Different Spectrophotometric and TLC-Densitometric Methods for Determination of Mesalazine in Presence of Its Two Toxic Impurities

Martha Moheb Morcoss; Nada Sayed Abdelwahab; Nouruddin Wagieh Ali; Mohammed Taha Elsaady

doi:10.1248/cpb.c16-00143

Abstract

Two selective spectrophotometric and TLC-densitometric methods were developed for determination of mesalazine (ME) and its two toxic impurities, 4-amino phenol (4AP) and salicylic acid (SA) without preliminary separation. The proposed methods are: ratio difference in the subtracted spectra (RDSS) {Method 1}, area under the curve (AUC) {Method 2} and TLC-densitometric {Method 3}. In method {1} combination of measuring the amplitude of the constant at 350 nm (using standard spectrum of 10 µg/mL ME as a divisor) and ratio difference in the subtracted ratio spectrum for determination of 4AP and SA using the ratio difference at 221.4 and 242.2 nm, 230 and 241.2 nm, respectively. In method {2} ME was determined by direct measuring the AUC in the wavelength range of 350–370 nm while the impurities could be determined by dividing their spectra by standard spectrum of 10 µg/mL ME then interference from ME was eliminated by subtracting the amplitude of the constant at 350 nm then multiplying by the divisor. AUC in the range of 220–230 and 235–245 nm was used for measuring concentrations of 4AP and SA. On the other hand, the third method {3} is TLC–densitometric method at which chromatographic separation was achieved using ethyl acetate–methanol–triethylamine (8.5 : 2 : 0.7, v/v/v) as a developing system with UV scanning at 230 nm. The validation of the proposed methods was performed according to International Conference on Harmonization (ICH) guidelines. No significant difference was found when these methods were compared to the reported one.

Mesalazine also named mesalamine, ME is 5-amino-2-hydroxybenzoic acid¹⁾ (Fig. 1). It is an anti-inflammatory drug structurally related to salicylates and active in inflammatory bowel diseases, such as ulcerative colitis and mild-to moderate Crohn’s disease.^2–4) It is considered to be the active moiety of sulfasalazine. It is thought to act locally on the inflamed intestinal tissue, rather than systemically. Two major impurities of ME have been reported in British Pharmacopoeia (BP)¹⁾ namely 4-amino phenol (4AP) and salicylic acid (SA) (Fig. 1). 4AP is known to cause nephro-toxicity in rats, in which it produces selective necrosis to renal proximal tubules and was also reported to have a significant teratogenic effect.^5–7) SA is known to have bacteriostatic, fungicidal, and keratolytic actions.⁸⁾ It is used as a caustic in preparations for the removal of warts.⁹⁾ I would like to mention that the selection of impurities was based on the most toxic impurities that could be found with the drug.^5–9) On searching on 2,5-dihydroxy benzoic acid and 2-hydroxy-5-nitrobenzoic acid, it was found that they are not toxic according to safety data sheets.

Fig. 1. Chemical Structure of (A) Mesalazine (ME), (B) Salicylic Acid (SA) and (C) 4-Amino Phenol (4AP)

Literature survey revealed that some analytical methods have been reported for the determination of ME. It was analyzed as a raw material in both BP¹⁾ and United States Pharmacopeia (USP)¹⁰⁾ pharmacopoeias by non-aqueous titration method with potentiometric detection of the end point. Also, the drug was analyzed in pharmaceutical dosage form and in biological samples by different colorimetric methods.^11–15) The drug was also determined by different UV-spectrophotometric,¹⁶⁾ and Vireodt’s method.¹⁷⁾ Spectrofluorimetric,^18,19) TLC,^20,21) HPLC,^22–25) ultra performance liquid chromatography (UPLC)^26,27) and LC-MS²⁸⁾ were reported for analysis of ME either in single or in combination with other drugs. Only one HPTLC method was published for determination of ME and its degradation products that depended on using toluene–methanol–ethyl acetate (6.5 : 2.5 : 1, v/v/v) as a developing system and scanning at 244 nm.

