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Nariman A. El-Ragehy Department of Analytical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini St. 11562, Cairo, Egypt

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Maha A. Hegazy Department of Analytical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini St. 11562, Cairo, Egypt

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Samia A. Tawfik Department of Analytical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini St. 11562, Cairo, Egypt

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Ghada A. Sedik Department of Analytical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini St. 11562, Cairo, Egypt

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Abstract

Sulfacetamide sodium is a widely prescribed sulfonamide drug due to its topical antibacterial action on eye and skin. Four impurities are stated in the British Pharmacopoeia among which are sulfanilamide and dapsone. This work presents two specific, accurate and precise chromatographic methods for the simultaneous determination of a mixture of sulfacetamide sodium, sulfanilamide and dapsone. The first method is an isocratic RP-HPLC where the separation of components was achieved on C18 column. A green mobile phase was used consisting of methanol:water (60:40, v/v). The flow rate was 1.0 mL/min and effluent was monitored at 273 nm. The second method is a TLC-spectrodensitometric one where good separation was achieved by using silica plates and a mobile phase consisting of chloroform:dichloromethane:acetic acid (6:2.5:1.5, by volume). Determination was done by densitometry in the absorbance mode at 273 nm. Both methods were validated in compliance with ICH guidelines. They were also successfully applied for the determination of sulfacetamide sodium and its impurities in Ocusol® ophthalmic solutions. The obtained results were statistically compared to the results obtained by applying the official methods of analysis of each component where no significant difference was found with respect to accuracy and precision.

Abstract

Sulfacetamide sodium is a widely prescribed sulfonamide drug due to its topical antibacterial action on eye and skin. Four impurities are stated in the British Pharmacopoeia among which are sulfanilamide and dapsone. This work presents two specific, accurate and precise chromatographic methods for the simultaneous determination of a mixture of sulfacetamide sodium, sulfanilamide and dapsone. The first method is an isocratic RP-HPLC where the separation of components was achieved on C18 column. A green mobile phase was used consisting of methanol:water (60:40, v/v). The flow rate was 1.0 mL/min and effluent was monitored at 273 nm. The second method is a TLC-spectrodensitometric one where good separation was achieved by using silica plates and a mobile phase consisting of chloroform:dichloromethane:acetic acid (6:2.5:1.5, by volume). Determination was done by densitometry in the absorbance mode at 273 nm. Both methods were validated in compliance with ICH guidelines. They were also successfully applied for the determination of sulfacetamide sodium and its impurities in Ocusol® ophthalmic solutions. The obtained results were statistically compared to the results obtained by applying the official methods of analysis of each component where no significant difference was found with respect to accuracy and precision.

1 Introduction

International Conference on Harmonization (ICH) and other national regulatory authorities focus on identification and quantification of possible impurities in drug substances [1]. Existence of impurities, even if in trivial quantities, may not only affect the efficacy of the drug but also the safety of pharmaceutical preparations [2]. Sulfacetamide sodium is chemically named as sodium acetyl[(4-aminophenyl) sulfonyl]azanide [3]. It is principally used in topical preparations as antibacterial agent [4]. It is described for treatment of conjunctivitis, corneal ulcers and other superficial infections [5]. Sulfanilamide is chemically designated as 4-aminobenzenesulfonamide [3]. It is reported to be impurity A for sulfacetamide sodium [3] as it is considered its major degradation product under the effect of light and temperature [6]. Dapsone is 4,4′-sulfonyldianiline and an official impurity for sulfacetamide sodium [3]. However, It is a medication used for the treatment of leprosy as it inhibits folic acid synthesis [3]. The structures and molecular weights of the studied components are illustrated in Fig. 1. Chromatographic techniques are considered the most suitable and commonly used analytical techniques for determination of drug substances in presence of impurities in pharmaceutical analysis [7–9]. Several chromatographic methods were reported for the simultaneous determination of sulfacetamide and sulfanilamide [10–14]. However, no method was reported in the literature for the simultaneous determination of ternary mixture of sulfacetamide sodium, sulfanilamide and dapsone. Therefore, the aim of this work is to develop and validate sensitive, economic, accurate and precise chromatographic methods for the simultaneous determination of sulfacetamide sodium together with sulfanilamide and dapsone.

Fig. 1.
Fig. 1.

Chemical structures and molecular weights of (a) sulfacetamide sodium, (b) sulfanilamide and (c) dapsone

Citation: Acta Chromatographica 34, 4; 10.1556/1326.2021.00921

2 Materials and methods

2.1 Instruments

2.1.1 RP-HPLC method

HPLC system consists of an Agilent pump 1,100 series, equipped with a variable wavelength detector (model 1,100 series; Agilent, USA), and a 20 μL volume injection loop using X-bridge ODS column (5 μm, 250 mm × 4.6 mm i.d.), Waters, USA; a degasser (model DGU-12A). The samples were injected by using a 50-µL Hamilton analytical syringe, pH-meter (Jenway model 3,505, UK) was also used.

