Determination of Chlorzoxazone, Diclofenac

Determination of Chlorzoxazone, Diclofenac Potassium, and Chlorzoxazone Toxic Degradation Product by Different Chromatographic Methods Maha M. Abdelrahman, Nada S. Abdelwahab, Ahmed A. Taha, and John M. Boshra*

Key Words: Diclofenac potassium Chlorzoxazone 2-Amino-4-chlorophenol High-performance liquid chromatography Thin-layer chromatography

Summary

1 Introduction

The presented study was intended to design two validated, simple, and precise chromatographic methods for the determination of chlorzoxazone (CHZ) and diclofenac potassium (DIC) in the presence of chlorzoxazone nephrotoxic degradation product, 2-amino-4-chlorophenol (ACP) which was reported to be its main impurity. Reversed-phase high-performance liquid chromatography (RP-HPLC) was the first method where chromatographic separation was performed on ZORBAX Eclipse Plus C8 column using methanol‒water‒phosphoric acid (75:25:0.05, by volume) as the mobile phase at a flow rate of 1 mLmin−1. CHZ, DIC, and ACP retention times were found to be 4.26, 7.94, and 3.17, respectively, using photodiode array detector (DAD) at 230 nm. The calibration curves showed good linear relationships in the concentration ranges of 3–45 µg mL−1 for CHZ, 3–40 µg mL−1 for DIC, and 5-45 µg mL−1 for ACP. The second method was thin-layer chromatography (TLC) at which chromatographic separation was carried out on Merck TLC silica gel 60 F254 aluminum plates followed by measurement of separated bands at 230 nm and using chloroform‒ethanol‒ triethylamine (9:1:0.1, by volume) as the developing system. The studied components were successfully separated with significantly different Rf values (CHZ, Rf = 0.63; DIC, Rf = 0.35; ACP, Rf = 0.42). Linearity was constructed in the range of 1.2–5 µg band−1 for CHZ, 0.5–4 µg band−1 for DIC, and 0.4–4 µg band−1 for ACP. The developed methods were applied to Declophen plus® capsules, and no interference from excipients was observed. The methods were validated as per the United States Pharmacopeia (USP) guidelines, and they were compared favorably with the reported method.

Chloroxazone (CHZ, Figure 1), chemically known as 5-chloro-2-benzoxazolinone, is a centrally acting skeletal muscle relaxant with sedative properties, and it is official in the United States Pharmacopeia [1–3]. It is claimed to inhibit muscle tone and tension and, thus, to inhibit spasm and pain by an effect primarily at the level of the spinal cord by depressing reflexes and subcortical areas of the brain [4, 5]. Lactone and lactam functional groups present in the structure of CHZ make it highly unstable and give 2-amino-4-chlorophenol by alkaline hydrolysis [2, 3, 6].

Figure 1 The chemical structures of chlorzoxazone (a), diclofenac potassium (b), and 2-amino-4-chlorophenol (c).

M.M. Abdelrahman and N.S. Abdelwahab, Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Beni‐Suef, 62514 Egypt; and A.A. Taha and J.M. Boshra, Analytical Chemistry Department, Faculty of Pharmacy, Nahda University, Beni‐Suef, 62514 Egypt. E-mail: [email protected] Journal of Planar Chromatography 29 (2016) 6, 453–461 Journal of Planar Chromatography 29 (2016) 3 0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest

Diclofenac potassium (DIC, Figure 1), chemically known as 2-[(2,6-dichlorophenyl)amino]benzeneacetic acid, monopotassium, is a non-steroidal anti-inflammatory drug (NSAID), and it is official in the British Pharmacopoeia [7]. It has analgesic and antipyretic actions. It is a potent inhibitor of prostaglandin bio-synthesis by inhibition of both leukocyte migration and DOI: 10.1556/1006.2016.29.6.8

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Determination of CHZ and DIZ in the Presence of ACP by Different Chromatographic Methods

