1. INTRODUCTION:

Macrolides are a group of drugs (typically antibiotics) whose activity stems from the presence of a macrolide ring, a large macrocyclic lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. The lactone rings are usually 14-, 15-, or 16-membered.The mechanism of action of macrolides is inhibition of bacterial protein biosynthesis, and they are thought to do this by preventing peptidyltransferase from adding the peptidyl attached to tRNA to the next amino acid(1) (similarly to chloramphenicolas well as inhibiting ribosomal translocation.

Clarithromycin. several methods have been developed for its determination, including spectrophotometric methds (2-5), high-performance liquid chromatography (HPLC) (6-8) electro chemical methods (9).

Lincosamide antibiotics are one of the classes of antibiotics most associated with pseudomembranous colitis caused by Clastridium difficile. Lincosamides prevent bacteria replicating by interfering with the synthesis of proteins. They bind to the 23s portion of the 50S subunit of bacterial ribosomes and cause premature dissociation of the peptidyl-tRNA from the ribosome (14).

From this group we study clindamycin. Several methods have been developed for its determination, including spectrophotometric methods (10-11), high-performance liquid chromatography (HPLC)(12-14), Electro chemical methods (15).

Ion association (ion pairing) is based on the formation of associates composed of colorless (or colored) analyte ion and the colored (or colorless) reagent ion of opposite charge (counter ion).The absorption of these associates can be measured either directly in the reaction medium provided that the absorption maximum is different enough from the absorption maximum of the reagent or after extraction from the aqueous solutions into an organic solvent immiscible with water.

In this part, two acidic dyes, bromocresol purple and Bromocresol green have been used to form ion pair extractable complexes with clarithromycin and clindamycin HCl.

2. EXPERIMENTAL:

2.1. Apparatus:

UV-VIS Labomed^® Spectro UV-VIS Double Beam (UVD-2950) Spectrophotometer with matched 1 cm quartz cells connected to windows compatible computer using UV Win 5 Software v5.0.5(USA).

2.2. Materials and reagents:

All solvents and reagents were of analytical grade and double distilled water was used throughout the work. Clarithromycin. Standard stock solution 500 µg.ml^-1were prepared by dissolving pure drug in 25ml of methanol then completing to 100 ml with bidistilled water and for molar ratio 1.8x10^-3M . Standard working solution (8 ml from stock solution in 100ml measuring flask and complete to 100 ml with bidistilled water to be 40 µg.ml^-1in case of bromocresol purple method) or (4 ml and complete to 100ml with bidistilled water to be 20 µg.ml^-1in case of bromocresol green method)

Clindamycin HCl. Standard stock solutions 500 µg m^l-1and for molar ratio 1.8x10^{-3 .}Standard working solution (8 ml from stock solution in 100ml measuring flask and complete to 100 ml with bidistilled water to be 40 µg.ml^-1in case of bromocresol purple method) or (6.4 ml and complete to 100ml with bidistilled water to be 32 µg.ml^-1in case of bromocresol green method).Phosphate buffer solution of pH values 3 – 7 were prepared as in recommended methods⁽¹⁶⁾. Bromocresol purple (Aldrich Chemical Co. Ltd., Dorset, England) 1.8x10^-3M in 30 % methanol solution as stock solution (stable for 2 weeks at least). Bromocresol Green(Aldrich Chemical Co. Ltd., Dorset, England) 1.8x10^-3M in 30 % methanol solution as stock solution (stable for 2 weeks at least). Methylene chloride (El-Nasr Pharmaceutical Chemicals).

2.3. Pharmaceutical preparations:

The following available preparations were analyzed: Clindam^® tablets labeled to contain 150 mg Clindamycin per tablet. Batch No. 11318 (Sigma, Egypt), Clarihro^® tablets labeled to contain 250 mg Clarithromycin per tablet. Batch No. 502102 (Amriya, Egypt).

