Table 1: Comparison between the reported methods for spectrophotometric determination of RZT.

Method	λ_max (nm)	Linear range (µg ml^-¹)	Ε (l mol^-¹ cm^-¹) x 10⁴	LOD	References
Bromophenol blue	425	0.8-16	1.76	0.13	13
Bromocresol purple	425	1-20	1.96	0.11
Bromothymol blue	420	1.2-24	1.63	0.09
NBS /janus green (JG)	620	0.5-8.0	3.03	0.28	14
NBS /calmagite (CMG)	540	1.5-30	1.15	0.48	14
(a) p-CA	530	14-245	0.13	1.36	15
(b) DDQ	590	4-70	0.522	0.6	15
(a) MO	420	10-50	1.02	-	16
(b) FeCl₃/ 2,2'- bipyridyl	490	4.0-20	1.0	-	16
a) FeCl₃/ 1,10-phenanthroline		2.0-10	3.85	-	17
b) Folin–Ciocalteu reagent/ NaOH	510 610	2.0-10	0.35	-
c) ARS	425	4.0-20	0.54
(a) DCQC	610	5–25	1.34	0.134	[18]
(b) NQS	480	15-75	0.43	0.412
c) Brucine/ sodium metaperiodate	530	8-40	0.81	0.22
TCNQ	744	10-100	-	-	12
UV	226	1.0-8.0	-	0.00368	19
UV	280	0.5-80	0.703		20
Vanillin	590	50-250	1.33	0.156	21
BCG	416	0.5-50	-	0.5	22
Sodium Nitro prusside-Acetaldehyde	560	2.0-10	1.09	0.145	23
Chloramine-B in HCl					24
Initial rate	490	0.01-0.1		0.244
Fixed rate		0.01-0.1		0.11
Variable time		0.01-0.1		0.09
Ammonium molybdate / H₂SO₄	590	2.0-10	24.2	0.073	25
p-dimethylaminobenzaldehyde	545	5.0-25	18.3	0.120	25
Cerium (IV) sulphate					Proposed methods
AM	518	1.0-10	1.0505	0.25
MB	663	0.5-8.0	1.7331	0.27
IC	609	1.0-14	1.3508	0.53

Cerium (IV) sulphate has been widely used as an effective analytical reagent in spectrophotometric methods for the determination of many pharmaceutical compounds31-35. Cerium (IV) sulphate is a strong oxidant, and it has not been applied for the assay of RZT in pure form and tablets.

This paper describes for the first time the application of acidic cerium (IV) sulphate to the spectrophotometric determination of RZT using amaranth (AM), methylene blue (MB) and indigo carmine (IC) as chromogenic agents. The proposed methods have the advantages of simplicity, sensitivity and rapidity besides being accurate precise and validated spectrophotometric method for the estimation of RZT in pure and dosage forms and can be adopted by the pharmaceutical laboratories for industrial quality control.

MATERIALS AND METHODS:

Apparatus:

All absorption spectra were made using Varian UV–Vis spectrophotometer (Cary 100 Conc., Australia) equipped with a 10 mm quartz cell was used for absorbance measurements. This spectrophotometer has a wavelength accuracy of ±0.2 nm with a scanning speed of 200 nm/min and a bandwidth of 2.0 nm in the wavelength range of 200–900 nm.

Materials and reagents:

All chemicals and reagents used were of analytical or pharmaceutical grade and all solutions were prepared fresh daily. Bidistilled water was used throughout the investigation.

Pure RZT drug and pharmaceutical formulations:

Pharmaceutical grade RZT was kindly supplied by Delta Pharmaceutical Industries, Cairo, Egypt. The commercial pharmaceutical formulations (Rizatriptane tablets (Sandoz Pharmaceutical Company, Cairo, Egypt), labeled to contain 10 mg RZT per tablet) were purchased from local market were subjected to the analytical procedure.

Stock standard solution:

A standard stock solution of RZT containing 100 μg mL^-1 was prepared by dissolving 10 mg of pure drug in 20 mL bidistilled water and was further diluted to 100 mL to obtain the working concentration. The standard solution was kept in the refrigerator and was found to be stable for at least one week if they had been stored in a cool (< 25 ºC) and dark place.

Reagents:

A stock solution of 5.0 × 10^-3 mol/L cerium(IV) sulphate (E-Merk, Darmstadt, Germany) was freshly prepared by dissolving 316.2 mg of cerium(IV) sulphate in the least amount of H₂SO₄ (2.0 mol/L) then completed to the mark in a 100 mL calibrated flask with the same acid and kept in a dark bottle and a refrigerator when not in use. A stock solution of 2.0 mol/L H₂SO₄ was prepared by adding 10.8 mL of concentrated acid (Merck, Darmstadt, Germany, 98%, Sp. Gr. 1.84) to bidistilled water, cooled to room temperature, transfer to 100 mL with measuring flask, diluted to the mark and standardized as recorded³⁶.