2.1.2 TLC-spectrodensitometric method

TLC system consists of Camag Linomat auto sampler (Muttenzl, Switzerland), Camag microsyringe (100 µL) and Camag TLC scanner 3S/N/30319 with winCATS software. A short wavelength UV lamp emitting at 254.0 nm was also used (Desaga, Wiesloch, Germany). TLC plates were pre-coated with silica gel F254 20 × 10 cm, 0.25 mm thickness (Merck, Darmstadt, Germany).

2.2 Materials

2.2.1 Pure standards

Sulfacetamide sodium monohydrate was kindly offered by the Egyptian International Pharmaceutical Industries Company (EIPICO), Egypt and its purity was checked and found to be 99.96% ± 0.722 according to the British Pharmacopoeia method (BP) (titrimetric method) [3]. Sulfanilamide was obtained from El-Nasr Pharmaceutical Chemicals Co., Egypt. Its purity was checked and found to be 99.86% ± 1.548 according to the United States Pharmacopeia (USP) method (titrimetric method) [15]. Dapsone was kindly supplied by The Nile for Pharmaceuticals & Chemical Industries, Egypt. Its purity was found to be 99.87% ± 1.046 according to USP method (HPLC method) [15].

2.2.2 Pharmaceutical formulations

Ocusol® 10% eye drops, batch No.5518007, labeled to contain 100 mg of sulfacetamide sodium per one mL. Ocusol® 20% eye drops, batch No.6519001, labeled to contain 200 mg of sulfacetamide sodium per one mL. Both are manufactured by Alexandria Co. for Pharmaceuticals & Chemical Industries, Egypt and were purchased from local pharmacies.

2.2.3 Chemicals and solvents

All chemicals used throughout this work were of analytical grade, and the solvents were of HPLC grade. They are as follows: methanol, chloroform, and dichloromethane (Sigma-Aldrich, Steinheim, Germany). Glacial acetic acid (El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt), double-distilled deionized water (Otsuka, Cairo, Egypt), Orthophosphoric acid (Adwic, Cairo, Egypt).

2.3 Standard solutions

2.3.1 RP-HPLC method

2.3.1.1 Stock standard solutions

Stock standard solutions of sulfacetamide sodium, sulfanilamide and dapsone (1.00 mg mL−1) were prepared by accurately weighing and transferring 100.00 mg of each pure drug into three separate 100-mL volumetric flasks in methanol. It was dissolved in methanol and completed to volume with the same solvent.

2.3.1.2 Working standard solutions

Working standard solutions of sulfacetamide sodium (50.00 μg mL−1), sulfanilamide and dapsone (25.00 μg mL−1) were prepared by transferring 5.0 mL of sulfacetamide sodium and 2.5 mL of sulfanilamide and dapsone stock standard solutions into three separate 100-mL volumetric flasks. The volume was then completed with methanol.

2.3.2 TLC-spectrodensitometric method

Standard solutions of sulfacetamide sodium (1.00 mg mL−1), sulfanilamide and dapsone (0.10 mg mL−1) were prepared in three separate 100-mL volumetric flasks by accurately weighing and dissolving 100.00 mg of sulfacetamide sodium, 10.00 mg of sulfanilamide and dapsone in methanol. The volume was completed with the same solvent.

2.4 Procedures

2.4.1 Construction of calibration graphs

2.4.1.1 RP-HPLC method

Aliquots of sulfacetamide sodium, sulfanilamide and dapsone equivalent to 10.00–300.0 µg, 5.00–80.00 µg and 10.00–160.00 µg, respectively, were accurately transferred from their corresponding working standard solutions, into three separate series of 10-mL volumetric flasks. The volumes were completed to the mark with the mobile phase (methanol:water (60:40, v/v); adjusted pH 5.0 by orthophosphoric acid). Conditioning of the stationary phase was performed for 1 h. A 20 μL aliquot of each solution was injected into X-bridge ODS column (5 μm, 250 mm × 4.6 mm i.d.) with the mobile phase. Flow rate was 1.0 mL min−1 and effluent was monitored at 273.0 nm. The calibration graphs of the studied drugs were constructed by plotting the integrated peak area against the corresponding concentrations in μg mL−1. The regression equations were then computed for each component.

2.4.1.2 TLC-spectrodensitometric method

Aliquots of sulfacetamide sodium, sulfanilamide and dapsone equivalent to 0.10–4.00 mg, 0.03–0.60 mg and 0.03–0.60 mg, respectively, were accurately transferred from their corresponding standard solutions, into three separate series of 10-mL volumetric flasks. The volume was then completed with methanol. 10 μL of each solution were separately applied in triplicates on TLC plates, using Camag Linomat autosampler. The bands were spaced 1 cm apart from each other and 1 cm from the bottom edge of the plates. The mobile phase was allowed to develop to 9 cm height. The chromatographic chamber was previously saturated with the mobile phase for 30 min and the plate was developed by ascending technique using chloroform:dichloromethane:acetic acid (6:2.5:1.5, by volume) as a mobile phase. The plate was air dried at ambient temperature, detected under UV lamp and scanned at 273.0 nm. The calibration graphs representing the relationship between the integrated peak area and the corresponding concentration in μg band −1 were plotted. The corresponding regression equations were then computed.

