Green Chemistry Approach for Determination of H₂-receptor Antagonists in Their Pharmaceutical Dosage Forms with an Optical Probe

 

Ahmed I. Hassan*

Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Al-Azhar University, Assiut branch, Assiut 71526, Egypt

 

ABSTRACT:

An environmentally safe, sensitive and simple spectrophotometric method has been developed for determination of H₂-receptor antagonists: cimetidine (CIM), famotidine (FAM), nizatidine (NIZ), and ranitidine hydrochloride (RAN). The method was depend on the reaction of the studied drugs with N- bromosuccinimide (NBS) , a green reagent, the excess NBS was determined by its reaction with metanil yellow (MY) and measuring the absorbance at 530 nm. The absorption intensity was correlated with drug concentrations. By using of the optimum conditions, linear calibration curves with good correlation coefficients (0.9991–0.9998) were found between the measured absorbance values and the corresponding drugs concentrations in the range of 2.0-25 μg mL^–1. Limits of detection were in the range 0.42-0.67 μg mL^–1. The proposed method was successfully applied for the analysis of H₂RAs in their pharmaceutical dosage forms and the results were comparable with that obtained by the official methods.

 

 

 

INTRODUCTION:

Histamine H₂-receptor antagonists (H₂RAs) are competitively inhibits the action of histamine on H₂-receptors of parietal cells and thus reduces the gastric acid secretion under daytime and nocturnal basal conditions. Therefore, these drugs are useful in treatment of active duodenal ulcer, gastric ulcer and Zollinger- Ellison Syndrome¹. Cimetidine (CIM), famotidine (FAM), nizatdine (NIZ) and ranitidine hydrochloride (RAN) are commonly used H₂-receptor antagonists in our community.

 

The chemical structures of these drugs are given in Fig. 1. An extensive literature survey revealed that: H₂RAs drugs have been estimated in their bulk and pharmaceutical dosage forms. These methods include titrimetry², electrochemical methods³, TLC⁴, HPLC^5,6, capillary electrophoresis⁷ and fluorimetry^8,9. These techniques were associated with some drawbacks as lack of sensitivity (titrimetry), time-consuming (TLC), requires expensive instruments that are not available in all quality control laboratories (HPLC and capillary electrophoresis).Spectrophotometric techniques provided practical (less-time consuming, simple, and more convenient) and significant economic advantages over other methods; therefore, they are a frequent choice for pharmaceutical analysis^10–16. Therefore, the present work was directed to the development of new simple, accurate, and economic spectrophotometric method that overcomes these drawbacks.

 

N-bromosuccinimide (NBS) is an environmental-friendly reagent which used as effective oxidizing/brominating agents. The spectrophotometric determinations involving NBS were based on direct measurement of the chromogenic derivative of the drug, or indirectly by measuring the remaining NBS with color-producing reagents^17-22. Metanil yellow (MY), acid yellow dye which gives a red color in acidic medium, is easily susceptible to oxidation by NBS causing bleaching of the red color¹⁷. The purpose of the present work is to describe, for the first time, the use of a NBS/MY combination for determination of H₂RAs. The analytical procedure based on oxidation of H₂RAs with excess NBS and subsequent measurement of unreacted NBS by its reaction with MY dye and measuring the absorbance at 530 nm. The increase in the absorption intensity at 530 nm was directly proportional to the amount of the drug in the sample solution.

 

 

Fig. 1: Chemical structures of the investigated H₂RAs

 

EXPERIMENTAL:

Apparatus:

UV-1601 PC (Shimadzu, Japan) and Lambda-3 B (Perkin-Elmer, USA) ultraviolet--visible spectrophotometers with matched 1-cm quartz cells were used for all measurements.

 

Reagents and standards:

Cimetidine and famotidine (Sigma Chemical Co., USA), nizatidine (Eli Lilly Co, USA), and ranitidine hydrochloride (Glaxo-Wellcome, UK) were obtained and used as received. Stock standard solutions (0.2 mg mL^–1) were prepared using distilled water as a green solvent. Working standard solutions were obtained by further dilution of the stock solution with water. N-bromosuccinimde (Merck, USA) was 0.15% (w/v) aqueous solution prepared fresh daily. Metanil yellow (Laba Chemie PVT Ltd., India) was 0.05% (w/v) prepared fresh daily by dissolving MY in 2.0% (v/v) hydrochloric acid. All solvents, acids, and other chemicals used throughout the study were of analytical grade. Doubly distilled water was used throughout the work.

