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The Use of Chloranilic Acid for the Spectrophotometric Determination of Three Macrolides through Charge Transfer Complex | OMICS International
ISSN: 2161-0444
Medicinal Chemistry

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The Use of Chloranilic Acid for the Spectrophotometric Determination of Three Macrolides through Charge Transfer Complex

Najma Sultana1, Saeed Arayne M2 and Saeeda Nadir Ali2*

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi-75270, Pakistan

2Department of Chemistry, University of Karachi, Karachi-75270, Pakistan

*Corresponding Author:
Saeeda Nadir Ali
Department of Chemistry
University of Karachi
Karachi-75270, Pakistan
E-mail: [email protected]

Received date: June 13, 2013; Accepted date: July 25, 2013; Published date: July 27, 2013

Citation: Sultana N, Saeed Arayne M, Ali SN (2013) The Use of Chloranilic Acid for the Spectrophotometric Determination of Three Macrolides through Charge Transfer Complex. Med chem 3:241-246. doi:10.4172/2161-0444.1000146

Copyright: © 2013 Sultana N, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

In the present study, simple and fast spectrophotometricmethod have been reported for the determination of three macrolides i.e., erythromycin, roxithromycin and clarithromycin through charge transfer complexes. The method involves the interaction of macrolides with chloranilic acid in acetonitrile medium. Stoichiometry was found to be 1:1 for all the complexes. Under the optimizedconditions, the complexes were found to be absorbed at 498, 496 and 491 nm with in the linearity range of 3-36, 4-40 and 8-40 μg mL -1 with minimum detection limit 190, 600 and 370 ng mL -1 for respectively. The corresponding molar absorptivity values were determined to be 2.07×10 4 , 1.81×10 4 and 1.67×10 4 Mol -1 cm -1 respectively. The data is discussed in terms of oscillator’s strength, dipole moment, ionization potential, energy of complexes, resonance energy, association constant and Gibb’s free energy changes. Benesi- Hildebrand plots for all complexes have been constructed. Furthermore, the methods were successfully applied for the determination of studied macrolides in pharmaceutical formulations. The interday and intraday precision and percent recovery values were evaluated. Results of analysis were validated successfully. Commonly present excipients did not show interference during analysis

Keywords

Charge transfer complexes; Macrolides; Chloranilic acid; Benesi-Hildebrand plots

Introduction

Macrolides (Figure 1), a broad spectrum antibiotic drugs, belong to polyketide class of natural products, consisting of usually 14, 15 or 16-membered macrocycliclactone ring attached with one or more deoxy sugars, usually cladinose and desosamine. They are primarily used against gram-positive cocci and intracellular pathogens such as mycoplasma, chlamydia, campylobacter, legionella and prescribed to treat infectionsof the respiratory tract, genital, gastrointestinal tract and soft tissue infections which occur by strains of bacteria susceptible to this class of antibiotics. Macrolides are the less toxic preparations among other antibacterial drugs [1].

medicinal-chemistry-Chemical-structures

Figure 1: Chemical structures of (I) ERY, (II) ROX and (III) CLR.

Various analytical methods have been reported for the determination of studied macrolides. Flurer reported the determination of erythromycin (ERY) and clarithromycin (CLR) by capillary electrophoresis [2]. Lalloo et al. reported the separation of ERY with other macrolides by capillary electrophoresis [3]. The have been determined by Spectrofluorimetry [4,5], liquid chromatography [6-9] by near infrared reflectance (NIR) spectroscopy [10,11]. A number of spectrophotometric methods have also been employed for the determination of macrolides [12]. ERY has been determined using 1, 2-naphthoquinone-4-sulphonate [13], gentiana violet [14] CLR has been determined spectrophotometrically by using bromothymol blue and cresol red [15], p-dimethylamino benzaldehyde [16]. Recently a review article has been published by Bekeke and Gebeyehu describing the reported analytical methods and microbial assay for the determination of studied macrolides [1].

In the last decade, a number of spectrophotometric methods for the determination of verapamil [17], gabapentin [18], quinolone antibiotics [19], metformin [20], ascorbic acid [21] and montelukast [22] have been developed by our research fellows. In the present study we aimed to describe the rapid and accurate spectrophotometric methods for the determinationof three macrolides; ERY, roxithromycin (ROX) and CLR. Since macrolides do not have sufficient chromophoric groups, which enable this group of compound to be determined directly by spectrophotometer, therefore the analysis has been carried out by charge transfer complexes of these macrolides with chloranilic acid (ChA). The optimum reaction conditions of the developed methods have been established, besides, the oscillator strength (f), dipole moment (μ), ionization potential (Ip), energy of charge transfer complex (ECT) and resonance energy (RN) and also the association constant (Kc) and standard free energy changes (ΔG°) have been evaluated. Furthermore, the method was successfully applied for the determination of studied macrolides in pharmaceutical formulations. Excipients of formulations did not found to interfere in the assay of macrolides in pharmaceutical formulations.