(COX-1 and COX-2) cylooxygenase enzymes [5, 8]. It is used in the treatment of signs and symptoms of osteoarthritis and rheumatoid arthritis, and it has been shown to be effective in relieving headache in migraine attacks [5]. The combination CHZ and DIC is indicated for the treatment, prevention, control, and improvement of muscle aches, pain and swelling, dental pain, back pain, sports injuries, menstrual cramps, and other conditions by reducing the substances in the body that cause inflammation and pain and by blocking pain sensations that are sent to the brain [4, 5, 8]. 2-Amino-4-chlorophenol (ACP, Figure 1) is the CHZ primary degradation product which is produced by alkaline hydrolysis [2, 3, 6], so it is considered as CHZ synthetic precursor, but also, ACP is reported in the United States Pharmacopeia (USP) as a related substance and potential impurity of CHZ with 0.5% maximum allowed limit [1]. It is considered a hazardous and harmful substance that causes irritation to the eye, skin, and respiratory system which is labeled in the Sigma-Aldrich Catalogue [9]. It can make renal toxicity [3] and also interfere the ability of the blood to carry oxygen after exposure to high level of ACP causing headache, dizziness, trouble in breathing, blue color of skin and lips, and death [10, 11]. A review in the literature revealed that CHZ and DIC have been determined together as a binary mixture or with other drugs by different analytical techniques. CHZ and DIC as a binary mixture have been determined by spectrophotometric [12, 13] and RP-HPLC [14]. Ternary mixtures of CHZ, DIC, and paracetamol have been determined by spectrophotometric [15], RP-HPLC [4, 16, 17, 18], TLC [19, 20], and supercritical fluid chromatographic (SCFC) [21] methods. CHZ, DIC, and tramadol hydrochloride have been determined by RP-HPLC [22]. Due to nephrotoxic and lethal effect of ACP, it was necessary to develop accurate methods for the detection and determination of this impurity in pure samples and pharmaceutical formulations of CHZ and DIC. The present research work aims at developing simple, accurate, precise, sensitive, and reproducible methods for the simultaneous determination of CHZ and DIC in the presence of chlorzoxazone nephrotoxic degradation product using HPLC and TLC–densitometry methods.

2 Experimental 2.1 Instruments

HPLC method was carried out on Agilent 1260 infinity instrument (Waldbronn, Germany), equipped with an Agilent 1260 infinity preparative pump (G1361A), Agilent 1260 Infinity Diode Array Detector VL (G131SD), Agilent 1260 Infinity Thermostatted column compartment (G1316A), and an Agilent 1260 Infinity Preparative Autosampler (G2260A). Separation and quantitation were performed on ZORBAX Eclipse Plus C8 column (250 mm × 4.6 mm i.d., 5 µm particle size) (Kansas City, MI, USA). TLC method was carried out on a CAMAG TLC Scanner 3 S/N 130319 with winCATS software (CAMAG, Muttenz, Switzer land). The following requirements were taken into consideration: source of radiation, deuterium lamp; scan mode absorbance mode; slit dimension, 3 × 0.45 mm; scanning speed, 20 mm s−1; output, chromatogram and integrated peak area. The other

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instruments included a Linomat 5 autosampler (CAMAG), a CAMAG microsyringe (100 µL), TLC aluminum plates (20 cm × 20 cm) coated with 0.25 mm silica gel 60 F254 (Merck, Darmstadt, Germany), with 0.1 mm thickness, and a Sonix TV SS-series ultrasonicator (Springfield, VA, USA). 2.2 Materials 2.2.1 Pure Standard

CHZ and DIC were received as a gift from Pharco Pharmaceuticals (Alexandria, Egypt) with claimed purities of 99.90% and 100.10%, respectively, according to manufacturer certificates of analysis. Pure standard of ACP was purchased from Sigma-Aldrich Co. (Cairo, Egypt) with purity of 99%. 2.2.2 Pharmaceutical Formulation

Declophen plus® capsules (batch No. BN129) was manufactured by Pharco Pharmaceuticals (Alexandria, Egypt) and claimed to contain 250 and 50 mg CHZ and DIC, respectively, per capsule. 2.2.3 Chemicals and Reagents