2.4.2 Application to pharmaceutical formulations

2.4.2.1 RP-HPLC method

Aliquots of 1.0 and 0.5 mL were accurately transferred from Ocusol® 10% and Ocusol® 20% eye drops, respectively, into two separate 100-mL volumetric flasks. The volume was then completed with methanol. These solutions were further diluted by transferring 0.5 mL to two separate 50-mL volumetric flasks. The volume was completed with the mobile phase to have concentration equivalent to 10 μg mL−1 sulfacetamide sodium. The general procedure previously described was followed. The concentration of sulfacetamide sodium was calculated using the corresponding regression equation.

2.4.2.2 TLC-spectrodensitometric method

Aliquots of 1.0 and 0.5 mL were accurately transferred from Ocusol® 10% and Ocusol® 20% eye drops, respectively, into two separate 100-mL volumetric flasks. The volume was then completed to the mark with methanol. A 1 µL aliquot of the solution claimed to contain 1 μg band–1 of sulfacetamide sodium was applied onto the TLC plate by the Camag micro syringe. The general procedure previously described was followed. The concentration of sulfacetamide sodium was calculated using the corresponding regression equation.

3 Results and discussion

Nowadays, the mainstream of drug-related impurity determinations is performed by HPLC as it offers the desired level of sensitivity with high degree of automation [16, 17]. Selection from wide variety of stationary phases, mobile phases and operation modes makes HPLC applicable to all drug classes [18]. Consequently, a RP-HPLC method is described for the simultaneous determination of sulfacetamide sodium with its BP stated related impurities; sulfanilamide and dapsone without prior separation. Furthermore, the development of an HPLC method which uses eco-friendly solvents would be favored to decline the harmful effects of used solvents on both human and environment. Despite all the mentioned advantages of HPLC methods, economic issues and the need for high operated instruments makes sometimes its implementation difficult. So, TLC would be found as a powerful tool for the screening and isolation of impurities owing to its simplicity and low cost [19]. Therefore, another TLC-spectrodensitometric method was developed for the quantitive determination of the studied ternary mixture.

3.1 Methods development

3.1.1 RP-HPLC method

Different green mobile phases were tried to approach the goal of green chemistry such as; ethanol:water (80:20, v/v), ethanol:water (60:40, v/v), methanol:water (70:30, v/v) and methanol:water (80:20, v/v). The best resolution with sharp and symmetric peaks was accomplished upon using mobile phase consisted of methanol:water (60:40, v/v), (pH 5.0 adjusted by orthophosphoric acid).The suggested mobile phase composition proved to be one of the most green solvents [20]. Different flow rates and detection wavelengths were tested; satisfactory resolution was obtained with isocratic mode at flow rate 1.0 mL min−1 and UV detection at 273.0 nm. The retention time obtained was 4.3, 3.1 and 6.0 min for sulfacetamide sodium, sulfanilamide and dapsone, respectively, under the described conditions, Fig. 2.

Fig. 2.
Fig. 2.

HPLC chromatogram of a ternary mixture containing 2.00 μg mL−1 of sulfanilamide (t R = 3.1), 10.00 μg mL−1 of sulfacetamide sodium (t R = 4.3), and 2.00 μg mL−1dapsone (t R = 6.0) using a mobile phase of methanol - water (60:40, v/v) pH adjusted to 5.0 by orthophosphoric acid, at flow rate of 1.0 mL min−1 and UV detection at 273 nm

Citation: Acta Chromatographica 34, 4; 10.1556/1326.2021.00921

3.1.2 TLC-spectrodensitometric method

Studying the optimum parameters for maximum separation was carried out by trying different two and three components developing systems with different ratios such as; chloroform:methanol (7:3, v/v), chloroform:methanol (9:1, v/v) and chloroform:dichloromethane (7:3, v/v), but highly tailed peaks were observed. Other systems were tried such as; chloroform:dichloromethane:acetic acid and chloroform:methanol:acetic acid, in different ratios, where incomplete resolution of the drugs was obtained with tailed peaks. Eventually, chloroform:dichloromethane:acetic acid (6:2.5:1.5, by volume) was the developing system of choice which permits the determination of the studied mixture with minimum tailing and maximum separation. The corresponding retardation factors (R f ) values were found to be 0.60, 0.41 and 0.75 for sulfacetamide sodium, sulfanilamide and dapsone, respectively, Fig. 3. UV detection at 273.0 nm and Slit dimensions (3.00 × 0.45 mm) offer best results; regarding sensitivity, peak symmetry and peak sharpness.

Fig. 3.
Fig. 3.

