INTRODUCTION:

Peptic ulcer disorder (PUD) is a major disorder of the gastro intestinal tract caused mainly by Helicobacter pylori a bacteria and NSAID’s although other factors like dietary habits, smoking and excessive intake of alcohol also responsible for PUD. Clarithromycin is a semisynthetic macrolide that exhibits broad-spectrum activity against gram-positive, negative aerobes and Helicobacter pylori. It is indicated for the treatment of duodenal ulcer disease due to H. pylori infection in adults and children¹. It is widely used in a standard eradication treatment of gastric H. pylori infections, where it is combined with an acid suppressing agent and a second antibiotic². Clarithromycin is a 14-membered semi synthetic macrolide antibiotic. Chemically, it is 6-0-methylerythromycin. The molecular formula is C₃₈H₆₉NO₁₃ (Fig.-1) and the molecular weight is 747.96. Clarithromycin is rapidly absorbed from the gastrointestinal tract after oral administration. The absolute bioavailability of 250 mg clarithromycin tablets was approximately 50%³ Clarithromycin has a t_1/2of 3.5±0.5 h with an oral bioavailability of 50% due to its hepatic first pass metabolism^{4, 5}.

It has a good stability in gastric acid, a better bioavailability and a more favorable pharmacokinetic profile than erythromycin. Clarithromycin exerts its antibacterial action by binding to the 50S ribosomal subunit of susceptible microorganisms resulting in inhibition of protein synthesis. Clarithromycin is mainly metabolized by cytochrome P450 (CYP) 3A enzymes (CYP3A4 and CYP3A5) to an active 14-hydroxy clarithromycin metabolite which has clinically significant antimicrobial activity⁶.The conventional treatment of PUD is for seven days. Clarithromycin at a dose of 500 mg bid, has been reported to inhibit H. Pylori in humans^7,8. Helicobacter pylori infections Dosage 500 mg BID for 14 days Clarithromycin is approved in dual therapy, with either omeprazole or ranitidine bismuth citrate, or in triple drug therapy, with either lansoprazole and amoxicillin or omeprazole and amoxicillin for triple-drug therapy for peptic ulcer disease treatment⁹. Clarithromycin chemical structure is given in figure -1 and its IUPAC name is (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R) -6-[(2S,3R,4S,6R)-4-dimethylamino-hydroxy-6-methyloxan -2-yl] oxy-14-ethyl-12,13-dihydroxy-4-[(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy-7-methoxy -3,5,7,9,11,13-hexamethyl-1-oxacyclotetradecane-2,10-dione¹⁰.

Figure 1: Chemical structure of Clarithromycin drug molecule.

Methods reported previously for the detection of clarithromycin have mainly used sample preparation procedures involving liquid–liquid extraction, solid-phase extraction, HPLC analysis and electrochemical methods. ² Further a sensitive liquid chromatographic method for the analysis of clarithromycin- a macrolide antibiotic- in human serum, using pre-column derivatization with 9-fluorenylmethyl chloroformate was developed by Amir Farshchi and his co workers¹¹. Several techniques such as HPLC, liquid chromatography – tandem spectrophotometry, pre column derivatization HPLC for the estimation of clarithromycin alone and with its major 14-OH metabolites had been reported^{12, 13}.Review of past literature given in sight that no UV spectrocolorimetric method is reported for the analysis of clarithromycin. Hence the present investigation was undertaken to develop a sensitive and robust UV Spectrophotometric method and its validation for the quantitation of clarithromycin in bulk fluids and Preformulation studies of the proposed drug candidate.

MATERIALS AND METHODS:

Instruments and Apparatus: Shimadzu UV 1700 double beam spectrophotometer (Japan) was used for all the spectrophotometric measurements. The absorption spectra of the reference and test solutions were carried out in quartz cells with 1 cm path length and 1 cm spectral band width over the range of 200 - 400 nm. Rotary shaker (konark instruments, Mumbai), Theil’s melting point apparatus was used for drug melting point determination. Dhona 200 D electronic balance (Mumbai) was used for weighing of all samples. All laboratory glassware like volumetric flasks and pipettes were calibrated and used for experimentation.

Active pharmaceutical ingredient, Chemicals and Reagents: Clarithromycin was kindly supplied by Micro labs, Bangalore, India) the drug was used without further purification. All the solvents and chemicals like n-octanol (INR Chemicals, Mumbai). Methanol (Pampasara distillaries). Potassium dihydrogen ortho phosphate and Sodium hydroxide pellets from S.d. Fine chemicals limited, Mumbai used in Spectrophotometric analysis were of laboratory reagent grade.

