How I can determine the concentration of tetracycline in an aqueous solution using a Shimadzu uv 1800 spectrophotometer?

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Research Article | Open Access

Volume 2013 |Article ID 305124 | 11 pages |https://doi.org/10.1155/2013/305124

Determination of Tetracycline in Pharmaceutical Preparation by Molecular and Atomic Absorption Spectrophotometry and High Performance Liquid Chromatography via Complex Formation with Au(III) and Hg(II) Ions in Solutions

Ahlam Jameel Abdulghani,1 Hadi Hassan Jasim,2and Abbas Shebeeb Hassan2

Academic Editor: Peter A. Tanner

Published18 Jun 2013

Abstract

UV-visible and atomic spectrophotometry and HPLC techniques were applied for the determination of tetracycline (TC) in pharmaceutical preparations via complexation of the drug with Au(III) and Hg(II) ions in solutions. The mole ratio of TC to metal ions was 1 : 1. Maximum peak absorption at λ 425 and 320 nm for the two ions, respectively, was optimized at heating temperature 75°C for 15 minutes at pH = 4 followed by the extraction with ethyl acetate. The percentage of extraction and stability constants for the two complexes was 95.247, 95.335% and , M−1, respectively. HPLC method was applied without extraction process. The analytical data obtained from direct calibration curves of UV-visible absorption, FAAS, and HPLC for Au(III) complexes were recovery (100.78, 104.85, and 101.777%, resp.); detection limits (0.7403, 0.0997, and 2.647 μg/ml, resp.); linearity (5–70, 5–30, and 10–150 μg/ml, resp.), and correlation coefficient (0.9991, 0.9967, and 0.9986, resp.). The analytical data obtained from direct calibration curves for Hg(II) complexes by UV-visible spectrophotometry and HPLC were recovery (100.95 and 102.000%, resp.); detection limits (0.5867 and 2.532 μg/ml, resp.); linearity (5–70 and 10–150 μg/ml, resp.); and correlation coefficients (0.9989 and 0.9997, resp.).

1. Introduction

Tetracyclines possess a wide range of antimicrobial activity against Gram-positive and Gram-negative bacteria. They have been used not only in human medicine for the treatment of infectious diseases but also as additives in animal feed to promote growth. Beside the pharmacological importance of tetracycline (TC), this molecule possesses many potential metal-binding sites. Therefore a wide range of research work has been reported on chelation of TCs with various metal ions which have been utilized successfully in pharmaceutical analysis [1–15].

Depending on the chosen experimental conditions such as solvent medium, pH, metal ion type, and ligand: metal ratio, the O(10), O(11), and O(12) on the BCD-ring and O(1), O(3), and N(4) in ring A and at the carboxamide group on the A-ring are the main coordination sites that were frequently proposed in the literature [1–6, 16]. Pharmacokinetics and bioavailability of TC are affected by its coordination with metal ions such as calcium in blood plasma and magnesium ion in the intracellular medium [4]. By combining with the copper, zinc, iron, and other trace metal elements in enzymes such as collagenase, tetracyclines inhibit the enzymatic destruction of tissues [5].

Complexes of (TC) with Co(Il), Ni(II), and Fe(III) ions showed higher activity against Bacillus subtilis, Serratia species, and Escherichia coli than that the original tetracycline hydrochloride [6]. Platinum(II) complexes of tetracycline and doxycycline were reported to bind to DNA and inhibit tumoral cell growth [15]. Although the Au(III) is as important as Pt(II) ions in this aspect, little attention has been paid to Au(III) complexes of TC. Environmental studies on TCs showed that complexation of these drugs with heavy metal cations in natural waters and soils may induce chronic toxic effects in aquatic plants [7, 8, 17].

In this work we study the determination of TC (see Figure 1) in pure and dosage form via complexation with gold(III) and mercury(II) ions in solution using UV-visible and atomic absorption spectrophotometry and HPLC techniques.

Figure 1

TC. HCl.

2. Experimental

UV-visible spectra were recorded on Varian Gary 100 UV-visible spectrophotometer supplied with UV-probe software. FTIR spectra were recorded on Shimadzu FT-IR 8400S Fourier transforms infra-red spectrophotometer. Flame atomic absorption spectrophotometric studies (FAAS) on Au(III) ion and its complexes were carried out using GBC 933 plus flame atomic absorption spectrophotometer supplied with hollow cathode lamp, D2 Lamp (GBC), flame autosampler, and air-acetylene flame. High performance liquid chromatography was performed on HPLC, Shimadzu, and Shimadzu LC 2010A, supplied with UV-visible detector. HPLC conditions for determination of Tc-Au(III) complexes were : stationary phase or column mm supelcosil LC-18, mobile phase acetonitrile : acetate buffer solution (40 : 60), flow rate (1 mL/min), injection volume 100 μL, detection wavelength (220 nm). HPLC conditions for determination of Hg(II)-TC complexes were : mobile phase acetonitrile : acetate buffer solution (20 : 80), stationary phase or column mm ODS C-18, flow rate (2 mL/min), injection volume 200 μL, detection wavelength (276 nm). pH of solutions were measured by using HANA, HI 98150 GLP PH/ORP-meter calibrated with reference pH solutions at 4.0 and 9.0.

