Spectrophotometric and spectrofluorimetric methods

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 192 (2018) 108–116

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Spectrophotometric and spectrofluorimetric methods for determination of certain biologically active phenolic drugs in their bulk powders and different pharmaceutical formulations Mahmoud A. Omar a,⁎, Kalid M. Badr El-Din a, Hesham Salem b, Osama H. Abdelmageed c a b c

Analytical Chemistry Department, Faculty of Pharmacy, Minia University, Minia, Egypt Pharmaceutical Chemistry Department, Faculty of Pharmacy, Deraya University, Minia, Egypt Pharmaceutical Chemistry Department, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 27 August 2017 Received in revised form 20 October 2017 Accepted 25 October 2017 Available online 8 November 2017 Keywords: Spectrophotometry Spectrofluorimetry Phenolic drugs Cerium (IV) Pharmaceutical analysis

a b s t r a c t Two simple and sensitive spectrophotometric and spectrofluorimetric methods for the determination of terbutaline sulfate, fenoterol hydrobromide, etilefrine hydrochloride, isoxsuprine hydrochloride, ethamsylate, doxycycline hyclate have been developed. Both methods were based on the oxidation of the cited drugs with cerium (IV) in acid medium. The spectrophotometric method was based on measurement of the absorbance difference (ΔA), which represents the excess cerium (IV), at 317 nm for each drug. On the other hand, the spectrofluorimetric method was based on measurement of the fluorescent of the produced cerium (III) at emission wavelength 354 nm (λexcitation = 255 nm) for the concentrations studied for each drug. For both methods, the variables affecting the reactions were carefully investigated and the conditions were optimized. Linear relationships were found between either ΔA or the fluorescent of the produced cerium (III) values and the concentration of the studied drugs in a general concentration range of 2.0–24.0 μg mL−1, 20.0–24.0 ng mL−1 with good correlation coefficients in the following range 0.9990–0.9999, 0.9990–0.9993 for spectrophotometric and spectrofluorimetric methods respectively. The limits of detection and quantitation of spectrophotometric method were found in general concentration range 0.190–0.787 and 0.634–2.624 μg mL−1respectively. For spectrofluorimetric method, the limits of detection and quantitation were found in general concentration range 4.77–9.52 and 15.91–31.74 ng mL−1 respectively. The stoichiometry of the reaction was determined, and the reactions pathways were postulated. The analytical performance of the methods, in terms of accuracy and precision, were statistically validated and the results obtained were satisfactory. The methods have been successfully applied to the determination of the cited drugs in their commercial pharmaceutical formulations. Statistical comparison of the results with the reference methods showed excellent agreement and proved that no significant difference in the accuracy and precision. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Terbutaline sulfate; 5-[2-[(1,1-Dimethylethyl) amino]-1– hydroxyethyl]-1,3–benzenediol (TER), fenoterol hydrobromide; 5-[1Hydroxy–2-[[2-(4–hydroxyphenyl)-1–methylethyl]amino]ethyl]-1,3– benzenediol (FEN) and isoxsuprine hydrochloride; 4-hydroxy-α-[1-[(1methyl-2-phenoxyethyl) amino]ethyl] benzenemethanol (ISO) are β2selectiveadrenoreceptor [1,2]. Terbutaline sulfate and fenoterol hydrobromide both are used in the treatment of bronchial asthma, while isoxsuprine hydrochloride is used to inhibit uterine contraction in cases of premature labor or fetal distress [1,2]. Etilefrine hydrochloride; α-[(Ethylamino)methyl]-3–hydroxybenzenemethanol (ETI) is a

⁎ Corresponding author. E-mail address: [email protected] (M.A. Omar).

https://doi.org/10.1016/j.saa.2017.10.065 1386-1425/© 2017 Elsevier B.V. All rights reserved.

selective α1 receptor agonist; it activates α receptors only at much higher concentrations. The drug causes marked arterial vasoconstriction. The major clinical effects of a number of sympathomimetic drugs are due to activation of α adrenergic receptors in vascular smooth muscle. As a result, peripheral vascular resistance is increased and blood pressure is maintained or elevated [2]. Doxycycline hyclate (DOX); is a semisynthetic derivative of tetracyclines. It is bacteriostatic antibiotic with activity against a wide range of aerobic and anaerobic gram-positive and gramnegative bacteria [1,2]. Ethamsylate; N-ethylethanamine-2,5dihydroxybenzenesulfate (ETH) is a synthetic compound which has a haemostatic action. It basically normalizes the balance between coagulants and anti-coagulants. This drug raises the formation of platelet plug by increasing the aggregation and adhesiveness of platelet. Also it augments fibrin formation, by increasing the generation of thromboplastin, which is the III coagulation factor and is able to accelerate the clotting of blood [1,2].

