Direct Determination of Tetracycline Hydrochloride and the Interaction with Bovine Serum Albumin by UV Spectra in Water

The UV fourth order derivative spectra were selected as the more reliable technique for the direct quantification of pure Tetracycline Hydrochloride in water, after the measurement of the zero, first, second, third and fourth order spectra, within the concentration range at (0.2885144.27)μg/ml with R =0.9998 and RSD=0.215 %. Bovine serum albumin was directly quantified by the fourth order UV derivative technique within the concentration range (6.7-2680) μg/ml with R = 0.9998 and RSD=0.324 %. Appreciable interaction between Tetracycline hydrochloride and Bovine serum Albumin was found in accordance with the fraction coefficient and the apparent binding constant. The UV–fourth order derivative technique appears to be in a good accuracy and precise method for the direct quantification of Tetracycline Hydrochloride and Bovine Serum Albumin (BSA) in water.


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INTRODUCTION Tetracycline hydrochloride is a yellow crystalline powder, soluble in water and alcohol, its molecular formula is C 22 H 24 N 2 O 8 .HCl and its structural formula is:

Tetracycline Hydrochloride
M.WT=480.9 It is one of the tetracyclines antibiotics which treats numerous types of gram positive and gram negative bacteria (Navigation, 2005), (Katzung et al., 2009).
This work aimed to show the direct determination of pure tetracycline hydrochloride in water, and to study their interactions with albumin by recording the fourth order derivative UV spectra in water at λ =282 nm and 310°K with a good accuracy and precision and the results were compared with the standard pharmaceutical method.

EXPERIMENTAL Material
Teracycline hydrochloride is supplied by the state company for drug industries and medical appliances in Nineveh, Iraq (N.D.I), and was used without any further purification. Bovine serum albumin is supplied by Fluka company.

Preparation of stock solution:
A stock solution of (0.01M) of pure tetracycline hydrochloride was prepared by dissolving 0.1202g in 25ml distilled water. Other concentrations were prepared from this solution by proper dilution.
A stock solution of (0.0001M) of pure bovine serum albumin was prepared by dissolving (0.1675)g in 25ml distilled water. Other solutions were prepared from this solution by proper dilution with distilled water.

Instrumentation
The main spectra measurements were performed by a double beam Shimadzu UV-1800 (UV-Visible) spectrophotometer connected to a computer with a Pentium 4 processor. The system is supplied with a temperature controller type Shimadzu TCC-240 A and TCC-240 CE, which is equipped with insulated constant temperature cell houses. The temperature deviation varied within the limits of ± 0.1 º C. The optimized conditions for spectrophotometric measurement were derivative modes at scan speed fast, slit width 2nm. Derivative UV spectra were recorded over wavelength range (1100-190) nm using teflon stopper quartz cuvette with dimensions of (1×1×4cm). Other used apparatus are mettler sartor us 2004 MP6 electronic semi-micro balance with five digits and Electrothermal melting point apparatus IA 9300.