Experimental methodology: Analytical method development for the estimation of Clarithromycin in bulk fluids or in dosage forms:

Preparation of stock solution of Clarithromycin: Stock solution of Clarithromycin was prepared by dissolving 100 mg of accurately weighed amount of Clarithromycin in 10 ml of dry ethanol and then the volume was adjusted to 100 ml with the same solution to get 1 mg / ml solution. The stock solution of drug was subsequently diluted with dry ethanol to get 2 μg, 4 μg, 6 μg, 8 μg and 10 μg, of drug per ml.

UV Wave length scan procedure: The above stock solution of drug was subsequently diluted with dry ethanol to get 2 μg, 4 μg, 6 μg, 8 μg and 10 μg, of drug per ml. Later, 5 ml of 10 μg / ml clarithromycin solution was pipetted out into quartz cuvettes of UV spectrophotometer (against blank of methanol), and scanned for maximum absorbance between 200 nm and 400 nm in a UV 1700 Shimadzu double beam Spectrophotometer, Japan. Average of triplicate readings was taken. The peaks (n=3) with their respective absorbance’s were noted and from the maximum absorbance, the UV absorption maximaof clarithromycin was obtained at 211 nm which corroborates to literature value 210 nm. ⁷(Table-1, Fig.2).

Further calibration curve of clarithromycin was plotted by measuring absorbances of 2 μg /ml, 4 μg/ml, 6 μg/ml, 8 μg/ml and 10 μg /ml solutions at 211 nm. Average of triplicate readings was taken and tabulated. Regression equation was derived from the slope of the curve (y = 0.0250*X -0.0010; (r² = 0.999). (Table-2)

Table 1. Spectrophotometric data for the estimation of clarithromycin at 211 nm.

Sl. No	Concentration (µg/ml)	Absorbance ± SD
1	0. 00	0.00±0.000
2	2. 00	0.051±0.002
3	4. 00	0.102±0.003
4	6. 00	0.151±0.001
5	8. 00	0.201±0.002
6	10. 00	0.251±0.002

Figure2. Calibration curve of Clarithromycin by UV Spectrophotometer in dry ethanol at 211 nm.

Table 2. Linearity studies data of Clarithromycin.

Sl. No	Conc. (µg/ml)	Absorbance (nm)	Regression Data
1	2	0.000	Slope (m) = 0.0250 Interept (c)= -0.0010 Regression (r) =0.999979
2	4	0.051
3	6	0.102
4	8	0.151
5	10	0.201

Table 3. Inter day and Intraday variation studies of clarithromycin analytical method.

Concentration (μg /ml)	Absorbances
Morning	Day 1 Avg	Day 2 Avg	Day 3 Avg	Avg±SD
0	0	0	0	0.000
2	0.049	0.049	0.054	0.051±0.003
4	0.099	0.104	0.104	0.102±0.003
6	0.149	0.15	0.147	0.149±0.002
8	0.201	0.189	0.196	0.195±0.006
10	0.252	0.249	0.251	0.251±0.002
Afternoon	Day 1 Avg	Day 2 Avg	Day 3 Avg	Avg±SD
0	0	0	0	0.000
2	0.045	0.053	0.043	0.047±0.005
4	0.093	0.11	0.105	0.103±0.009
6	0.145	0.149	0.147	0.147±0.002
8	0.195	0.202	0.199	0.199±0.004
10	0.246	0.248	0.248	0.247±0.001
Evening	Day 1 Avg	Day 2 Avg	Day 3 Avg	Avg±SD
0	0	0	0	0.000
2	0.045	0.046	0.048	0.046±0.002
4	0.097	0.103	0.095	0.098±0.004
6	0.144	0.145	0.148	0.146±0.002
8	0.193	0.199	0.196	0.196±0.003
10	0.244	0.245	0.246	0.245±0.001
Concentration (μg /ml)	Day 1 Morning Average	Day 2 Afternoon Average	Day 3 Evening Average	3 Days Average ±SD
0	0	0	0	0.000
2	0.051	0.047	0.046	0.048±0.003
4	0.102	0.103	0.098	0.101±0.003
6	0.149	0.147	0.146	0.147±0.002
8	0.195	0.199	0.196	0.197±0.002
10	0.251	0.247	0.245	0.248±0.003

Linearity studies:¹⁴ The analytical method so developed was validated for linearity. To establish linearity of the proposed method six different concentrations of drug solutions (0 - 10 μg/ml) was prepared from clarithromycin stock solution and analysed for their absorbance at 211 nm. The obtained data was subjected for linear regression analysis by using method of least squares.