Experimental

Materials and reagents

Pure ERY and CLR were obtained from Abbott Laboratories Pakistan Ltd and ROX was a kind gift from Aventis Pharma Pakistan Ltd. Erythrocin® 100 mg tablets (Abbott Laboratories Pakistan Ltd), Rithmo® 250 mg (Sami Pharmaceuticals (PVT) Ltd) and Claritek® 250 mg (Getz Pharma Pakistan (PVT) Ltd) were purchased from local market. ChA was purchased from Merck Schuchardt OHG, Darmstadt, Germany. Analytical grade acetonitrile was used throughout.

Instrument

Shimadzu model 1800 double beam UV-visible spectrophotometer provided with 1 cm quartz cells connected with Pentium IV computer loaded with version 2.32 software.

Stock standard solutions

1 mg mL-1 stock solution of each drug was separately prepared in analytical grade acetonitrile. Working standard solutions were prepared by further dilution of these solutions with same solvent. 0.1% ChA solution was prepared in acetonitrile.

Calibration curves

Serial volumes of stock solutions ranging from 0.3-3.6, 0.8-4.0 and 0.4-4.0 mL ERY, ROX and CLR were transferred to 10 ml volumetric flasks. To each flask 0.5 ml ChA was added and the volume was brought to mark by adding acetonitrile. The absorbance was measured against reagent blank prepared similarly. Calibration graph in each case was prepared by plotting absorbance vs. concentration of each macrolides.

Pharmaceutical formulation

Twenty tablets of each formulationwere separately weighed and finely powdered into pestle and mortar. An accurately weighed portion of powder equivalent to 100 mg of drug was dissolved in acetonitrile and shaken well for proper mixing. The contents were allowed to stand for 30 min and then sonicated for complete extraction of drugs. The residue were filtered and washed. Finally, the volume was made up to 100 mL with same solvent. The measurements were carried out according to the procedure described under the preparation of calibration graphs.

Results and Discussion

Absorbance spectra

Macrolides belongs to an interesting drug class called antibiotics. They are reported to take part in many of the chemical reactions [23,24]. In the present study, we aimed to develop charge transfer complexes of macrolides with ChA. ChA showed strong red color, giving maximum absorbance at 430 nm in the acetonitrile medium [25]. The interaction of studied macrolides with ChA show wavelength apart from reagents alone. The newly formed complexes showed pink color for ERY and CLR and light purple color for ROX which gives absorptionmaxima at 498, 496 and 491 nm respectively against reagent blank prepared under identical conditions. Figure 2 represents the electronic absorption spectra of complexes and the proposed reaction is illustrated in Scheme 1.

medicinal-chemistry-UV-Spectra

Figure 2: UV Spectra of ERY (−), ROX (…) and CLR (---).

medicinal-chemistry-Schematic-diagram

Scheme 1: Schematic diagram of reaction of macrolides with ChA.

Optimization of experimental conditions

In order to establish the optimum reaction conditions suitable for the complexation, various analytical parameters were studied. The effect of each parameter was observed by altering one parameter at a time while keeping others constant. A number of analytical solvents were checked like methanol, acetonitrile, dichloromethane, acetone, dimethyl sulfoxideand water, acetonitrile was found to give high sensitivity and maximum absorbance. To study the optimum reaction time the absorbance of complexes were measured 0, 2, 5 and 10 min. It was noticed that complete color development was achieved instantaneously at ambient temperature (25 ± 2°C) and there was no effect on absorbance at different time interval (Figure 3). The formed color complexes were found to be stable for 24 h for all three complexes. To determine the effect of ChA concentration, the concentration of each macrolides was kept constant and the concentration of ChA was varied by varying the mL of stock solution. Above 0.28, 0.24 and 0.27 mL of ChA for ERY, ROX and CLR respectively, there was no effect on absorbance of complex, therefore, 0.5 mL ChA was found to be sufficient for complete complexation (Figure 4).

medicinal-chemistry-complexation

Figure 3: Effect of time on ChA complexation with (1) ERY (15 μg mL-1), (2) ROX (14 μg mL-1) and (3) CLR (16 μg mL-1).

medicinal-chemistry-reagent-concentration

Figure 4: Effect of reagent concentration on ChA complexes with (1) ERY (15 μg mL-1), (2) ROX (12 μg mL-1) and (3) CLR (20 μg mL-1).