2.4. General spectrophotometric procedures and construction of calibration curves using Bromocresol purple method:

Aliquot portions of Clarithromycin and Clindamycin HCl ranging from (0,3 -0.7 ml) in case of clarithromycin and (0,2 – 1 ml) in case of clindamycin were transferred into a series of 125-ml separating funnels. To these, 1 ml of phosphate buffer in case of clarithromycin and 2ml in case of clindamycin then 1 ml of bromocresol purple dye was in case of clarithromycin and 2ml in case of clindamycin added. The pH of phosphate buffer adjusted to be 4 in case of clarithromycin and 6 in case of clindamycin and 5 ml of Methylene chloride was added twice. The contents were shaken vigorously for 5 minutes. The two phases were allowed to separate and the organic layer was collected in 10 ml flask completed to the mark and the absorbance of the yellow colored extract was measured at 390 nm and 397 nm (in case of clarithromycin and clindamycin HCl respectively) against a reagent blank.

2.5. General spectrophotometric procedures and construction of calibration curves using Bromocresol Green method:

Aliquot portions of Clarithromycin and Clindamycin HCl ranging from (0,2-1 ml) for clarithromycin or (0.5-1 ml) for clindamycin were transferred into a series of 125-ml separating funnels. To these, 1 ml of buffer solution in case of clarithromycin and 1ml in case of clindamycin than1ml of bromocresol green dye in case of clarithromycin and 2ml in case of clindamycin were added. The pH of phosphate buffer is 6 in case of clarithromycin and 4 in case of clindamycin and 5 ml of methylene choloride was added twice. The contents were shaken vigorously for 5 minutes. The two phases were allowed to separate and the organic layer was collected in 10 ml flask completed to the mark and the absorbance of the yellow colored extract was measured at 413 nm against a reagent blank.

2.6. Procedures for pharmaceutical preparations:

For Clarithro^® tablets:

10 tablets were weighed and powdered. A accurate weight of the powder equivalent to 134.6 mg of clarithromycin were dissolved in 25 ml of Methanol, filtered into 100-ml measuring flask and completed to volume with bidistilled water and this is the stock standard solution. From the stock solution we make the standard working solution and complete the procedures as previously mentioned under materials and reagents and the general procedures.

For Clindam^®tablets:

10 tablets were weighed and powdered. A accurate weight of the powder equivalent to 76.5 mg of Clindamycin HCl were dissolved in bidistilled water, filtered into 100-ml measuring flask and completed to volume with bidistilled water and this is the stock standard solution. From the stock solution we made the standard working solution and procedures as previously mentioned under materials and reagents and the general procedures.

2.7. Job’s method Procedures for determination of molar ratio:

For Bromocresol Purple method:

Clarithromycin and clindamycin HCl and bromocresol purple solutions of equimolar concentrations (1.8x10^-3 M) were prepared. Aliquots of each solution were added in different ratios so that the total volume of both was 5 ml in presence of the recommended buffer (pH 4 for clarithromycin and pH6 for clindamycin). Absorbance of the yellow colored extract was measured against reagent blank at the appropriate wavelength as shown in (fig.1).

Figure 1. Job’s method for molar ratio estimation of 1.9x10^{-3M bromocresol} purple with 1.8x10^-3M Clarithromycin and Clindamycin HCl.

For Bromocresol Green method:

Clarithromycin and clindamycin HCl and bromocresol green solutions of equimolar concentrations (1.8x10^-3M) were prepared. Aliquots of each solution were added in different ratios so that the total volume of both was 5 ml in presence of the recommended buffer (pH 6 for clarithromycin and pH 4 for clindamycin). Absorbance of the yellow colored extract was measured against reagent blank at the appropriate wavelength as shown in (fig. 2) .

Figure 2. Job’s method for molar ratio estimation of 1.4x10^{-3M Bromocresol} green with 1.8x10^-3M clarithromycin and clindamycin HCl.

3. RESULTS AND DISCUSSION:

The studied macrolides contain terminal nitrogen atom in pyrrolidine moiety for clindamycin or in dimethylamine group in clarithromycin. In proper acidic medium, this nitrogen atom is protonated to give positively charged quaternary ammonium group which in turn forms an ion pair complex with negatively charged dye containing sulphonic acid group. This complex is readily extractable in methylene chloride and measured at the appropriate wavelength.

