Tetracycline hydrochloride, fig 1, is chemically 4-(dimethyl amino)-1, 4, 4a, 5, 5a, 6, 11, 12a-octa hydro-3, 6, 10, 12, 12a-penta hydroxy-6-methyl-1, 11-dioxo-2-naphthacene-carboxamide [1]. Tetracycline (TC) is a widely used antibiotic and it has high activity against nearly all Gram–positive and Gram–negative bacteria [2].

C₂₂H₂₄N₂O₈, HCl 480.9??

Fig 1: Chemical Structure of Tetracycline Hydrochloride

Various methods have been developed for the determination of tetracycline hydrochloride in pharmaceutical preparations and biological samples including fluorimetry [3,4] electrochemical method [5-8], atomic Absorption Spectrophotometry [9], liquid chromatography [10-14] capillary electrophoresis [15] and chemiluminescence [16-20].UV-Visible spectrophotometry is still considered to be a convenient and low cost method for the analytical determination of tetracycline in Pharmaceuticals formulations. A number of spectrophotometric and colorimetric procedures for the determination of tetracyclines in bulk material and dosage forms are reported in the literature [21-25].  The aim of this work is to develop a very simple, accurate and sensitive spectrophotometric method for the determination of tetracycline hydrochloride.

Apparatus

A spectra scan 50 UV- visible spectrophotometer with 1.0 cm quartz cells was used.

Reagents

All chemicals used were of analytical grade and the tetracycline hydrochloride standard material was provided from AL-Hokamaa Company for pharmaceutical industries (HPI) Mosul-Iraq.

Tetracycline hydrochloride stock solution (1.5%) was prepared by dissolving1.5g of tetracycline hydrochloride in 100ml distilled water in a volumetric flask.                                 

Recommended Procedure  

An aliquots of standard solution of tetracycline hydrochloride (50-750mg) were transferred into a series of 25 ml calibrated flasks, dilute the solution to the mark with distilled water. The absorbance of the product was measured at 360 nm against a reagent blank (distilled water).

Assay Procedure for Pharmaceutical Preparations

Capsule

An amount of finely ground capsule powder equivalent to 1.5 gm. of tetracycline hydrochloride was accurately weighed into a 100ml calibrated flask, 60ml of distilled water added and shaken for 20 min. Then, the volume was made up to the mark with distilled water, mixed well, and filtered using what man No.42 filter paper. This solution measured as mentioned under recommended procedure.

Ointments

Prepare a composite sample by mixing the contents of five containers and accurately weigh a sample equivalent to 1.0 gm. of tetracycline hydrochloride into a 100-ml beaker. Add 80- ml of hot distilled water and heat on a water-bath for 20 min, cool in freezer, then filter into 100 ml calibrated flask, dilute to 100-ml with distilled water, mixed well . Treat this solution as mentioned under recommended procedure.           

Procedure for Spiked Industrial Wastewater

To demonstrate the practical applicability of the proposed method, industrial waste water sample from AL-Hokamaa Company for pharmaceutical industries (HPI) Mosul-Iraq. Were analyzed, since industrial waste water sample was found to be free from tetracycline hydrochloride, synthetic samples were prepared by spiked known amount of tetracycline hydrochloride with the concentrations ranging from 5-25 mg.ml^-1 and aliquot of this solution was treated as described above for recommended procedure.    

UV- Visible spectrophotometry is still considered to be a convenient and low cost method for the estimation of pharmaceuticals [26-31]. This method used for the estimation of Tetracycline hydrochloride in pharmaceutical preparations and environmental wastewater samples was found to be high sensitive, simple, accurate, and reproducible. Tetracycline hydrochloride  which absorbs maximally at 360nm as shown in Figure (2), the colorless reagent blank has practically negligible absorbance at this wavelength and this wavelength was recommended for determination. Beer’s law is obeyed over the concentration range 2- 30 mg /ml Figure [3].

Figure 2: Absorption Spectra of Tetracycline. HCL (15mg/Ml) Product against Distilled Water.  

Figure 3: Calibration Graph of Tetracycline Hydrochloride.

A regression analysis of Beer 's law plot at 360 nm revealed a good correlation (r=0.9992, n=10) the graph of the absorbance versus the concentration of tetracycline hydrochloride showed a low intercept and slope (0.0335) .The apparent molar absorptivity was 1.6113×10⁴ l. mol^-1 .cm^-1     

Accuracy and precision

The accuracy and precision of the method was established by analyzing the pure drug solution at three different levels. The average recovery which is a measure of accuracy is 100 ± 0.55 revealing high accuracy of the method. The relative standard deviation (RSD), which is an indicator of precision is less than ±2%. The results are compiled in Table [1].                    

Table 1: Optical Characteristics and Statistical Data for Regression Equation of the Proposed Method.

Effect of Interferences

The interfering effect of foreign species often accompanied with tetracycline   hydrochloride in the pharmaceutical preparations were studied by adding different  amounts of foreign species to 20µg/25ml of tetracycline hydrochloride in solution and the recommended procedure for the determination of tetracycline hydrochloride  was followed . The species are considered to interfere seriously if the cause aching of more than 2% in the absorbance obtained for tetracycline hydrochloride a lone [32]. Results of the recovery analysis are presented in Table [2]. Excipients at the concentration show in Table [2] do not interfere with the assay .In addition recoveries in most cases were around 100%.

Table 2: Determination of Tetracycline Hydrochloride in Presence of Excipients.

* Average of five replicate determinations.

Application of the Proposed Method

The proposed method was successfully applied to the analysis of tetracycline hydrochloride in capsules, Skin and Eye ointments and industrial waste water sample. The result of analysis for pharmaceutical formulations revels that there is close agreement between the results obtained by the proposed method and the label claim table (3), and the results of water samples table (4) show that the recovery values obtained were close to100%.

Table 3: Assay of Tetracycline Hydrochloride in Pharmaceutical Formulations.

*Mean of ten determinations.

Table 4: Determination of Tetracycline .HCL in Spiked Industrial Wastewater.

 *Mean of ten determinations.

Application of the Proposed Method to Content Uniformity

Content uniformity or the uniformity of dosage unit was defined as the degree of uniformity in the amount of active substance among dosage units. The risk assessment strategy underlying content uniformity testing is the assumption that some pre-specified limits exist where safety and efficacy outcomes may change if content uniformity fails. The proposed method proved to be suitable for the content uniformity test, where a great number of assays on individual capsules are required. Data presented in Table.4 indicate that the proposed method can accurately and precisely quantities of tetracycline hydrochloride in its commercially available. The mean percentage with (RSD) of the labeled claim found in ten capsules was (0.822%) which falls within the content uniformity limits specified by the by the United State Pharmacopeia 33-NF28USP 33 [33-35].

Table 4: Content Uniformity Testing of Tetracycline Hydrochloride Capsules.

In this work, a simple, rapid, precise and accurate spectrophotometric method was developed and validated for the determination of tetracycline hydrochloride in pharmaceutical preparations and industrial waste water samples. The method free from such experimental variables as heating or solvent extraction step. The method rely on the use of simple and cheap chemicals and techniques and can be used for rapid routine determination and quality control of tetracycline hydrochloride in pure form, bulk sample ,pharmaceutical preparations and real industrial waste water sample.

Acknowledgments

The author wishes to express gratitude to his former company [AL-Hokamaa Company for pharmaceutical industries (HPI) Mosul-Iraq. for providing gift samples of tetracycline hydrochloride standard materials and pharmaceutical preparations (capsules and ointments).

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