Objective: Simple and rapid methods have been developed for the determination of azithromycin, sparfloxacin and cephalexin monohydrate in bulk and pharmaceutical formulations. Method: These methods were based on the formation of bluish-green ion pair complexes of these antibiotics with bromophenol blue (BPB). Acetonitrile was used as a solvent for azithromycin and sparfloxacin whereas methanolacetonitrile medium for cephalexin monohydrate. Results: 2:1 complexes were formed between the drug and reagent almost instantaneously with absorption maxima 595 nm, 620 nm, 600 nm for the three drugs respectively. Different parameters such as effect of time, effect of reagent concentration were optimised. Under optimum conditions, calibration curves were found to be linear over the range of 0-50 μg mL-1 for azithromycin, 10-80 μg mL-1 for sparfloxacin and 10-170 μg mL-1 for cephalexin monohydrate respectively. The detection limits were found to be 0.10 μg mL-1, 0.21 μg mL-1 and 1.69 μg mL-1 with Sandell’s sensitivity 0.0559 μg cm-2, 0.1034 μg cm-2 and 1.3920 μg cm-2 respectively for the three drugs. Stability constant (log K) was found to be 6.19 ± 0.04, 5.00 ± 0.07 and 4.05 ± 0.05 showing high stability of the complexes. Molar absorptivity was found to be 1.369×104 L mol-1 cm-1, 3.774×103 L mol-1 cm-1 and 2.620×102 L mol-1 cm-1 with Gibb’s free energy change -2.725×103 kJ mol-1, -2.393×103 kJ mol-1 and -1.938×103 kJ mol-1. These methods were subjected to analytical quality control. Accuracy, precision, recovery and interference studies have been carried out. Conclusion: The proposed methods were successfully applied to the determination of these drugs in their pharmaceutical formulations and human urine samples.
Azithromycin is an important member of macrolide class of antibiotics. It is European Pharmacopoeia recommended antibiotic . Macrolides are a group of antibiotics that belongs to the polyketide class of natural products. Their activity stems from the presence of a macrolide ring, a 15-membered macrocyclic lactone ring to which one or more deoxy-sugars may be attached . Sparfloxacin is third generation quinolone antibiotic. It is official in Martindale extra Pharmacopeia . Due to the presence of fluorine atom at C-6 position of quinolone, this clinically useful quinolone is described as fluoroquinolone. It is active against both gram-positive and gram-negative bacteria. It is widely used to treat human and veterinary diseases [4,5]. Cephalexin monohydrate is first generation cephalosporin antibiotic. Mode of action of cephalexin monohydrate is same as that of β- lactam antibiotics; it inhibits the synthesis of peptidoglycan layer of bacterial cell wall . It is antibacterial, used as alternative to penicillin. It is useful for bone joint infections, pneumonia, urinary tract infection and its common side effect is intestinal upset. It is European Pharmacopoeia recommended antibiotic .
Bromophenol blue (BPB) is a triphenylmethane dye and is commonly used as indicator and spectrophotometric reagent. The structures of the three antibiotics and bromophenol blue are shown in Figure1.
¨Several methods have been reported in literature for the analysis of azithromycin, sparfloxacin and cephalexin monohydrate such as HPLC , spectrofluorometry , capillary electrophoresis , voltammetry , etc.
Spectrophotometric method is based on the formation of coloured (charge transfer or ion-pair) complex between drug and reagent which can be estimated by visible spectrophotometer. In ion-pair complex, ions of opposite electric charge are held together in solution by Coulomb attraction to form a distinct chemical entity. It behaves as a single unit. Ion pair formation, initially investigated by the physical chemistry has been found extremely interesting for the chemical analysis, including pharmaceutical analysis. Reported spectrophotometric methods of these three antibiotics includes complex formation with quinalizarin , eosin Y , rose Bengal , chloranilic acid and 7,7,8,8-tetracyanoquinodimethane , bromocresol purple and bromecresol green , ammonium vanadate , p-nitro phenol , Mo(V)-thiocyanate , p- dimethyl aminobenzaldehyde , molybdenum blue , Sodium 1,2-napthoquinone-4-sulphonate , p-benzaquinone , iodine, 2,3-dichloro-5,6-dicyano-1,4- benzoquinone and 7,7,8,8-tetracyanoquinodimethane .
The proposed methods are based on formation of ion pair complexes with bromophenol blue. Bromophenol blue has been used for the first time with significantly low detection limit, high sensitivity and wider dynamic range. An important feature of these methods is that no extraction is required and it is feasible at room temperature. These methods could be applied to the analysis of pharmaceutical formulations and urine sample.
