determination of dantrolene sodium in the presence of its ... thin-layer...

462

SummaryDantrolene sodium (DAN) is widely used as a muscle relaxant drug for the treatment of malignant hyperthermia. 5-(4-Nitro-phenyl)-2-furaldehyde is reported to be DAN-related compound C (DC), and it is the synthetic precursor of DAN that may be present-ed as process-related impurity in a pharmaceutical formulation. Also, it is considered to be the acidic and photolytic degradation product of DAN. The synthesis of DC has been performed from p-nitroaniline with good yield and high purity; structure elucida-tion has been confirmed by infrared (IR), mass spectrometry (MS), and nuclear magnetic resonance (NMR) analyses. In the present study, high-performance thin-layer chromatography (HPTLC) technique was developed and validated for the separation and quantification of DAN along with DC. Separation was carried out on HPTLC silica gel 60 F254 plates using a developing system con-sisting of chloroform–ethylacetate–acetic acid (10:0.5:0.01, by vol-ume) with ultraviolet (UV) scanning at 380 nm. The method was highly sensitive and could be used for the determination of DAN and DC in the range of 0.1–1.5 μg band−1 and 0.1–2.0 μg band−1, respectively. The method has been validated in compliance with the International Conference on Harmonization (ICH) guidelines and was successfully applied to a pharmaceutical formulation. The method was compared favorably with the reported British Phar-macopoeia high-performance liquid chromatography (BP HPLC) method.

1 Introduction

Dantrolene sodium (DAN), 1-(5-p-nitrophenylfurfurylidene-amino)hydantoin sodium [1] (Figure 1A), is a muscle relax-ant that is commonly used to relieve spasticity in neuromus-cular disorders as spinal cord injury, cerebral palsy, stroke,

N.S. Abdelwahab, Pharmaceutical Analytical Chemistry, Faculty of Pharma-cy, Beni-Suef University, Beni-Suef, Egypt; M.T. Elsaady and A.G. Korany, Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt; and M.A. Hgazy, Pharmaceutical Analytical Chemistry, Faculty of Phar-macy, Cairo University, Cairo, Egypt.E-mail: [email protected]

and multiple sclerosis [2]. It acts specifically and effectively on treating malignant hyperthermia [3]. DAN is a pharma-copeial drug in the United States Pharmacopeia (USP), the British Pharmacopoeia (BP), and the Japanese Pharmacopeia (JP) [4]. It was reported to have more than one related com-pounds including related compound C [5] which is chemi-cally known as 5-(4-nitrophenyl)-2-furaldehyde (Figure 1A). This related compound was considered DAN synthetic pre-cursor [6]. It was also reported to be the acidic and photol-ytic degradation product of DAN [7, 8]. After an extensive literature review, it was found that DAN has been analyzed in BP [1], USP [5], and JP [9] by a reversed-phase high-per-formance liquid chromatography (RP-HPLC) method. The drug was also determined by different HPLC [10–15] and ultra-performance liquid chromatographic (UPLC) methods [7, 8] either in biological fluids, pharmaceutical formulation, or in the presence of its reported impurities or metabolites. In addition, the drug was determined in the presence of its metabolites by colorimetry [16], pulse polarography [17], and spectrophotofluorometry [18]. There is no report, however, on the synthesis of DC along with HPTLC technique for the separation and determination of DAN and DC. Our aim in this study was to prepare DC with high yield and purity using a simple preparation method and to develop and validate an

Determination of Dantrolene Sodium in the Presence of its Process-Related Impurity by High-Performance Thin-Layer Chromatography–Spectrodensitometry

Nada S. Abdelwahab, Mohammed T. Elsaady, Aml G. Korany*, and Maha A. Hgazy

Key Words:

DantroleneDantrolene impurityHigh-performance thin-layer chromatographyValidationSynthesis

Journal of Planar Chromatography 29 (2016) 6, 462–468 DOI: 10.1556/1006.2016.29.6.90933-4173/$ 20.00 © Akadémiai Kiadó, Budapest

Figure 1

(A) Chemical structures of dantrolene and related compound and (B) synthesis scheme of 5-(4-nitrophenyl)-2-furaldehyde.

Determination of Dantrolene Sodium by HPTLC–Densitometry

Journal of Planar Chromatography 29 (2016) 6 463

HPTLC technique for the selective determination of DAN and DC. This method has advantages of being sensitive and less time- and money-consuming. It did not need high-cost chemicals or sample pretreatment steps.