A stock solutions (1000 μg/mL) AM, MB, and IC were first prepared by dissolving accurately weighed 112 mg of each dye (Sigma-aldrish, 90 % dye content) in bidistilled water and diluting to volume in a 100 mL calibrated flask. The solution was then diluted 5-fold to get the working concentration of 200 μg/mL of each dye.

General procedures:

Different aliquots (0.2-2.0 mL), (0.1-1.6 mL), and (0.2-2.8 mL) of a standard 50 μg/mL RZT solution using AM, MB, and IC methods, respectively were transferred into a series of 10 mL calibrated flasks by means of a micro burette and the total volume was adjusted to 5.0 ml by adding adequate quantity of water. To each flask 2.0 ml of 2.0 mol/L H₂SO₄ and 1.0 mL of (5.0 × 10^-3 mol/L) CAS solution were added, respectively. The flasks were stoppered, contents were mixed well, and the flasks were kept aside for 5.0 min with occasional shaking. Finally, 1.0 mL of (200 μg/ mL) AM, MB, or IC dye solution was added to each flask and mixed well, and then the volume was diluted to the mark with bidistilled water. The color intensity of dyes was measured after 5.0 min against reagent blank solution treated similarly omitting the drug, at their corresponding λ_max 518, 663, and 609 nm, respectively. The concentration of unknown was determined in each case from calibration graph or computed from the regression equation derived using Beer’s law data.

Assay procedure for pharmaceutical formulations:

The content of twenty tablets each containing 10 mg RZT was finely powdered using an agate mortar and weighed accurately. An accurately weighed quantity of the powder equivalent to 10 mg RZT were transferred into 100 mL calibrated flask and dissolved in 25 mL methanol. The content of the flask was shaken and sonicated for about 10 min, mixed well and then filtered using Whatman No.42 filter paper. The first portion of the filtrate was rejected, and the solution was then completed to volume with bidistilled water to prepare a stock solution of 100 μg/mL. Aliquots covering the working concentration ranges for each method were transferred into a series of 10 mL volumetric flasks and the proposed methods were applied. The nominal content of the tablets was determined using the corresponding regression equations or the calibration graphs.

RESULTS AND DISCUSSION:

Absorption spectra:

The proposed spectrophotometric methods for the determination of RZT is indirect and involves two steps namely:

1. Oxidation of RZT with a known excess of cerium (IV) sulphate in acidic medium at room temperature (25 ±2 °C).

2. Determination of the residual cerium (IV) sulphate by reacting it with a fixed amount of AM, MB, or IC dyes and measuring the increase in absorbance at λ_max 518, 663, or 609 nm, respectively (Scheme 1) (Fig. 2).

Figure 2: Absorption spectra for the unreacted cerium (IV) sulphate oxidant that determined by reacting with a fixed amount of AM, MB and IC dyes and measuring the absorbance at 518, 663 and 609 nm for (200 µg/mL) AM, MB and IC methods, respectively.

Optimization of the reaction conditions:

The optimum conditions for the assay procedures and color development for each method have been established by varying the parameters one at a time, keeping the others fixed and observing the effect produced on the absorbance of the colored species.

Effect of acid type and concentration:

Different types of acids were examined (HCl, H₂SO₄, H₃PO₄, HNO₃ and CH₃COOH) to achieve maximum yield of redox reactions. The results indicated that the sulphuric acid (H₂SO₄)(2.0 mol/L) was the most suitable acid with cerium (IV) sulphate as oxidant. Moreover, different volumes (0.25–3.0 mL) of 2.0 mol/L H₂SO₄ were tested, keeping the concentrations of oxidant and drug fixed. The results indicated that, at (1.5-2.5 mL) of H₂SO₄ (2.0 mol/L), there were almost same absorbance values were obtained in the presence of the studied drugs. At the acid volumes less than 1.5 mL, reaction led to go slower and incomplete. Therefore, 2.0 mL of H₂SO₄ (2.0 mol/L) was the optimum volume for subsequent studies (Fig. 3).

Figure 3: Effect of volume of H₂SO₄ (2.0 mol/L) on the absorbance of RZT (8.0 µg/mL) with cerium (IV) sulphate (5.0 x 10^-3 mol/L) and (200 µg/mL) dye.