The theoretical basis of this method is that the dissociation equilibrium of BA-type (which is dissociated in aqueous medium) can be shifted toward the left (association) if the ion pair is removed by extraction using a solvent immiscible with water⁽¹⁷⁾.

BA B⁺ + A^-

Where, B⁺is the protonated amine drug and A^-is the dye anion form.

3.1. Absorption spectra:

Absorption spectra of the reagents with clarithromycin and clindamycin HCl were studied over range of 200-800 nm. Bromocresol Purple reacts with clarithromycin, clindamycin HCl to yield an extractable yellow colored complex exhibiting maximum absorption at 390 nm and 397 nm respectively (Fig. 3). Also bromocresol green yields a yellow colored extractable complex with these drugs exhibiting maximum absorption at 413 nm (Fig. 4).

3.2. Effect of pH:

Figure 5. Effect of pH on ion-pair complex between bromocresol purple and 20 µg/ml clarithromycin and clindamycin HCl .

Variation in pH from 3.0 to 7.0 was investigated on the reaction of bromocresol purple and bromocresol green with concerned drugs. Maximum sensitivity is obtained at pH 4 (for clarithromycin) and pH 6 (for clindamycin) in case of bromocresol purpleand vice versa in case of bromocresol green (Fig. 5 and 6).

Figure 6.Effect of pH on ion-pair complex between bromocresolgreen and 20 µg/ml clarithromycin and clindamycin HCl .

3.3. Effect of Dye Concentration::

Effect of dye concentration on the intensity of absorption was studied by varying the dye concentration while other factors were held constant (Fig. 7 and 8).

Figure 7. Effect of dye volume of bromocresol purple on absorbance with 20 µg/ml clarithromycin and clindamycin HCl.

Figure 8. Effect of dye volume of bromocresol green on absorbance with 20µg/ml clarithromycin and clindamycin HCl.

3.4. Effect of Extracting solvent:

Different solvents have been tried in order to achieve maximum sensitivity and product stability such as Dichloromethane, chloroform, ethyl acetate, petroleum ether and toluene.

Dichloromethane and chloroform showed the highest sensitivity but dichloromethane was preferred as it is less toxic (10 times) and cheaper than chloroform⁽¹⁸⁾. To achieve maximum extraction, 5 ml of dichloromethane was added twice on two portions.

3.5. Effect of buffer volume;

The effect of buffer volume was studied using different volumes fig (9) and (10) .

Figure 9. Effect of buffer volume on ion-pair complex between bromocresol purple and 25 µg/ml clarithromycin and clindamycin HCl .

3.6. Effect of addition sequence:

Addition sequences were studied and results revealed that the most appropriate sequence for drugs with both dyes was drug, buffer and finally dye addition.

Figure 10. Effect of buffer volume on ion-pair complex between bromocresol green and 20 µg/ml clarithromycin and clindamycin HCl .

4. METHOD VALIDATION:

The developed methods were validated according to international conference on harmonization guidelines⁽¹⁹⁾.The linearity range of absorbance as a function of drug concentration (Tables 1 and 2) provides good indication about sensitivity of reagents used. Calibration curves have correlation coefficients (r) higher than 0.999 indicating good linearity. The accuracy of the methods were determined by investigating the recovery of drugs at concentration levels covering the specified range (three replicates of each concentration). The results showed excellent recoveries (Tables 3and 4). Also, the Limit of detection (L.O.D.), Limit of quantitation (L.O.Q.), Sandell’s sensitivity (S.S.) and Molar absorbitivity were calculated. Intra-day precision was evaluated by calculating standard deviation (SD) of five replicate determinations using the same solution containing pure drug. The SD values revealed the high precision of the methods. For inter - day reproducibility on a day - to - day basis, a series was run, in which the standard drug solutions were analyzed each for five days as shown in Table (7,8). The robustness of the methods was evaluated by making small changes in the pH of buffer, volume of buffer and dye volume where the effect of the changes was studied on the percent recovery of drugs. The changes had negligible influence on the results as revealed by small SD values as shown in Table (9,10).