Stoichiometric ratio of complexes

The stoichiometric ratio of macrolides and ChA was established by applying Job’s method of continuous variation using equimolar solutions [26] by taking absorbance of complex solutions of different ratios (0:10, 1:9, ....10:0) (donor:acceptor). The graph was plotted between the mole fractions of each drug vs. absorbance. Figure 5 indicates that all the macrolides interacted with ChA in stoichiometric ratio of 1:1.

medicinal-chemistry-plot-complexes

Figure 5: Job’s plot for complexes of ERY (♦), ROX (?) and CLR (?) with ChA.

Linearity, accuracy and precision

Under the described analytical conditions, standard calibration curves for each macrolides with ChA were constructed over the range 3-36, 4-40 and 8-40 μg mL-1 for ERY, ROX and CLR respectively by plotting absorbance verses concentration of drug. The correlation coefficient in each case is greater than 0.998 indicating good linearity. The corresponding molar absorptivity values have been calculated, which were determined to be 2.07×104, 1.81×104 and 1.67×104 Mol-1 cm-1 respectively. Linearity, correlation coefficient, slope, intercept, molar absorptivity, limit of detection and quantitation are listed in Table 1. The high values of molar absorptivity of the resulting colored complexes indicate the high sensitivity of the methods. High value of correlation coefficient confirms the best linearity of the calibration curve.

Parameters Linearity
(µgmL-1)
εmax x104 Slope x10-2 Intercept R2 LOD (µgmL-1) LOQ (µgmL-1)
ERY-ChA 3-36 2.07 2.78 0.0032 0.9990 0.19 0.59
ROX-ChA 4-40 1.81 2.14 0.0034 0.9995 0.60 1.82
CLR-ChA 8-40 1.67 2.39 -0.0226 0.9984 0.37 1.12

Table 1: Analytical data for the reaction of the macrolides with ChA.

In order to study the accuracy and precision of the proposed method, five concentration of each drug within the linearity range were selected and analyzed. Precision of the method was evaluated by inter-day and intra-day percent relative standard deviation which was found to be in the range of 0.23-1.71, 0.17-0.77 and 0.07-0.38 respectively. The results indicate that the methods have good repeatability and reproducibility (Table 2). Accuracy of the method was established in terms of percent recovery values and percent error in dosage formulation. The percent relative error was found to be in the range of 0.23-1.96, 0.18-0.52 and 0.04-0.28 respectively (Table 3).

Complex Added (µgmL-1) Found (µgmL-1) % Rec % Err %RSD
ERY-ChA 3 3.06 101.96 1.96 1.71
6 6.01 100.23 0.23 0.23
10 10.10 101.04 1.04 0.95
15 15.08 100.54 0.54 0.56
27 27.09 100.33 0.33 0.42
ROX-ChA 6 5.97 100.34 -0.52 0.50
8 7.97 99.48 -0.35 0.77
10 9.98 99.65 -0.18 0.35
12 11.94 99.82 -0.49 0.17
14 13.95 99.51 -0.35 0.58
CLR-ChA 8 8.01 100.17 0.17 0.15
12 12.00 99.96 -0.04 0.07
16 16.03 100.18 0.18 0.19
20 20.06 100.28 0.28 0.38
24 24.04 100.18 0.18 0.16

Table 2: Evaluation of accuracy and precision of the method in pure drug.

Pharmaceutical preparations Taken (µg/mL) Found (µg/mL) Mean % Rec
Erythrocin 100 mg 15 15.03 100.45
19 18.98 99.82
23 23.14 100.84
Rulid® 50 mg 8 8.23 101.82
12 12.37 101.86
16 15.82 99.40
Claritek 250mg 24 23.96 99.49
28 27.81 99.37
32 31.97 99.72

Table 3: Recovery of macrolides in pharmaceutical formulations by the proposed method.

Sensitivity

Limit of detection and quantitation were determined to establish the sensitivity of method. These were found to be 0.19, 0.60, 0.37 and 0.59, 1.82, 1.12 for ERY, ROX and CLR respectively (Table 1).

Interference of excipients

Accurately weighed 10 mg lactose monohydrate, microcrystalline cellulose, magnesium stearate, starch and talc were separately transferred into 50 mL volumetric flask and small amount of acetonitrile was added, the contents were sonicated for complete mixing, then the volume was finally brought to the mark with the same solvent and filtered. Aliquot of excipient solutions were spiked with drug solutions and absorbance were measured. The results were not affected in presence of commonly encountered excipients. Good recovery values (Table 4) obtained confirms the sensitivityof method.

Excipients ERY ROX CLR
Lactose monohydrate 98.08 102.09 100.25
Microcrystalline cellulose 99.27 101.98 98.54
Magnesium stearate 99.52 101.96 98.71
Starch 99.30 102.09 98.09
Talc 99.54 99.78 101.24

Table 4: Recovery of macrolides in presence of common excipients.