TLC-densitogram of a resolved mixture of sulfanilamide (0.20 μg band−1, R f = 0.41), sulfacetamide sodium (2.00 μg band−1, R f = 0.60) and dapsone (0.20 μg band−1, R f = 0.75) using a developing system of chloroform-dichloromethane- acetic acid (6:2.5:1.5, by volume) and UV detection at 273 nm

Citation: Acta Chromatographica 34, 4; 10.1556/1326.2021.00921

3.2 Methods validation

Calculation of system suitability parameters is necessary to verify the performance of the proposed RP-HPLC and TLC-spectrodensitometric systems [21, 22]. Good results were obtained as shown in Tables 1 and 2. ICH guidelines of the proposed chromatographic methods were followed by measuring linearity, range, LOD, LOQ, accuracy, precision, specificity and robustness [23]. Results are shown in Tables 3 and 4.

Table 1.

System suitability parameters for the proposed HPLC method for the simultaneous determination of sulfacetamide sodium, sulfanilamide and dapsone

Parameter Sulfanilamide Sulfacetamide sodium Dapsone Reference values [21]
Retention time (tR) 3.1 4.3 6.0
Resolution factor (Rs)a 6.65 11.63 Rs > 1.5
Tailing factor (T) 0.81 0.96 0.83 T = 1 ± 0.2 for a typical symmetric peak
Capacity factor (K′)b 2.87 4.37 6.5 1–10 acceptable
Selectivity factor (α)c 1.52 1.48 α > 1
Number of theoretical plates (N)d 5,870 6,522 6,995 Increase with the efficiency of the separation
Height equivalent to theoretical plate (HETP) [mm]e 0.0425 0.0038 0.0359 The smaller the value, the higher the column efficiency

a R s = [2 (t R 2−t R 1)]/(W1 + W2), where t R is the retention time and W is peak width.

b k’ = (t R t 0)/t 0, where t 0 is the retention time of unretained substance (mobile phase).

c α = k’2/k’1, where k’ is the capacity factor.

d N= 16 (t R /W) 2.

e HETP = L/N, where L is column length in mm.

Table 2.

System suitability parameters of the proposed TLC-spectrodensitometric method for the simultaneous determination of sulfacetamide sodium, sulfanilamide and dapsone

Parameters Sulfanilamide Sulfacetamide sodium Dapsone Reference values [22]
Retardation factor (R f ) 0.24 0.32 0.46
Resolution (R s )a 1.91 1.71 >1
Capacity factor (K′) 1.43 0.67 0.34 1 < K′ < 5
Selectivity factor (α)b 2.13 1.97 >1
Tailing factor (T) 0.97 1.09 1.16 =1 for typical symmetric peak
Theoretical plates number (N)c 2,843 4,442 6,400 high N indicates better separation
Height equivalent to theoretical plate HETP (mm)d 0.0028 0.0018 0.0013 Low HETP indicates better separation

a R s = [2 (R f 2−R f 1)]/(W1 + W2), where R f is retardation factor and W is peak width.

b α = k’2/k’1, where k’ is the capacity factor; k’ = (1−R f )/R f .

c N = 16 (z/w)2, where z is the migration length of the spot and w is the spot width.

d HETP = z/N, where z is the migration length of the spot.

Table 3.

Regression and validation parameters of the proposed HPLC method for the determination of pure sulfacetamide sodium, sulfanilamide and dapsone

Parameter Sulfacetamide sodium Sulfanilamide Dapssone
Linearity
Linearity range (µg mL−1) 1.00–30.00 0.10–8.00 0.50–16.00
Slope 82.869 70.583 70.997
Intercept 35.237 7.745 7.0395
Correlation coefficient (r) 0.9998 1.0000 0.9999
Accuracy (mean ± SD) 100.63 ± 0.643 100.90 ± 1.708 99.37 ± 1.339
Precision (RSD%)
Repeatabilitya 0.421 0.912 0.425
Intermediate precisionb 0.623 1.992 0.826
Specificity (mean ± SD) 99.76 ± 1.299 99.67 ± 1.816 99.68 ± 0.932
Robustness (RSD%) 1.570 1.123 1.940
LOD (µg mL−1)c ___ 0.028 0.153
LOQ (µg mL−1)c ___ 0.084 0.469

a Intra-day (n = 3), average of three concentrations of sulfacetamide sodium (4.00, 12.00 and 24.00 μg mL−1), sulfanilamide (2.00, 4.00, 8.00 μg mL−1) and dapsone (4.00, 8.00 and 12.00 μg mL−1), repeated three times within the same day.

b Inter-day (n = 3), average of three concentrations of sulfacetamide sodium (0.50, 2.00 and 3.00 μg mL−1), sulfanilamide (2.00, 4.00, 8.00 μg mL−1) and dapsone (0.20, 0.40 and 0.60 μg mL−1), repeated three times in three consecutive days.

c Calculated from equation [LOD = 3.3 (SD/S), LOQ = 10 (SD/S); where SD is the standard deviation of regression residuals and S is calibration curves slope.

Table 4.

