0.0504 Involves heating in a boiling
water bath for 5 min and cooling
23
NED (Bratton-Marshall
reagent)
536
545
Sulfathiazole
Sulfadiazine
Sulfacetamide
Sulfadoxine
Sulfanilamide
Sulfadiazine
Sulfamethizole
Sulfamerazine
Sulfabenzamide
Sulfamethazine
Sulfaquinoxaline
Sulfachloropyridazine
Sulfamethoxazole
Sulphathiazole
Sulphamerazine
Sulphamethoxazole
Sulphaquinoxaline
Sulfadoxine
Sulfamethoxazole
Sulfanilamide
Sulfathiazole
Sulfadoxine
Sulfamethoxazole
Sulfamethoxazole
Sulfacetamide
Sulfadiazine
Sulfaguanidine
Sulfamerazine,
Sulfamethazine
Sulfamethoxazole
Sulfathiazole
Sulfanilamide
Sulfaguanidine
Sulfacetamide
sodium
Sulfamerazine
Sulfadimidine sodium
Sulfadiazine
Sulfathiazole
Sulfasomidine
Sulfacetamide sodium
Sulfadiazine
Sulfadimidine
Sulfathiazolene
Sulfathiazole
Sulfadiazine
Sulfacetamide
Sulfamethoxazole
Sulfamerazine
Sulfaguanidine
Sulfadimidine
Sulfadiazine
Sulfanilamide
Sulfamethoxazole
Sulfadiazine
Sulfacetamide Sodium
Sulfadimidine
Sulfaguandine
Sulfanilamide
Sulfathiazole
Sulfathiazole
Sulfadiazine
Sulfacetamide
Sulfamethoxazole
Sulfamerazine
Sulfaguanidine
Sulphacetamide
Sulphadiazine
Sulphamerazine
Sulphadimidine
Sulphasomidine
Sulphamethoxazole
Sulphamethoxypyridazine
1.0–5.0
2.0–20
0.160
–
–
0.6–1.1
38
39
0.6–7.0
Up to 0.2
4.0–12
0.49–7.25
0.50–7.6
0.48–7.3
0.57–8.64
5.0–25
2.0–10
5.0–100
–
–
0.177
–
–
0.05
–
–
0.025
0.040
–
–
–
–
4.0–8.0
2.0–10
1.0–5.0
0.1–7.0
0.110
0.004
0.127
0.125–0.145
–
–
–
0.03–0.05
50–250
0.2–2.5
0.2–2.5
0.5–3.5
–
–
–
–
46
47
0.0086
0.0085
0.0108
0.0089
0.075–0.197
–
Preliminary heating on water bath
18
for 5 min and later at 40 °C for 25 min
Low sensitivity
0.033–0.087
The coupling agent (Iminodibenzyl)
is too expensive
(US 48 $/mg)
17
0.192–0.998
10–240
High concentration and
detection range
48
–
High concentration range
49
545
545
536
546
Diphenylamine
524
516
480–550
Resorcinol
496
430
502
500
8-Hydroxyquinoline
530
500–504
Acetylacetone-Formaldehyde 400
Iminodibenzyl
570–580
o-phthalaldehyde
340
Alizarine derivatives
452–645
(I) alizarine
(II) alizarine blue
(III) alizarine red
(IV) quinalizarine
Dopamine–molybdate ions
490–510
p-benzoquinone
500
0.5–3.5
1.0–3.5
0.2–3.5
1.0–4.0
1.0–4.0
4.0–80
4.0–72
4.0–60
4.0–80
0.05–6.0
10–100
0.24–240
16–144
Comment
Ref
Low sensitivity and detection
40
41
36
42
Require 20 min for color
development
38
36
43
38
44
36
45
Low sensitivity
Cooling in an ice bath
is needed.