Spectrophotometric studies were carried out with Spectronic 20D+ (Thermo-Spectronic) visible spectrophotometer. A Mettler balance H 51AR (Ner-Parma instrument Corp. L.C. = 0.01mg) was used for weighing purpose.
Materials and reagents
All chemicals and reagents used were of analytical grade. Azithromycin, sparfloxacin and cephalexin monohydrate were obtained from ZIM laboratories. Bromophenol blue, HPLC grade acetonitrile and methanol were obtained from LOBA Chemie.
Preparation of solutions
Stock solutions of azithromycin or sparfloxacin were prepared as 0.01M in acetonitrile while 0.01M cephalexin monohydrate solution was prepared in methanol whereas 0.01M bromophenol blue solution was prepared in acetonitrile. The solutions were further diluted as per requirement.
Procedure for calibration curve
Suitable aliquots of azithromycin or sparfloxacin solutions in acetonitrile or cephalexin monohydrate in methanol were transferred into 10 ml volumetric flasks. To it, 2 ml of 8x10-5 M bromophenol blue solution for azithromycin, 5ml of 8x10-5 M bromophenol blue solution for sparfloxacin and 6ml of 5×10-5 M bromophenol blue solution for cephalexin monohydrate was added and volume was made up to 10 ml with respective solvents. This made the final concentration of bromophenol blue to 16 μM, 40 μM and 30 μM respectively. After 15, 10 and 5 minutes, for three drugs respectively, the absorbance of bluish-green solution was measured at 595 nm, 620 nm and 600 nm against the appropriate reagent blank.
Procedure for dosage form
For analysis of tablets or capsules, five tablets or capsules were weighed and average weight of one tablet or capsule was determined. They were powdered and 0.05 g of azithromycin and 0.04 g of cephalexin monohydrate exactly weighed and shaken with 30 ml of acetonitrile and methanol respectively for 30 minutes. These solutions were filtered with Whatmann filter paper no. 40 and made up to 50 ml with respective solvents. The same procedure was applied for oral suspension of azithromycin using 1ml suspension. Suitable aliquots were analysed using general procedure.
Procedure for urine sample
The urine samples were collected from healthy volunteer. In order to analyse urine samples, 10 ml of urine samples were spiked with azithromycin and sparfloxacin separately. The drugs were extracted with dichloromethane and evaporated to dryness. The residue was dissolved in respective solvents and analysed using general procedure.
Effect of solvent
Various solvents like methanol, ethanol, acetone, dichloromethane, dichloroethane, dimethylsulphoxide, chloroform and acetonitrile were used to check the solubility, complex formation, to achieve maximum sensitivity and product stability. Acetonitrile for azithromycin, sparfloxacin and bromophenol blue and methanol for cephalexin monohydrate were found to be most suitable solvents.
Solutions of azithromycin or sparfloxacin and bromophenol blue in acetonitrile and cephalexin monohydrate in methanol were prepared. Absorption spectra of these solutions were recorded individually. When the drug solutions were mixed with BPB solution, bluishgreen complexes were formed with absorption maxima at 595 nm for azithromycin, 620 nm for sparfloxacin and 600 nm for cephalexin monohydrate respectively (Figure 2). Under experimental conditions, the reagent as well as the drug showed negligible absorbance while the complexes showed maximum absorbance at these wavelengths. Hence, it was concluded that the studies for quantitative analysis could be carried out at these wavelengths.
Stoichiometric relationship and stability studies
Composition and stability constants of these complexes were established by applying Job’s method of continuous variation. Equimolar solutions of the drug and the reagent were mixed in various proportions and absorbance of each mixture was recorded. The results indicated that the complexes are formed in the ratio of 2:1 (D:R) (Figure 3). Mechanism of formation of such complexes with composition (DH)+2 (R)2- has been discussed by Gainza and Konyeaso . The suggested structure of ion pair complex of azithromycin can be shown as in Figure 4. Similar structures can be suggested for sparfloxacin as well as for cephalexin monohydrate. The stability constants (log K) values were found to be 6.19 ± 0.04 for azithromycin, 5.00 ± 0.07 for sparfloxacin and 4.05 ± 0.05 for cephalexin monohydrate respectively showing high stability of the complexes. The large negative values of Gibb’s free energy change for complex formation show spontaneity of process (Table 1).
|Molar ratio (D:R)||2:1||2:1||2:1|
|Linear range (μg mL-1)||0-50||10-80||10-170|
|Sandell’s sensitivity (μg cm-2)||0.0559||0.1034||1.3920|
|Stability constant (log K)||6.19 ± 0.04||5.00 ± 0.07||4.05 ± 0.05|
|Molar absorptivity (L mol-1 cm-1)||1.369×104||3.774×103||2.620×102|
|LODa(μg mL-1 )||0.10||0.21||1.69|
|LOQb (μg mL-1 )||0.35||0.71||5.63|
|%RSDc(at 10 μg mL-1)||0.55||0.33||3.06|
aLimit of detection
bLimit of Quantitation
cRelative standard deviation
dFree energy change
Table 1: Qualitative and statistical parameters for proposed methods.