Till now no published method has been found for determination of ME and its toxic impurities. Therefore, the objective of this work is to develop three accurate, selective and reliable spectrophotometric and TLC-densitometric methods for determination of ME in co-existence with its toxic impurities with high sensitivity and selectively and to validate the developed methods according to International Conference of Harmonization (ICH) guidelines.²⁹⁾

Experimental

Instruments

A double beam UV-visible spectrophotometer (Shimadzu, Japan) model UV-1601 PC with quartz cell of 1 cm and UV-PC personal software version 3.7 was used.

CAMAG TLC Scanner 3 S/N 130319 with WINCATS software.

These requirements were taken into consideration:

Scan mode: absorbance mode
Scanning speed: 20 mm s⁻¹
Source of radiation: deuterium lamp
Slit dimension: 3×0.45 mm
Band width: 6 mm
Output: chromatogram and integrated peak area

A sample applicator for TLC Linomat IV with a 100-µL syringe (Camag, Muttenz, Switzerland) was utilized for this work. Also, an ultrasonic bath by Bandelin Songrex (Sigma-Aldrich, St. Louis, MO, U.S.A.) and a Jenway 3305pH=mv Meter with double junction glass electrode (Fisher, U.S.A.) were also employed in this work.

Materials

Pure Samples

Meslazine was provided from El Pharonia (Borg El Arab City, Alexandria, Egypt). Its purity was certified to be 99.84. Pure standard 4AP and SA were purchased from Sigma-Aldrich Co. (Chemie GmbH, Germany) with claimed purities of 99.56 and 99%, respectively, according to the manufacturer certificates.

Pharmaceutical Dosage Forms

Pentasa^® tablets (Batch No. L13362A) labeled to contain 500 mg ME [manufactured by FERRING International Center SA, St. Prex, Switzerland].

Solvents

Ethanol (El NASR Pharmaceutical Chemicals Co., Abu-Zabaal, Cairo, Egypt).

Standard Solutions

Stock standard solutions of ME, 4AP, and SA (1 mg/mL) were prepared in ethanol for methods {1 and 2} and in methanol for method {3}.

Working standard solutions of ME, 4AP, and SA (0.1 mg/mL). Ten milliliters of ME, 4AP, and SA stock standard solutions (1 mg/mL) was transferred accurately into three separate volumetric flasks. The volume was then completed to the mark with either ethanol for methods {1 and 2} or with methanol for method {3}.

Laboratory-Prepared Mixtures

Mixtures containing different ratios of ME, 4AP, and SA were prepared using their respective working standard solutions (0.1 mg/mL) and using either ethanol for methods {1 and 2} or methanol for method {3} as a solvent.

Application to Pharmaceutical Formulation

Ten tablets of Pentasa^® were grinded well and then an accurately weighed amount equivalent to 100 mg ME was transferred into 100-mL volumetric flask. Seventy five milliliters either ethanol for methods {1 and 2} or methanol for method {3} was added and the solution was ultrasonicated for 30 min, filtered and then the volume was completed with the same solvent to prepare sample stock solution (1 mg/mL). Sample working solution (0.1 mg/mL) was then prepared using the corresponding solvents.

Procedure

Spectral Characteristics of ME, 4AP, and SA

Zero order absorption spectra of 5 µg/mL each of ME, 4AP, and SA were recorded from 200 to 400 nm using ethanol as a solvent (Fig. 2).

Fig. 2. Zero Order Absorption Spectra of 5 µg/mL Each of ME (—), 4AP (- - -), SA (…) and Mixture Containing 5 µg/mL of Each (— · —) Using Ethanol as a Solvent and the Selected AUC at 350–370 nm for Determination of ME

Construction of Calibration Curves

Aliquots in the range of 3–20, 2–20 and 2–20 µg/mL were accurately transferred from ME, 4AP, and SA working standard solutions (0.1 mg/mL), respectively and then the volume was completed with ethanol. The prepared solutions were scanned in the range of 200–400 nm.