5.0–100
5.0–80
10–110
5.0–130
0.04–8.0
0.0089–0.0204
0.0069–0.0163
0.0059–0.0096
0.0089–0.0204
0.074–0.123
Low sensitivity
0.023–0.068
Diazotization coupling, product
is complexed with molybdate
Low sensitivity
10–50
0.0121–0.024
–
Involves heating in a thermostatically 50
controlled water-bath for 10 min
at 90 °C and cooling
20
(continued on next page)
282
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Table 2 (continued)
λmax
(nm)
Coupling agent
Derivative studied
Beer’s law range Sensitivity = Slope LODa
(μg mL−1)
(Abs μg−1 mL)
Orcinol
390
Sulphathiourea
Sulphamethoxydiazine
Sulphaphenazole
Sulphamoxole
Sulphadimethoxine
Sulphaethidole
Sulphadoxine
Sulphamethoxazole
β-naphthol
477
470
Sulfamethoxazole
Sulfadoxine
5.0–25
4.0–60
0.0489
2.1 × 10−3
__
0.51
NED (Modiﬁed
Bratton-Marshall reaction)
536
Sulfanilamide
Sulfadiazine
Sulfamerazine
Sulfacetamide
Sulfathiazole
Sulfadimethoxine
Sulfamamethoxazole
Sulfamerazine
0.1–1.0
0.307
0.170
0.173
0.250
0.218
0.258
0.213
0.198
0.019–0.05
a
2.0–10
0.005
–
Comment
Ref
Cooling in an ice bath is needed
Low sensitivity
Only for higher concentration
of sulfonamides
Low sensitivity
Highly sensitive, involves no heating
or cooling and reaction is rapid
Low concentration and
detection range
Simple method
Successfully coupled to solid phase
extraction method to reach a
concentration level of ng mL−1
51
36
52
Present
work
LOD: Limit of detection.
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.
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 method of Salinas et al., [42] as indicated in
Table 2. The optimized method gives a high sensitivity, low detection
and quantiﬁcation limits.
3.8. Comparison of the reported spectrophotometric methods and the proposed method for sulfonamides determination
3.9. Analytical applications
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
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 and
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
Recovery of sulfamethoxazole in spiked water samples.
Water sample
Amount of drug added
(μg mL−1)
(Theoretical)
A
Amount of drug found
(μg mL−1)
(Practical)
Ba
Recovery (%) = (Practical/theoretical) × 100
RSD (%)a
Bias (%) B−A
A
Mineral Water 1
Mineral Water 2
Mineral Water 3
Tap water
0.25
0.25
0.25
0.25
0.248 ± 0.008
0.253 ± 0.005
0.280 ± 0.005
0.278 ± 0.007
99.2
101.2
112.0
111.2
3.2
1.9
1.7
2.5
−0.8
+1.2
+12.0
+11.2
a
Average of three determinations ± SD.
Table 4
Determination of sulfonamide derivatives in commercial drug samples.
Commercial Sample
COTRIM®
TRIMETO-SULFA®
a
Label claim (mg) A
800 mg
83,5 mg mL−1
Average of six determinations ± SD.
Amount of drug found (mg) Ba
Proposed method
Recovery (%)
RSD (%)a
Bias (%) B−A
A
797 ± 90 mg
81 ± 6 mg mL−1
99.6%
97%
11%
7%
−0.37%
+2.9%
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283
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 be the appropriate solvent towards extraction. Since then, it
was chosen as the extraction solvent for further experiments.
3.10.3. Solid Phase Extraction using Oasis HLB columns
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.
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 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).
3.10.3.1. 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.
3.10.3.2. 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 = 0.353C (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 values for seawater samples which were comprised between 99%
and 104% for the range of concentration tested 0.19 and 126 ng mL−1.
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
Table 5
Recovery studies of seawater samples.
Type of sample
Analyte
Cartridge
Concentration of seawater
fortiﬁed (ng mL−1)
Volume of Seawater required
for extraction (mL)
Final volume of concentrate
(mL)
Concentration
factor
Recovery (%)
Bias (%)
Distilled water
Seawater 1
Seawater 2
Seawater 3
Seawater 4
Seawater 5
SMX
Blank
SMX
SMX
SMX
SMX
Oasis HLB
Oasis HLB
Oasis HLB
Oasis HLB
Oasis HLB
Oasis HLB
126
Blank
126
2.4
1.2
0.19
500
500
500
500
1000
1000
10
10
10
10
6
1.2
50
50
50
50
166
838
90%
NDa
99%
96%
98%
104%
+11.0%
NDa
−0.99%
−4.0%
−1.6%
−4.0%
a
ND: Not detectable.
284
S.A. Errayess et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 181 (2017) 276–285
Fig. 7. UPLC-MS chromatogram of sulfamethoxazole.
Table 6
Comparison between the results obtained using UPLC-MS and Spectrophotometry.