Effect of time
Mixtures of drug and reagent were prepared; the optimum reaction time was determined by recording the absorbance of the formed complexes at different time intervals. The variation has been shown in Figure 5. It was found that the complexes were formed instantaneously at room temperature. The absorbance was found to be steady after 15, 5 and 20 minutes for azithromycin, sparfloxacin and cephalexin monohydrate respectively. Hence, in order to remove time effect, all observations were made after respective time interval of complex formation. The sample solutions were kept in air tight flasks to avoid any evaporation losses.
Effect of reagent concentration
The optimum concentration of bromophenol blue was determined by adding various concentrations of bromophenol blue to the drugs. The colour intensity was found to increase with addition of bromophenol blue up to a particular concentration and then either decrease or remain steady. The absorbance was found to be maximum at bromophenol blue concentration of 16 μM for azithromycin, 40 μM for sparfloxacin and 30 μM for cephalexin monohydrate. Therefore, these concentrations were used to prepare calibration curve (Figure 6).
All the observations were made in triplicate and mean of the three values have been plotted in each graph.
Calibration curves for azithromycin, sparfloxacin and cephalexin monohydrate were plotted between absorbance and concentration. A linear absorbance-concentration correlation was found to be 0-50 μg mL-1, 10-80 μg mL-1 and 10-170 μg mL-1 with correlation coefficients 0.9871, 0.9967 and 0.9992 respectively for the three drugs. The molar absorptivity values were found to be 1.369×104, 3.774×103 and 2.620×102 L mol-1 cm-1 respectively. The limit of detection and limit of quantitation were calculated in accordance with equations,
LOD = 3σ/S
LOQ = 10σ/S
The σ is the standard deviation of the response and S is the slope of calibration graph. The detection limits were found to be 0.10, 0.21 and 1.69 μg mL-1 respectively with Sandell’s sensitivity 0.0559, 0.1034 and 1.3920 μg cm-2, respectively. Under optimum conditions, various analytical parameters were obtained (Table 1). The value of correlation coefficient indicates good linearity for all the three systems. Values of molar absorptivities and Sandell’s sensitivities reflect high sensitivity of these methods
Recovery studies were carried out for all the three drugs using calibration curve at three different concentrations over the linear range. The recoveries were found to be in vicinity of 100% (Table 2).
|Drug||Taken (μg mL-1)||Founda(μg mL-1)||%Recovery||Mean±SD|
|Azithromycin||10.0||10.5||105.0||103.6 ± 1.3|
|Sparfloxacin||25.0||26.0||104.0||100.6 ± 4.4|
|Cephalexin monohydrate||55.0||53.0||96.4||98.6 ± 2.3|
aAverage of three determinations.
Table 2: Recovery studies of the three drugs.
Accuracy and precision
Accuracy expresses the closeness between the reference value and the found value. It was evaluated as percentage relative error between the measured concentrations and taken concentrations of these three drugs. The precision of these methods were calculated in terms of intermediate precision (intra-day and inter-day). Three concentrations of all the three drugs were analysed in three replicates during same day (intra-day precision) and three consecutive days (inter-day precision). RSD (%) values of intra-day and inter-day studies show high degree of precision (Table 3).
|Drug||Taken (µg mL-1)||Intra-day (n=3)||Inter-day (n=3)|
|Founda (µgmL-1)||% RSD||%RE||Founda (µg mL-1)||% RSD||%RE|
aAverage of three determinations
% RSD: Relative standard deviation
%RE: Relative error
Table 3: Evaluation of intraday and interday precision and accuracy.
Robustness and ruggedness
The robustness of all the three systems were evaluated by making small incremental changes in time (25 ± 5 min) and the effect of the change of absorbance were studied on the ion-pair complex. It was found that the changes had negligible influence on the results and expressed as % RSD values. The ruggedness of these methods were evaluated by performing analysis using two different cuvettes and expressed in % RSD values as shown in Table 4.
|Sample||Taken (μg mL-1)||Robustness Reaction timea (n=3)% RSD||Ruggedness Inter cuvettes (n=2)% RSD|
a Reaction time- 20,25 and 30 min
Table 4: Evaluation of robustness and ruggedness.