(a) Methanol and chloroform of HPLC (Chromasolv®, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). (b) Ethanol and orthophosphoric acid of analytical grade were obtained from El-Nasr Pharmaceutical Chemicals Co. (El-Saida Zainab, Cairo, Egypt). (c) Triethylamine of analytical grade was purchased from El-Goumhouria Company for Trading Chemicals and Medical Appliances (Cairo, Egypt). (d) Deionized water was purchased from SEDICO Pharmaceuticals Co. (Cairo, Egypt). 2.3 Standard Solutions

(a) A stock standard solution of CHZ, DIC, and ACP was prepared in methanol at concentration of 1 mgmL−1 by accurately weighing 0.1 g of each into three separate 100 mL volumetric flasks. (b) Working standard solutions of CHZ, DIC, and ACP (0.1 mg mL−1) were prepared by suitable dilutions of their respective stock standard solutions either with the HPLC mobile phase (for HPLC method) or with methanol (for TLC method). 2.4 Methods 2.4.1 Chromatographic Conditions 2.4.1.1 HPLC Method

Chromatographic separation was achieved on ZORBAX Eclipse Plus C8 column (250 mm × 4.6 mm i.d., 5 µm particle size) using a mobile phase consisting of a mixture of methanol–water–phosphoric acid (75:25:0.05, by volume) in isocratic mode. The used temperature was 25°C at 1 mL min−1 flow rate and 9 min run time. The injection volume was 20 µL, and the photodiode array detector was adjusted at 230 nm. 2.4.1.2 TLC Method

The mobile phase used was chloroform–ethanol–triethylamine (9:1:0.1, by volume). It was poured into the TLC chromatographic tank and covered with a lid, then left to stand Journal of Planar Chromatography 29 (2016) 6


for saturation with the mobile phase at room temperature for 15 min. The bands were applied in TLC plates (20 cm × 12 cm) 20 mm apart and 15 mm from the bottom edge. The sample-loaded TLC plates were transferred to the chromatographic tank, and then, linear ascending development was done for not less than 100 mm from the lower edge of the plate. The developed TLC plates were air-dried and densitometrically scanned at 230 nm under the specified instrumental conditions. 2.4.2 Linearity and Construction of the Calibration Curves 2.4.2.1 HPLC Method

Series of dilutions ranging from 3 to 45 μg mL−1 of pure CHZ, DIC, and ACP were prepared by transferring accurately measured standard solutions (0.3, 0.5, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 mL) from their respective working standard solutions (0.1 mg mL−1) to a series of 10 mL of volumetric flasks and diluted to the mark with the mobile phase. Triplicate 20 µL injections were made for each prepared sample, and chromatographic separation was made under the previously mentioned HPLC conditions. The calibration graphs were obtained as the peak area at 230 nm plotted against the corresponding concentrations.

involved the use of RP-C8 column with isocratic elution and methanol‒water‒phosphoric acid (75:25:0.05, by volume) as the mobile phase at a flow rate of 1 mL min−1. CHZ, DIC, and ACP displayed typical peak characteristics at 230 nm with acceptable retention times, tR = 4.26 ± 0.01, 7.94 ± 0.02, 3.17 ± 0.02, respectively (Figure 2). One of the advantages of this method is that the mobile phase is not complicated with the need for pH adjustment. The developed method was able to resolve pharmaceutical formulation drugs from CHZ degradation product in a single run without any interference from excipients. An advantage of the developed HPLC method over the published RP-HPLC method [14] for the binary mixture or RP-HPLC methods [4, 16, 17, 18] for the ternary mixture with paracetamol is its ability to separate and quantify the active drugs in the presence of CHZ nephrotoxic impurity. All of the previously reported HPLC methods employing different mobile phases have not been capable of achieving good resolution among the studied components.

2.4.2.2 TLC Method

Accurate aliquots equivalent to 0.2–5 mg of CHZ, DIC, and ACP, were separately transferred from their respective standard stock solutions (1 mg mL−1) into a series of 10 mL volumetric flasks, and then the volume was completed with methanol till the mark. Then, an amount of 10 μL of each solution applied in triplicate to Merck TLC silica gel 60 F254 aluminum sheets as bands and chromatographic separation was made under the previously mentioned TLC conditions. The calibration curves for each component were calculated related to the integrated area under the peak against the corresponding concentration.