5. APPLICATIONS:

Some Pharmaceutical formulations containing stated drugs have been successfully analyzed by the proposed methods. Results obtained were compared to those obtained by applying reported reference methods⁾ where Student’s t-test and F-test were performed for comparison. The reported reference method of clarithromycin⁽²⁰⁾ depends on formation of yellow colored chloroform extractable ion-association complexes of clarithromycin with bromo thymol blue (BTB) and cresol red (CR) in buffered aqueous solution at pH 4.

Table (1). Analytical parameters for the determination of Clarithromycin and Clindamycin HCl using bromocresol purple method.

Parameters	Bromocresol purple
Parameters	Clarithromycin	Clarithromycin	Clarithromycin
Wave length, nm	390	390	390
Volumof dye, ml	1	1	1
pH	4	4	4
Beer's law limits, µg/ml	12-28	12-28	12-28
Regression equation	y=0.020x-0.043	y=0.020x-0.043	y=0.020x-0.043
Correlation Coefficient	0.999	0.999	0.999
Molar Ratio	1:1	1:1	1:1
Volume of buffer ml	1	1	1

y = a + bx, where y is the absorbance, a is the intercept, b is the slope and x is the concentration in μg/ml.

Table (2). Analytical parameters for the determination of Clarithromycin and Clindamycin HCl using bromocresol green method.

Parameters	BromoCresol Green
Parameters	Clarithromycin	Clarithromycin	Clarithromycin
Wave length, nm	413	413	413
Volumof dye, ml	1	1	1
pH	6	6	6
Beer's law limits, µg/ml	4-20	4-20	4-20
Regression equation	y=0.034x-0.004	y=0.034x-0.004	y=0.034x-0.004
Correlation Coefficient	0.999	0.999	0.999
Molar Ratio	1:1	1:1	1:1
Volume of buffer ml	1	1	1

y = a + bx, where y is the absorbance, a is the intercept, b is the slope and x is the concentration in µg/ml.

Table (3). Results of the analysis for determination of, Clarithromycin and Clindamycin HCl using bromocresol purple method.

Parameters	Clarithromycin*			Clarithromycin*
Parameters	Taken µg/ml	Taken µg/ml	Taken µg/ml		Taken µg/ml	Taken µg/ml	Taken µg/ml
	12	12	12		12	12	12
	16	16	16		16	16	16
	20	20	20		20	20	20
	24	24	24		24	24	24
	28	28	28		28	28	28
Mean			100.19				100.19
±SD			0.3				0.911
±RSD			0.3				0.909
±SE			0.14				0.407
Variance			0.092				0.830
Slope			0.02				0.014
L.D.			3.93				2.65
L.Q.			11.8				7.95
S.S.			0.035				0.035
Apparent Molar absorbitivity L.Mol^-1.cm^-1			1.32X10⁷				1.31X10⁷

* Average of three independent procedures.

Table (4). Results of the analysis for determination of Clarithromycin and Clindamycin HCl using bromocresol green method.

Parameters	Clindamycin HCl *			Clarithromycin *
Parameters	Taken µg/ml	Found µg/ml	*Recovery %**	Taken µg/ml	Found µg/ml	*Recovery %**
	4	4	100	16	16.12	100.75
	8	8.12	101.47	20	20.28	101.4
	12	11.97	99.75	24	24.16	100.67
	16	16.15	100.92	28	28.16	100.57
	20	20.12	100.59	32	32.36	101.13
Mean			100.55			100.9
±SD			0.69			0.35
±RSD			0.69			0.35
±SE			0.31			0.16
Variance			0.48			0.12
Slope			0.034			0.025
L.D.			1.3			5.3
L.Q.			3.9			15.8
S.S.			0.018			0.039
Apparent Molar absorbitivity L.Mol^-1.cm^-1			2.5x10⁵			1.1x10⁵

* Average of three independent procedures.

Table (5). Statistical analysis of results obtained by the proposed methods applied on Clindamycin in the Clindam tablets compared with reference method.

Parameters	Bromocresol purple method	Bromocresol green method	Reported method⁽²¹⁾
N	5	5	5
Mean	100.44	100.06	99.84
S D	0.523	0.596	1.226
RSD	0.521	0.595	1.226
SE	0.234	0.243	0.550
Variance	0.274	0.355	1.051
Student-t	1.006 (2.57) a	0.360 (2.57) a
F-test	3.836 (6.256) b	2.960 (6.256) b

a and b are the Theoretical Student t-values and F-ratios at p=0.05.