The effect of common excipients and other additives were tested for possible interferences in the assay. Various amount of excipients such as lactose, dextrose, cellulose, magnesium stearate, talc, starch, gelatine were added to known amount of three drugs and were examined using developed procedure. It was found that these compounds have negligible solubility in acetonitrile and methanol therefore; they did not interfere in the determination of these three drugs even when present 100 times in excess. To evaluate the selectivity of the proposed method of analysis in pharmaceutical formulations, placebo blank was compared with synthetic mixture and the results are incorporated as % RSD (Table 5).
|Excipients||Amount added (mg)||Azithromycina
% Recovery ± SD
% Recovery ± SD
|Lactose||20||99.3 ± 0.7||98.2 ± 0.3|
|Dextrose||20||98.4 ± 0.9||99.5 ± 0.7|
|Cellulose||20||98.5 ± 0.8||101.1 ± 0.6|
|Magnesium stearate||20||101.2 ± 0.4||99.4 ± 0.4|
|Talc||20||98.4 ± 0.4||100.2 ± 0.4|
|Starch||20||99.5 ± 0.2||99.4 ± 0.7|
|Gelatine||20||100.2 ± 0.5||98.8 ± 0.6|
a40 (μg m L-1) of azithromycin
b100 (μg m L-1) of cephalexin monohydrate
Table 5: Determination of drugs in presence of excipients.
The proposed methods have been successfully applied for the determination of azithromycin and cephalexin monohydrate in their pharmaceutical formulation such as tablets, capsules and oral suspension. Calibration curve method and standard addition method were adopted for quantitative analysis (Table 6). Urine samples were successfully analysed with azithromycin and sparfloxacin. Quantitation was carried out using calibration graph (Table 7).
|Pharmaceutical Preparation||Labelled Amount (mg)||Found Amount (mg)a|
|Lazithro tablets b||250||249.7 ± 2.7||247.6 ± 3.4|
|Azee tablets c||250||260.7 ± 6.9||249.3 ± 1.1|
|AzithralLiquidd||20||20.2 ± 1.2||21.1 ± 1.8|
|Phexincapsulese||500||493.1 ± 3.0||499.1 ± 5.9|
|Cefftabletsf||250||247.6 ± 3.0||251.7 ± 1.9|
|Phexin oral suspensiong||250||254.5 ± 1.6||245.2 ± 2.7|
a(Avg ± SD) of three determinations
dAlembic Pharmaceutical Limited
e,g GlaxoSmithKline Limited
f Lupin Limited
Table 6: Analysis of pharmaceutical formulations.
|Drug||Taken (μg mL-1)||Founda (μg mL-1)||% Recovery||% RSD|
aAverage of three determinations
%RSD Relative standard deviation
Table 7: Analysis of spiked urine samples.
The developed methods were found to be versatile and have many advantages over the previously reported methods. A comparison of these methods with reported methods have been presented in (Table 8). It has been observed that, bromophenol blue methods have wider linear ranges as compared to most of the reported methods [11,12,14-16,20-23]. These proposed methods are more sensitive compared to some of the established method as shown by the molar absorptivity . The detection limits are lower than the reported methods [11,13,18,19,23].
|Drug||Reagent used||Linear range
(L mol-1 cm-1)
|Eosin Y||1-10||--||--||Human urine, plasma||13|
|Rose Bengal||4-20||0.31||3.78× 104||Tablets, capsules, syrup||14|
|Bromophenol blue||0-50||0.10||1.369×104||Tablets, syrup, Human Urine||Present work|
|Ammonium vanadate||0.8-28||--||--||Tablets, Human Urine||17|
|p- dimethyl amino-benzaldehyde||2-80||0.22||4.9×103||Tablets, Human urine, Blood serum||20|
|Bromophenol blue||10-80||0.21||3.774×103||Human Urine||Present Work|
|Cephalexin monohydrate||Molybdenum blue||0-45||--||--||Capsules||21|
Table 8: Comparison of present work with reported methods.
These methods require only dye and solvents which are comparatively cheaper and readily available. These methods are simple as they do not involve adjustment of critical conditions like temperature, pH or tedious sample preparation. These methods have many advantages over other analytical methods due to its simplicity, sensitivity, rapidity, low cost instrumentation, accuracy, free from interference by common additives and excipients. Due to these advantages these methods can be used for quality control and routine analysis.
The authors are thankful to UGC, New Delhi for financial assistant under Start-up- Grant and RTM Nagpur University for University Research Scheme.