Effect of cerium (IV) sulphate concentration:

The influence of the concentration of cerium (IV) sulphate on the absorbance of the colored products was investigated using different volumes of 5.0 × 10-3 mol/L cerium (IV) sulphate solution from (0.25-3.0 mL). The results indicate that the maximum and constant absorbance was obtained using 1.0 mL of 5.0 × 10-3 mol/Lcerium (IV) sulphate solution and the color intensity constant or decreased (Fig. 4).

Figure 4: Effect of volume of cerium (IV) sulphate (5.0 x 10^-3 mol/L) on the reaction product of RZT (8.0 µg/mL) and dyes in H₂SO₄ medium.

Effect of dye concentration:

The effect of dye concentration on the intensity of the color developed was carried out to obtain the optimum concentration of dyes that produces the maximum and reproducible color intensity by reducing the residual of cerium (IV) sulphate. The effect dye concentration was studied using different volumes (0.25–3.0 mL) of the studied dyes (200 μg/mL). It was observed that maximum color intensity of the oxidation products was achieved with 1.0 ml of each dye solution. The color was found to be stable up to 10 h.

Effect of temperature and mixing time:

The effect of temperature was studied by heating a series of sample and blank solutions at different temperatures ranging from 20 to 60 °C in water bath. It was found that raising the temperature does not accelerate the oxidation process and does not give reproducible results, so maximum color intensity was obtained at room temperature (25±2 °C). The effect of mixing time required completing oxidation of RZT and for reducing the excess oxidant was studied by measuring the absorbance of sample solution against blank solution prepared similarly at various time intervals 2.0–20 min. It was found that the contact times gave constant and reproducible absorbance values at 5.0 min. After oxidation process, 5.0 min standing time was found necessary for the complete bleaching of the dye color by the residual cerium (IV) sulphate and the absorbance of the unreacted dye was stable for at least 10 h, thereafter (Fig. 5).

Figure 5: Effect of time on the absorbance of the reaction product: (RZT (8.0 µg/mL); cerium (IV) sulphate (5.0 x 10^-3 mol/L) and dyes (200 μg/mL) in H₂SO₄ acidic medium).

Effect of sequence of addition:

After optimizing all other experimental variables, further experiments were performed to ascertain the influence of sequence of addition of reactants on the color development by measuring the absorbance. The optimum sequence of addition was RZT–H₂SO₄–cerium(IV) sulphate–dye. Other sequences gave lower absorbance values under the same experimental conditions.

Validation of the proposed methods:

The validity of the methods was tested regarding linearity, specificity, accuracy, repeatability and precision according to International Conference on Harmonization (ICH)³⁷ guidelines.

Linearity, detection, and quantification limits:

Following the proposed experimental conditions, linear regression equations were obtained. The regression plots showed that there was a linear dependence of the absorbance to the concentration of RZT in the range 11.0-10, 0.5-8.0 and 1.0-14 μg/mL using AM, MB and IC methods, respectively. Linear regression analysis of the data gave the following equations. For AM, A = -0.0039+ 0.0285C, r²= 0.9993, A= 0.0419 + 0.003C, r²= 0.9997 using MB and A= 0.0055 + 0.0322C, r²= 0.9996 using IC, where A is the absorbance, C is the concentration of RZT, and r² is the correlation coefficient.

The limits of detection (LOD) were determined by establishing the minimum level at which the analyte can be reliably detected, and the limit of quantification (LOQ) was determined by establishing the lowest concentration that can be measured with acceptable accuracy and precision according to ICH^37,38. The results are also summarized in Table 2. LOQ and LOD were calculated according to the following equations:

LOD=3.3s /k

LOQ=10s /k

Where s is the standard deviation of replicate determination values under the same conditions as for the sample analysis in the absence of the analyte. k: is the slope of the calibration graph. In accordance with the formula, the LOD were found to be 0.25, 0.27 and 0.53 µg/mL and LOQ were found to be 0.83, 0.90 and 1.77 µg/mL sing AM, MB and IC, respectively.

Table 2: Analytical and regression parameters of proposed oxidation spectrophotometric methods for determination of RZT.