Regression and validation parameters of TLC-spectrodensitometric method for the simultaneous determination of sulfacetamide sodium, sulfanilamide and dapsone samples

Parameter TLC-spectrodensitometric method
Sulfacetamide sodium Sulfanilamide Dapsone
Linearity a
Range (µg band−1) 0.10–4.00 0.03–0.60 0.03–0.60
Slope Slopea–1022.1 14,636 20,389
Slopeb 9796.1
Intercept 2912.8 588.84 1050.4
Correlation coefficient (r) 0.9999 0.9999 0.9999
Accuracy (mean ± SD) 99.03 ± 1.776 100.02 ± 1.102 98.73 ± 1.594
Precision (RSD%)
Repeatabilityb 1.702 0.858 0.746
Intermediate precisionc 1.913 1.537 1.601
Specificity (mean ± SD) 100.29 ± 1.828 100.59 ± 1.691 100.15 ± 1.548
Robustness (RSD%) 1.791 1.123 1.941
LOD (µg band−1) d ___ 0.008 0.008
LOQ (µg band−1) d ___ 0.025 0.024

a Slope a and slope b are the coefficients of X 2 and X, respectively. Following a polynomial regression.

A = ax2 + bx + c Where, A is the peak area, x is the concentration in (μg/band), a and b are coefficients 1 and 2, respectively and c is the intercept.

b Intra-day (n = 3), average of three concentrations of sulfacetamide sodium (0.50, 2.00 and 3.00 µg band−1), sulfanilamide and dapsone (0.20, 0.40 and 0.60 µg band−1), repeated three times within the same day.

c Inter-day (n = 3), average of three concentrations of sulfacetamide sodium (0.50, 2.00 and 3.00 µg band−1), sulfanilamide and dapsone (0.20, 0.40 and 0.60 µg band−1), repeated three times in three consecutive days.

d Calculated from equation [LOD = 3.3 (SD/S), LOQ = 10 (SD/S); where SD is the standard deviation of regression residuals and S is the slope of the calibration curves.

3.2.1 Linearity

3.2.1.1 RP-HPLC method
A linear relationship was obtained between the integrated peak areas and the corresponding concentrations of sulfacetamide sodium, sulfanilamide and dapsone in the ranges of 1.00–30.00 μg mL−1, 0.10–8.00 μg mL−1 and 0.50–16.00 μg mL−1, respectively. The regression equations were computed and found to be:
A = 82 . 269 C + 35 . 237 r = 0 . 9998 for sulfacetamide sodium
A = 70 . 583 C + 7 . 7446 r = 1 . 0000 for sulfanilamide
A = 70 . 997 C + 7 . 0395 r = 0 . 9999 for dapsone
Where, A is the integrated peak area, C is the concentration in μg mL−1 and r is the correlation coefficient.
3.2.1.2 TLC-spectrodensitometric method
For sulfacetamide sodium, a polynomial relationship was found to exist between the integrated peak area and the corresponding concentrations in the range of 0.10–4.00 μg band−1. The second order polynomial regression was conducted to improve the sensitivity level, concentration range and the regression coefficient of the method. For sulfanilamide and dapsone, a linear relationship was established between the integrated peak areas and the corresponding concentrations in the range of 0.03–0.60 μg band−1 for both components. The regression equations were computed and found to be:
A = 1022 . 1 C 2 + 9796 . 1 C + 2912 . 8 r = 0 . 9999 for sulfacetamide sodium
A = 14636 C + 588 . 84 r = 0 . 9999 for sulfanilamide
A = 20389 C + 1050 . 4 r = 0 . 9999 for dapsone
Where A is integrated peak area, C is the concentrations in µg band−1 and r is the correlation coefficient.

3.2.2 Detection and quantitation limits

The BP monograph of sulfacetamide sodium specifies a range between 99 and 101% for sulfacetamide contents and a limit for sulfanilamide and total related substances not exceeding 0.2% and 0.5% of the declared content of sulfacetamide sodium, respectively [3]. The LOD and LOQ values of both impurities were calculated by using the following calculations: LOD = (SD of regression residuals/slope) × 3.3; LOQ = (SD of regression residuals/slope) × 10. The low LOD and LOQ values demonstrate the high sensitivity of the proposed methods.

3.2.3 Accuracy

The accuracy of the results was checked by applying the proposed RP-HPLC method for determination of different samples of sulfacetamide sodium (2.00, 6.00, 10.00, 16.00, 25.00 μg mL−1), sulfanilamide (0.80, 3.00, 5.00, 7.00, 8.00 μg mL−1) and dapsone (3.00, 5.00, 6.00, 8.00, 10.00 μg mL−1). Other five different concentrations of sulfacetamide sodium (0.20, 0.60, 1.50, 2.50, 4.00 μg band−1), sulfanilamide and dapsone (0.05, 0.15, 0.25, 0.35, 0.45 μg band−1) were analyzed to study the accuracy of TLC-spectrodensitometric method. The calculated percentage recoveries indicate good accuracy of both chromatographic methods.

3.2.4 Precision

To study the precision of the RP-HPLC method, three different concentrations of pure samples of sulfacetamide sodium (4.00, 12.00, 24.00 μg mL−1), sulfanilamide (2.00, 4.00, 8.00 μg mL−1) and dapsone (4.00, 8.00, 10.00 μg mL−1) were analyzed in triplicate on a single day (intraday precision) and on three consecutive days (interday precision). For TLC-spectrodensitometric method, three different concentrations of pure samples of sulfacetamide sodium (0.50, 2.00, 3.00 µg band−1), sulfanilamide and dapsone (0.20, 0.40, 0.60 µg band−1) were also analyzed in triplicate on a single day (intraday precision) and on three consecutive days (interday precision). The percentage relative standard deviations (RSD %) for the mentioned drugs were calculated. Low RSD % shows that the developed methods are precise enough.