Analyte
Cartridge
Nominal concentration in
spiked seawater (ng mL−1)
Initial Volume
of seawater
Concentration found by
UPLC-MS method (ng mL−1)a
Concentration found by
Spectrophotometry method (ng mL−1)a
t-value
F-value
SMX
SMX
Oasis HLB
Oasis HLB
2.4
0.19
500
1000
4.1 ± 0.14
0.32 ± 0.011
2.1 ± 0.003
0.27 ± 0.003
26.1
2.65
516
31
Theoretical t-value =2.776; Theoretical F-value =19.0.
a
Mean ± SD; n = 3.
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.
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 No. 613844.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.saa.2017.03.061.
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Available online at www.ijpsdr.com
International Journal of Pharmaceutical Sciences and Drug Research 2010; 2(3): 204-209
Research Article
ISSN 0975-248X
Development and Validation of Spectrophotometric Methods for
Estimating Sulfamethoxazole in Pharmaceutical Preparations
Sangita Sharma*, M. Neog, Dipti Dabhi
Department of Chemistry, Hemchandracharya North Gujarat University, Patan-384265, India
ABSTRACT
Four simple, sensitive, accurate, and rapid visible spectrophotometric methods (A, B, C and D) have been developed for
the estimation of sulfamethoxazole in pharmaceutical Preparation. They are based on the diazotization of sulfamethoxazole
with sodium nitrite and hydrochloric acid followed by coupling with N-(1-naphthyl ethylenediamine dihydrochloride
(Method A) to form pink coloured chromogen, diphenylamine (Method B) to form pink coloured chromogen, β-napthol (in
alkaline medium) (Method C) to form a orange yellow coloured chromogen and Resorcinol (in alkaline medium) (Method
D) to form orange red coloured chromogen and exhibiting absorption maxima λmax at 536 nm, 516 nm, 477 nm and 502 nm
respectively. The coloured chromogens formed are stable for more than 2 h. Beer’s law was obeyed in the concentration
range of 4 -12 µg mL-1 in method A , 2 – 10 µg mL-1 in method B, 5 – 25 µg mL-1 in method C and 1 – 5 µg mL-1 in
method D respectively. The Results of the four analysis have been validated statistically and by recovery studies. The
results obtained in the proposed methods are in good agreements with labeled amounts, when marketed pharmaceutical
preparations are analyzed.
Keywords: Sulfamethoxazole, Diazotization, Visible spectrophotometric, Chromogen.
INTRODUCTION
Sulfamethoxazole [1] is chemically 4-amino-N-(5methylisoxazol-3-yl)-benzenesulfonamide (Molecular mass
253.279 g mol-1). It is a sulfonamide bacteriostatic antibiotic.
It is most often used as part of a synergistic combination with
trimethoprim in a 5:1 ratio in co-trimoxazole, which is also
known as Bactrim, Septrin, or Septra (also abbreviated
SMX/TMP). Its primary activity is against susceptible forms
of Streptococcus, Staphylococcus aureus, Escherichia coli,
Haemophilus influenzae, and oral anaerobes. It is commonly
used to treat urinary tract infections. In addition can be used
as an alternative to amoxicillin-based antibiotics to treat
sinusitis. It can also be used to treat toxoplasmosis. It is
official in United State Pharmacopoeia, British
Pharmacopoeia and European Pharmacopoeia. Literature
survey reveals the estimation of Sulfamethoxazole in
pharmaceutical formulations by various spectrophotometric
[2-7]
, HPLC [8-11], HPTLC [12], Capillary zone electrophoresis
[13]
, Micellar electrokinetic chromatography [14], Derivative
ratio spectrometry [15], Flow injection sensor [16],
Sulfamethoxazole-imprinted polymer [17], Spectrofluorometry
[18]
, Fluorescence Spectrophotometric [19] and NMR [20]
methods.
*Corresponding author: Mrs. Sangita Sharma,
Department Of Chemistry, Hemchandracharya North Gujarat
University, Patan-384265, India;
E-mail: researchsharma@yahoo.com
The present work deals with the development of four simple
and sensitive visible spectrophotometric methods for the
quantitative estimation of Sulfamethoxazole in bulk and
pharmaceutical preparations.
The aromatic amino group present in Sulfamethoxazole is
diazotized [21] with nitrous acid (NaNO2 / HCl) at room
temperature and diazonium salt thus formed is coupled with
the N-(1-napthyl) ethylenediamine dihydrochloride (0.2%
w/v; Bratton Marshall Reagent) in method A, diphenylamine
in method B, β-naphthol (in alkaline medium) in method C
and resorcinol (in alkaline medium) in method D to fo…
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