2 Experimental

2.1 Instruments

1. CAMAG TLC Scanner 3 S/N 130319 with winCATS software (CAMAG, Muttenz, Switzerland).

2. The following requirements were taken into consideration:

‒ Source of radiation: deuterium lamp;

‒ Scan mode: absorbance mode;

‒ Slit dimension: 6.00 × 0.3 mm, Micro;

‒ Scanning speed: 20 mm s–1;

‒ Output: chromatogram and integrated peak area.

3. Linomat 5 autosampler and CAMAG microsyringe (100 μL).

4. Sonix TV ss-series ultrasonicator (USA).

5. Aluminum sheets (20 cm × 20 cm) coated with 0.25 mm silica gel 60 F254 (Merck, Darmstadt, Germany).

6. Proton nuclear magnetic resonance (1H-NMR) spectra were recorded in CDCl3 and DMSO-d6 on a Varian Mercury spec-trometer (400 MHz) (Bruker AG, Fällanden, Switzerland).

7. Infrared (IR) spectra were made on a BRUKER Vector 22 (Japan) infrared spectrophotometer and were expressed in wavenumber (cm−1) using a potassium bromide disk.

8. Mass spectra were recorded on Finnigan MAT, SSQ 7000, mass spectrometer at 70 eV (EI).

9. IA 9100MK-Digital Melting Point Apparatus.

2.2 Materials

2.2.1 Pure Samples

Standard DAN was obtained from Sigma-Aldrich (St. Louis, MO, USA), with certified purity of 98%.

2.2.2 Pharmaceutical Formulation

Dantrelex® capsules, batch No. 150728A, were manufactured by Chemipharm Pharmaceutical Industries (6th of October City, Egypt). Each tablet is claimed to contain 25 mg of DAN and was obtained from a local market.

2.2.3 Reagents

1. For the synthesis of dantrolene process impurity:

‒ p-Nitroaniline was obtained from Belami Fine Chemicals (Mumbai, India), with certified purity of 98%.

‒ Sodium nitrite was obtained from Loba Chemie (Mumbai, India), with certified purity of 97%.

‒ 2-Furaldehyde was obtained from Sigma-Aldrich (St. Louis, MO, USA), with certified purity of 99%.

‒ CuCl2 was obtained from Laboratory Rasayan (Anand, India), with certified purity of 99%.

– Acetone and absolute ethanol were obtained from El‐Nasr Pharmaceutical Chemicals Company Abu‐Zabaal (Cairo, Egypt).

2. For HPTLC:

All reagents and chemicals used were of analytical grade and include:

‒ Methanol HPLC grade (Tedia, Fairfield, OH, USA).

‒ Chloroform, HCl, ethyl acetate and glacial acetic acid (El‐Nasr Pharmaceutical Chemicals Company Abu‐Zabaal, Cairo, Egypt).

2.2.4 Solutions

Stock solutions at 0.5 mg mL−1 and 1.0 mg mL−1 in methanol were prepared for DAN and DC, respectively. Working solu-tions were prepared for each component in methanol having the concentration of 100.00 µg mL−1. DAN stock and working solu-tions were stored at 5°C and protected from light.

2.3 Procedures

2.3.1 Synthesis of Dantrolene Process Impurity

5-(4-Nitrophenyl)-2-furaldehyde was synthesized as it is the key precursor for DAN as reported by Snyder et al. [6]. Syn-thesis steps are shown in Figure 1B. These steps are as follows:

1. Formation of nitro-phenyl diazonium chloride salt:

To a suspension of p-nitrophenol (1 g, 0.5 mole), 1.5 mL of conc. HCl was added and then heated until solid dissolved. An additional 3 mL of conc. HCl was added to stabilize the formed diazonium salt [19]. The solution was cooled to 0°C. A solution of (0.5 g, 0.5 mole) NaNO2 was added dropwise and stirred for 30 min, and then diazonium salt was precipi-tated immediately.

2. Coupling of diazonium salt with 2-furaldehyde to produce DC:

To the formed diazonium salt, solution of 2-furaldehyde (0.695 g, 0.5 mole) in acetone was added, followed by addi-tion of CuCl2 (0.2 g) in aqueous solution as catalyst. The temperature was allowed to rise to 15–20°C. Solution was stirred for 4 h, and reddish brown precipitate was formed. The obtained precipitate was filtered, washed with water, and recrystallized from absolute ethanol. The formed product was structurally elucidated by:

(a) Measuring melting point: by open capillary tube method using an IA 9100MK-Digital Melting Point Apparatus.