3.2.5 Specificity

The specificity was accomplished by the analysis of several laboratory prepared mixtures containing the studied components in different ratios within the linearity range using the previously mentioned procedure under each method. The specificity of the proposed methods was checked by the calculated percentage recoveries and small SD value.

3.2.6 Robustness

Robustness of the developed RP-HPLC method was assessed through deliberately changing experimental conditions. Three different concentrations of each drug were analyzed under a variety of conditions, such as small changes in mobile phase ratio ±2%, pH ± 0.2, flow rate± 0.1 and the detection wavelength ±1.0 nm. For TLC-spectrodensitometric method, the changed experimental conditions were small modifications in proportions of the developing system ±1% and detection wavelength ±1.0 nm. For both methods, the RSD % for the studied compounds were calculated and found to be less than 2% which confirms the robustness of the chromatographic methods.

3.3 Application to pharmaceutical formulations

The developed chromatographic methods were successfully applied for the determination of sulfacetamide sodium in both Ocusol® 10% and 20% eye drops. Results presented in Table 5 proved the applicability of the suggested methods for the determination of sulfacetamide sodium in its ophthalmic solutions without any interference of the excipients or impurities that may be found in the pharmaceutical formulations.

Table 5.

Quantitative determination of sulfacetamide sodium in pharmaceutical formulations by the proposed chromatographic methods

Pharmaceutical formulation RP-HPLC method TLC‐spectrodensitometric method
Claimed (µg mL−1) Founda % ± SD Claimed (µg band−1) Found* % ± SD
Ocusol® 10% eye drops

Claimed to contain 100 mg of sulfacetamide sodium per one mL

Batch No.5518007
10 99.45 ± 1.598 1 100.10 ± 1.005
Ocusol® 20% eye drops

Claimed to contain 200 mg of sulfacetamide sodium per one mL

Batch No.6519001
10 99.73 ± 1.070 1 101.77 ± 0.623

* Average of five experiments.

3.4 Statistical analysis

The results obtained from the analysis of sulfacetamide sodium by the suggested chromatographic methods were statistically compared to those obtained by applying the USP official method for the determination of sulfacetamide sodium in its ophthalmic solutions [15]. Sulfanilamide and dapsone were analyzed in their pure forms by the established chromatographic methods and the USP official ones [15], where the results acquired by the mentioned techniques were statistically compared [24]. The calculated t and F-values were less than the corresponding theoretical ones indicating that there is no significant difference between the proposed methods and the official ones regarding accuracy and precision, Tables 6 and 7.

Table 6.

Statistical analysis of the results obtained by the proposed HPLC method and the official methods for the determination of sulfacetamide sodium in its eye drops, sulfanilamide and dapsone in their pure forms

Item HPLC Official methods [15]
Sulfacetamide Sodium Sulfanilamide Dapsone Sulfacetamide sodiuma Sulfanilamideb Dapsonec
(Ocusol® 10%) (ocusol® 20%) (Ocusol® 10%) (ocusol® 20%)
Mean 99.40 99.70 100.63 99.37 101.04 100.63 99.86 99.87
SD 1.598 1.070 0.643 1.339 0.772 1.559 1.548 1.046
Variance 2.554 1.145 0.413 1.793 0.596 2.430 2.396 1.094
n 5 5 5 5 5 5 5 5
Student's t-test (2.306) d 2.066 1.110 1.027 0.658
F value (6.39) d 4.285 2.122 5.801 1.639

a HPLC method using C18 column with mobile phase containing a mixture of water:methanol:glacial acetic acid (89:10:1, by volume) with flow rate 1.5 mL min−1 and UV detection wavelength at 254.0 nm.

b Potentiometric titration method using 0.1M sodium nitrite as a titrant and detection of end point by starch iodide paste as an external indicator.

c HPLC method using silica column with mobile phase containing a mixture of isopropyl:acetonitrile:ethyl acetate:hexane (10:10:10:70, by volume), flow rate 1.0 mL min−1 and UV detection wavelength at 254.0 nm.

d Figures in parentheses are the corresponding tabulated values for t and F at P = 0.05.

Table 7.

