Determination of Doxycycline as Pure and in Pharmaceutical Preparation (Capsule) Using 4-Aminoantipyrine in Presence of Potassium Periodate

A simple spectrophotometric method for the determination of doxycycline in aqueous medium using oxidative coupling reaction has been described. The method was based on the reaction of doxycycline with 4-aminoantipyrene (4-AAP) which gives soluble and stable red colored product that gives the highest absorption at the wavelength 515 nm, and the linearity of Beer's law was in the concentration range from 5 to 110 µg/ml, and the value of the determination coefficient for the standard curve was 0.9995, which statistically indicates that it has excellent linear characteristics. The molar absorptivity was calculated and found to be 1.387x10 3 l.mol -1 .cm -1 . Sandell's sensitivity value, the Limit of detection (LOD), and ‎‏ limit of quantitation (LOQ) were calculated and equal to 0.3334 μg/cm 2 , 0.114 and 0.38 µg/ml respectively the method was successfully applied for the determination of doxycycline in its pharmaceutical preparation.


INTRODUCTION
Doxycycline is one of the tetracycline-series antibiotics that occupies an important place among the broad-spectrum antibiotics. They overpower the imitation of Gram-negative and positive, and a lot of viruses (Korolkovas and Burckhalter, 1981;Al-Abachi and Al-Nedawi, 2015;Mamani et al., 2006), and is a good-looking handling choice for COVID-19 (Malek et al., 2020;Tommy, 2020). Doxycycline monohydrate (C 22 H 24 N 2 O 8 .H 2 O, M.wt. = 462.5 g/mol) yellow crystalline powder, slightly soluble in alcohol and water, also dissolved in dilute mineral acids and alkaline solutions. Doxycycline has the following structure in scheme 1 (British Pharmacopoeia, 2016).

EXPERIMENTAL Apparatus
Spectral measurements and absorbance readings were carried out using a JASCOV-630 spectrometer, and glass cells with a light path of 1 cm were used. The acidity of the solutions was measured using TRANSE BP3001 professional pH meter

Chemicals and prepared solutions
The chemicals used in this research were of high purity and did not require any purification process.

4-aminoantipyrine solution (1.23 x 10 -3 M).
This solution was prepared by dissolving 0.0250 g of pure 4-aminoantipyrine in distilled water, then transferred to a 100 ml volumetric flask, and the volume was completed to the mark with distilled water.

Potassium periodate solution (1.5 x 10 -2 M)
0.3448 g of potassium periodate (Fluka Company) was weighed and dissolved in distilled water, then transferred to a 100 mL volumetric flask and the volume was completed to the mark with distilled water.

Sodium hydroxide solution (4 M)
Sodium hydroxide solution in 4M concentration was prepare by diluting an ampoule of 10 mol/L (100 ml) supplied by Fluka Company with distilled water in a 250 ml volumetric flask, and then kept in a non-glass (plastic) container. Doxycycline solution (500 µg/ml): This solution was prepared by dissolving 0.0500 g of pure doxycycline (SDI) in 100 ml of distilled water in a volumetric flask. Pharmaceutical preparation solutions (500 µg/ml) 5 capsules formulation (Saudi Arabia/ Tabuk), were carefully weighed and mixed well, amount of the powder equivalent to 0.0500 g of pure doxycycline was weighed and dissolved in distilled water then filtered into a volumetric flask of 100 ml and supplemented with distilled water up to the mark. 5 capsules formulation (India / Ajanta pharma limited), were carefully weighed and mixing well, and the same procedure above has been followed.