(b) Microanalysis includeing 1H-NMR, IR, and mass spectroscopy.

2.4 HPTLC–Spectrodensitometric Determination of Dantrolene in the Presence of Its Process Impurity

2.4.1 Linearity and Construction of Calibration Curves

Aliquots equivalent to 1–15 μg mL−1 and 1–20 μg mL−1 of DAN and DC, respectively, were transferred from their working solu-tion (100.00 μg mL−1), and 10 μL of each solution was applied in duplicate to HPTLC plates (20 cm × 10 cm) as bands with 5 mm width using a CAMAG Linomat IV applicator. The bands


464 Journal of Planar Chromatography 29 (2016) 6

were spaced 5 mm from each other and 15 mm apart from the bottom edge of the plate. Linear ascending development was performed in a chromatographic chamber previously saturated with chloroform–ethyl acetate–glacial acetic acid (10:0.5:0.01, by volume) as the developing system for 30 min, at room tem-perature to a distance of 9 cm. The plates were air-dried and scanned at 380 nm using a slit dimension of 6.00 × 0.3 mm, Micro. The calibration curves for the studied components were constructed by plotting the integrated peak area × 10−4 versus the corresponding concentrations, and the regression equation was computed.

2.4.2 Application to Pharmaceutical Formulations

The content of ten capsules of Dantrelax® was accurately weighed and mixed. An accurately weighed portion equivalent to 50.00 mg of DAN was transferred into a 50-mL volumetric flask, and then, an amount of 25 mL methanol was added. The prepared solution was sonicated for 30 min, then cooled, and completed to volume with methanol. The solution was filtered and diluted to obtain a solution having a final concentration of 100.00 µg mL−1.

3 Results and Discussion

In this work, the synthesis of DAN main impurity was achieved along with the determination of DAN. The first part of this work is concerned with simple, safe, and economic synthesis for DC, while the second part is to establish a sensitive, selective, accurate, and validated method for the quantitation of DAN in the presence of its process-related impurity, according to lit-erature; only two UPLC methods have been reported for the determination of DAN in the presence of its related compound [7, 8]. In order to develop a simpler method that is available to be performed in quality control laboratories, an HPTLC tech-nique with spectrodensitometric detection was developed and validated for the determination of DAN in the presence of DC; it is not only the first HPTLC technique for the determination of DAN in the presence of process-related impurity, but also the first method that includes the synthesis of this impurity with high purity and good yield.

3.1 Synthesis of Dantrolene Process Impurity

According to USP, 5-(4-nitrophenyl)-2-furaldehyde was de- scribed as DAN related compound C [5] . It is a key precursor for DAN preparation [6]. Also, it is an acidic and a photolytic degradation product of DAN [8, 7]. The synthesis of DC was carried out as shown in Figure 1. Highly purified product was obtained which was confirmed by several ways:

1. Measuring melting point that was 202–204°C, agreeing with the reported value [20].

2. Microanalysis which include:

(a) 1H-NMR (DMSO-d6, 400 MHz): the spectrum (Figure 2A) shows characteristic chemical shifts 9.6 (s, 1H, CHO), 8.1–8.3 (dd, 4H, J = 8 Hz, aromatic protons), and 7.5–7.7 (s, 2H, proton of furan ring), and there is no disappearance of any chemical shifts upon deuteration which approves the absence of exchangeable protons (Figure 2B).

(b) IR spectrometry: the IR spectrum (Figure 2C) shows two characteristic bands at about 1679, indicating car-bonyl group; 2850 and 2900, indicating CH aldehydic; and 3400, indicating OH group of absorbed water to which IR spectrophotometer is sensitive.

(c) Mass spectrometry: Mass chart of DC (Figure 2D) shows parent peak identified at m/z 217 (corresponding to the molecular weight of DC).

3.2 HPTLC–Spectrodensitometric Determination of Dantrolene in the Presence of Its Process Impurity

Thin-layer chromatography (TLC) has made a great progress and it has a wide acceptance as a major tool for both quali-tative and quantitative analyses; it has become a well-estab-lished method for the assay of drugs in mixtures. HPTLC offers the advantages of automatic application, high separa-tion power, using smaller amount of solvent, and providing dense and compact bands with properly and significantly dif-ferent retention factor (Rf) values in addition to high sensi-tivity [21].