All chemicals used were of analytical reagent grade, and tetracycline hydrochloride was provided from state company for drug industries, and medical appliance (SDI), Samarra Iraq.

2.1. Antibiotic Standard Solutions

A stock solution of tetracycline hydrochloride (TC. HCl) (1000 μg/mL) was prepared by dissolving 0.100 g of TC. HCl standard powder in 100 mL distilled deionized water (DDW). Working standard solutions of TC. HCl (100–150 μg/mL) were prepared by diluting 10–15 mL of stock solution to 100 mL with DDW in 100 mL volumetric flask.

2.2. Metal Ions Standard Solutions (100 μg/mL)

A stock solution of Hg(II) ions (1000 μg/mL) was prepared by dissolving 0.1350 g of HgCl2 in a 100 mL (DDW) in 100 mL volumetric flask. Working solutions (100 μg/mL) were prepared by diluting 10 mL of the stock solution to 100 mL with DDW in a 100 mL volumetric flask. Working solutions of Au(III) ions (100 μg/mL) were prepared from 1000 μg · mL−1 Au(III) solution provided from suppliers for atomic absorption spectrophotometric analysis.

2.3. UV-Visible Spectrophotometry Complex Formation

To 1 mL aliquots of metal ion solutions ( M) in a series of 10 mL volumetric flasks were added 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, and 4.0 mL of TC. HCl solution ( M) and volumes were completed to the marks. All experimental conditions were set to their optimum values, and resulting complexes were extracted from aqueous solutions with ethyl acetate (5 : 1 v/v aq : EA) before measuring the absorbance.

2.3.1. Determination of Antibiotic Complexes by Direct Method

Solution mixtures of 5–70 μg/mL TC. HCl and 10 μg/mL Au(III) ion or 15 μg/mL of Hg(II) standard solutions were prepared by adding different volumes of antibiotics’ standard solutions (100 μg/mL) to 1 ml or 1.5 mL, respectively, of metal ion standard solutions (100 μg/mL) in 10 mL volumetric flasks. The volumes were completed to the mark, and the experimental conditions were adjusted to the respective optimum values of concentration, pH and temperature, and liquid extraction with ethyl acetate (EA) (5 : 1 v/v aq : EA). The absorbance of complexes in each case was recorded at the recommended and plotted against the concentration of the cited antibiotic

2.3.2. Determination of Antibiotic Complexes in Dosage Form by Direct Method

0.1 g of TC powder obtained from 20 capsules of samacycline 250 mg was weighted and dissolved in 100 mL distilled water in volumetric flask. Ten mL of the resulted solution was diluted to 100 mL by DDW in volumetric flasks. Then 1, 2, and 3 mL of the end solution were transferred to 10 mL volumetric flasks containing 1 mL of Au(III) or 1.5 mL of Hg(II) standard solutions (100 μg/mL), and the volumes were completed to the marks with DDW. After adjusting to the optimum conditions, the absorbance of these solutions was measured against blank solution, and the concentration of the studied analyst was calculated depending upon the respective standard direct calibration curve.

2.3.3. Determination of Antibiotic Complexes in Dosage Form by Standard Addition Method

To a series of solutions containing 1 mL of Au(III) or 1.5 mL of Hg(II) standard solutions (100 μg/mL) and various amounts 0.5–6.0 mL or 0.5–5.5 mL, respectively, of TC. HCl standard solutions (100 μg · mL−1) was added 1 mL of dosage antibiotic (samacycline) solutions (100 μg · mL−1) in 10 mL volumetric flasks. The volumes were completed to the marks with DDW. After adjusting the optimum conditions in each case, the absorbance was measured, and the relationship between absorbance and concentration was plotted to construct standard addition curves.

2.4. Flame Atomic Absorption Spectrometry FAAS for Au(III) Complexes

This method was worked out for the determination of dosage TC at the same optimum conditions fixed in the UV-visible method except that the suitable concentration of Au(III) ion was 6 μg/mL.

2.4.1. Determination of TC-Au(III) Complexes by Direct Method

Solution mixtures containing 1–30 μg/mL of TC. HCl and 6 μg · mL−1 of Au(III) ion were prepared by adding different volumes of antibiotics’ standard solutions (100 μg/mL) to 0.6 mL of Au(III) ion standard solution (100 μg/mL) in 10 mL volumetric flasks. The volumes were completed to the mark and the experimental conditions were adjusted. The absorbance was measured, and the relationship between absorbance and concentration was plotted to produce standard direct calibration curve

2.4.2. Determination of TC-Au(III) Complexes in Dosage Form by Direct Method

From a prepared solution of dosage TC (100 μg/mL) 0.6, 0.8, and 1.0 mL were transferred to 10 mL volumetric flasks, containing 0.6 mL of Au(III) standard solution (100 μg/mL). The volumes were completed to the marks, and all optimum conditions were adjusted. The absorbance of these solutions against blank solution was measured, and the concentrations of solutions were calculated depending on standard calibration curve.