Statistical analysis of the results obtained by the proposed TLC-spectrodensitometric method and the official methods for the determination of sulfacetamide sodium in its eye drops, sulfanilamide and dapsone in their pure forms

Item TLC- spectrodensitometric method Official methods [15]
Sulfacetamide sodium Sulfanilamide Dapsone Sulfacetamide sodiuma Sulfanilamideb Dapsonec
(Ocusol® 10%) (ocusol® 20%) (Ocusol® 10%) (Ocusol® 20%)
Mean 100.20 101.70 99.59 99.83 101.04 100.63 99.86 99.87
SD 1.005 0.623 0.749 1.371 0.772 1.559 1.548 1.046
Variance 1.010 0.388 0.561 1.880 0.596 2.430 2.396 1.094
n 5 5 5 5 5 5 5 5
Student's t-test (2.306) d 1.482 1.425 0.188 1.337
F value (6.39) d 1.695 6.265 4.271 1.718

a HPLC method using C18 column with mobile phase containing a mixture of water:methanol:glacial acetic acid (89:10:1, by volume) with flow rate 1.5 mL min−1and UV detection wavelength at 254.0 nm.

b Potentiometric titration method using 0.1M sodium nitrite as a titrant and detection of end point by starch iodide paste as an external indicator.

c HPLC method using silica column with mobile phase containing a mixture of isopropyl:acetonitrile:ethyl acetate:hexane (10:10:10:70, by volume), flow rate 1.0 mL min−1 and UV detection wavelength at 254.0 nm.

d Figures in parentheses are the corresponding tabulated values for t and F at P = 0.05.

4 Conclusion

Ultimately, the presented RP-HPLC and TLC-spectrodensitometric methods provide a quantitative approach for the simultaneous determination of sulfacetamide sodium and its official impurities; sulfanilamide and dapsone. The presented eco-friendly RP-HPLC method shows high sensitivity, accuracy and precision. From the economic point of view, the shorter run time (∼6 min) without affecting the resolution of separation leads to low mobile phase consumption and makes it a feasible method for the routine analysis of the drugs. The suggested TLC-spectrodensitometric method has the merits of running several samples simultaneously, using simple composition of mobile phase, and offering high sensitivity and specificity with relatively low cost. The proposed chromatographic methods have been validated in accordance with ICH guidelines. Both chromatographic methods can be applied in routine and quality control analysis of sulfacetamide sodium in its pure powdered forms and in its commercially available ophthalmic formulations without the interference of commonly encountered dosage form additives. In addition, the successful application of the suggested techniques to detect trace amounts of sulfanilamide and dapsone reveals the usefulness of these methods for impurity profiling.

Conflict of interest

The authors have declared no conflict of interest.

References

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  • 1.

    Rahman, N. ; Azmi, S.N.H. ; Wu, H.F. The importance of impurity analysis in pharmaceutical products: an integrated approach. Accreditation Qual. Assur. 2006, 11, 6974.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Bari, S.B. ; Kadam, B.R. ; Jaiswal, Y.S. ; Shirkhedkar, A.A. Impurity profile: significance in active pharmaceutical ingredient. Eurasian J. Anal. Chem. 2007, 2, 3253.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    The British Pharmacopoeia . The Stationary Office; London, 2016.

  • 4.

    Brunton, L. ; Chabner, B. ; Knollman, B. Goodman & Gilman’s the Pharmacological Basis of Therapeutics. 12th ed.; The McGraw-Hill Companies: New York, 2011.

    • Search Google Scholar
    • Export Citation
  • 5.

    Agarwal, R. Textbook on Clinical Ocular Pharmacology & Therapeutics; JP Medical Ltd, 2014.

  • 6.

    Sweetman, S.C. Martindale the Complete Drug Reference; The Pharmaceutical Press: London, 2011.

  • 7.

    Zhang, K. ; Ma, P. ; Jing, W. ; Zhang, X. A developed HPLC method for the determination of Alogliptin Benzoate and its potential impurities in bulk drug and tablets. Asian J. Pharm. Sci. 2015, 10, 152158.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Ragab, M.A. ; Eman, I. High performance liquid chromatography with photo diode array for separation and analysis of naproxen and esomeprazole in presence of their chiral impurities: enantiomeric purity determination in tablets. J. Chromatogr. A 2017, 1497, 110117.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Al Alamein, A.M.A. ; Saad, A.S. ; Galal, M.M. ; Zaazaa, H.E. A comparative study of different chromatographic techniques for determination of toxic impurities of some commonly used anesthetics. JPC-Journal of Planar Chromatography-Modern TLC 2018, 31, 280289.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Xu, S. ; Wu, L. Determination of sulfacetamide sodium and sulfanilamide in shao tang ling ointment by high performance liquid chromatography. Chin. J. Chromatogr. 1999, 17, 206-207.

    • Search Google Scholar
    • Export Citation
  • 11.

    Guo, X.G. ; Guo, X.J. ; Guo, H.B. Simultaneous determination of sulfacetamide sodium and impurity sulfanilamide in sulfacetamide sodium eye drops by HPLC. China Pharm. 2013, 25.

    • Search Google Scholar
    • Export Citation
  • 12.

    Gruber, M.P. ; Klein, R.W. TLC determination of sulfanilamide as a degradation product in pharmaceutical preparations containing sodium sulfacetamide. J. Pharm. Sci. 1968, 57, 12121214.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Penner, M.H. Assay of sulfacetamide sodium ophthalmic solutions by high‐pressure liquid chromatography. J. Pharm. Sci. 1975, 64, 10171019.

  • 14.