 

Pharmaceutical formulations:

Famotin® tablets (Memphis, Egypt), Antodine® tablets (Amoun Pharmaceutical Industries, Egypt), Famotak® tablets (South Egypt Industries Company, Egypt), and Antodine® ampoules (Amoun Pharmaceutical Industries, Egypt) are labeled to contain 40 mg of FAM per tablet or ampoule. Nizatin® capsules (Hi Pharm, Egypt) are labeled to contain 300 mg of NIZ per capsule. Ranitidol® tablets (El-Nasr Pharmaceutical Chemicals, Egypt) are labeled to contain 150 mg of RAN per tablet. Ranitak® tablets (South Egypt Industries Company, Egypt) are labeled to contain 300 mg of RAN per tablet. Zantac® tablets (Glaxo-Simthkline) are labeled to contain 300 mg of RAN per tablet. Zantac® ampoules (Glaxo-Simthkline) are labeled to contain 50 mg of RAN per ampoule. Cimetidine tablets were simulated in the laboratory according to the reported formulation and were labeled to contain 300 mg of CIM per tablet.

 

General recommended procedure:

One milliliter of the standard or sample solution containing 20–200 μg mL^–1 of the active material was transferred into a 10-mL calibrated flask. One milliliter of NBS solution (0.15%, w/v) was added, and the reaction was allowed to proceed at room temperature (25 ± 5°C) for 15 min. One milliliter of MY (0.05%, w/v) was added. The contents of the flask were mixed and allowed to stand at room temperature (25 ± 5 °C) for 5 min. The reaction mixtures were made up to volume with water and absorbance was measured at 530 nm against blank solutions prepared in the same manner without the drug. Calibration curves were constructed by plotting the absorbance values versus the corresponding drug concentration.

 

Determination of accuracy:

Standard addition method was used for determination of the accuracy of the proposed method. Three concentrations of the authentic materials were added for each analyte; 5.0, 10.0 and 15.0 mg were added to a dosage form containing a fixed amount of the active ingredient, and the recovery percentage was determined for the added amount. Famotin®, Ranitidol® and Cimetidine tablets as well as Nizatin capsules were used.

 

Interferences study:

The general recommended procedure of the proposed method was used for determination of CIM samples prepared by mixing a known amount (300 mg) of CIM with common excipients: starch, sucrose, lactose, magnesium stearate, and ascorbic acid (added as stabilizer in the formulation of the ampoule). The recovery values were determined in order to determine the effects of these excipients.

 

Analysis of dosage forms:

Tablets and capsules:

Twenty tablets or contents of 20 capsules were weighed accurately and ground into a fine powder. A quantity of powder equivalent to 200 mg of the active ingredient was accurately into a 100-mL calibrated flask and dissolved in about 50 mL of water. The content was shaken for about 20 min; the volume was diluted to the mark with the water mixed, and filtered. First, 10 mL portion of the filtrate was discarded, and a convenient aliquot was taken, and the assay was completed according to the procedure described earlier.

 

Ampoules:

The contents of five ampoules were quantitatively transferred into a 250-mL calibrated flask, the volume was diluted to the mark with water mixed, and the resulting solution was used for analysis by the recommended procedure.

 

RESULTS AND DISCUSSIONS:

The use of molecular bromine as oxidizing and brominating agent has several drawbacks as it is harmful and there are difficulties in handling and maintaining the stoichiometric ratio during the reaction. From the green chemistry point of view, the replacement of such harmful reagents with non-toxic, inexpensive, commercially available and non-polluting reagents is an important goal. Recently, NBS has gained much attention as oxidation and bromination agent in determination of a variety of organic compounds including those of pharmaceuticals ^17-22. NBS can be considered a convenient source of molecular bromine, or it can also act as a source of hypobromous acid which is the actual oxidizing agent.