2.4.3. Determination of TC-Au(III) Complexes in Dosage Form by Standard Addition Method

In this method 0.6 mL of dosage TC (100 μg/mL) solutions was added to aqueous solutions containing a mixture of 0.6 mL Au(III) standard solutions (100 μg/mL) and different volumes of TC. HCl standard solutions to form 1–25 μg/mL solutions. After all optimum conditions have been adjusted, the relationship between absorbance and concentration was plotted to produce standard addition curves for determination of TC in dosage form.

2.5. High Performance Liquid Chromatography (HPLC)

Solutions were prepared as was mentioned in the direct UV-spectrophotometric method but with different concentration ranges of standard TC. HCl (10-150 μg/mL) and without solvent extraction. The peak area of complexes was plotted against antibiotic concentration to construct the direct calibration curves.

2.5.1. Determination of Dosage TC Au(III) and Hg(II) Complexes by Direct HPLC Method

A powder of 20 capsules (0.1 g) was weighted and dissolved in (100 mL) DDW in a volumetric flask. Twenty-five mL of this solution was diluted to 100 mL with DDW in a 100 mL volumetric flask. Then 1.2, 2.0, and 4.0 mL of the end solution were transferred to (10 mL) volumetric flasks containing 1.0 mL of Au(III) or 1.5 mL of Hg(II) standard solutions (100 μg/mL). The volumes were completed to the marks, and all optimum conditions were adjusted. Peak area of these solutions was measured, and the concentration of solutions was calculated depending on standard direct calibration curve.

3. Result and Discussion3.1. UV-Visible Spectrophotometry

The UV-visible spectra of TC. HCl, metal ions, and their complexes (50 μg · mL−1) in aqueous solutions are shown in Figure 2. The spectrum of the drug exhibited a multiplet with maximum absorption peaks at λ 235, 270, and 370 nm corresponding mainly to transitions [18]. The spectrum of the yellow gold(III) aqueous solution exhibited two absorption bands, a doublet appeared at λ 240 and 295 nm and a low intensity band at 385 nm and were assigned to ligand to metal charge transfer and transitions of square planar tetrachloroaurate(III) anion [19]. The spectrum of the Hg(II) ion exhibited a single high intensity band at λ 285 nm and was assigned to charge transfer [19]. The spectrum of TC. HCl with the Au(III) ion exhibited hypsochromic shift of the transitions band and the appearance of two new absorption bands at 350 and 425 nm assigned to and transitions of square planar gold(III) complexes, respectively [16, 19, 20]. The spectrum of the Hg(II) complex solution exhibited shifts of ligand bands to shorter wavelengths and the appearance of additional band at 320 nm attributed to ligand to metal charge transfer transition [16, 19, 20].

(a)

(b)

(c)

(d)

(e)

(a)

(b)

(c)

(d)

(e)

Figure 2

UV-visible spectra of (a) gold(III) ion, (b) mercury(II) ion, (c) TC. HCl, (d) TC-Au(III), and (e) TC-Hg(II) complexes in aqueous solutions (50 μg/mL each).

3.1.1. Optimisation of the Experimental Conditions

Figure 3 shows the effect of metal ion concentration, pH of solution, temperature, and heating time on the absorbance of the TC-Au(III) and TC-Hg(II) complex solutions at 425 and 320 nm, respectively, using 50 μg/mL of drug solutions. The optimum concentration of the ions that gave maximum absorbance was 10 μg/mL of Au(III) ions and 15 μg/mL of Hg(II) ions. The best values of pH recorded for the highest absorbance values were 2–4. At high pH values formation of metal hydroxides took place [21, 22]. It is also important to mention that tetracycline is a very adaptive molecule, capable of easily modifying itself through tautomerism in response to various chemical environments [10, 23–25]. Therefore protonation and deprotonation of Tcs in acidic and basic aqueous solutions have important effects on their coordination behavior and their absorption spectra [23, 24]. However the hypsochromic shift of the transitions band in this study supports the suggested structures of Au(III) and Hg(II) complexes [16]. The extraction efficiency and the choice of extracting solvent have also an important effect on absorbance values [26, 27]. Optimum experimental conditions for TC. HCl complexes with Au(III) and Hg(II) ions are described in Table 1.

ComplexIon conc. (µg/mL)pHHeating temp. (°C)Heating time (min.)Phase ratio

(EA : aq)Extraction time (min.)Extraction% (E%)Distribution

ratioTC-Au10475151 : 5195.242100.196TC-Hg152–475151 : 51-295.335102.181

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