RESULTS AND DISCUSSION
The zero order UV spectrum of pure tetracycline hydrochloride solution in distilled water was measured against distilled water as a blank. The spectrum showed two bands at 276 nm with max ε =15425 lit.mol -1 .cm -1 and another band at 356nm with max ε =14925 lit.mol -1 .cm -1 Fig. (1). The effect of temperature and time on these spectra showed that ,there is no effect of temperature on the absorbance of pure tetracycline spectra in water within the temperature range (298.16-313.16 °K) as shown in (Table 1). The effect of time on the zero-order spectrum of tetracycline hydrochloride solution in water shows that the solution is stable up to more than 24 hours, as shown in (Table 2). The quantification of pure tetracycline hydrochloride in water was accomplished according to the absorbance at max λ =356 nm and at 298.16°K, for this purpose, the spectra of a series of different concentration solutions were recorded and the absorbance at max λ =356 nm plotted versus the molar concentrations. The results showed a linear relationship obeying Beer's-Lambert law within the concentration range (0.385-48.090) µg/ml , R 2 = 0.9993 and RSD=0.125 Fig. (2), (Table 3).  The first order derivatives UVspectra of tetracycline hydrochloride water solution were recorded for a series of solution of different concentrations (0.385-144.270) µg/ml. The spectra showed a positive peak at λ =(238-276) nm crossing the zero axis at 276 nm and a negative peak at λ =(276-314) nm, and another positive peak at λ =(314-356) nm crossing the zero axis at 356nm and a negative peak at λ =(356-429) nm Fig. (3). The positive peak at λ =(314-356) nm was chosen for the direct quantification of tetracycline by measuring the peak height of this peak for each solution and their plot versus the solution concentration. The results showed a straight line obeying Beer's -Lambert law within the range (0.385-48.090) µg/ml with R 2 = 0.9999 and RSD=0.381, (Table 4)   The second order derivative of tetracycline hydrochloride in water was recorded for a series of solutions of different concentrations ranging from (0.192-96.180) µg/ml. The spectrum shows two main negative peaks ( 1 λ =274nm ) and ( 2 λ =364nm), with two satellites one at the both sides of each peak Fig. (5). The quantification was achieved according to the peak height at ( λ =364nm) which was estimated for each concentration of tetracycline solution in water. The plot of these absorbances versus the molar concentrations result in a straight line relation obeying Beer's-Lambert law within the concentration range (0.385-96.180) µg/ml with R 2 =0.9998 and RSD=0.593 (Table 5), Fig. (6).   The third order derivatives UV spectra were recorded for a series of different concentrations of tetracycline hydrochloride in water. The spectrum consists of a negative peak in the region (258-274) nm crossing the zero axis at 274 nm and a positive peak at (274-292) nm, and a second negative peak at (338-364) nm crossing the zero axis at λ =364nm and a positive peak at λ =(364-393) nm Fig. (7). The quantification of tetracycline was accomplished according to the positive peak at λ =(364-393) nm through the estimation of their peak height and their plot against the concentration which results in a straight line relation obeying Beer's-Lambert law within the range (0.385-48.090) µg/ml with R 2 =0.9995 and RSD=0.612, Table (6), Fig. (8).   The fourth order derivatives UV spectra of pure tetracycline hydrochloride in water were recorded for a series of different concentrations. The spectrum showed two main positive peaks at λ=274nm and λ=368nm with two satellites at both sides of each peak Fig. (9). The quantification was accomplished according to the peak height at λ =368nm which was plotted versus the concentrations and resulted in a straight line relation obeying Beer's-Lambert law within the concentration range of (0.289 -144.270) µg /ml with R 2 =0.9998 and RSD=0.215, (Table 7) Fig. 10: UV -absorption of fourth order spectra of (19.236) µg /ml of tetracycline hydrochloride in water at 298.16°K at 368 nm in water.
According to the results obtained, it is obvious that the fourth order derivative UV technique is the more reliable technique for the direct quantification of pure tetracycline hydrochloride in water, due to their measurable larger range of concentration.
For the examination of the accuracy and precision of the fourth order derivative technique for the quantification of pure tetracycline hydrochloride in water, the absorbance of three samples at three different concentrations were measured at λ =368nm and the results reveal a good accuracy and precision as show in Table (8).

Bovine Serum Albumin (BSA):
The zero order UV spectrum of BSA in distilled water showed a peak at max λ =278 nm with max ε =43325 lit.mol -1 .cm -1 . The absorbance values measured at this wavelength remain unchanged for more than 2hours within a temperatures range 298.16-313.16°K Tables (9 and 10).  The fourth order derivative spectrum of BSA in water showed a main positive peak at λ =282nm with two satallites at each side of the peak. The peak height of the band at λ =282nm was measured for a series of different concentration solutions of pure BSA in water and plotted versus the concentration. The result is linear relation obeying Beer's -Lambert law within a concentration range (6.7-2680) µg/ml with R 2 =0.9998 and RSD= 0.0836 % (Table 11), Fig.  (11,12).   The accuracy and precision of the quantification of BSA in water was accomplished by the measurement of the peak height at λ =282nm of three samples at different concentrations of accurately prepared solutions, which indicate a good accuracy and precision for this method, (Table 12). The interaction of pure tetracycline hydrochloride with BSA : The addition of successive amount of pure tetracycline hydrochloride in water of concentration (9x10 -5 ) M to a solution of (BSA )(1X10 -5 M) in water, causes a gradual decrease in the measured absorbance at nm 282 = λ  Where K is the apparent binding constant, A° is the absorbance at nm 282 = λ before the addition of tetracycline hydrochloride, A the absorbance after each addition under the assumption of reversible complex of (BSA Tetracycline hydrochloride) is 1:1 association complex. Then the plot of Log(1/conc[TCH] )versus Log (A/A°-A) result in a linear relation with the intercept of log K, Fig.  (14), from which the K value =9.772x10 3 was calculated indicated that an appreciable interaction between BSA and tetracycline hydrochloride do exist. The determination of tetracycline hydrochloride by HPLC method at their optimum experimental conditions accomplished at the state company of drug industries and medical appliance in Nineveh, Iraq (NDI), and comparison with our present method (fourth order derivative) indicates that the fourth order derivative method is of a very good recovery percent as shown in (Table 14).