Parameters	AM	MB	IC
Beer’s law limits, µg/mL	1.0-10	0.5-8.0	1.0-14
Ringboom limits, µg/mL	2.0-8.0	2.0-7.0	3.0-12
Molar absorptivity, x 10⁴ L/mol. cm	1.0505	1.7331	1.3508
Sandell sensitivity, ng cm^-2	37.26	22.58	28.98
Regression equation ^a
Intercept (a)	-0.0039	0.0419	0.0055
Standard deviation of intercept (S_a)	0.064	0.045	0.06
Slope (b)	0.0285	0.003	0.0322
Standard deviation of slope (S_b)	0.05	0.07	0.059
Correlation coefficient, (r)	0.9993	0.9997	0.9996
Mean ± SD	99.40±0.70	99.30±0.80	99.90±0.95
RSD%	0.70	0.81	0.95
RE%	0.74	0.85	1.0
Limit of detection, µg/mL	0.25	0.27	0.53
Limit of quantification, µg/mL	0.83	0.90	1.77
Calculated t-value^b	0.56	0.71	0.33
Calculated F-value ^b	1.92	1.47	1.04

^a A = a + bC, where C is the concentration in µg/mL, A is the absorbance units, a is the intercept, b is the slope.

^b The theoretical values of t and F are 2.57 and 5.05, respectively at confidence limit at 95% confidence level and five degrees of freedom (p= 0.05).

Accuracy and precision:

To evaluate the accuracy and precision of the proposed methods, intraday and inter-day determination of RZT at three different concentrations for each method were prepared and analyzed. The intraday studies were performed in one day and inter-day studies in five days (for each level n=6). The accuracy and precisions expressed as percent relative error (RE%) and relative standard deviation (RSD%) values, respectively and found to be within -1.50-0.50% and 0.65–1.80%, respectively for intraday analysis and within -1.20-1.0% and 0.75-1.90%, respectively for inter-day analysis (Table 3). The data proved good accuracy and precision for the developed methods.

Ruggedness and robustness:

Robustness of the proposed method was assessed by evaluating the influence of small variation of experimental variables, including concentration of analytical reagents and reaction time, on the analytical performance of the proposed method. In these experiments, one experimental parameter was changed while the other parameters were kept unchanged, and the recovery percentage was calculated each time. The analysis was performed with altered conditions by taking three different concentrations of RZT and it was found that the small variations in any of the variables did not significantly affect the results. The RSD% values were in the ranges 0.60 – 2.50% (Table 4). This indicated the reliability of the proposed method during its routine application for the analysis of RZT. The ruggedness of the proposed method was assessed by applying the procedures using two different instruments in three different laboratories (instruments) at different times and three different analysts. The inter-analysts RSD% were in the ranges 0.50-2.20%, whereas the inter-instruments RSD% ranged from 0.80-2.50%, these results were found to be reproducible because the RSD did not exceed 3.0% (Table 4).

Table 3: Results of intra-day and inter-day accuracy and precision study obtained by the proposed methods.

Method	Taken (μg/mL)	Recovery %	Precision RSD % ^a	Accuracy RE %	Confidence Limit ^b
	Intra-day
AM	3.0	99.20	1.0	-0.80	2.976 ± 0.031
	6.0	99.70	1.15	-0.30	5.982 ±0.072
	9.0	98.50	1.40	-1.50	8.865 ± 0.13
MB	2.0	99.00	0.80	-1.0	1.98 ± 0.017
	4.0	99.70	0.65	-0.30	3.988 ± 0.027
	6.0	100.50	1.80	0.50	6.03 ± 0.114
IC	4.0	99.70	1.10	-0.30	3.988 ± 0.046
	8.0	100.30	0.90	0.30	8.024 ± 0.076
	12	99.10	1.60	-0.90	11.892 ± 0.20
AM	Inter-day
AM	3.0	100.60	0.75	0.60	3.018 ± 0.024
	6.0	101.0	0.90	1.00	6.06 ± 0.057
	9.0	99.40	1.50	-0.60	8.946 ± 0.141
MB	2.0	100.0	0.90	1.0	2.0 ± 0.019
	4.0	99.30	1.50	-0.70	3.972 ± 0.063
	6.0	99.60	1.90	-0.40	5.976 ± 0.119
IC	4.0	99.50	0.80	-0.50	3.98 ± 0.033
	8.0	98.80	1.60	-1.20	7.904 ± 0.133
	12	100.90	1.70	0.90	12.108 ± 0.216

^a RSD%, percentage relative standard deviation; RE%, percentage relative error.

^b Mean ± standard error.

Table 4: Results of method robustness and ruggedness (all values in RSD%) studies.