Method {1} Ratio Difference in the Subtracted Spectra (RDSS)

For determination of ME the scanned spectra were divided by standard spectrum of 10 µg/mL of ME and then the amplitude of the constant at 350 nm was recorded and used for construction of the calibration graph. While for determination of 4AP and SA the stored absorption spectra of each component were divided by the standard spectrum of 10 µg/mL ME as a divisor to obtain ratio spectra. Calibration curves were constructed relating the ratio difference at 221.4 and 242.2 nm (ΔA_{221.4–242.2 nm}), 230 and 241.2 nm and (ΔA_{230–241.2 nm}) to the corresponding 4AP and SA concentrations, respectively and the regression equations were computed.

Method {2} Area under the Curve (AUC)

In the range of 350–370 nm of ME scanned spectra were recorded and used for construction of the corresponding calibration curve and computing the regression equation.

On the other hand, for determination of 4AP and SA, the AUC in the range of 220–230 nm (λ₁–λ₂) and 235–245 nm (λ₃–λ₄) of the previously scanned spectra of their pure samples were measured. The absorptivity (Y) value was then calculated for each component in the selected wavelength ranges where Y=the recorded AUC of the component from (220–230 or 235–245 nm)/concentration of the component in µg/mL. Cramer’s rule was then applied and the following equations were used:

A₁, A₂ are the AUC in the range of 220–230 and 235–245 nm, respectively. Y_x1, Y_x2 are the absorptivity values of 4AP at (λ₁–λ₂) and (λ₃–λ₄), respectively. Y_z₁, Y_z₂ are the absorptivity values of SA at (λ₁–λ₂) and (λ₃–λ₄), respectively. C_x and C_z are the concentrations of 4AP and SA, respectively.

Method {3} TLC-Densitometric Method

Accurate and separate aliquots equivalent to 50–400, 50–300 and 50–250 µg/band were separately transferred from their respective stock standard solutions (1 mg/mL) and then the volume was completed with methanol. Ten microliters of each sample was applied in triplicates to TLC plates to obtain concentrations in the range of 0.5–4, 0.5–3, and 0.5–2.5 µg/band for ME, 4AP, and SA, respectively. Prepared samples were applied as bands of 6 mm width on TLC plates (20×10 cm with 250 µm thickness) using a Camag Linomat IV applicator. The bands were applied at 5 mm intervals and 10 mm from the bottom edge of the plate. Linear ascending development was performed to a distance 8 cm in a chromatographic jar previously saturated for 30 min with a developing system consisted of ethyl acetate–methanol–triethylamine (8.5 : 2 : 0.7, v/v/v) and the separated bands were UV-scanned at 230 nm. The calibration curves were constructed by plotting the integrated peak area versus the corresponding concentrations of ME, 4AP, and SA and the regression equations were computed.

Analysis of Laboratory Prepared Mixtures for Methods {1 and 2}

Different synthetic mixtures containing different ratios of ME, 4AP, and SA were prepared in ethanol and UV scanned in the range of 200–400 nm and then the scanned spectra were used for calculating ME, 4AP, and SA concentrations.

For Method {1}: The stored spectra were divided by standard spectrum of 10 µg/mL ME and then the amplitude of the constant at 350 nm was measured and used for calculating ME concentrations by applying in the corresponding regression equation. Subtraction of the amplitude of the constant was carried out and the ratio difference at 221.4 and 242.2 nm (ΔA_{221.4–242.2 nm}), 230 and 241.2 nm (ΔA_{230–241.2 nm}) was calculated and used for measuring 4AP and SA concentrations using the previously computed regression equations.

