New spectrofluorimetric method for determination of cephalosporins in pharmaceutical formulations
Shazalia M. Ali Ahmed, Abdalla A. Elbashir, Fakhr Eldin O. Suliman and Hassan Y. Aboul-Enein, Luminescence 2012,
Published online in Wiley Online Library
LOGO
Abstract
LOGO
Results
A simple, accurate, precise spectrofluorimetric method has been proposed for the determination of three cephalosporins,
namely, cefixime (cefi), cephalexine (ceph), and cefotaxime sodium (cefo) in pharmaceutical formulations. This method is
based on a reaction between cephalosporins with 8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt (HPTS) in alkaline
medium, at pH 12.0 for cefi and 13.0 for ceph and cefo to give highly fluorescent derivatives extracted with chloroform
and subsequent measurements of the formed fluorescent products at 520, 500 and 510nm after excitation at 480, 470
and 480 nm for cefi, ceph and cefo respectively. The optimum experimental conditions have been studied. Beer’s law is
obeyed over concentrations of 10–60 ng/mL, 5–35ng/mL and 10–60ng/mL for cefi, ceph and cefo, respectively. The
detection limits were 4.20ng/mL, 2.54ng/mL and 4.09 ng/mL for cefi, ceph and cefo, respectively, with a linear regression
correlation coefficient of 0.99783, 0.99705 and 0.9978 and recoveries in ranges 96.96–105.77, 96.13–102.55 and 95.45–
105.39% for cefi, ceph and cefo, respectively. This method is simple and can be applied for the determination of cefi,
ceph and cefo in pharmaceutical formulations in quality control laboratories.
Absorption spectra
According to the procedure the absorption spectrum of products produced by the reaction of cefi, ceph and cefo with
HPTS are recorded in Figs 2–4. The maximum absorption wavelength peak (lmax) at 480, 470, and 480 nm, for cefi,
ceph and cefo, respectively and the lmax of HPTS was 400nm. The derivatives
exhibit maximum fluorescence intensities lmax at 520, 500 and 510nm after excitation at 480, 470 and 480nm for cefi,
ceph and cefo respectively (Figs 5–7).
Determination of stoichiometric ratio
Under optimum conditions (Table 2), the stoichiometric ratio between HPTS and each of the investigated cephalosporins
was1 : 1 (Fig. 8). Based on this ratio, the reaction pathways were postulated as shown in Scheme 1, indicating that
cephalosporins used in this study are susceptible to reaction with HPTS producing fluorescent products.
Optimization of reaction variables
To optimize the reaction conditions between the HPTS and cephalosporins the following parameters were investigated:
pH of the buffer, reaction time, and temperature and HPTS concentration.
Effect of pH. The effect of pH on the reaction of cefi, ceph, and cefo with HPTS were examined by varying the pH from
4.0 to 13.0, the results reveal that cefi, ceph and cefo are difficult to react with HPTS in acidic media (Fig. 9). This is due
to the existence of the amino group of cefi, ceph and cefo in the form of hydrochloride salt; thus, it loses its nucleophilic
substitution capability. As the pH increased, the readings increased rapidly, as the amino group of cefi, ceph and cefo (in
the hydrochloride salt) turns into the free amino group, thus facilitating the nucleophilic substitution. The maximum
readings were attained at pH values of 12.0 for cefi and 13.0 for ceph and cefo. At pH values more than 12.0 for cefi and
more than 13.0 for ceph and cefo, a decrease in the readings occurred. This was probably attributed to the increase in
the amount of hydroxide ion that suppresses the reaction of cefi, ceph and cefo with HPTS.
Introduction
Cephalosporins are antibacterial agents commonly used to control Gram-positive and Gram-negative bacteria. Cephalosporins are the second most
important b-lactams after penicillin for treating infectious diseases (1). Many of these manifestations, such as urticaria and exanthema, are cutaneous, but
anaphylactic reactions
have also been reported (2).
Cephalosporins are derivatives of 7-aminocephalosporanic acid composed of a b-lactam ring fused with a dihydrothiazine ring (Fig. 1) but differ in the nature
of substituent at the 3- and/or 7-positions of the cephem ring as shown in Table 1 (3,4).
Several methods have been described for the quantitative determination of cephalosporins, including spectrophotometry (5–8), spectrofluorimetry (9,10),
high-performance liquid chromatography (11–18), and voltammetry (19). These methods were time-consuming, tedious, and used sophisticated and
expensive analytical instruments. Spectrophotometric methods are the most convenient techniques because of their inherent simplicity, high sensitivity, low
cost, and wide availability in quality control laboratories. Unfortunately, the spectrophotometric methods that have been reported for determination
of cephalosporins in their pharmaceutical formulations were associated with some major disadvantages such as the lack of selectivity, tedious extraction
procedures and were timeconsuming.
The official procedures in pharmaceutical preparations use high-performance liquid chromatography(20).
Therefore, the development of new alternative spectrofluorimetricmethod for the determination of cephalosporins that can overcome the disadvantages of
existing methods is essential.