M.A. Omar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 192 (2018) 108–116

Several methods have been reported for the determination of the studied drugs in their pure forms, pharmaceutical preparations or biological fluids. These methods include spectrophotometric [3–19], spectrofluorimetric [19–20], chromatographic [21–30], electrophoresis [31], immunoassay [32–33], flow injection [34–36] and electrochemical methods [37–40]. According to the literatures, it has been reported that ISO has been determined fluorimetrically after oxidation with cerium (IV) [18]. Therefore the objective of the current work is to expand the use of cerium (IV) as oxidizing agent in order to develop not only spectrofluorimetric method for the rest of the cited compounds but also another spectrophotometric method for all the cited drugs. Both methods were utilized for the construction of calibration graphs to determine the concentration of the studied drugs either in their pure forms or in their pharmaceutical formulations. This study also involves the investigation of the structure variations of the selected compounds on sensitivities of both methods. In addition, the stoichiometry as well as the reaction mechanism of the reaction of cerium with the cited drugs was also studied.

109

Fig. 1. Absorption spectra of cerium ammonium sulfate in presence and absence of terbutaline sulfate as a representative example (4.0 μg mL−1).

2. Experimental 2.1. Instrumentation and Apparatus Spectronic™ Genesys™ UV/Visible spectrophotometer (Milton Roy Co, Westhaven, USA) connected with IBM computer loaded with Winspec™ application software and Jenway® 6505, UV/Visible spectrophotometer (Jenway Co, London, UK) with matched 1 cm quartz cell were used for all measurements. Perkin-Elmer UK model LS 45 Luminescence spectrometer, equipped with a 150 W Xenon arc lamp, grating excitation and emission monochromators were set at 10 mm. A 1 cm quartz cell was used, connected to an IBM computer loaded with the FL Winlab™ application software. Thermostatically controlled water bath (Salvis AG Emmenbruck, Luzern, Germany), digital analytical balance (AG 245, MetlerTaledo, Switherland) and sonicator (Bender + Hobein, B-220, Germany) were also used. 2.2. Pharmaceuticals and Reagents Samples of the investigated drugs were generously supplied by their respective manufacturers with overall general estimated purities 99.44– 99.82% and were used without further purification. Those are Terbutaline sulfate, fenoterol hydrobromide and etilefrine hydrochloride (TER, FEN, ETI; CID Co., Cairo, Egypt). Isoxsuprine hydrochloride (ISO; Pharco Co., Alexandria, Egypt). Ethamsylate (ETH; Memphis Co., Cairo, Egypt). Doxycycline hyclate (DOX; El-Nile Co., Cairo, Egypt). Ceric ammonium sulfate (Riedel De-Haen, AG, Seelze - Hamnover, Germany). Perchloric acid 70% aqueous solution, sulfuric acid (Sigma Co., St. Louis, USA). Nitric acid, hydrochloric acid and acetic acid (El-Nasr Co, Cairo, Egypt). Two molar solutions of ceric ammonium sulfate were prepared, 2.0 × 10−3 M and 5.0 × 10−4 M, in different solvents. 2.0 × 10−3 molar solution was prepared by dissolving 126.5 g in 100 mL of 2 molar perchloric acid solution. 5.0 × 10−4 molar solution was prepared by dissolving 31.6 g in 100 mL of 2 molar sulfuric acid solution. Both standards were freshly prepared daily and kept in brown glass bottles to avoid photochemical reaction. All solvent used throughout this study such as absolute methanol, absolute ethanol, n-propanol, acetone and acetonitrile (El-Nasr Co, Cairo, Egypt) were of analytical grade. In addition, double distilled water was also used. The following available commercial dosage forms were analyzed; Bricanyl® tablets and syrups (CID Co., Cairo, Egypt) are labeled to contain 2.5 mg of TER per tablet or 1.5 mg per 5 mL of syrup. Berotec® tablets and syrups (CID Co., Cairo, Egypt) are labeled to contain 2.5 mg of FEN per tablet or per 5 mL syrup. Effortil® tablets and oral drops (CID Co., Cairo, Egypt) are labeled to contain 5 mg of ETI per tablet or 5 mg per 10 drops. Duvadilan® tablets (Pharco Co., Alexandria, Egypt) are labeled to contain 20 mg of ISO per tablet. Dicynon®

tablets and ampoules (Memphis Co., Cairo, Egypt) are labeled to contain 250 mg of ETH per tablet or ampoule. Doxymycin® capsules (El-Nile Co., Cairo, Egypt) are labeled to contain 100 mg of DOX per capsule. 2.3. Preparation of Standard Solutions Stock solutions containing 1.0 mg mL−1 of the investigated drugs were prepared in double distilled water. The working standard solutions containing 40.0 μg mL−1 for spectrophotometric determination or 0.4 μg mL−1 for spectrofluorimetric determination, were prepared daily by suitable dilution of stock solutions with double distilled water. 2.4. Preparation of Sample Solution for Pharmaceutical Formulation 2.4.1. Tablets An accurately weighed amount equivalent to 10.0 mg of each drug from composite of 20 powdered tablets was transferred into a 100 mL volumetric calibrated flask, dissolved in about 20 mL of double distilled water and then diluted to the volume with same solvent. The resultant mixture was sonicated for 10 min, filtered off and the first portion was rejected. Further dilutions with double distilled water were made to yield working solutions of 40.0 μg mL−1 and 0.4 μg mL−1 for spectrophotometric and spectrofluorimetric methods respectively. Then the general procedure was continued, as described under Section 2.5. 2.4.2. Capsules The contents of 20 capsules were evacuated, mixed well, and then an accurately weighed amount equivalent to 10.0 mg of each drug was

Fig. 2. Excitation and emission spectra of cerium ammonium sulfate (5.0 × 10−4 M) in presence of fenoterol hydrobromide as a representative example (100 ng mL−1).