For Method {2}: AUC of the scanned spectra in the range of 350–370 nm was calculated and used for determination of ME concentrations using the previously computed regression equation. To eliminate the interference due to ME, by dividing the spectra of mixtures by standard spectrum of 10 µg/mL ME and then getting the value of the amplitude of the constant at 350 nm. Subtraction of the amplitude value and then multiplying the resulted subtracted ratio spectra by the divisor (10 µg/mL ME) resulted in the ordinary spectra of 4AP and SA from which the AUC in the ranges of 220–230 or 235–245 nm were calculated and used in the following equations:

(1)

(2)

Where C_4AP and C_SA are the concentrations of 4AP and SA in µg/mL, respectively. 0.5298 and 0.5247 are the absorptivity (Y value) of 4AP at (λ₁–λ₂) and (λ₃–λ₄), respectively. 0.5131 and 0.2737 are absorptivity (Y value) of SA at (λ₁–λ₂) and (λ₃–λ₄), respectively. A₁ and A₂ are the areas under curve of sample spectrum at the wavelength range (λ₁–λ₂) and (λ₃–λ₄), respectively. Then concentrations of 4AP and SA in the ternary mixture could be obtained.

For Method {3}: Different laboratory mixtures of the studied components containing different ratios of ME, 4AP and SA were mixed into a two series of 10 mL volumetric flasks. The procedures previously mentioned under construction of calibration curves were followed then the peak areas of the obtained chromatograms were measured for each component and the concentrations were calculated from their corresponding regression equations.

Analysis of Pharmaceutical Formulation

The procedure under laboratory prepared mixtures for each method was followed on different prepared samples of Pentasa^® tablets and the concentration of ME was calculated from the corresponding regression equation. The validity of the methods was further assessed by applying the standard addition technique.

Results and Discussion

The aim of this work is to validate three simple methods of analysis to determine ME in pure form and in pharmaceutical formulation. As for quality control analysis of drugs, simple and fast methods are desirable. Spectrophotometric methods are still widely used because they are time and money saving and easy to be performed. On the other hand, TLC-densitometric method provides sensitivity, time and money saving method of analysis. Till now no reported method has been published for determination of ME and its two toxic impurities in their ternary mixture.

Method Development and Optimization

Methods {1 and 2}

Zero order absorption spectra of the drug and its two toxic impurities showed intensive spectral overlap as shown in Fig. 2, which hindered their direct determination. On the other hand, ME showed extended spectrum where no interference from 4AP and SA in the range of 325–370 nm. Measuring ME absorbance at 330 nm resulted in bad sensitivity and hence poor reproducibility, hence, different spectrophotometric methods have been tried in order to resolve these spectral overlap. There are many factors that affect selectivity and sensitivity of the proposed methods such as the used solvent, the divisor concentration and the chosen wavelength.

Different solvents were tried (methanol, ethanol, water, 0.05 N HCl, 0.05 N NaOH). Regarding sensitivity and selectivity, it was found that ethanol was the best solvent for the developed spectrophotometric methods.

The divisor concentration has a great effect on the method selectivity and analytical parameters such as correlation coefficients, slopes and intercepts of the calibration equations. So different concentrations of ME were tested (5, 10, 15 µg/mL). Ten micrograms/milliliters of ME was used as a divisor for all the developed spectrophotometric methods as it resulted in optimum selectivity with minimum noise.

In method {2}, selection of wavelength ranges is a critical step in method optimization as it affects selectivity of the method. So different wavelength ranges were examined and it was found that wavelength ranges of 350–370, 220–230 or 235–245 nm were the most suitable ranges for selective determination of ME, 4AP, and SA.

Method {1}

ME can be determined by measuring the amplitude of the constant at 350 nm obtained after dividing by standard spectrum of 10 µg/mL ME where no interference from 4AP and SA was observed (Fig. 3). The peak amplitude was plotted versus the corresponding concentration of ME in the range of 3–20 µg/mL and the calibration curve was constructed.