    Hall, L. ; Chadwick, V. Quantitative determination of sulfanilamide in sodium sulfacetamide raw material and ophthalmic solutions by high-performance liquid chromatography. J. Chromatogr. A 1989, 478, 438445.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    The United States Pharmacopeia and National Formulary; USP 34-NF 29; U.S. Pharmacopeial Convention: Rockville, USA, 2011.

  • 16.

    Seshachalam, U. ; Haribabu, B. ; Chandrasekhar, K.B. Development and validation of a stability‐indicating liquid chromatographic method for determination of emtricitabine and related impurities in drug substance. J. Separat. Sci. 2007, 30, 9991004.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Nageswara Rao, R. ; Narasa Raju, A. Simultaneous separation and determination of process‐related substances and degradation products of venlafaxine by reversed‐phase HPLC. J. Separat. Sci. 2006, 29, 27332744.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Rao, R.N. ; Nagaraju, V. An overview of the recent trends in development of HPLC methods for determination of impurities in drugs. J. Pharm. Biomed. Anal. 2003, 33, 335377.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Sherma, J. ; Fried, B. Hand Book of Thin-Layer Chromatography, 2nd ed.; Mercel Dekker: New York, 2003.

  • 20.

    Capello, C. ; Fischer, U. ; Hungerbuhler, K. What is a green solvent? A comprehensive framework for the environmental assessment of solvents. Green. Chem. 2007, 9, 927934.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Weston, A. ; Brown, P.R. HPLC and CE; Principles and Practice; Academic Press: USA, 1997.

  • 22.

    Variyar, P.S. ; Chatterjee, S. ; Sharma, A. High-performance thin-layer chromatography (HPTLC), Chapter 2: Fundmentals and Theory of HPTLC-Based Separation, Springer: New York, 2011.

    • Search Google Scholar
    • Export Citation
  • 23.

    International Conerence on Harmonaization, Q2(R1), Validation of Analytical Procdures: Text and Methodology; Geneva, Switzerland, 2005.

    • Search Google Scholar
    • Export Citation
  • 24.

    Spiegel, M. ; Stephens, L. Schaum’s Outline of Theory and Problems of Statistics; The McGraw-Hill Companies: New York, 1999.

  • Collapse
  • Expand

Senior editors

Editor(s)-in-Chief: Sajewicz, Mieczyslaw, University of Silesia, Katowice, Poland

Editors(s)

  • Danica Agbaba, University of Belgrade, Belgrade, Serbia
  • Łukasz Komsta, Medical University of Lublin, Lublin, Poland
  • Ivana Stanimirova-Daszykowska, University of Silesia, Katowice, Poland
  • Monika Waksmundzka-Hajnos, Medical University of Lublin, Lublin, Poland

Editorial Board

  • Ravi Bhushan, The Indian Institute of Technology, Roorkee, India
  • Jacek Bojarski, Jagiellonian University, Kraków, Poland
  • Bezhan Chankvetadze, State University of Tbilisi, Tbilisi, Georgia
  • Michał Daszykowski, University of Silesia, Katowice, Poland
  • Tadeusz H. Dzido, Medical University of Lublin, Lublin, Poland
  • Attila Felinger, University of Pécs, Pécs, Hungary
  • Kazimierz Glowniak, Medical University of Lublin, Lublin, Poland
  • Bronisław Glód, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland
  • Anna Gumieniczek, Medical University of Lublin, Lublin, Poland
  • Urszula Hubicka, Jagiellonian University, Kraków, Poland
  • Krzysztof Kaczmarski, Rzeszow University of Technology, Rzeszów, Poland
  • Huba Kalász, Semmelweis University, Budapest, Hungary
  • Katarina Karljiković Rajić, University of Belgrade, Belgrade, Serbia
  • Imre Klebovich, Semmelweis University, Budapest, Hungary
  • Angelika Koch, Private Pharmacy, Hamburg, Germany
  • Piotr Kus, Univerity of Silesia, Katowice, Poland
  • Debby Mangelings, Free University of Brussels, Brussels, Belgium
  • Emil Mincsovics, Corvinus University of Budapest, Budapest, Hungary
  • Ágnes M. Móricz, Centre for Agricultural Research, Budapest, Hungary
  • Gertrud Morlock, Giessen University, Giessen, Germany
  • Anna Petruczynik, Medical University of Lublin, Lublin, Poland
  • Robert Skibiński, Medical University of Lublin, Lublin, Poland
  • Bernd Spangenberg, Offenburg University of Applied Sciences, Germany
  • Tomasz Tuzimski, Medical University of Lublin, Lublin, Poland
  • Adam Voelkel, Poznań University of Technology, Poznań, Poland
  • Beata Walczak, University of Silesia, Katowice, Poland
  • Wiesław Wasiak, Adam Mickiewicz University, Poznań, Poland
  • Igor G. Zenkevich, St. Petersburg State University, St. Petersburg, Russian Federation

 

SAJEWICZ, MIECZYSLAW
E-mail:mieczyslaw.sajewicz@us.edu.pl

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Acta Chromatographica
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