3.3 Method Development and Optimization

In order to optimize HPTLC technique, the following variables were tested:

3.3.1 Developing System

Different developing systems of different compositions and ratios were tried such as chloroform–methanol (10:1, by vol-ume), (10:0.5, by volume), chloroform–methanol–glacial acetic acid (10:0.5:0.1, by volume), chloroform–acetone–glacial acetic acid (10:0.3:0.01, by volume), and chloroform–ethyl acetate–glacial acetic (10:0.5:0.01, by volume). The best developing sys-tem was found to be chloroform–ethyl acetate–glacial acetic (10:0.5:0.01, by volume), where the good separation between DAN and DC was shown by the difference in the Rf values and symmetrical peaks; the Rf values were 0.22 ± 0.01 for DAN and 0.83 ± 0.01 for DC as shown in Figure 3. The presence of glacial acetic acid in the developing system is essential for the separation and prevention of DAN tailing.

3.3.2 Scanning Wavelength

Different scanning wavelengths were tried (254, 350, and 380 nm) in order to enhance the sensitivity of the method, where scanning at 380 nm gave the best sensitivity for the studied components. The peaks were sharp, well separated, and sym-metrical with minimum noise, as shown in Figure 3.

3.3.3 Band Dimensions and Slit Dimensions of Scanning Lightbeam

The band width and interspaces between bands should be selected carefully to avoid the spread of bands outside the tracks and interference between adjacent bands [21]. Different band dimensions were tried to obtain sharp and symmetrical peaks. The optimum band width chosen was 5 mm, and the interspace between bands was 5 mm. Moreover, different slit dimensions were tried where 6.00 × 0.3 mm, Micro, proved to be the slit dimensions of choice which provided the highest sensitivity.



The method offered high sensitivity and selectivity for the anal-ysis of DAN in the presence of DC. A relationship between the integrated peaks area × 10−4 versus the concentrations of DAN

and DC in the range of 0.1–1.5 μg band−1 and 0.1–2.0 μg band−1 for DAN and DC, respectively, is shown in Table 1; the regres-sion equations were computed using polynomial order as shown in Figure 4 and found to be:

A = −0.4049C2 + 1.2415C + 0.2168 r = 0.9992 for DAN

A = −0–0.241C2 + 1.4158C + 0.3283 r = 0.9993 for DC

where A is the integrated peak area ×10−4, C is the concentra-tions in μg band−1, and r is the correlation coefficient. Regres-sion equation parameters are given in Table 1. The validity of the proposed methods for the analysis of DAN was studied by assaying Dantrelax® capsules (Figure 5 and Table 2). It was further assessed by applying standard addition technique, which showed that there was no interference from excipients (Table 2).

3.4 Method Validation

Method validation was carried out according to the International Conference on Harmonization (ICH) guidelines [22].

Figure 2

(A) 1H-NMR spectra of DC, (B) D2O spectra of DC, (C) IR spectra of DC, and (D) mass spectra of DC.

Figure 3

HPTLC densitogram of 1 μg band–1 of DAN (Rf = 0.22) and DC (R

f = 0.83)

using chloroform–ethyl acetate–glacial acetic (10:0.5:0.01, by vol-ume) as the developing system and scanning at 380 nm.



3.4.1 Linearity

Under optimum experimental conditions, the linearity of DAN and DC was evaluated by analyzing several concentra-tions determined in duplicates ranging between 0.1–1.5 μg band−1 and 0.1–2.0 μg band−1 for DAN and DC, respectively; working ranges and regression equations parameters are listed in Table 1. The correlation coefficient near to the unit, besides residuals mean, equals zero (Table 1), and random pattern of residual plots (Figure 6) proved the goodness of the regres-sion equation.

3.4.2 Accuracy

The accuracy of the method was assured by applying the developing method for the determination of different con-centrations of pure DAN and DC samples and then applying the corresponding regression equations (the results, given in Table 1, confirmed the good accuracy of the reported method). The accuracy of the method was also checked by applying the standard addition technique, involving analysis of marketed samples (Dantrelax® capsules) to which certain amounts of pure DAN were added. The resulting mixtures were assayed, and recoveries (%) were calculated. Good recoveries were obtained, confirming the good accuracy of the proposed method and revealing no interference from excipients, as shown in Table 2.

Figure 4

Calibration curves showing a relationship between peaks area × 10–4 versus the concentrations using polynomial equation in the range of 0.1–1.5 μg band–1 for DAN and 0.1–2.0 μg band–1 for DC.