Table (6). Statistical analysis of results obtained by the proposed methods applied on Claritromycin in the Clarithro^® tablets compared with reference method.

Parameters	Bromocresolpurple method	Bromocresol green method	Reported method⁽²⁰⁾
N	5	5	5
Mean	100.466	100.529	100.01
S D	0.446	0.553	1.353
RSD	0.444	0.550	1.353
SE	0.199	0.247	0.605
Variance	0.199	0.306	1.210
Student-t	0.720 (2.57) a	0.781 (2.57) a
F-test	6.080 (6.256) b	3.954 (6.256) b

a and b are the Theoretical Student t-values and F-ratios at p=0.05.

Table (8). The intraday and interday precision for the determination of clarithromycin and clindamycin HCl using bromocresol purple method.

Bromocresol purple				conc.ug/ml	Drug
Interday		Intraday
RSD	Mean± SD	RSD	mean + SD
0.44	100.09 ± 0.44	0.65	100.27 ± 0.65	20	Clarithromycin
0.51	100.01 ± 0.51	0.37	100.03 ± 0.37	20	ClindamycinHCl

Table (9( The intraday and interday precision for the determination of clarithromycin and clindamycin HCl using bromocresol green method.

Bromocresol green				Conc.ug/ml	Drug
Interday		Intraday
RSD	mean ±SD	RSD	mean + SD
0.85	99.99 ± 0.85	0.39	100.12 ± 0.39	20	Clarithromycin
0.68	100.12 ± 0.68	0.66	100.06 ± 0.66	20	ClindamycinHCl

Table (10). Robustness for the determination of clarithromycin and clindamycin HCl using bromocresol purple method.

% of recovery ± SD		Parameters
Clindamycin	Clarithromycin	Parameters
99.5 ± 0.3	100.3 ± 0.5	pH 5.05
100.88 ±0.31	99.5 ± 0.9	pH 4.95
100.54 ±0.53	100 ±0.5	buffer 0.95
99.81±0.23	99.6 ± 0.2	buffer 1.05
99.92 ±0.61	100.2 ± 0.5	dye 0.95
100.54±0.53	100.7 ± 0.6	dye 1.05

Table (11). Robustness for the determination of clarithromycin and clindamycin HCl using bromocresol green method.

% of recovery ± SD		Parameters
Clindamycin	Clarithromycin	Parameters
99.5 ± 0.3	100.3 ± 0.12	pH 5.05
100.54 ±0.78	100.12 ± 0.34	pH 4.95
100.41 ±0.67	100.54 ±0.53	buffer 0.95
100.41 ±0.67	99.61 ± 0.77	buffer 1.05
100.02 ±1.2	99.96 ± 0.76	dye 0.95
99.95 ±0.81	100.52± 0.62	dye 1.05

The extracted complexes showed maximum absorbance at 410 and 415 nm for BTB and CR, respectively. The reported reference method of clindamycin⁽²¹⁾ depends on oxidation of the sulfur atom in this drug with potassium iodate in acidic medium with the liberation of iodine and subsequent extraction with cyclohexane followed by measuring the absorbance at 520 nm. Results are shown in Tables 19 and 20 where the calculated t and F values were less than tabulated values which in turn indicate that there is no significant difference between proposed methods and reference ones relative to accuracy and precision.

6. CONCLUSION:

Unlike GC and HPLC techniques, spectrophotometry is simple and inexpensive. The proposed methods require only dyes as reagents which are cheaper and readily available and the procedures do not involve any critical reaction conditions or tedious sample preparation. Morever, both methods are simple, fast, accurate and adequately sensitive. The amounts obtained by the proposed methods are between 99.80% and 100.37%, within the acceptance level of 95% to 105%.

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Received on 09.11.2014 Accepted on 27.11.2014

Asian J. Res. Pharm. Sci. 4(4): Oct.-Dec. 2014; Page 179-186