Figure 2 HPLC chromatogram of 40 µg mL –1 each of (a) 2-amino-4-chlorophenol, (b) chlorzoxazone, and (c) diclofenac potassium using methanol–water–phosphoric acid ( 75:25:0.05, v/v) as the mobile phase.

3.1.1 HPLC Method Development and Optimization 2.5 Application to a Pharmaceutical Formulation

The content of ten Declophen plus capsules was separately emptied, mixed, and weighed, and an amount of this formulation powder equivalent to 0.25 g CHZ (containing 0.05 g DIC) was accurately transferred to a 100-mL volumetric flask. An amount of 75 mL of methanol was added, and the solution was sonicated for 15 min and allowed to cool well, and then, the volume was adjusted to 100 mL with methanol and then filtered to prepare stock solutions of 1 mg mL−1 CHZ and 0.2 mg mL−1 DIC. The working sample solutions and different concentrations of CHZ and DIC were then prepared by suitable dilutions with the mobile phase for HPLC method and methanol for TLC method, and then, the procedures were followed under the previously mentioned chromatographic conditions. Concentrations and percentage recoveries of CHZ and DIC were then calculated using the corresponding regression equation for each drug. ®

3 Results and Discussion 3.1 HPLC Method

A HPLC–DAD method was developed to give rapid and reliable quality control analysis of CHZ and DIC in the presence of CHZ nephrotoxic degradation product: ACP. The method Journal of Planar Chromatography 29 (2016) 6

During method development and optimization, many parameters must be evaluated and optimized in order to develop the optimum separation of CHZ, DIC, and ACP in a single run without any interference. In order to select the appropriate mobile phase, several mobile phases were tried using various proportions on the basis of trials and error, taking the retention time, the system suitability parameters, the tailing factor, the number of theoretical plates, and HETP into consideration. Initially, methanol and water in different proportions were tried. However, CHZ and ACP could not be separated; therefore, phosphoric acid (0.05 m) was added. A mixture of methanol– water treated by phosphoric acid (0.05 m) was used in different ratios, and it was found that using a mixture of methanol– water–phosphoric acid (75:25:0.05, by volume) as the mobile phase at 1 mL min−1 flow rate gave an acceptable separation with suitable retention times, theoretical plates, and a good resolution between CHZ, DIC, and ACP. Different phosphoric acid concentrations were tried (0.025 and 0.075 m) and also substituted by acetic acid in some trials. In all these trials, the chromatograms showed broad asymmetric peaks and/or increased retention times and/or no separation was obtained. Different columns were tested, such as ZORBAX Eclipse Plus C8 and C18, but the separation had a better resolution using ZORBAX Eclipse Plus C8. Different flow rates (0.8, 1, and 1.5 mL min−1) of the mobile phase were tried in order to obtain the maximum resolution separation within the shortest

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analysis time. Pumping the mobile phase at 1 mL min−1 flow rate resulted in a good resolution separation within 9 min analysis time. Quantitation was achieved using photodiode array detector (DAD) which was set at different detection wavelengths (215, 230, 254, and 280 nm) in order to obtain maximum resolution and increasing method sensitivity. It was found that scanning at 230 nm gave the highest sensitivity for the detected signals and gave better results for the measured parameters. Using the thermostatted column compartment, different temperatures (20, 25, and 30°C) were tested. It was found that column temperature does not affect chromatographic separation or peak shapes. Finally, the chromatographic separation of CHZ, DIC, and ACP was carried out on ZORBAX Eclipse Plus C8 column with a mixture of methanol‒water‒phosphoric acid (75:25:0.05, by volume) as the mobile phase delivered at 1 mL min−1 flow rate, adjusting the column temperature at 25°C and the detection at 230 nm.

the nephrotoxic ACP impurity with low LOD and LOQ values, so it can be more useful as a substitute to the RP-HPLC technique. The reported TLC–densitometric method for the determination of chlorzoxazone and paracetamol in the presence of their toxic impurities 2-amino-4-chlorophenol and 4-aminophenol, respectively [3], and all of the reported TLC–densitometric methods for the determination of mixtures of CHZ, DIC, and paracetamol as ternary mixture [19, 20] employed different developing systems to separate the studied components CHZ, DIC, and ACP. None of these mobile phases has been capable of achieving good resolution for the separation of the studied components. 3.2.1 Development and Optimization of a TLC–Densitometric Method