 

Method Development:

The proposed method using NBS/MY is based on oxidation of the investigated H₂RAs with a known excess amount of NBS and determination of the residual NBS by its reaction with MY. The increase in the absorption intensity at λ_max 530 nm was corresponding to the drug content in its sample solution (Fig. 2). It was observed that the color increased by increasing the concentration of MY dye (Fig. 3). The optimum absorbance (≈0.9) was obtained at a concentration of 0.05% (w/v) prepared fresh daily by dissolving MY in 2.0% (v/v) hydrochloric acid, and thus this concentration was used in all further experiments. Similar series of experiments were performed to establish the optimum concentration of NBS reagent. The results revealed that the optimum concentration was 0.15% (w/v) (Fig. 4). The reaction was completed within 10–20 min at room temperature (25 ± 5 °C); therefore measurements were carried out after 15 min in order to achieve high precision (Fig. 5). Water was preferred as a solvent for economic reasons and safe- environment considerations (green solvent). After dilution with water, the absorbance values at λ_max 530 nm were found to be stable for at least 30 min.

 

Molar ratio and the reaction mechanism:

The molar ratio of the reaction between the investigated H₂RAs and NBS in ratio 1:4 (1 mole of drug reacted with 4 mole of NBS) as reported previously in our work; I. A. Darwish et al.^{14, 15} (Fig. 6). NBS oxidize and decompose MY causing disappearance of its red color in acidic medium ¹⁷.

 

 

Fig. 2: Absorption spectra of 20 µg/ml of CIM (1), 0.05% (w/v) MY in the absence (2) and in the presence (2) of NBS (0.15% w/v) and CIM (10 µg/ml).

 

 

Fig. 3: Effect of MY concentration.

 

Fig. 4: Effect of NBS concentration NBS using MY (0.5 mg/ml).

 

 

Fig. 5: Effect of time on the reaction NBS with CIM (-□-), FAM (-■-), NIZ (-▲-), and RAN (-●-). The concentrations of drugs were 10µg mL^-1.

 

 

Fig. 6: The proposed reaction mechanism of RAN with NBS.

 

Method validation:

Linearity, limits of detection and quantitation:

The calibration graphs were linear with good correlation coefficients (0.9991–0.9998) between the absorbance at λ_max 530 nm, and concentration ranges of drugs (Table I). The molar absorptivity and together with the limits of detection and quantification were also summarized in Table 1. The limits of detection (LOD) and limits of quantitation (LOQ) were determined ²³ using the formula: LOD or LOQ = kSDa/b, where k = 3.3 for LOD and 10 for LOQ, SDa is the standard deviation of the intercept, and b is the slope. The results obtained by determination of H₂RA using the proposed NBS/MY method showed an improvement of the sensitivity that previously reported^{14, 15}.

 

Accuracy and Precision:

Evaluation of the accuracy of the proposed method was carried out by using the standard addition method. The obtained recovery values were in the range 98.9-100.8% (Table 2). This indicated high accuracy of the proposed method. Five separate solutions of the working standards at three concentration levels were analyzed to evaluate the precision (repeatability) of the proposed method. Relative standard deviations were not exceeded 2.5 % indicating good precision (Table 3).

 

Interferences:

Recoveries obtained from the interferences study of the proposed method showed that; there is no interference was found from the commonly used excipients (starch, sucrose, lactose, and magnesium stearate). Ascorbic acid (added as stabilizer in the formulation of the ampoule) was found to interfere with the assay procedure. This interference could be eliminated by adding 1 mL of 0.1% (m/V) aqueous solution of potassium bromate to the ampoule samples prior to their analysis ¹⁸.

 

Robustness and ruggedness:

Robustness was examined by changing one parameter of the method whereas the others were kept unchanged, and the recovery percentage was observed each time. It was found that small variations in these variables did not affect the method significantly (Table 4). Results obtained from lab-to-lab and day-to-day variations in order to evaluate the ruggedness of the proposed method were found to be reproducible and RSD values were less than 2.5%.