Fig. 3. Ratio Spectra of ME (—), 4AP (…) and SA (- - -), Using 10 µg/mL of ME as Divisor and Ethanol as a Blank

For determination of 4AP and SA, zero order absorption spectra of each solution were recorded then divided by the standard spectrum of 10 µg/mL ME as a divisor to obtain ratio spectra. Calibration curve was constructed relating the ratio difference in absorbance of the resultant ratio spectra at 221.4 and 242.2 nm (ΔA_{221.4–242.2 nm}), 230 and 241.2 nm (ΔA_{230–241.2 nm}) to the corresponding 4AP and SA concentrations, respectively and the regression equations were computed (Table 1). The selectivity of the method was tested by applying on the laboratory prepared mixtures and the results illustrated in Table 2 showing good percentage recoveries.

Table 1. Regression and Analytical Parameters of the Proposed Methods for Determination of Mesalazine (ME), 4-Amino Phenol (4AP) and Salicylic Acid (SA) by the Proposed Methods

Parameters	ME			4AP			SA
Parameters	Amplitude of the constant at 350 nm	AUC method	TLC	Ratio difference at 221.4–242.2 nm	AUC method	TLC	Ratio difference at 230–241.2 nm	AUC method	TLC
Range (µg/mL) or (µg/band)	3–20	3–20	0.5–4	2–20	1–20	0.5–3	2–20	1–20	0.5–2.5
Correlation coefficient (r)	0.9997	0.9998	0.9999	0.9999	A_220–230=0.5298 C_4AP+0.5131 C_SAA_235–245=0.5247 C_4AP+0.2737 C_SA	0.9999	0.9999	A_220–230=0.5298 C_4AP+0.5131 C_SAA_235–245=0.5247 C_4AP+0.2737 C_SA	0.9999
Slope	0.0595	0.1153	3096.398	0.0582		5232.28	0.0301		2437.097
Intercept	0.1086	0.1514	189.2066	−0.0141		111.7249	0.0439		535.4441
Accuracy (% found)	101.33	101.85	100.33	99.19	99.09	100.45	100.36	98.59	100.35
Specificity (Mean±%RSD)	99.66±1.286	100.77±1.106	—	99.49±1.419	98.29±0.797	—	99.71±0.884	99.55±1.532	—
Precision (%RSD)
Repeatability*	1.099	1.265	0.737	0.766	0.776	0.566	0.571	0.798	0.765
Intermediate**	1.770	1.160	0.884	1.235	0.958	0.628	0.919	1.141	0.731
LOD (µg/mL)***	0.95	0.9	0.15	0.62	0.3	0.13	0.63	0.31	0.16
LOQ (µg/mL)***	2.85	2.7	0.45	1.85	0.9	0.4	1.90	0.95	0.48

* The intraday (n=3), average of three different concentrations repeated three times within day. ** The interday (n=3), average of three different concentrations repeated three times in three successive days. *** Limit of detection and quantitation are determined LOD=3.3×(S.D. of the intercept/slope); LOQ=10×(S.D. of the intercept/slope).

Table 2. Assay Results for the Determination of ME, 4AP and SA in Synthetic Mixtures Using the Proposed Methods

Mixture No.	Claimed taken (µg/mL)			Found* (%)
	Claimed taken (µg/mL)			Method {1}			Method {2}
	ME	4AP	SA	ME	4AP	SA	ME	4AP	SA
1	15.00	5.00	5.00	101.34	99.40	97.40	100.66	97.45	99.67
2	15.00	3.00	2.50	99.72	98.00	101.20	98.64	100.77	99.77
3	12.00	4.00	3.00	101.08	97.50	99.33	99.78	101.03	100.50
4	5.00	3.00	4.00	102.79	97.33	101.25	101.92	100.73	99.67
5	11.00	3.00	5.00	100.10	98.33	97.80	98.46	98.74	98.01
6	8.00	5.00	3.00	99.59	99.20	100.33	98.54	98.19	100.78
Mean±RSD				100.77±1.106	98.29±0.797	99.55±1.532	99.66±1.286	99.49±1.419	99.71±0.884
Mixture No.	Claimed taken (µg/band)			Found* (%)
	Claimed taken (µg/band)			TLC-densitometric method
	ME	4AP	SA	ME	4AP	SA
1	1.5	1.5	1.5	99.62	101.78	97.98
2	3	0.1	0.1	100.99	98.89	99.40
3	2	1	1	98.21	99.77	101.22
Mean±RSD				99.61±1.135	100.15±1.210	99.53±1.326