Method optimization was made to achieve good chromatographic resolution among the three components and to get better quantitation and detection limits for the studied chlorzoxazone nephrotoxic impurity. 3.2.1.1 Developing System

3.2 TLC–Densitometric Method

The present work is concerned with investigating a highly sensitive, selective, and accurate TLC‒densitometric technique for the determination of CHZ and DIC along with CHZ nephrotoxic impurity, ACP, in their pure forms and capsules. The method involves the use of chloroform‒ethanol‒triethylamine (9:1:0.1, by volume) as the developing system and chromatographic separation carried out on Merck TLC silica gel 60 F254 aluminum sheets followed by densitometric measurement of the separated bands at 230 nm to achieve good chromatographic separation with significantly different Rf =values (CHZ, Rf = 0.63 ± 0.01; DIC, Rf = 0.35 ± 0.02; ACP, Rf = 0.42 ± 0.02) (Figure 3). The developed TLC–densitometric method has an advantage over the published RP-HPLC method [14], that is, the developed method is more cost-effective and less time-consuming and it has the capacity to separate and quantify the active drugs and

Different developing systems and preliminary trials were performed to achieve good chromatographic separation with non-tailed symmetric peaks, such as chloroform–methanol (8:2, by volume), chloroform–methanol–glacial acetic acid (9.5:0.5:0.25, by volume) and chloroform–methanol–triethylamine (9.5:0.5:0.1, by volume). When using the first system, CHZ, DIC, and ACP had similar retardation factor (Rf) values. When using the second system, DIC and ACP had similar retardation factor (Rf) values. Using triethylamine instead of glacial acetic acid in the third system slightly enhanced the resolution but with tailing peaks. Using ethanol instead of methanol in the third system enhanced the resolution and gave symmetric non-tailed peaks; hence, different ratios of chloroform–ethanol (5:5 to 9.5:0.5) with different amounts of triethylamine (0.1–0.4) were tested to enhance the resolution. Using a developing system consisting of chloroform‒ethanol‒triethylamine (9:1:0.1, by volume) gave the best resolution with symmetric non-tailed peaks. 3.2.1.2 Saturation Time

The saturation time of the TLC tank with the developing system was optimized and found to be 15 min; it is important to give homogenic atmosphere saturated with the mobile phase. 3.2.1.3 Scanning Wavelength

Different scanning wavelengths (220, 230, 254, and 280 nm) were attempted to improve the detection of the studied components. Scanning at 220 nm gave the highest signal-to-noise ratio. Scanning at 230 nm gave the optimum sensitivity for all the studied components. 3.3 Method Validation

Figure 3 TLC densitogram of a mixture of standard (a) diclofenac potassium (1 µg band –1), R f = 0.35; (b) 2-amino-4-chlorophenol (1 µg band –1), R f = 0.42; (c) chlorzoxazone (2 µg band –1), R f = 0.63, using the developing system of chloroform–ethanol–triethylamine (9:1:0.1, by volume).

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Method validation was conducted according to USP guidelines [23]. 3.3.1 Linearity

Under the optimum experimental chromatographic conditions, the evaluation of the linearity of the developed methods was made by measuring the area under the peak integrated to Journal of Planar Chromatography 29 (2016) 6

Determination of CHZ and DIZ in the Presence of ACP by Different Chromatographic Methods Table 1 Regression and analytical parameters of the proposed methods for the determination of chlorzoxazone, diclofenac potassium, and 2-amino-4-chlorophenol.