110


Table 1 Quantitative analytical parameters for the reaction of the studied drugs with cerium (IV) after measuring the absorbance difference at 317 nm. Drug

Linear range (μg mL−1)

a

b

r

r2

LOD (μg mL−1)

LOQ (μg mL−1)

Terbutaline sulfate Fenoterol hydrobromide Isoxsuprine hydrochloride Etilefrine hydrochloride Ethamsylate Doxycycline hyclate

2.0–14.0 2.0–12.0 3.0–24.0 2.0–16.0 3.0–18.0 2.0–12.0

0.0041 0.0104 −0.0005 −0.0055 0.0004 0.0014

0.0538 0.0596 0.0331 0.0458 0.0324 0.0604

0.9991 0.9999 0.9995 0.9998 0.9990 0.9996

0.9983 0.9997 0.9990 0.9996 0.9980 0.9993

0.496 0.190 0.574 0.242 0.787 0.313

1.654 0.634 1.914 0.808 2.624 1.043

a: intercept, b: slope, r: correlation coefficient, r2: determination coefficient, LOD: limit of detection, LOQ: Limit of Quantitation calculated as reported [49].

transferred into a 100 mL calibrated flask, dissolved in about 20 mL of double distilled water and then diluted to the volume with same solvent. Then the procedure was continued as described under Section 2.4.1 for tablet preparation starting from the resultant mixture was sonicated to the end of the procedure.

was added. The resultant solution was allowed to stand for 10 min, then diluted to the volume with double distilled water, and mixed well. The relative fluorescence intensity was measured at emission wavelength 354 nm (λexcitation 255 nm) against reagent blank treated similarly.

2.4.3. Syrups and Oral Drops An accurately measured portion of the content of syrup or oral drop bottles equivalent to 10 mg of the each drug was transferred into a 100 mL calibrated volumetric flask, and then diluted to the volume with double distilled water. Further dilutions with double distilled water were made to obtain sample working solutions of 40.0 μg mL−1 and 0.4 μg mL−1 for spectrophotometric and spectrofluorimetric methods respectively. Then the general procedure was continued, as described under 2.5.

3. Results and Discussion

2.4.4. Ampoules The content of two ampoules equivalent to 500 mg of ethamsylate was quantitatively transferred into 100 mL calibrated volumetric flask, and then diluted to the volume with double distilled water. Further dilutions with double distilled water were made to obtain sample working solutions of 40.0 μg mL−1 and 0.4 μg mL−1 for spectrophotometric and spectrofluorimetric methods respectively. Then the general procedure was continued, as described under Section 2.5. 2.5. General Procedure 2.5.1. Spectrophotometric Method An accurately measured aliquot volumes of the working standard or sample solutions containing 200.0–240.0 μg mL−1 were transferred into 10.0 mL volumetric calibrated flasks. 2.0 mL of 2.0 × 10−3 molar solution of ceric ammonium sulfate, dissolved in 1.5 M solution of perchloric acid for TER, ISO and ETH or in 2.0 M solution of perchloric acid for FEN, ETI and DOX was added. The resultant solution was allowed to stand for 10–15 min, then diluted to the volume with double distilled water, and mixed well. The resultant absorbance difference readings were recorded at 317 nm against reagent blank treated similarly.

Oxidation–reduction reactions have been used as the basis for the development of simple and sensitive spectrophotometric methods for the determination of many pharmaceutical compounds [41–43]. Ceric, because of its high oxidation potential and excellent solution stability, has been widely used as an effective analytical reagent in some of the reported methods [42,44–45]. On the other hand, ceric (IV) has been used as an oxidizing agent for the determination of some pharmaceutical compounds by monitoring the fluorescence of cerium (III) produced from their oxidation reaction [18,46]. For these reasons, these reactions presented the basis behind the development of sensitive spectrophotometric and fluorimetric methods for the determination of the cited compounds. 3.1. Absorption and Fluorescence Characteristics of the Proposed Method 3.1.1. Absorption Spectra A solution of Ce (IV) dissolved in 1.5–2.0 molar solution of perchloric acid has a faint yellow color of maximum absorbance at 317 nm. The decrease in the absorption intensity at this λmax, caused by the presence of any of the investigated phenolic drugs, is directly proportional to the amount of the drug that has reacted (Fig. 1). 3.1.2. Fluorescence spectra The investigated phenolic drugs were oxidized using a solution of 5.0 × 10−4 M cerium ammonium sulfate dissolved in 2.0 M solution of sulfuric acid. The fluorescence intensity of the formed cerium (III) at λex/ em 255/354 nm was found to be directly proportional to the concentration of the investigated drugs (Fig. 2). 3.2. Optimization of Reaction Variables

2.5.2. Spectrofluorimetric Method An accurately measured aliquot volumes of the working standard or sample solutions containing 0.20–2.40 μg mL−1 were transferred into 10.0 mL volumetric calibrated flasks. 2.0 mL of 5.0 × 10−4 molar solution of ceric ammonium sulfate, dissolved in 2.0 M solution of sulfuric acid,

The optimum conditions for attaining the maximum stable absorbance difference or maximum stable fluorescence intensity were established. This study was carried out by varying the variable under study, while the other variables are kept constant using fixed and the