* Average of three determinations.

Method {2}

ME can be determined by AUC at 350–370 nm (Fig. 2). Calibration curve was constructed in the range of 3–20 µg/mL. The regression equation parameters are found in Table 1.

The AUC of the absorption spectra each of 4AP and SA in the wavelength ranges 220–230 nm (λ₁–λ₂) and 235–245 nm (λ₃–λ₄) were calculated for different concentrations in the range of 1–20 µg/mL. The absorptivity ‘Y’ values of 4AP and SA were calculated at each wavelength range (Fig. 4). The concentrations of 4AP and SA can be obtained by applying Cramer’s rule and equations presented in Table 1.

Fig. 4. (a) Mixture Contains 5 µg/mL Each of ME, 4AP and SA Using ME 10 µg/mL as Divisor (b) after Subtraction the Plateau at 350 (c) Then Multiplying by Divisor (d) Mixture Binary of 4AP and SA Overlaid with the Resulted Spectrum

The developed methods were successfully used for determination of the three components in their laboratory prepared mixtures. On applying method {2} on the laboratory prepared mixtures and in order to calculate ME concentrations, AUC in the range of 350 nm was used while for measuring 4AP and SA concentrations, the scanned spectrum of each mixture was divided by standard spectrum of 10 µg/mL ME and the value of the amplitude of the constant at 350 nm was subtracted. The resulted subtracted ratio spectrum of each mixture was then multiplied by the divisor. The original absorption spectrum of both 4AP and SA was then resulted (Fig. 4) from which AUC in the ranges of 220–230 and 235–245 nm were calculated. By applying in equations in Table 1 concentrations of both 4AP and SA in the mixture could be obtained. The obtained mean percentage recoveries were given in Table 2, those confirmed method selectivity.

Method {3}

Series of trials were established to select the most appropriate system for the separation process, starting with ethyl acetate–chloroform (8 : 2, v/v) which did not yield the desired separation as ME was highly retained on the stationary phase and appeared on the base line close to the spot of SA. While, 4AP eluted rapidly with the developing system. By addition of acetic acid no improving in the chromatographic separation has been noticed. Changing the developing system with ethyl acetate–acetone–ammonia solution (9.5 : 0.5 : 0.3, v/v/v) gave good separation but with SA peak near the front line. Suitable chromatographic separation of the three components was observed upon using ethyl acetate–methanol–triethylamine (8.5 : 2 : 0.7, v/v/v) in Fig. 5. That gave compact peaks at Rf values of 0.1, 0.25, and 0.7 for ME, SA, and 4AP, respectively. Linearity was achieved in the ranges of 0.5–4, 0.5–3, and 0.5–2.5 µg/band for ME, 4AP, and SA, respectively (Table 1).

Fig. 5. TLC Densitogram of Mixture of (1.5 µg/Band) Each of (a) ME, (b) 4AP and (c) SA Using Developing System of Ethyl Acetate–Methanol–Triethylamine (8.5 : 2 : 0.7, v/v/v)

Application to Pharmaceutical Formulations

The proposed methods were also applied for determination of ME in Pentasa^® tablets and satisfactory results were obtained (Table 3). In addition, applying standard addition technique which also confirmed the accuracy of the developed methods in Table 3.