Figure 5

HPTLC densitogram of 0.5 μg band–1 of Dantrelax® capsule in the presence of DAN.

Table 1

Regression and validation parameters of the proposed method for the determination of dantrolene in the presence of its process impurity.

DCDANParameter

0.1–2.00.1–1.5Range (µg band−1)

−0.2411.4158

−0.40491.2415

Slopea):Coefficient aCoefficient b

0.32830.2168Intercepta)

0.99930.9992Correlation coefficient (r)

0.0000.000Residual mean

100.25 ± 0.33799.518 ± 0.644Accuracy (mean ± SD)

Precision

0.2190.319Repeatability (% RSD)

0.4880.672Intermediate precision (% RSD)

0.0320.033LODb) (µg band−1)

0.0960.099LOQc) (µg band−1)

Robustness parameters (% RSD)

1.701.27Chloroform (10 ± 0.2 mL)

1.711.59Ethyl acetate (0.5 ± 0.05 mL)

0.8460.645Saturation time (30 ± 5 min)

a)Following a polynomial regression, A = aX2 + bX + C, where A is the integrated peak area, X is the concentration in μg band−1, a and b are coefficients 1 and 2, respectively, and C is the interceptb)LOD = 3.3 × SD/slopec)LOQ = 10 × SD/slope

Table 2

Determination of dantrolene in the presence of its process impurity in its pharmaceutical formulation by the proposed method and ap-plication of a standard addition technique.

Dantrelax® capsules

Taken (µg band−1) Found% ± SD Pure added

(µg band−1)Recovery %

0.5 99.84 ±0.866 0.300.501.00

99.219100.72298.89

Mean ± SD (99.61 ± 0.9766)



3.4.3 Precision

Precision was studied with respect to both repeatability and intermediate precision.

3.4.4 Repeatability

Three concentrations (0.5, 0.8, and 1.0 µg band−1) of DAN and (0.4, 0.6, 1.0 µg band−1) of DC were determined in triplicates on the same day to estimate intra-day variation. Acceptable relative standard deviation (RSD %) values were obtained, confirming the repeatability of the method as given in Table 1.

3.4.5 Intermediate Precision

The previous procedure was repeated on the same concentra-tions on three different days to determine the intermediate pre-cision. Acceptable RSD % values were obtained as shown in Table 1.

3.4.6 Limits of Detection and Quantitation (LOD and LOQ)

LOD and LOQ have been calculated for DAN and DC using ICH equations [22]. The low values of both LOD and LOQ indi-cated the high sensitivity of the developed methods (Table 1).

3.4.7 Robustness

Robustness measures the capacity to remain unaffected by minor deliberate variations in the method parameters and indi-cates the reliability of the method under normal usage condi-tions. The proposed method was found to be robust where delib-erate minor changes in the studied chromatographic conditions (e.g., change in ethyl acetate amount ±0.05, chloroform ±0.2 and saturation time ±5 min) did not lead to significant changes

in the Rf values. However, concentration of glacial acetic acid proved to be critical where, upon decreasing or increasing its amount ±0.01, dramatic changes in Rf values, and hence, chro-matographic resolution occurred (Table 1).

3.5 System Suitability

System suitability tests are a fundamental part of liquid chro-matographic analyses according to ICH. They determine whether the operating systems were performing properly for the intended analysis. System suitability testing parameters for the developed method were calculated, and acceptable results were obtained as shown in Table 3.

Table 3

System suitability testing parameters of the proposed method for the determination of dantrolene in the presence of its process im-purity.

Parameter Obtained value (DAN)

Obtained value (DC)

Reference value [22]

Symmetry factor 1.125 1.093 <1.5

Capacity factor (K´) 1.1 7.3 1–10

Resolution (R) 4.679 >1.5

Selectivity factor (α) 6.636 >1

The results obtained by the proposed method were statistically compared with those obtained by the reported BP HPLC method [1] using student’s t- and F-tests at p < 0.05. The obtained val-ues are less than the theoretical ones, indicating that there is no significant difference between the proposed methods and the reported method. This statistical comparison clarifies the proposed method and is as precise and accurate as the reported method (Table 4).

Table 4

Statistical comparison of the results obtained by the proposed method and the reported method in BP for the determination of dantrolene in the presence of its process impurity.