Table 2 Quantitative parameters for the reaction of the studied drugs with cerium (IV) measuring the RFI at λex/em = 255/354 nm. Drug

Linear range (ng mL−1)

a

b

r

r2

LOD (ng mL−1)

LOQ (ng mL−1)

Terbutaline sulfate Fenoterol hydrobromide Etilefrine hydrochloride Ethamsylate Doxycycline hyclate

20.0–160.0 20.0–160.0 20.0–200.0 40.0–240.0 20.0–160.0

4.50 −5.25 −4.13 3.07 −6.96

2.917 3.567 2.350 1.972 3.772

0.9990 0.9991 0.9990 0.9992 0.9993

0.9980 0.9981 0.9981 0.9983 0.9985

5.51 5.37 5.73 9.52 4.77

18.30 17.91 19.10 31.74 15.91

a: intercept, b: slope, r: correlation coefficient, r2: determination coefficient, LOD: limit of detection, LOQ: Limit of Quantitation calculated as reported [49].


perchloric acid. The reaction of Ce (IV) in 2 M sulfuric acid (E° = 1.44 V) or nitric acid (E° = 1.61 V) as oxidant for organic compounds proved to be extremely slow and failed to be stoichiometric. The greater oxidation potential of Ce (IV) in perchloric acid medium (E° = 1.75 V) overcomes both the slowness of the oxidation process and the inexact stoichiometry encountered in both sulfuric and nitric acid. Its solution in hydrochloric acid (E° = 1.28 V) is unstable owing to the oxidation of chloride ion to chlorine gas [48].

Table 3 Evaluation of accuracy of the analytical procedure using spectrophotometric method. Drug

Concentrated studied (μg mL−1)

% Recoverya

Terbutaline hydrochloride

4.0 8.0 14.0 4.0 8.0 12.0 9.0 18.0 24.0 6.0 12.0 16.0 6.0 12.0 18.0 4.0 8.0 12.0

100.38 ± 1.31 100.27 ± 0.69 100.05 ± 0.67 100.08 ± 1.27 100.08 ± 0.92 100.28 ± 1.12 99.34 ± 1.25 99.87 ± 0.90 99.45 ± 0.98 99.49 ± 1.03 99.70 ± 0.67 99.58 ± 0.89 100.10 ± 1.62 100.21 ± 0.76 99.73 ± 0.76 99.19 ± 0.87 99.92 ± 0.68 99.73 ± 0.61

Fenoterol hydrobromide

Isoxsuprine hydrochloride

Etilefrine hydrochloride

Ethamsylate

Doxycycline hyclate

a

3.2.1. Type of Acid Used For spectrophotometric method, different types of acids such as acetic, hydrochloric, nitric, sulfuric and perchloric acid were tried. The results of such study were in agreement with the previous reported methods [43–44], where perchloric acid was found to be the optimum acid for maximum absorbance for all the investigated phenolic drugs. The optimum concentration of perchloric acid was determined by following the absorbance development using 2 mL of ceric solution dissolved in different molarities of perchloric acid ranging from 0.5–4.0 M solution at 25 ± 5°C. 2 mL of ceric solution dissolved in 1.5 M solution of perchloric acid for TER, ISO and ETH or in 2.0 M solution of perchloric acid for FEN, ETI and DOX were found to be optimal for maximum absorbance difference at 317 nm. For fluorescence investigation, the reaction was carried out in presence of perchloric acid and sulfuric acid and the reaction as well as the fluorescence of Ce (III) were found to be high. However, perchloric acid gave high blank readings, so sulfuric acid was selected in this study. The fluorescence intensities were found to be increased with the increase in the molarity of sulfuric acid to reach the maximum intensity at 1.5 M and be constant in the range of 1.5–3.0 M. Therefore, 2.0 M solution of sulfuric acid was found to be optimum for maximum fluorescence intensity for all investigated drugs.

Mean of 5 replicates ± SD.

Table 4 Evaluation of accuracy of the analytical procedure using spectrofluorimetric method. Drug

Concentrated studied (ng mL−1)

% Recoverya


60.0 120.0 160.0 60.0 120.0 160.0 80.0 140.0 200.0 80.0 160.0 240.0 60.0 120.0 160.0

99.61 ± 0.60 99.66 ± 0.73 100.13 ± 0.85 99.30 ± 0.90 99.86 ± 0.48 100.14 ± 1.30 99.69 ± 1.08 99.75 ± 0.73 99.28 ± 0.44 100.25 ± 1.46 100.00 ± 1.08 99.54 ± 0.74 100.10 ± 1.36 99.44 ± 1.06 100.17 ± 0.58



Ethamsylate

Doxycycline hyclate

a

111

Mean of 5 replicates ± SD.

same concentrations of drugs under study. In each study the general procedure described under Section 2.5 was applied. First of all, the step of oxidation has to be conducted in acid medium, to prevent the precipitation of hydrated ceric oxide, CeO2·XH2O [47]. The reaction of the studied compounds with Ce (IV) proceeds quantitatively only in the presence of

3.2.2. Volume of Ceric Molar Solution For spectrophotometric method, the reaction progress increases substantially with increasing the volume of 2.0 × 10−3 molar solution of cerium (IV). Maximum absorbance difference was obtained when 1.5– 2.5 mL was used, after that the absorbance difference may be decreased. Thus, the use of 2.0 mL of cerium (IV) proved to be optimum for the maximum absorbance difference for all investigated drugs. For spectrofluorometric method, the reaction progress increases with increasing the volume of 5.0 × 10−4 molar solution of cerium (IV). Maximum fluorescence intensity was obtained in the range of 1.0–2.0 mL (in case of TER, FEN, and ETI), 1.0–2.5 mL (in case of ETH) or in the range of 1.5–2.0 mL (in case of DOX). Thus the use of 1.5 mL (in case of TER, FEN, ETI, and ETH) and 2.0 mL (in case of DOX) of cerium (IV) were optimal.