HPLC method

Parameters

CHZ

DIC

ACP

CHZ 1.2–5 µg band

ACP

3–45 µg mL

Slope

38.42

44

58.28

2.006

2.134

3.707

Intercept

1.274

8.126

29.29

1.908

1.873

1.332

Correlation coefficient

0.9999

0.9998

0.9999

0.9997

0.9999

0.9999

Residual

0.60

1.24

8.47

2.09

1.92

1.48

Accuracy % Recovery

99.61

100.10

100.09

100.24

99.30

99.40

Precision (% RSD) Repeatabilitya) Intermediate precisionb)

0.630 1.146

0.257 0.544

1.210 1.643

0.723 1.097

1.220 1.722

0.751 1.318

LODc) LOQc)

0.93 2.83

0.94 2.85

1.56 4.75

0.32 0.98

0.14 0.45

0.12 0.37

Flow rate change (±0.1 mL min−1)

1.41

1.82

1.62

Change of working wavelength (±0.2 nm)

0.56

1.57

0.61

Change composition of mobile phase ±2%

0.92

0.36

0.87

0.96

1.24

0.59

Two different analysts

1.01

1.61

1.10

0.78

0.39

0.74

Using methanol from different manufactures

1.19

1.87

1.71 0.34

0.42

0.63

−1

5–45 µg mL

DIC

Range

−1

3–40 µg mL

TLC–densitometric method

−1

0.5–4 µg band

−1

−1

0.4–4 µg band−1

Robustness

Ruggedness

Using chloroform from different manufactures##

The intra-day precision (n = 3), determined by average of three different concentrations repeated three times within a day The inter-day precision (n =3), determined by average of three different concentrations repeated three times on three successive days c) Limit of detection and quantitation are determined by calculations LOD = (SD of the intercept/slope) × 3.3; LOQ = (SD of the intercept/slope) × 10 a)

b)

Table 2 Results of analysis of Declophen plus ® capsules and application of standard addition.

Pharmaceutical formulation

Founda) HPLC method CHZ

Declophen plus® Capsules % Founded ± RSD

DIC

106.26 ± 99.01 ± 0.852 1.284

HPLC method Added (µg mL−1)

TLC method CHZ

DIC

102.28 ± 100.09 ± 1.019 1.444

Added (µg band−1)

% Foundb)

% Foundb)

CHZ DIC

CHZ

DIC

CHZ DIC

CHZ

DIC

5

5

102.15

101.19

1.5

0.5

101.02

100.29

10

10

100.74

100.05

2

1

101.91

100.14

15

15

102.35

101.04

2.5

1.5

101.02

100.28

101.75 ± 1.106

100.76 ± 0.670

101.32 ± 0.522

100.24 ± 0.147

Mean ± SD a)

TLC method

Average of six determinations Average of three determinations

b)

Journal of Planar Chromatography 29 (2016) 6

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different concentrations each of CHZ, DIC, and ACP and then plotting the calibration graphs related to the peak area against the corresponding concentration for each component. The obtained results for regression equations and linearity ranges for both HPLC and TLC methods are listed in Table 1. 3.3.2 Accuracy

Accuracy values were calculated as the percentage recoveries of pure CHZ, DIC, and ACP, and were assessed by determining seven concentrations covering the working ranges of the drugs. By using relative peak area and the regression equation for each component, the mean percentage recoveries were calculated and found to be 99.6%, 100.109%, and 100.09% for CHZ, DIC, and ACP, respectively, for HPLC method and 100.24%, 99.301%, and 99.408% for CHZ, DIC, and ACP, respectively, for TLC method. Accuracy was further calculated by the application of the standard addition technique at three levels, and the results obtained at each level proved that the developed HPLC and TLC methods are accurate and reliable (see Tables 1 and 2).

Figure 4 HPLC chromatogram of Declophen plus ® capsules (25 µg mL–1 CHZ–5 µg mL–1 DIC).

3.3.3 Precision

Precision was examined with respect to testing the intra-day (repeatability) and inter-day (intermediate) variations and was calculated using standard solutions of 5, 8, and 25 μg mL−1 for CHZ; 10, 20, and 30 μg mL−1 for DIC; and 10, 20, and 25 μg mL−1 for ACP for HPLC method and using standard solutions of 1.2, 2, and 4 µg band−1 for CHZ; 1.2, 3.6, and 4 µg band−1 for DIC; and 0.4, 2.8, and 4 for ACP for TLC method. For the intra-day precision, each concentration was tested three times on the same day, and precision was calculated as the relative percent standard deviation (% RSD, n = 9) of the total peak areas of CHZ, DIC, and ACP. In the same way, inter-day precision was calculated by testing the same standard solutions (n = 9) on three successive days and calculating % RSD. The obtained results of % RSD were less than 2%, proving that the developed HPLC and TLC methods progress good precision (see Table 1). 3.3.4 Limits of Detection and Quantitation (LOD and LOQ)