Validation of analytical method of Clarithromycin developed in the laboratory by accuracy and precision study: ¹⁴ Further the analytical method will be validated for other parameters like accuracy, precision in prospective work. Repeatability of method was determined by using different concentration levels of drug solutions was prepared from independent stock solution and analysed. Inter-day and intra-day variation was taken to determine precision of the proposed method. Different levels of drug concentrations (0 – 10 μg/ml) in triplicates were prepared three different times in a day and studied for intra-day variation. Same protocol was followed for three different days to study inter-day variation. The predicted concentrations relative standard deviation from the regression equation was taken as precision. The analytical method developed in the laboratory was validated for linearity, accuracy and precision. Inter day and Intraday variation was also studied. (Table-3).

Limit of Quantitation studies (LOQ) of clarithromycin:

LOQ studies were conducted by taking concentrations of 0.25 μg /ml, 0.50 μg/ml, 0.75 μg/ml, 1.00 μg/ml and 1.50 μg /ml for lower limit and 15.00 μg /ml, 18.00 μg/ml, 20.00 μg/ml, 25.00 μg/ml and 30.00 μg /ml for upper limit to find out the deviation from beers lamberts law. The clarithromycin obeyed beers law in the concentration range of 2.00 μg /ml to 10.00 μg /ml and the LOQ was found to be 1.5 μg/ml in UV spectrophotometer (UV 1700, Shimadzu, Japan). (Table-4).

Table 4. Limit of Quantitation studies of clarithromycin.

Sl. no.	Conc. (μg /ml)	I	II	III	Avg ± SD
Sl. no.	Conc. (μg /ml)	Absorbance
1	0.00	0	0	0	0.000
2	0.25	0.003	0.004	0.003	0.003±0.001
3	0.50	0.011	0.013	0.009	0.011±0.002
4	0.75	0.019	0.021	0.017	0.019±0.002
5	1.00	0.023	0.024	0.023	0.023±0.001
6	1.50	0.038	0.039	0.036	0.038±0.002
7	2.00	0.051	0.050	0.051	0.052±0.001
8	4.00	0.102	0.102	0.103	0.101±0.002
9	6.00	0.151	0.150	0.152	0.150±0.002
10	8.00	0.201	0.201	0.202	0.200±0.001
11	10.00	0.251	0.252	0.252	0.250±0.002
12	15.00	0.333	0.337	0.332	0.334±0.003
13	18.00	0.370	0.375	0.369	0.371±0.003
14	20.00	0.411	0.407	0.411	0.410±0.002
15	25.00	0.420	0.418	0.423	0.420±0.004
16	30.00	0.431	0.434	0.430	0.432±0.002

Table 5. Melting point determination studies of clarithromycin (n=3).

Sl. no	Melting point (⁰C)	Average Melting point(⁰C)
1	222	222.66
2	224
3	222

Table 6. Solubility studies of Clarithromycin in various solvents (n=3).

Sl. no	Solvent/Buffer	Absorbance				Solubility (mg/ml)
Sl. no	Solvent/Buffer	I	II	III	Avg Abs± SD	Solubility (mg/ml)
1	Distilled water	0.083	0.084	0.081	0.083±0.002	0.0331
2	Dry ethanol	0.18	0.181	0.18	0.180±0.001	0.720
3	Methanol	0.144	0.142	0.143	0.143±0.001	0.572
4	Acetone	0.084	0.085	0.084	0.084±0.001	33.601
5	pH 1.2 buffer	0.129	0.128	0.131	0.129±0.002	0.5173
6	pH 4.0 buffer	0.178	0.176	0.177	0.177±0.001	0.0708
7	pH 7.4 buffer	0.155	0.157	0.155	0.156±0.001	0.0623
8	pH 9.0 buffer	0.109	0.111	0.112	0.111±0.002	0.0443

Table 7. Partition coefficient studies of Clarithromycin (n=3).

Drug	Solvent phase	Absorbance			Average Abs ± SD	Concentration (mg/ml)	K _o/w	Log K
		I	II	III
Clarithromycin	Distilled water	0.030	0.03	0.029	0.029± 0.002	0.0012	14.833	1.17
	n-Octanol	0.446	0.447	0.445	0.446± 0.001	0.0178

Methods used for Preformulation studies of clarithromycin:

Melting point determination: Melting point of the drug was determined by taking a small amount of drug in a capillary tube closed at one end and was placed in theil’s melting point apparatus and the temperature at which the drug melts was noted. Average of triplicate readings was noted. (Table-5).