Table 5 Spectrophotometric determination of the studied drugs (8 μg mL−1) in the presence of the studied excipients. Excipient

Excipient add % Recovery ± SD (n = 5) (μg mL−1)

Glucose

25 50 100 Lactose 25 50 100 Sucrose 25 50 100 Starch 25 50 100 Gum acacia 25 50 100

Terbutaline hydrochloride Fenoterol hydrobromide Ethamsylate

Etilefrine hydrochloride Doxycycline hyclate Isoxsuprine hydrochloride

98. 16 ± 1.60 100.20 ± 1.40 104.20 ± 1.90 97. 20 ± 1.30 101.40 ± 1.80 100.15 ± 1.20 99. 20 ± 1.70 100.20 ± 1.30 102.15 ± 1.40 97.60 ± 1.20 99.20 ± 1.50 98.15 ± 1.10 96.80 ± 1.30 99.00 ± 1.10 99.15 ± 1.80

97. 14 ± 1.30 100.20 ± 1.60 105.00 ± 1.70 99. 20 ± 1.60 98.60 ± 1.80 101.00 ± 1.40 99. 60 ± 1.30 101.20 ± 1.60 100.60 ± 1.40 96.70 ± 1.80 99.70 ± 1.90 100.00 ± 1.20 98.00 ± 1.80 97.00 ± 1.60 95.10 ± 1.40

95. 20 ± 1.10 99.20 ± 1.20 102.60 ± 1.90 99. 40 ± 1.90 100.30 ± 1.60 99.20 ± 1.80 99. 80 ± 1.50 101.60 ± 1.30 100.70 ± 1.80 98.90 ± 1.42 100.50 ± 1.10 97.60 ± 1.90 99.50 ± 1.82 96.90 ± 1.10 97.20 ± 1.80

96. 16 ± 1.34 101.20 ± 1.50 101.50 ± 1.60 98. 20 ± 1.60 100.60 ± 1.70 101.20 ± 1.60 99. 00 ± 1.50 102.20 ± 1.80 99.60 ± 1.50 95.60 ± 1.60 98.70 ± 1.80 98.00 ± 1.40 96.00 ± 1.90 98.00 ± 1.70 96.10 ± 1.80

95. 80 ± 1.50 99.40 ± 1.80 103.70 ± 1.90 99. 80 ± 1.70 101.70 ± 1.90 102.60 ± 1.80 99. 70 ± 1.40 100.80 ± 1.70 101.60 ± 1.50 95.60 ± 1.60 99.70 ± 1.90 100.40 ± 1.10 97.70 ± 1.30 99.10 ± 1.70 100.60 ± 1.50

99. 20 ± 1.90 98.20 ± 1.50 103.60 ± 1.60 98. 80 ± 1.80 101.60 ± 1.40 100.40 ± 1.50 97. 00 ± 1.40 99.20 ± 1.50 99.70 ± 1.30 95.60 ± 1.60 98.70 ± 1.80 98.00 ± 1.40 99.00 ± 1.70 98.90 ± 1.40 98.10 ± 1.80

112


Table 6 Spectrofluorimetric determination of the studied drugs (100 ng mL−1) in the presence of the studied excipients. Excipient

Glucose

Lactose

Sucrose

Starch

Gum acacia

Excipient add

% Recovery SD (n = 5)

(ng mL−1)



Ethamsylate


Doxycycline hyclate

250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000

97. 30 ± 1.70 100.20 ± 1.40 99.20 ± 1.60 100. 20 ± 1.40 101.20 ± 1.70 99.20 ± 1.40 99. 30 ± 1.80 99.60 ± 1.70 100.6 ± 1.50 99.70 ± 1.40 99.40 ± 1.60 98.60 ± 1.40 98.00 ± 1.60 99.00 ± 1.40 99.6 ± 1.30

98. 20 ± 1.40 99.50 ± 1.50 100.20 ± 1.60 99. 70 ± 1.60 100.40 ± 1.50 99.20 ± 1.40 99. 30 ± 1.30 100.60 ± 1.50 99.70 ± 1.60 97.80 ± 1.40 100.00 ± 1.60 98.70 ± 1.70 99.00 ± 1.70 97.40 ± 1.30 98.60 ± 1.50

99. 40 ± 1.70 98.20 ± 1.50 99.60 ± 1.90 98. 20 ± 1.30 99.70 ± 1.50 98.90 ± 1.40 99. 60 ± 1.60 100.20 ± 1.70 98.60 ± 1.40 97.60 ± 1.70 99.80 ± 1.90 99.00 ± 1.60 98.00 ± 1.30 99.00 ± 1.10 97.20 ± 1.50