Table 3. Determination of Studied Drugs in Pentasa^® Tablets by the Developed Methods and the Application of Standard Addition Technique

Pharmaceutical formulation	Method {1}
	Component	Taken (µg/mL)	Found%±%RSD*	Standard addition
	Component	Taken (µg/mL)	Found%±%RSD*	Added (µg/mL)	Found (µg/mL) or (µg/band) **	Found %
Pentasa^® tablets claimed to contain 500 mg of ME (Batch No. L13362A).	ME	5.00	99.73±1.372	5.00	5.02	100.36
				7.50	7.37	98.25
				10.00	10.14	101.35
				Mean±RSD 99.99±1.295
	Method {2}
	ME	5.00	99.12±1.322	5.00	5.07	101.31
				7.50	7.42	98.91
				10.00	10.02	100.24
				Mean±RSD 100.15±0.981
	Method {3}
	ME	1.00	106.34±1.414	0.80	0.80	99.65
				1.00	1.01	100.65
				1.20	1.21	101.22
				Mean±RSD 100.51±0.791

* Average of six determination. ** Average of three determination.

The results obtained by applying the developed methods were statistically compared with those obtained by applying the reported HPLC method for determination of ME.²⁵⁾ The values of the obtained F and Student’s t-tests were less than the calculated ones which indicated that there is no significant difference between the developed methods and the reported one (Table 4).

Table 4. Statistical Comparison between the Results Obtained by the Developed Methods and the Reported Methods for the Determination of ME

	Method {1}	Method {2}	Method {3}	Reported method*²⁵⁾
Component	ME	ME	ME
Mean	101.85	101.33	100.33	100.79
%RSD	1.174	0.759	1.079	1.357
N	7	7	7	7
Student’s t-test (2.179)**	1.431	0.853	0.655	—
F-Test (4.284)**	1.308	3.163	1.863	—

* HPLC determination of ME using acetonitrile–0.05 M KH₂PO₄ (35 : 65, v/v) as developing system and detection at 258 nm.²⁵⁾ ** Figures between parenthesis represent the corresponding tabulated values of t and F at p=0.05.

Method Validation³⁰⁾

Method validation was carried out according to ICH guidelines.²⁹⁾ The results presented in Table 1 showed that the developed methods possess good accuracy and precision.

In addition, selectivity of the methods has been checked by application of the methods to different prepared mixtures containing different ratios of ME, 4AP, and SA where the obtained percentage recoveries proved the good selectivity of the developed methods in Table 2. Moreover, results of dosage form confirmed that tablets additives did not interfere, in Table 3. In method {3} robustness and system suitability parameters are represented in Tables 5 and 6.

Table 5. Experimental Results of Robustness for Determination of ME, 4AP and SA by the Developed TLC-Densitometric Method

Parameters (%RSD)	TLC-densitometric method
Parameters (%RSD)	ME	4AP	SA
Methanol±1 mL	1.124	1.578	1.288
Saturation time±5 min	0.547	0.723	0.968

Table 6. System Suitability Testing Parameters of the Developed TLC-Densitometric Method

Parameters	TLC-densitometric method			Reference value
Parameters	ME	4AP	SA	Reference value
Tailing factor (T)	1.12	1.09	1.13	<1.5–2 or <2
Selectivity (α)	2.5	2.36		>1
Resolution	2.61	6.31		R >1.5

Conclusion

Three methods were used for the first time for determination of ME, 4AP and SA in their ternary mixture without preliminary separation. The developed methods are simple, precise and selective for determination of the studied drug either in their laboratory prepared mixtures or in their pharmaceutical formulation. The advantages of TLC-densitometric method is its ability to determine the cited drugs using the same developing system and scanning wavelength, several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC, thus lowering analysis time and cost. While the developed spectrophotometric methods are highly selective and simple. They do not need any derivatization steps or complex algorithms. They are easily applied.

Conflict of Interest

The authors declare no conflict of interest.

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