LOD and LOQ determine the sensitivity of the methods. The concentrations of CHZ, DIC, and ACP in the lower part of the linear range of the calibration curves for each component and the equations of LOD = 3.3 × N/B and LOQ = 10 × N/B were used to calculate these two parameters, where N is the standard deviation of response and B is the slope of the corresponding component calibration (see Table 1). 3.3.5 Specificity

Specificity of the HPLC and TLC methods was determined by comparing the chromatograms attained from the standard solutions with those obtained from the sample solutions of Declophen plus® capsules. The same retention time tR and Rf values of the standard drugs and the drugs in the capsules test solution indicated that the methods were specific. On the other hand, in the case of HPLC method, the use of a photodiode array detector assessed the confirmation of the specificity of the method by comparison with the reference drug spectrum (see Figures 2 and 4). In addition, in the case of TLC method, the peak purities of CHZ, DIC, and ACP were confirmed by comparing their respective spectra at the peak start, apex, and peak end positions of the spot (see Figures 3 and 5). The good results obtained

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Figure 5 TLC densitogram of Declophen plus ® capsules (2.5 µg band –1 CHZ– 0.5 µg band –1 DIC).

by applying the method to Declophen plus® capsules (Table 2) assessed that the additives in the capsules did not interfere with any of the three separated components. 3.3.6 Robustness

The robustness of an analytical procedure is a measure of the performance of a method when small but deliberate changes are made in the method parameters. It was evaluated during HPLC and TLC method development and optimization in order to identify critical conditions for the successful application of the methods. In the case of HPLC method, this was performed by making small changes in the composition of the mobile phase (±2% from the initial composition of methanol and water), working wavelengths (±2 nm), flow rate (±0.1 mL min−1), and column temperature (±2°C). These variations did not show any significant effect on the chromatographic separation or the measured responses. In the case of TLC method, small changes in the studied chromatographic conditions such as change in the triethylamine amount, ±0.02 mL; methanol, ±0.05 mL; and time for saturation of the development chamber, ±5 min, led to no significant change in the Rf values and symmetry of the peaks or peak area. The low values of the SD, as shown in Table 1, indicated the robustness of the two proposed methods. 3.3.7 Ruggedness

Ruggedness is the degree of reproducibility of the results obtained under a variety of conditions. Ruggedness is assessed by performing the analysis by two different analysts and using methanol and chloroform from different manufactures Journal of Planar Chromatography 29 (2016) 6


(Chromasolv® Sigma-Aldrich, Germany and Fisher Scientific, UK). No significant changes were observed in the peak area of the components. The low values of the SD, as shown in Table 1, indicated that the method is rugged.

After optimization of the methods, the second step was their application to determine CHZ, DIC, and ACP in their pure forms and to determine CHZ and DIC in their pharmaceutical

formulation. Under the previous experimental chromatographic conditions, linear relationships were shown between the integrated peak area and the related concentration of CHZ, DIC, and ACP. The performance data and statistical parameters including concentration ranges, correlation coefficients (r), and other statistical parameters such as intercept, slope, percentage recovery of accuracy and precision, limit of detection, and limit of quantitation are listed in Table 1 for both the HPLC and the TLC method. Regression analysis for the calibration curves showed good linear relationships in the concentration ranges of

Figure 6

Figure 7

Calibration curves of CHZ, DIC, and ACP by HPLC method.

Calibration curves of CHZ, DIC, and ACP by TLC method.