98. 20 ± 1.40 99.20 ± 1.60 100.40 ± 1.50 99. 20 ± 1.70 98.60 ± 1.50 100.00 ± 1.40 98. 60 ± 1.40 99.20 ± 1.70 97.60 ± 1.40 98.40 ± 1.80 99.30 ± 1.70 100.20 ± 1.80 98.60 ± 1.70 98.00 ± 1.40 96.60 ± 1.50

96. 70 ± 1.40 99.60 ± 1.60 98.70 ± 1.50 99. 80 ± 1.50 101.60 ± 1.70 99.40 ± 1.30 99. 00 ± 1.60 100.60 ± 1.80 98.60 ± 1.20 98.40 ± 1.40 99.40 ± 1.50 98.30 ± 1.60 98.40 ± 1.20 99.60 ± 1.40 98.50 ± 1.30

3.2.3. Effect of Diluting Solvent Substitution of double distilled water by different other solvents, such as methanol, ethanol, acetonitrile, n-propanol and acetone resulted in significant decrease in either the absorbance differences or fluorescence intensities of the proposed methods. Therefore, double distilled water was chosen because it afforded maximum stability and intensity.

3.2.4. Effect of Temperature The influence of different heating temperatures, from 25 to 100 °C, was studied using a thermostatically controlled water bath. It was found that both the absorbance differences or the fluorescence

intensities remained constant even with the increase in temperature, thus the reaction was carried out in the room temperature. 3.2.5. Reaction Time and Stability of the Measured Response The reaction between the investigated drugs and Ce (IV) was completed within 5–10 min and remained stable for more than 30–40 min at 25 ± 5 °C, for both methods. Therefore a standing time of 15 min for all the investigated drugs was chosen to be the optimal time for either the maximum absorbance differences or the maximum fluorescence intensity. The yellow color of the remaining cerium (IV) or the fluorescence intensity of the produced cerium (III) was found to be completely stable for at least 30 min at 25 ± 5 °C.

Table 7 Determination of the studied drugs in their pharmaceutical dosage forms using spectrophotometric and spectrofluorimetric methods. Drug

Pharmaceutical dosage form

Terbutaline

Bricanyl® tablets

Terbutaline

Bricanyl® syrups

Fenoterol

Berotec® tablets

Fenoterol

Berotec® syrups

Isoxsuprine

Duvadilan® tablets

Etilefrine

Effortil® tablets

Etilefrine

Effortil® oral drops

Ethamsylate

Dicynon® tablets

Ethamsylate

Dicynon® ampoules

Doxycycline

Doxymycin® capsules

a b

Tabulated value at 95% confidence limit; t = 2.31 and F = 6.39. Reference of reported method.

Proposed methods ± SD (n = 5)

Reported method ± SD (n = 5)

Spectrophotometric method

Spectrofluorimetric method

98.68 ± 1.72 t = 0.52a F = 4.67a 100.16 ± 1.27 t = 2.13 F = 2.56 99.64 ± 1.62 t = 1.11 F = 1.84 100.03 ± 0.90 t = 2.08 F = 1.77 97.63 ± 1.21 t = 0.51 F = 1.14 99.10 ± 1.73 t = 0.09 F = 3.02 99.61 ± 1.42 t = 0.75 F = 2.04 98.15 ± 1.29 t = 1.20 F = 2.21 100.45 ± 1.31 t = 2.08 F = 2.26 99.13 ± 1.54 T = 0.94 F = 1.20

97.73 ± 1.24 t = 1.52 F = 2.43 99.53 ± 1.05 t = 1.36 F = 1.75 98.49 ± 1.57 t = 0.18 F = 1.73 99.92 ± 1.55 t = 1.46 F = 1.70 _________________

98.73 ± 0.80 [6]b

98.22 ± 0.63 t = 1.54 F = 2.48 99.99 ± 1.53 t = 1.18 F = 2.38 98.02 ± 1.25 t = 1.42 F = 2.05 100.09 ± 1.04 t = 1.82 F = 1.43 97.28 ± 0.64 t = 1.43 F = 4.88

99.03 ± 1.00 [4]b

98.73 ± 0.80 [6]b

98.64 ± 1.19 [3]b

98.64 ± 1.19 [3]b

98.01 ± 1.14 [18]b

99.03 ± 1.00 [4]b

98.99 ± 0.87 [5]b

98.99 ± 0.87 [5]b

98.26 ± 1.41 [12]b


3.3. Quantitation and Validation of the Proposed Methods The developed procedures were fully validated according to international conference on Harmonisation (ICH) guidelines [49] and USP XXVIII [50] validation guidelines. The following validation parameters were studied. 3.3.1. Linearity and Range Six to ten concentrations within the specified range for each drug were tested by applying the general procedure described under Section 2.5. For spectrophotometric and spectrofluorimetric methods, the regression equations representing these linear correlations can be written as: ΔA = a + bC, and RFI = a + bC respectively. ΔA is the absorbance difference and RFI is the relative fluorescence intensity. In both equations; a is the intercept of the standard curve, b is the slope of the curve used as a measure of the sensitivity of the proposed method and C is the concentration of the analyzed drug in μg mL−1 for spectrophotometric method or ng mL−1 for spectrofluorimetric method. The obtained results are summarized in Tables 1 and 2. Linearity was well assessed by the excellent correlation between the concentrations of the studied drugs and either the Δ A or and the RFI as indicated by the high correlation coefficients obtained. The calculated detection and quantitation limits for both methods are lower enough, indicating good sensitivity of the proposed methods. According to results provided in Tables 1–2, it was clear, in both methods, that DOX showed the highest sensitivity possible, followed by FEN while ETH showed the least one. Thus it was concluded that the presence of electron withdrawing group, e.g. SO3H, render the phenolic group less susceptible to oxidation by the reagent due to low electron density on the phenyl ring. On the other hand since DOX exists in