The pH-sensitive fluorescent dye 1-hydroxy-3,6,8-pyrenetrisulfonic acid trisodium salt also known as pyranine (HPTS) has distinct absorption band in the
visible light region, and has thus
been used in many aqueous-phase CO2 optical sensors. However, this is the first time that HPTS has been used asa fluorogenic reagent for the
determination of cephalosporins in their pharmaceutical formulations; HPTS contains three sulfonate groups at positions, 1, 3, and 6, which make it suitable
for nucleophilic substitution reactions with cephalosporins. This work describes a rapid spectrofluorimetric
method for determining the content of cefi, ceph and cefo in pharmaceutical formulations, which is based on the reaction of HPTS with amino group of cefi,
ceph and cefo molecules to form fluorescent derivatives (21–26).
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Figure_2.cephalexin spectrum
Figure_3effect of temperature
Methods and Materials
Figure_1.cephalosporins
Apparatus
The spectrofluorimetricmeasurements were made with Shimadzu spectrofluorophotometer RF-1501 with xenon lamp. In addition, a temperature controller was used for
the spectrofluorimetric measurements. A pH meter model
pH 211 (HANNA, Camisano Vicentino, Italy) was used for adjusting pH.
Reagents and solutions
All reagents were of analytical reagent grade. Double distilled water was used in all experiments.
The standards of cefi, ceph and cefo were supplied by (Orchid Chemicals and Pharmaceuticals Ltd., Chennai, India).
Pharmaceutical formulation. The following available pharmaceutical preparations were analyzed:
(1) cefi capsules (AMIPHARMA Laboratories, Khartoum-North, Sudan), labeled to contain 200mg cefi per capsule.
(2) ceph monohydrate capsules (AMIPHARMA Laboratories, Sudan), labeled to contain 500mg ceph per capsule.
(3) cefo for injection (Kilitch Drugs, Mumbai, India) labeled to contain 1000mg cefo per injection.
Stock standard solution of cefi, ceph and cefo (1000mg/mL). An accurately weighed 0.1 g standard sample of the three drugs was dissolved in methanol for cefi and in
double distilled water for ceph and cefo, transferred into a 100mL standard flask and diluted to the mark with methanol for cefi and with double distilled water for ceph
and cefo and then mixed well. This stock solution was further diluted to obtain working solutions in the ranges 10–35, 10–60 and 20–45 ng/mL for cefi, ceph and cefo,
respectively.
8-Hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt solution(0.0.02%, 0.025% w/v).
An accurately weighed 0.02 g and 0.025 g of HPTS (Sigma-Aldrich, St. Louis, MO) was dissolved in double distilled water, transferred into a 100mL standard flask and
diluted to the mark with double distilled water and mixed well to prepare 0.02% and 0.025% w/v, respectively. The solution was freshly prepared and
protected from light during use in a brown calibrated flask. Buffer solutions. A buffer solution of pH 12.0 was prepared by mixing 25 mL of 0.2 M KCl with 12 mL of 0.2 M
NaOH, and a buffer of pH 13.0 was prepared by mixing 25 mL of 0.20 M KCl solution with 65 mL of 0.20 M NaOH solution, in a 100-mL volumetric flask and adjusted by
a pH meter.
Buffer solutions of different pH values were also prepared according to the method in the literature (27).
Sample solutions. The contents of 20 capsules or 20 injection powders were carefully evacuated, weighed and finely powdered. Then an accurately weighed amount
equivalent to 100mg was transferred into a 100-mL calibrated volumetric flask, and dissolved in about 40 mL in methanol for cefi and in double distilled water for ceph
and cefo.The contents of the flask were swirled, sonicated for 5 min, and then filled to volume with methanol for cefi and with double distilled water for ceph and cefo.
The contents were mixed well and filtered. This prepared solution was diluted quantitatively with methanol for cefi and with double distilled water for ceph and cefo to
obtain a suitableconcentration for the analysis.
General recommended procedure. About 1mL of 100–600, 50–350 and 100–600 ng/mL for cefi, ceph and cefo, respectively, were transferredto a 10-mL calibrated
volumetric flask subsequently, 3mL of buffer solution pH 12.0 for cefi and 3mL of buffer solution pH 13.0 for ceph and cefo were added followed by 1mL of 0.025%
HPTS for cefi and 1mL of 0.02% HPTS solution was added for ceph and cefo. The solution was heated in a thermostat at 80 _C for 15 min and at 85 _C for 10 min and
at 60 _C for 15 min for cefi , ceph and cefo, respectively. The contents of the flasks were transferred quantitatively in to separating funnels and acidified with 0.5 mL of
0.1 M HCl for cefi and with 1mL of 0.1 M HCl for ceph and cefo. The solutions were extracted with two portions (5 mL) of chloroform. The chloroform extract was diluted
to volume with chloroform and the fluorescence intensities of the resulting solutions were measured at 520, 500 and 510nm after excitation at 480, 470 and 480nm for
cefi, ceph and cefo respectively against a reagent blank prepared in the same manner. The extraction step was necessary because the aqueous HPTS possesses
fluorescence properties, and it was essential to avoid interference(22).
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Figure_4.cefo and cefi spectrum
Conclusions
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Figure_5.Excitation and emission spectrum of the drug
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A novel spectrofluorimetric method for the analysis of three cephalosporins is described using HPTS as a derivatizing
reagent. The proposed method is simple, sensitive, accurate and cost-effective. The described method has advantages
over previously reported spectrofluorimetric methods in terms of the simplicity and sensitivity. The proposed method is
fully validated and successfully applied for the analysis of several cephalosporin pharmaceutical formulations. This
method can be recommended for the analysis of cefi, ceph and cefo in quality control laboratories.
Figure_.6Effect of pH and job method
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