Methods	Nominal amount concentration (μg/mL)	RSD%
		Robustness		Ruggedness
		Variable alerted ^a
		Acid volume (n=3)	Reaction time (n=3)	Different analysts (n=3)	Different instruments (n=3)
AM	3.0	0.65	1.0	0.90	0.80
	6.0	1.30	1.70	1.50	1.40
	9.0	1.70	2.50	1.90	2.0
MB	2.0	0.80	0.70	0.60	1.10
	4.0	1.20	1.40	1.20	1.50
	6.0	2.20	1.90	2.10	2.40
IC	4.0	1.40	0.90	0.50	1.20
	8.0	1.90	1.70	1.80	1.60
	12	2.30	2.50	2.20	2.50

^a Volume of (2.0 mol/L) H₂SO₄ is (±0.2 mL) and reaction time is (±2.0 min) (after adding cerium(IV) sulphate) were used.

Recovery studies:

The specificity of the proposed method was investigated by observing any interference encountered from the common capsule's excipients. The standard addition method was applied by adding known amounts of pure RZT to a previously analyzed tablet solution. This study was performed by spiking three different levels of pure RZT (50, 100 and 150% of the level present in the tablet) to a fixed amount of drugs in tablet powder (pre-analysed) and the total concentration was found by the proposed methods. The determination with each level was repeated three times and the percent recovery of the added standard was calculated from:

Recovery % = [C_F – C_T] / C_p x 100

Where C_F is the total concentration of the analyte found, C_T is a concentration of the analyte present in the tablet preparation; C_P is a concentration of analyte (pure RZT) added to tablets preparations. The results were recorded in Table 5. The high recovery values of the proposed methods indicated that the excipients did not interfere with the proposed methods indicating the high selectivity of the proposed methods.

Table 5: Results of recovery experiments by standard addition method for the determination of RZT in Riztriptan tablets (10 mg) using the proposed methods.

Method	Taken drug in tablet (μg/mL)	Pure drug Added (μg/mL)	Total found (μg/mL)	Recovery^a (%) ± SD
AM	4.0	2.0	5.96	99.40 ± 0.60
	4.0	4.0	8.04	100.50 ± 0.75
	4.0	6.0	10.08	100.80 ± 1.20
MB	4.0	2.0	5.94	99.00 ± 0.80
	4.0	4.0	7.94	99.20 ± 1.0
	4.0	6.0	9.94	99.40 ± 1.30
IC	4.0	2.0	5.95	99.10 ± 0.50
	4.0	4.0	8.04	100.50 ± 0.9
	4.0	6.0	9.90	99.00 ± 1.50

^aAverage of six determinations.

Analysis of the pharmaceutical preparations:

The proposed methods were applied to the determination of RZT in pharmaceutical formulations (Rizatriptan tablets, 10 mg RZT per tablet). The results of Recovery ± SD values of the proposed methods agree well with the label claim and also were in agreement with the results obtained by the reported method ¹³ and were statistically compared with those obtained using the reference methods¹³. Statistical analysis of the results, using Student’s t-test and the variance ratio F-test at 95% confidence level revealed no significant difference between the performance of the proposed and reference methods regarding the accuracy and precision, respectively (Table 6)³⁸. It is evident from these results that the proposed methods are applicable to the analysis of RZT in its dosage forms with comparable analytical performance.

Table 6: Application of the proposed methods for the determination of RZT in Riztriptan tablets (10 mg) and statistical comparison with the reference method.

Samples	Recovery^a (%) ± SD
	Proposed Methods			Reported method¹³
	AM	MB	IC
X ± SD	100.20 ± 0.80	99.70 ± 1.20	99.80 ± 0.90	100.80 ± 1.03
t-value (2.57)^b	1.03	1.56	1.63
F-value (5.05)^b	1.66	1.36	1.31

^aAverage of six determinations.

^b Theoretical values of t and F at confidence limit at 95% confidence level and five degrees of freedom (p = 0.05).

CONCLUSIONS:

A new, simple, rapid, useful and cost-effective spectrophotometric methods have been developed for determination of RZT in pure form and tablets using cerium (IV) sulphate as oxidizing agent and validated as per the current ICH guidelines. The present spectrophotometric methods are characterized by simplicity of operation, high selectivity, comparable sensitivity, low-cost instrument, they do not involve any critical experimental variable and are free from tedious and time-consuming extraction steps and use of organic solvents unlike many of the previous methods reported for RZT. The proposed methods have some additional advantages involve less stringent control of experimental parameters such as the stability of the colored system, accuracy, reproducibility, time of analysis, temperature independence and cheaper chemicals. These advantages encourage the application of the proposed methods in routine quality control analysis of RZT in pure and dosage forms.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 06.12.2018 Modified on 18.01.2019

Research J. Pharm. and Tech. 2019; 12(5):2123-2130.

DOI: 10.5958/0974-360X.2019.00352.4