Development and Validation of a Spectrofluorimetric Method for the Assay of Tetracycline in Capsules

The purpose of this study is to develop and validate a method for the analysis of tetracycline capsules by spectrofluorimetry. A pH 9 borate buffer was used as diluent of tetracycline after reaction with magnesium salt at the excitation wavelength of 372 nm and 516 nm of emission. A linear response was observed between 0.25 μg/mL and 1.5 μg/mL with a correlation coefficient (R) of 0.9998. The detection and quantification limits found are 0.0125 μg/mL and 0.0412 μg/mL respectively. The proposed method proved trueness with a recovery between 99.88% and 101.10%. The relative standard deviations of repeatability and intermediate precision found ≤2.88% reflected a good precision of the method. The proposed method is therefore valid within the limits of 90% to 110%. The proposed method was applied to the quality control of 9 tetracycline samples from market and gave results in accordance with the pharmacopoeia standards.


Introduction
the bacterial 50S ribosomal subunit and can alter the cytoplasmic membrane causing leakage of intracellular components of bacterial cells [1].
A large number of analytical techniques for the determination of tetracycline and its degradation products have been reported, particularly in biological fluids and pharmaceuticals such as spectrophotometry [2] [3] which is of limited utility due to its non-specificity.More efficient separative methods, such as capillary electrophoresis [4] and high performance liquid chromatography [5] [6] using UV and fluorescence detectors require a lot of time, proven expertise and a very high operating cost.This limits their use in routine quality control and especially in developing countries with limited resources.The aim of this study is to validate a spectrofluorometric method for tetracycline assay in capsules by using the accuracy profile approach [7].This method has proved to be simple, sensitive, fast, efficient and cheap.with the FLwinlab® application software was used.
Purified water was produced in situ with a Milli-Q Ultrapure Water System (Millipore, Molsheim, France).

Preparation of Solutions for the Determination of the Calibration
Curve 10.00 mg of THC are dissolved in 100 mL of distilled water to obtain the standard solution (SS).0.5-1-1.5-2-2.5 and 3 mL of the SS are transferred respectively into the 200 mL volumetric flasks and diluted to the mark.5 mL of the pH9 buffer solution are added to each 5mL of dilution followed by 2.5 mL of the MgSO 4 solution (0.75 M).

Accuracy Profile
Accuracy is the total error after the sum of systematic error (trueness) and random errors (precision).It is represented by an accuracy profile for the area of measures (75% -125%).Accuracy of the method is established through the accuracy profile described by Feinberg.
The accuracy profile approach allows to determine simultaneously the recovery and precision of the method using standard and validation solutions as previously described [8].

1) Preparation of Standard Solution
Three solutions of concentration of 0.75-1-1.25 μg/mL are prepared.5 ml of each dilution, 5 mL of the pH 9 buffer solution and 2.5 mL of the MgSO 4 solution (0.75 M) are mixed in a conical tube.The reading of the fluorescence intensity is carried out after 25 min at 372 nm of excitation and 516 nm of emission.

2) Preparation of Test Solution
The reference sample for validation is reconstituted from the active ingredient (tetracycline) and the matrix (Lactose, Mannitol, magnesium stearate and starch), thus the stock solution is made of 10 mg of tetracycline and 10 mg of the matrix dissolved in 100 mL of water and filtered.Solutions of concentrations of 0.75-1-1.25 μg/mL are prepared.5 mL of the buffer solution at pH 9 and 2.5 mL of the MgSO 4 solution (0.75 M) are added to 5 mL of each solution.The reading of the fluorescence intensity is carried out after 25 min at 372 nm of excitation and 516 nm of emission.

Limit of Detection (LOD) and Limit of Quantification (LOQ)
The limit of detection is estimated by diluting a solution of known concentration until the lowest near detectable signal is different from that of blank, while the limit of quantification is 3.3 times the limit of detection [9].They were determined as specified in the ICH (International Conference on Harmonisation) protocol [10].

Application of the Method
A test sample of 10 mg of tetracycline is carried out on the powder emptied from 5 capsules (taking into account the average weight).After dissolution in 100 mL of distilled water, homogenization with ultrasound for 10 minutes and filtration, 2 mL of the filtrate are diluted to 200 mL to obtain a solution of 1 μg/mL of THC.To 5 mL of the 1 μg/mL solution are added 5 mL of Borate buffer at pH 9

Effect of Solvent on Fluorescence Intensity
A solution with a cloudy appearance is obtained when the magnesium sulfate is put into the THC solution in basic medium.This solution is unusable due to the formation of insoluble compounds.On the other hand, in the water a weak signal is observed as it appears in Table 1.

Influence of pH
Signals are stronger with basic buffers than acidic buffers (Figure 2).An excellent and stable signal is observed at pH 9. The use of MgSO 4 at pH 5.6 helped to boost the fluorescence of tetracycline dissolved in water (non fluorescent).The complex formed between THC and Mg gave a good stability in function of time.

Study of Selectivity
The comparison of data found during the analysis of THC pure product and when it is with the other substances which accompany it in galenic presentation (the excipients) gave two spectra (Figure 3 and Figure 4).

Graph Calibration
Employing the conditions described in the procedure, a calibration curve was drawn in Figure 5. Table 1.Effect of magnesium sulfate on the bypass.

Accuracy Profile
The recovery data, precision and confidence interval are shown in Table 2 and Figure 6.For trueness, recoveries were between 99.88% and 101.10%.For intraday and inter-day precision the RSD (relative standard deviation) between 1.57% and 2.88% were lower than 5%.

Application of the Validated Method for the Determination of TCH in Pharmaceutical Preparations
To make the application of the method effective, it has been used for the direct determination of TCH in pharmaceutical preparations.Nine lots of THC capsules were separately selected for quantitative determination of THC.The THC content in samples collected from the market is determined qualitatively by the validated method and results are presented in Table 3.

Discussion
Although Tetracycline is soluble in aqueous solutions, water and methanol, it is far from stable in these solvents.Most often, it is transformed into Epitétracycline hydrochloride (ETC).This epimerization reaction is reversible.In the presence of divalent cations the reaction is not made [11].
Dissolved in water, no signal was found for TCH; on the other hand dissolved in water in the presence of Mg at a pH of 5.6, a signal was found at 380 nm of Tetracycline hydrochloride (THC) is a broad-spectrum polyketide antibiotic produced by the genus Streptomyces.It exerts a bacteriostatic effect on bacteria by reversibly binding the 30S ribosomal subunit of bacteria and blocking the binding of tRNA to the ribosome acceptor site.It also binds to some extent to L. M. Namegabe et al.DOI: 10.4236/ajac.2018.93014163 American Journal of Analytical Chemistry

2. 2 . 1 .
Analytical Parameters Optimum reaction conditions have been studied such as: The solvent, Effect of magnesium sulphate, Stability of tetracycline in water and Influence of pH.

and 2 .
5 ml of the 0.75 M MgSO 4 solution.The reading of the fluorescence inten-L.M. Namegabe et al.DOI: 10.4236/ajac.2018.93014165 American Journal of Analytical Chemistry sity is carried out after 25 min at 372 nm of excitation and 516 nm of emission.

Figure 2 .
Figure 2. Effect of pH on the fluorescence intensity.

Figure 4 .
Figure 4. Spectrum of TCH in the matrix.

Figure 6 .
Figure 6.Diagram of the accuracy profile of the proposed method.

Table 2 .
Summary data for establishing the accuracy profile.