Solubility studies: ¹⁵ The solubility of clarithromycin was determined in distilled water, different buffers, viz., pH 1.2, pH 4.0, pH 9.0 and in acetone, ethanol and methanol according to the method proposed by Diez et. al.The drug concentration was measured in a UV spectrophotometer at 211 nm Triplicate readings were taken and average was calculated. An excess amount of the drug was taken and dissolved in a measured amount of distilled water in a volumetric flask to get a saturated solution. The solution was shaken on rotary shaker for 24 h to assist the attainment of equilibrium with the un-dissolved drug particles. Then measured quantity of the filtered drug solution was withdrawn after 24 hours and successively diluted with distilled water suitably and the concentration was measured in a UV spectrophotometer at their respective absorbance maxima. Similarly the solubility of drug determined in dry ethanol, methanol, acetone and in different buffers i.e. pH 1.2, pH 4.0, pH 7.4 and pH 9.0. (Table-6).

Partition coefficient determination¹⁶: The partition coefficient of the drug was determined by taking equal volumes of n-octanol and aqueous phases in a separating funnel. A drug solution was prepared and 1ml of the solution was added to octanol: water (50:50) was taken in a separating funnel and shaken for 10 minutes and allowed to stand for 1 h and is continued for 24 h. Then aqueous phase and octanol phase was separated, centrifuged for 10 min at 2000 rpm. The aqueous phase and octanol phase were assayed before and after partitioning using UV Spectrophotometer at their respective λ max to get partition coefficient. Triplicate readings (n=3) were taken and average was calculated. (Table-7).

RESULTS AND DISCUSSION:

The UV absorption maxima of was 211 nm which corroborates to the reported literature value 210 nm.⁷The slope (m) of calibration curve of clarithromycin was 0.02506. Linearity studies indicated that estimation of the drug between 2.00 μg / ml to 10.00 μg /ml was found to be linear with slope (m) 0.02506, intercept (c) is -0.0010 (r = 0.999). (Table-8). The SD values of Inter day and Intraday variation studies indicated that the variation is minimum. Limit of Quantitation of clarithromycin was found to be of 1.50 μg /ml in UV spectrophotometer (UV 1700, Shimadzu, Japan). The melting point of clarithromycin was 222.6°C which corroborates with the literature value 217- 220°C¹⁷. The solubility of drug follows the order acetone > ethanol >methanol > pH 1.2 > pH 4.0 > pH 7.4 > pH 9.0. The log p value of clarithromycin was found to be 1.170 which corroborates to the reported literature value 1.172.¹⁷ The Preformulation studies showed that the bulk sample obtained was pure and analytical work for the estimation of clarithromycin was found to obey beer’s limit of 2-10 μg / ml and the curve was found to be linear. The developed analytical technique can be employed for quantitation of drug candidate in bulk fluids of dosage forms.

Table 8. Analytical parameters of developed Clarithromycin UV Spectrophotometric method.

Sl. no	Analytical Parameter	Value
1	UV absorption maxima (nm)	211nm
2	Beer’s law limits (μg / ml)	1-10
3	Relative standard deviation	0.0018
4	Slope (m)	0.02506
5	Intercept (c)	-0.0010
6	Correlation coefficient(r)	0.999
7	Regression equation (y)	0.0250*X -0.0010; ( r = 0.999)
8	Linearity range	2-10 μg / ml
9	Limit of quantitation	1.5 μg / ml

CONCLUSION:

The literature review encompassed the literature reports on various analytical methods of clarithromycin estimation useful in the study. The corroborating experimental values suggest the bulk sample drug obtained is pure. The solubility a study of clarithromycin indicates as the pH of the medium decreases the solubility of clarithromycin is increased. The analytical method developed using UV spectrophotometer is linear and LOQ is 1.5 μg /ml and the inter-day and intra-day variation is minimum. Although there are reports and publications of either colorimetric and HPLC methods for clarithromycin estimation, but there is no report or publication corresponding to the intended investigation, development and validation of UV Spectrophotometric method of clarithromycin. Therefore it could conclude that the proposed investigation is a novel work and the investigation would help in estimation of drug candidate spectrophotometrically in the bulk fluids and experimental in vitro dissolution studies.

ACKNOWLEDGEMENTS:

One of the authors Putta Rajesh Kumar was thankful to Management and Principal V.L. College of Pharmacy, Raichur, Karnataka, India for providing necessary research and laboratory facilities.

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Received on 27.08.2010 Modified on 04.09.2010

Research J. Pharm. and Tech. 4(2): February 2011; Page 242-246