Procedure and standard curve for the determination of doxycycline
After the optimum conditions for the determination of doxycycline were established, the standard curve was prepared by adding 1.5 ml of 4-aminoantipyrene reagent (1.25x10 -3 M) to increasing volumes of doxycycline solution at a concentration of 500 μg/ml added firstly to volumetric flasks of 10 ml, then adding 1.25 ml of the oxidizing agent (potassium periodate,1.5x10 -2 M) and finally adding 0.75 mL of sodium hydroxide solution (4 M), then placing the volumetric flasks in the water bath at a temperature of 50 ° C for 15 minutes, leave them for 5 min. after dilution with distilled water to the mark. The absorbance was measured at a wavelength of 515 nm. After drawing the standard curve for the estimation of doxycycline. It was found that the linear range of the concentrations ranged from 5 to 110 µg / mL, and the value of the determination coefficient for the standard curve was 0.9995, which statistically indicates that it has excellent linear specifications Fig. (1). The molar absorbance was calculated and found to be equal to 1.387x10 3 l/mol.cm. Sandell's index value equals to 0.3334 µg/cm 2 , which indicates the sensitivity of the method. The detection limits and quantitation limits were calculated, and they were 0.114 and 0.38 µg/ml, respectively.

RESULTS AND DISCUSSION
All factors affecting absorbance of the formed colored product have been studied:

Preliminary study
The method includes the formation of the colored product via coupling doxycycline with 4aminoantipyrine in the presence of potassium periodate in alkaline medium (4 M of sodium hydroxide), the reaction needs heating in a water bath at a temperature of 40°C for ten minutes. The colored product gave the highest absorption spectrum at the wavelength of 515 nm, so it was used in the subsequent measurements.

Setting the optimum conditions for the determination of doxycycline:
Subsequent experiments were performed using 1 mL of a 500 µg/mL of pure doxycycline solution in a final volume of 10 mL and the absorbance of the colored product at the wavelength of 515 nm was measured against its blank solution.

Selection of the oxidizing agent
A number of oxidizing agents were tested, such as potassium periodate, sodium periodate, Nbromosuccinimide, and N-chlorosuccinimide, by adding 1 ml of them separately to solutions containing equal concentrations of doxycycline (50 µg/mL) in the presence of fixed amounts of 4-4-aminoantipyrine (1 ml of 1.25 x 10 -3 M) and a solution of sodium hydroxide 1 mL at a concentration of (4 M) and then heated for 10 minutes at 40° C in a water bath (Table 1). The results in (Table 1) indicate that the maximum absorbance was obtained when using potassium periodate, in addition to that the value of the color contrast (∆λ) was good and potassium periodate was recommended to be used in subsequent experiments.

Effect of the amount of potassium periodate solution
The effect of increasing volumes from 0.5 to 2.0 ml of potassium periodae solution (1.5x10 -2 M) on the absorbance of the colored product from various amount of doxycycline (25-75 µg /mL) was studied, the results shown in (Table 2). The results in (Table 2) show that the best volume is 1.25 mL as it gave maximum absorbance and the highest determination coefficient value. Therefore, this volume was used in subsequent experiments.

Effect of the amount of 4-aminoantipyrine reagent solution
Different amounts (1.0 to 2.5 ml) of 4-aminoantipyrine reagent ((1.25x10 -3 M)) were added to different concentrations of doxycycline ranging from 10 to 100 µg/ml with the addition of 1.25 ml of potassium periodate and heating for 10 minutes in a water bath at 40°C, the results are shown in (Table 3). The results illustrated in )Table 3( show that using 1.5 ml of 4-aminoantipyrine reagent gave the highest absorption and the highest value of the determination factor, so it was confirmed in subsequent experiments.

Base type effect
The effect of different types of bases at a concentration of 4 M and a volume of 1 ml on the absorbance of the formed product was studied by adding 1.5 mL of 4-aminoantipyrine reagent and 1.25 mL of the oxidizing agent KIO 4 to 1 ml of doxycycline solution with a concentration of 500 µg/mL and then heating in the. water bath at 40°C for 10 minutes. The obtained results were illustrated in (Table 4). From the results listed in (Table 4), it was found that the reaction needs a strong base medium that is better than the weak, and the base sodium hydroxide gives the highest absorbance, so it has been confirmed in subsequent experiments.

Effect of base quantity
The effect of different volumes of sodium hydroxide on the oxidation and coupling of doxycycline with 4-AAP was studied and the results are shown in (Table 5). It was noted from the results in (Table 5) that the volume of 0.75 mL gave the highest absorbance of the colored product, and therefore it was used in subsequent experiments.