Parameter HPTLC–densitometric method

Reported method [1]

Mean 99.52 100.88

SD 0.645 0.939

Variance 0.346 0.735

n 6 6

Student’s t-testa) (2.228) 0.015

F-value (5.050) 2.12

a)Figures in parenthesis are the corresponding tabulated values at p = 0.05

Figure 6

Residual plots showing a relationship between the concentration ranges of DAN and DC versus their residuals showing random pat-tern.



4 Conclusion

The proposed work involved simple, economic, and safe syn-thesis for DAN process-related impurity. Furthermore, the main advantages of the proposed HPTLC method are high sensitivity and selectivity. Dantrolene and its impurity were determined in the range starting from 0.1 μg mL−1 (Table 1), while in other HPLC method, determination started from 0.5 μg mL−1 [8], in addition to the high resolution (R = 4.679) mentioned in Table 3, besides using one single scanning wavelength for the two studied compounds, saving time and effort, and running several samples simultaneously using a small quantity of developing system. On the other hand, the application of this method to a pharmaceutical preparation showed that the excipients did not interfere with the determination of the studied drug. Finally, the suggested method is simple, accurate, rapid, highly sensitive, and reproducible; hence, it can be used for routine and qual-ity control analysis either in bulk powder or in pharmaceutical formulations.

References

[1] British Pharmacopoeia, Dantrolene Sodium Monograph, 2013.

[2] A. Ward, M.O. Chaffman, E.M. Sorkin, Drugs 32 (1986) 130–168.

[3] G.G. Harrison, Br. J. Anaesth. 47 (1975) 62–65.

[4] S.C. Sweetman, Martindale: The Complete Drug Reference, Pharmaceutical Press, London, 2014, 1896–1897.

[5] United States Pharmacopeia (USP), Dantrolene Sodium Mono-graph, USP 38–National Formulary 33, 2015.

[6] H. Snyder, C. Davis, J. Med. Chem. 10 (1967) 807–810.

[7] S.R. Khan, M.A. Tawakkul, V.A. Sayeed, P.J. Faustino, J. Phar-macol. Pharm. 3 (2012) 281–290.

[8] M.A. Tawakkul, P.J. Faustino, V.A. Sayeed, M.A. Khan, S.R. Khan, Clin. Res. Regul. Aff. 27 (2010) 21–29.

[9] Japanese Pharmacopoeia, Dantrolene Sodium Monograph, 2013, 691–692.

[10] Y. Katogi, N. Tamaki, M. Adachi, J. Terao, M. Mitomi, J. Chro-matogr. B 228 (1982) 404–408.

[11] M. Lalande, P. Mills, R.G. Peterson, J. Chromatogr. B 430 (1988) 187–191.

[12] S.J. Saxena, I.L. Honigberg, J.T. Stewart, G.R. Keene, J.J. Vall-ner, J. Pharm. Sci. 66 (1977) 286–288.

[13] S.J. Saxena, I.L. Honigberg, J.T. Stewart, J.J. Vallner, J. Pharm. Sci. 66 (1977) 751–753.

[14] E.W. Wuis, A.C.L.M. Grutters, T.B. Vree, E. Van Der Kleyn, J. Chromatogr. B 231 (1982) 401–409.

[15] E.W. Wuis, M.G.A. Janssen, T.B. Vree, E. Van Der Kleijn, J. Chro-matogr. B 526 (1990) 575–580.

[16] J. Conklin, R. Sobers, J. Pharm. Sci. 62 (1973) 1024–1025.

[17] P.L. Cox, J.P. Heotis, D. Polin, G.M. Rose, J. Pharm. Sci. 58 (1969) 987–989.

[18] R.D. Hollifield, J.D. Conklin, J. Pharm. Sci. 62 (1973) 271–274.

[19] B.S. Furniss (ed.), Vogel’s Textbook of Practical Organic Chemis-try, 5th edn., School of Chemistry, Thames Polytechnic, London, 1989, 945–946.

[20] ChemSpider Search and Share Chemistry, http://www.chemspi-der.com/Chemical-Structure.73597.html.

[21] E.A. Naguib, E.A. Abdelaleem, H.E. Zaazaa, E.A. Hussein, Eur. J. Chem. 5 (2014) 219–226.

[22] ICH, Q2 (R1), Validation of Analytical Procedures, Proceedings of the International Conference on Harmonization, Geneva, 2005.

Ms received: April 30, 2016Accepted: August 2, 2016

determination of dantrolene sodium in the presence of its ... thin-layer...

Documents