Application of the proposed method

The proposed method was applied successfully for the determination of studied macrolides in commercial tablets by five replicate determinations. The results summarized in Table 3, agree well with the proposed method confirming the good accuracy and precision of the method. Satisfactory recovery data in the range of 99.82-100.84, 99.40-101.86 and 99.37-99.72 indicated the reliability of method. Commonly present excipients did not found to interfere during the assay.

Spectral characteristics

From the absorption spectra of each complex, different spectral characteristics were determined. Oscillator’s strength (f) [27] and transition dipole moment (μ) [28] were calculated using the formulae f = (4.319 x 10-9) εmax.?1/2 and μ = 0.0958 (εmax.?1/2 /?max)1/2. The ionization potential (Ip) of free donor [27] in acetonitrile medium was determined using the equation Ip = 5.76 + 1.53 x 10-4 ?CT. Resonance energy (RN) [29] and energy of charge transfer complexes (ECT) [30] were calculated by employing the equations εmax = 7.7×10-4/ [h?CT/ RN-3.5] and ECT = 1243.667/λCT, where εmax is the molar extinction coefficient at maximum absorbance, ?1/2 is the band-width at half absorbance in cm-1, ?max and ?CT are wave number in cm-1 and λCT is the wavelength of charge transfer band. The obtained data is summarized in Table 5.

Parameters λmax (nm) f µ IP(eV) ECT (eV) Rn K (L.mol-1) -ΔG° (KCal)
ERY-ChA 498 11.18 34.39 8.83 2.50 0.71 2.07 × 103 7.54
ROX-ChA 496 10.29 15.57 8.84 2.51 0.72 4.20 × 102 5.97
CLR-ChA 491 11.95 35.31 8.88 2.53 0.72 2.14 × 102 5.30

Table 5: Spectrophotometric results of macrolides-ChA complexes in ACN solvent at 25°C.

The association constant of the complexes (Table 5) was determined by using Benesi-Hildebrand equation [31], [A0]/A = 1/K [D0]. ε + 1/ε for cells with 1 cm optical path length, where, [A0] and [D0] are the initial concentrations of the acceptor and donor respectively, A is absorbance of definite charge transfer band, ε is molar extinction coefficient and K is the association constant. The concentration of acceptor is much greater then that of donor. On plotting the values of [A0]/A against 1/ [D0], sharp straight lines were obtained as shown in Figure 6. The data obtained throughout this calculation is given in Table 6.

Complex D x 10-5 (M) A x 10-3 (M) Abs 1/D x 105 A/Abs x 10-2
ERY-ChA 0.81 4.78 0.1707 1.22 2.80
1.36 4.78 0.2871 0.73 1.67
2.04 4.78 0.4068 0.49 1.18
2.59 4.78 0.5387 0.39 0.89
3.10 4.78 0.6345 0.32 0.75
3.60 4.78 0.7589 0.27 0.63
ROX-ChA 0.47 4.78 0.1720 2.09 2.78
0.59 4.78 0.2167 1.67 2.21
0.71 4.78 0.2621 1.39 1.82
0.83 4.78 0.3009 1.19 1.59
0.95 4.78 0.3465 1.04 1.38
2.39 4.78 0.8211 0.41 0.58
CLR-ChA 1.07 4.78 0.0882 0.94 2.73
1.60 4.78 0.1346 0.62 1.80
2.14 4.78 0.1720 0.47 1.39
2.67 4.78 0.2167 0.37 1.05
3.21 4.78 0.2621 0.31 0.86
3.74 4.78 0.3009 0.27 0.73

Table 6: The values of [A0]/Abs and 1/[D0] for macrolides complexes.

medicinal-chemistry-BH-plot

Figure 6: BH plot for complexes of (1) ERY, (2) ROX and (3) CLR with ChA.

The standard free energy changes (ΔG°) associated with macrolides complexes were calculated from the association constants by applying equation [32] ΔG° = -2.303RT log KC, where R is the gas constant (1.987 cal mol-1 deg-1), T is temperature in Kelvin and Kc is the association constant of drug-acceptor complexes. The values of ΔG° are given in Table 5.

Conclusion

The present work describes validated spectrophotometric methods based on charge transfer complexation for the determination of macrolides with ChA in bulk drug and in pharmaceutical formulations. The proposed method is simple, sensitive, inexpensive and reproducible for the determination of ERY, ROX and CLR. Spectral characteristics including oscillator’s strength, dipole moment, ionization potential, energy of complexes, resonance energy, association constant and Gibb’s free energy changes have been determined. The statistical parameters and the recovery data reveal good accuracy and precision of the methods. The method is free from interferences of the common excipients. Benesi-Hildebrand plots for each complex have been constructed.

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