Addition of components sequence effect
In order to choose the best sequence of the reacting components, the sequences shown in (Table 6) were chosen with the addition of the reaction components in the quantities mentioned previously and heating for 10 minutes in a water bath at 40° C. The results of the (Table 6) show that all the addition sequences give close values of absorbance, and the sequence I was chosen to give maximum absorbance.

Effect of temperature
The results listed in (Table 7) show the effect of different temperatures from 25 to 60 °C on the absorbance of the colored product, and it was found that the temperature of 50 °C is optimal for coupling doxycycline with -4-aminoantipyrine in the presence of potassium periodate in the sodium hydroxide medium. Therefore, this degree was fixed in the subsequence experiments.

Effect of heating time on the formation of the colored product
The time required to complete the coupling process between doxycycline and the reagent 4aminoantipyrine at 50 °C was studied, and the results in (Table 8) show that 15 minutes gave the highest absorbance of the colored product, and therefore its use was proven in subsequent experiments.

Stability time of the colored product
The effect of time on the stability of the colored product was studied by taking two different concentrations 25 and 50 µg/L and proceed according to the procedure mentioned before, the absorbance was read every 5 or 10 minutes for 60 minutes (Table 9). From the results cited in Table 9, the colored product has good stability at least for a period of 55 minutes.

Final absorption spectrum
After creating the optimal conditions shown in (Table 10), the absorption spectrum of the colored product was plotted, which consisted of the reaction of 50 μg/mL of Dox with 1.5 mL of 4-AAP, and according to the optimal conditions, a colored product gave the highest absorption at the wavelength 515 nm whereas the blank has lowest absorbance at this wavelength and the wavelength 515 nm was recommended in the subsequent measurements Fig. (2).

Accuracy and precision
Under the optimum conditions shown in the method of work mentioned in (Table 10), the recovery percentage, which expresses accuracy and the relative standard deviation (RSD%), which expresses the precision for two different concentrations of doxycycline 30 and 50 μg / ml were calculated. The results in (Table 11), which indicate that the method has good accuracy and precision.

The nature of the formed colored product
The two methods of continuous variation and mole ratios (Job, 1971) were applied to study the molar structural ratio of the colored product formed between Dox and 4-AAP at a concentration of 1.08 10 -3 mol/l for each solution, that the final volume of flasks is equal for all solution Fig. (3).

Fig. 3: The plot of continuous variation method for colored product.
From Fig. (3), we infer that the reaction ratio is 1:1, and to verify that this ratio is correct, the method of mole ratio was applied. Fig. (4) shows the mole ratio plot obtained from adding increasing volumes from 0.3 to 4.0 ml of 4-AAP solution at a concentration of 1.08 x 10 -3 mol./l to a constant volume of 1 ml of 1.08x10 -3 mol./l of Dox solution. Based on the results of figures 3 and 4, the proposed chemical structure for the formed colored product was suggested (Scheme 2).

Application of the method
The suggested method was applied in estimation of Dox in its pharmaceutical preparation's doxycycline capsule. The method was applied by taking different volumes of the stock solution of 500 µg/ml of the pharmaceutical preparation (capsule) to obtain concentrations of 10,30 and 50 µg/mL of pure Dox and treated according to the procedure for pure solution of doxycycline. The obtained results are summarized in (Table 12). It can be concluded from the results shown in (Table 12) that the recovery% for the analysis of the doxycycline capsule for Tabuk company was 100.07% and for Limassol was 99.75%, which indicates that the method has good efficiency and accuracy in estimation.

Standard addition method
In order to prove that the proposed method is credible and successful in the quantitative determination of doxycycline in pharmaceutical preparations and that it is free from the interferences of standard additives that used in preparation drugs, the standard addition method was applied on concentrations of 10 and 20 μg/mL Dox for the two pharmaceutical preparations, as shown in Fig. (5, 6) and (Table 13).

Comparison with other method
A comparison has been made for the most important analytical variables of the current proposed method with its counterparts in other spectroscopic method (Table 14).  (1981). "Química Farmacêutica, Editora Guanabara Koogan". S.A., Rio de Janeiro, 1ª ed., 595 p.