 

Application to dosage forms:

The validation of proposed method gave satisfactory results in determination of the investigated drugs in their pure forms. The pharmaceutical dosage forms of H₂RAs were subjected to the analysis by the proposed method and the official titrimetric method stated in the British Pharmacopoeia²⁴. The recovery values ranged from 98.5–102.5%. A statistical comparison of the results obtained from proposed and the official methods are presented in Table 2. When the results were statistically compared with those of the reference method by applying the t-test and F-test, the calculated values at 95% confidence level did not exceed the tabulated values. Hence, no significant difference exists between the proposed and reference method with respect to accuracy and precision in the analysis of the investigated drugs in their dosage forms.

 

 

CONCLUSIONS:

The assay results demonstrate that it is possible to use NBS, an environmentally friendly, reagent for sensitive indirect spectrophotometric determination of H₂-receptor antagonists using metanil yellow reagent. The proposed method was advantageous over the previously reported spectrophotometric methods in terms of sensitivity, simplicity, cost-effectiveness and applicability to analysis of four H₂RAs.

 

 

Table 1: Quantitative parameters and statistical data for the analysis of H₂RAs by the proposed NBS/MY method

Drug	ε x 10 3(Lmole-1cm-1)	Range (µg/ml)	LOD (µg/ml)	LOQ (µg/ml)	Slope± SD	Intercept± SD	Correlation coefficient (r)
CIM	6.960	2-25	0.67	2.01	0.0212 ± 0.0004	0.0093 ± 0.0030	0.9998
FAM	11.240	2-20	0.55	1.65	0.0344 ± 0.0042	0.0027 ± 0.0060	0.9991
NIZ	10.194	2-22	0.61	1.83	0.0265 ± 0.0003	0.0068 ± 0.0067	0.9995
RAN	16.500	2-15	0.42	1.26	0.0450 ± 0.0006	0.0041 ± 0.0075	0.9992

LOD: limit of detection and LOQ: limit of quantitation

 

Table 2: Results of standard addition method for the proposed NBS/MY method for determination of H₂RAs

 

Table 3: Precision of the NBS/MY proposed method for determination of H₂RAs

SD: Standard deviation.

RSD: Relative standard deviation.

 

Table 4: Results of evaluation of robustness of the proposed NBS/MY method for determination of H₂RAs

^a Values are the mean of three determinations ± SD.

^b The optimum conditions were; MY conc. of 0.11% (w/v), NBS conc. of 0.15% (w/v), HCl conc. of 2% (v/v) and reaction time of 15 min for NBS/drug and 2.0 min for NBS/MY at room temperature (25±5 ºC).

^c Performed without drugs.

 

 

Table 5: Analysis of H₂RAs-containing dosage forms by the proposed NBS/MY and official methods 

Dosage form	Ingredient (Content, mg)	% Recovery + SD		F-value b	t-value b
		Proposed method	Official method a
Cimetidine (Tablets)	Cimetidine(300)	100.5±0.96	98.9 ±0.64	2.25	2.45
Famotine® (Tablets)	Famotidine(20)	100.2 ±0.78	99.3±0.69	1.27	1.60
Famotak® (Tablets)	Famotidine(20)	100.7 ± 1.03	99.4± 0.69	2.22	2.03
Antodine® (Tablets)	Famotidine(20)	99.5 ± 0.76	98.6± 0.73	1.08	1.29
Antodine® (Ampoule)	Famotidine(20)	102.5 ± 0.79	101.5± 0.57	1.88	1.62
Nizatin® (Capsules)	Nizatidine(300)	100.3± 1.33	98.1± 1.25	1.13	2.36
Zantac® (Tablets)	Ranitidine(300)	99.5 ±1.12	97.3±0.79	2.00	2.53
Ranitak® (Tablets)	Ranitidine(300)	98.5±1.03	97.6 ± 0.69	2.22	1.56
Ranitidol® (Tablets)	Ranitidine(150)	99.5±1.49	97.2 ± 1.5	1.15	1.09
Zantac® (Ampoule)	Ranitidine(50)	101.6 ± 1.63	100.1± 1.25	1.71	1.92

^a Ref. ²⁴.

^b Theoretical values for t- and F-values at 95% confidence limit (n = 5) were 2.78 and 6.39, respectively.

 

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Received on 21.01.2019       Accepted on 21.02.2019     

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Asian J. Pharm. Ana. 2019; 9(1):19-24.