3.4 Application of the Methods


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3–45 µg mL−1 for CHZ, 3–40 µg mL−1 for DIC, and 5–45 µg mL−1 for ACP as judged by the correlation coefficient value for CHZ and ACP (r = 0.9999) and for DIC (r = 0.9998) for HPLC method and over the concentration range of 1.2–5 µg band−1 for CHZ, 0.5–4 µg band−1 for DIC, and 0.4–4 µg band−1 for ACP as judged by the correlation coefficients value for CHZ (r = 0.9997) and for DIC and ACP (r = 0.9999) for TLC method (see Figures 6 and 7). In order to determine the ability of the methods to quantify CHZ and DIC in the marketed pharmaceutical formulations, they were applied to determine CHZ and DIC in Declophen plus® capsules and no interference from excipients was observed. There were good percentage recoveries as well, as shown in Table 2. Also, the chromatograms in Figures 4 and 5 showed a complete resolution between the peaks of the CHZ and DIC drugs from all the excipients. Also, placebo samples were applied for both methods and no foreign peaks were observed. Results of the recovery studies for HPLC and TLC methods were found to be acceptable to all tested levels, proving the high accuracy of the proposed methods. HPLC and TLC methods can determine CHZ and DIC in the presence of CHZ nephrotoxic degradation product simultaneously, and

from the given data in this work, HPLC method is more selective and reproducible. The developed HPLC and TLC methods can quantify down to around 10% ACP in CHZ. The maximum allowed limit in USP is 0.5%, but due to the high toxic effect of ACP, as it is nephrotoxic and lethal, it was necessary to develop methods that can determine CHZ and DIC in the presence of ACP simultaneously. The results of the analysis obtained by applying the HPLC–DAD and TLC–densitometric methods for the determination of CHZ and DIC in bulk powder were compared statistically to results obtained by a reported RP-HPLC method [14] using the Student’s t-test and variance ratio F-test, and no significant difference was found between the methods results (Table 3). 3.5 System Suitability Testing

System suitability parameters were calculated for both the HPLC and the TLC method to fulfill the suitability and effectiveness of the developed systems such as selectivity, resolution, and symmetry factors, and the obtained results were in the acceptable ranges, as shown in Table 4.

Table 3 Statistical comparison of the results obtained by applying the proposed methods and the reported HPLC method for the determination of chlorzoxazone and diclofenac potassium in pure form.

Methods

HPLC method

TLC method

Reported methoda) [14]

Items

CHZ

DIC

CHZ

DIC

CHZ

DIC

Mean

105.94

99.87

103.68

99.83

104.65

100.94

SD

1.07

1.67

0.95

0.73

1.39

1.36

Variance

1.14

2.79

0.91

0.54

1.94

1.85

N

7

7

7

7

7

7

Student’s t-test (2.18)b)

1.94

1.31

1.52

1.91

–

–

F-value (4.28)b)

0.58

0.66

2.13

3.40

–

–

The reported HPLC method used the mobile phase consisting of a mixture of phosphate buffer (0.02 m KH2PO4, pH adjusted to 3 using orthophosphoric acid), acetonitrile, and methanol (30:30:40, v/v) at a flow rate of 1.0 mL min−1, on ACE 5 C18 column. Detection was at 279 nm b) Figures between parenthesis represent values of t and f at p = 0.05 a)

Table 4 System suitability testing parameters of the developed TLC–densitometric and HPLC methods.

Parameters

TLC

HPLC

Reference value [24, 25]

DIC

ACP

CHZ

ACP

CHZ

DIC

Tailing factor (T)

1.04

0.97

1

1.28

1.37

1.39

1.

Column efficiency (N)

883.33

1583.6

1672.6

Increase with efficiency of the separation

HETPa)

0.28

0.15

0.14

The smaller the value the higher the column efficiency

a)

3.03

1.59

1.33 2.16

Resolution (RS)

3.03

1.5

HETP, height equivalent to theoretical plate (cm plate−1)

460



4 Conclusion The primary task of this work was to build up and validate an RP-HPLC and TLC–densitometric methods that have advantages over the reported HPLC method; they have the ability for detection and quantitation of chlorzoxazone nephrotoxic and lethal impurity with high sensitivity. Moreover, the methods were capable of the determination of the pharmaceutical dosage form without any interference from additives or excipients. Quality control laboratories can use TLC method as it needs a minimal volume of solvents and several samples could be run at the same time, so it is time- and cost-effective.

Acknowledgment The authors would like to express their thanks and appreciation to Pharco Pharmaceuticals, Alexandria, Egypt, for the provision of the necessary materials to perform this work.

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