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poly-rigid rings structure, it facilitates the electron transfer to cerium (VI) which is confirmed by the higher values of slope. 3.3.2. Accuracy The accuracy was checked at three concentration levels within the specified range, five replicate measurements were recorded at each concentration level. The results were recorded as percentage recovery ± standard deviation (Tables 3, 4). The accuracy of the determinations of pure drug substances was further assessed by comparison of the results of the proposed analytical procedures with those of a second well-characterized procedure(s) [3–5,6,12,18] by means of t- and F-tests. The results obtained show the close agreement between the measured and true values. 3.3.3. Precision The precision was checked at three concentration levels within the specified range including 100% of the test concentration. Sex replicate measurements were recorded at each concentration level. The calculated relative standard deviations were all below 2% indicating excellent precision of the proposed procedures at both level of repeatability and intermediate precision. 3.3.4. Specificity and Interference The interferences effect of different excipients commonly used in pharmaceutical formulations such as glucose, lactose, sucrose, starch and gum acacia were investigated and evaluated. Thus solutions containing each of the studied drug and one of the excipients taken in different proportions were analyzed by the proposed methods and data of such analysis are presented in Tables 5, 6. According to this study, no interference could be observed from the studied excipients during

Fig. 3. a. Plot of log RFI against log drug used. b. Plot of log RFI against log Ceric used.

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Fig. 4. Possible reaction pathways between the cited drugs and cerium (VI).


the analysis of the cited drugs. Therefore the dilution effect not only improved the sensitivities of developed methods but also rendered the concentration of studied excipients behind the interference limits. In addition, the experimental procedures were carried at room temperature in absence of any catalyzing reagent. For these reasons the developed methods were considered highly selective for the determination of the cited drugs in presence these excipients with reducing properties. The spectrofluorimetric method is considered more superior in regarding the analysis of any interfering additive because the concentration range studied is hundred time less than those used for the spectrophotometric method. Interference from some lubricant such magnesium stearate and talc powder was also studied. These compounds are insoluble in water; therefore they are removed by filtration after mixing with aqueous sample of the studied drug. No significance difference could be observed upon comparison of results obtained after analysis of samples of the studied drugs mixed with different proportions of lubricant with control one. To confirm the specificity of the developed method, the pharmaceutical preparations extract were analyzed after spiking with known concentration of the investigated drugs. The obtained results from the recovery of the added amount(s) showed that, no interference is encountered from the matrix after applying the extraction method. 3.3.5. Robustness Robustness was well checked during the development phase, for both methods, by evaluating the influence of small variations in the following experimental variables on the analytical performance of the method: molarity of perchloric acid, volume of cerium (IV) and standing time. In these experiments, one experimental parameter was changed while the other parameters were kept unchanged and the recovery percentage was calculated each time. The obtained recoveries and standard deviations indicated that small variations in any of the variables did not significantly affect the results of the proposed procedures. This assessment provided an indication of the reliability of the proposed methods during routine work. 3.3.6. Ruggedness The analysis of the investigated drugs by spectrophotometric method was done using two different instruments, then the results were evaluated and no significant difference was observed. 3.3.7. Application to Pharmaceutical Dosage Forms The proposed methods have been tested on commercial dosage forms. The concentration of investigated drugs was computed from its responding regression equations. The results of the proposed methods were statistically compared with those of reported methods [3–5,6,12,18], in respect to accuracy and precision. In the F- and t-tests, no significant differences were found between the calculated and theoretical values of both the proposed and reported methods at 95% confidence level. This indicates good precision and accuracy in the analysis of the investigated in pharmaceutical dosage forms (Table 7). 3.4. Stoichiometry and Reaction Mechanism The molar ratio between the investigated drugs and ceric was established by the Limiting Logarithmic method [51–52] using the proposed spectrofluorimetric method. The relative fluorescence intensity (RFI) of the produced cerium (III) was measured in presence of excess reagent. Plots of log RFI against log [Drug] (Fig. 3a) and log RFI against log [Ceric] (Fig. 3b) gave straight lines correlation. The values of their slopes (b) are shown in Fig. 3a, b. Therefore, it was concluded that the reaction proceeds in the ratio 1:1 for all drugs except FEN the ratio was 1:2 (drug:ceric). Under the described experimental condition, the secondary or tertiary nitrogen, exist in the structure of the studied compounds, are fully

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protonated, thus they are not undergo oxidation by ceric. On the other hand, the phenolic group is more reliable to undergo oxidation by ceric rather than secondary alcohol group due to high electron density of the phenyl ring. Therefore based on these facts and the molar ratio of the suggested procedure, the reactions of the cited drugs with cerium (VI) were proposed to proceed according to the pathways illustrated in Fig. 4. 4. Conclusion Simple, sensitive, accurate, reliable, low cost spectrophotometric and spectrofluorimetric methods for the determination of the cite drugs have been successfully developed and validated. These methods were based on the oxidation of the cited drugs with cerium (VI) under acid conditions. The proposed methods do not require elaborate treatment of the samples and/or tedious extraction of the absorbing or the fluorescent species produced. Furthermore, the proposed methods use a routine spectrophotometer and spectrofluorimeter that are usually available in all quality control laboratories. These methods can be easily applied to the determination of the cited drugs in their pharmaceutical formulations. The proposed methods are sensitive enough to enable determination of lower amounts of the drug, and compared favorably with the previously reported methods in terms of accuracy and precision. These advantages encourage the application of the proposed methods in routine analysis of the cited drugs in quality control laboratories, as alternatives for the existing methods. References [1] A.C. Moffat, M.D. Osselton, B. Widdop, Clarke's Analysis of Drugs and Poisons, fourth ed. Pharmaceutical Press, London, UK, 2011. [2] L.L. Brunton, J.S. Lazo, K.L. Parker, Goodman and Gilman's the Pharmacological Basis of Therapeutics, Eleventh edition McGraw-Hill, New York, USA, 2006. [3] M.A. Abounassif, E.A. Abdel-Moety, Spectrophotometric quantification of fenoterol hydrobromide in tablets and inhalation aerosol, Acta Pharm. Jugosl. 39 (1989) 359–363. [4] R.S. Bakry, A.F.M. El-Walily, S.F. Belal, Spectrophotometric determination of etilefrine hydrochloride, prenalterol hydrochloride and ritodrine hydrochloride in pharmaceutical dosage form through nitrosation and subsequent copper chelation, Anal. Lett. 29 (1996) 409–422. [5] N. El-Enany, F. Belal, M. Rizk, Kinetic spectrophotometric determination of ethamsylate in dosage forms, J. AOAC.Int. 90 (2007) 679–685. [6] Y. Kumar, Y.K.S. Rathore, S.C. Mathur, N. Murugesan, P.D. Sethi, Spectrophotometric determination of terbutaline sulphate through cobalt (II) and copper (II) chelation, Indian Drugs 29 (1992) 416–418. [7] R.S. Bakry, A.F.M. El-Walily, S.F. Belal, Spectrophotometric determination of some phenolic sympathomimetic drugs through reaction with 2,6-dihaloquinone chlorimides, Mikrochim. Acta 127 (1997) 89–93. [8] Y. El-Shabrawy, F. Belal, M. Sharaf El-Din, S. Shalan, Spectrophotometric determination of fenoterol hydrobromide in pure form and dosage forms, Farmaco 58 (2003) 1033–1038. [9] H.A. Al-Malaq, A.A. Al-Majed, F. Belal, A stability-indicating spectrophotometric method for the determination of fenoterol in pharmaceutical preparations, Anal. Lett. 33 (2000) 1961–1974. [10] F. Salinas, J.J. Berzas-Nevado, A. Espinosa, Determination of oxytetracycline and doxycycline in pharmaceutical compounds, urine and honey by derivative spectrophotometry, Analyst 114 (1989) 1141–1145. [11] P.K. Hon, W.K. Fung, Identification of tetracyclines by second-derivative ultra-violet spectrophotometry, Analyst 116 (1991) 751–752. [12] U. Saha, Colorimetric determination of tetracycline derivatives in pharmaceutical preparations, J. AOAC Int. 72 (1989) 242–244. [13] K.B. Vinay, H.D. Revanasiddappa, O.Z. Devi, K. Basavaiah, Permangano-metric determination of etamsylate in bulk drugs and in tablets, Chem. Ind. Chem. Eng. Q. 15 (2009) 149–157. [14] M.Y. Dhamra, T.N. Al-Sabha, T.S. Al-Ghabsha, Spectrophotometric determination of terbutaline sulphate and tetracycline hydrochloride via ion pair complex formation using eosin Y, Pak. J. Anal. Environ. Chem. 15 (2014) 4–92. [15] O.A. Tarkhanova, S.A. Vasyuk, Spectrophotometric assay of ethalmsylate using a tetrazolium salt, Pharm. Chem. J. 44 (2010) 161–165. [16] M.M. Ayad, H.E. Abdellatef, M.M. Hosny, Y.A. Sharaf, Determination of etilefrine hydrochloride, fenoterol hydrobromide, salbutamol sulphate and estradiol valerate using surface plasmon resonance band of silver nanoparticles, Int. J. Pharm. Sci. 7 (2015) 327–333. [17] V. Sreeram, A.V.D. Nagendrakumar, S.R. Karumuri, M. Madhu, UV assay method for the determination of doxycycline hyclate in bulk and pharmaceutical formulation, Chem. Sci. Trans. 4 (2015) 69–74. [18] N.A.A. Alarfaj, Fluorimetric determination of isoxsuprine hydrochloride in pharmaceuticals and biological fluids, J. Pharm. Biomed. Anal. 28 (2002) 331–335.

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