Accepted Manuscript Title: HPTLC and RP-HPLC Methods for Simultaneous Determination of Paracetamol and Pamabrom in Presence of Their Potential Impurities Author: Eglal A. Abdelaleem Ibrahim A. Naguib Eman S. Hassan Nouruddin W. Ali PII: DOI: Reference:
S0731-7085(15)00276-9 http://dx.doi.org/doi:10.1016/j.jpba.2015.04.043 PBA 10079
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
5-3-2015 28-4-2015 30-4-2015
Please cite this article as: E.A. Abdelaleem, I.A. Naguib, E.S. Hassan, N.W. Ali., HPTLC and RP-HPLC Methods for Simultaneous Determination of Paracetamol and Pamabrom in Presence of Their Potential Impurities, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.04.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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HPTLC and RP-HPLC Methods for Simultaneous Determination of
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Paracetamol and Pamabrom in Presence of Their Potential Impurities
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Eglal A. Abdelaleem, Ibrahim A. Naguib, Eman S. Hassan *, and Nouruddin W. Ali. Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef
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University, Alshaheed Shehata Ahmad Hegazy St., 62514, Beni-Suef, Egypt.
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*Corresponding author information: Corresponding author: Eman Sherif Hassan Khairy
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Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef
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Email address: [email protected]
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Tel: +2/ 01009158403
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Fax: +082/2317950
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University, Alshaheed Shehata Ahmad Hegazy St., Beni-Suef, 62514, Egypt.
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Abstract Two chromatgraphic methods were developed for determination of Paracetamol (PCM) and Pamabrom (PAM) in presence of P-aminophenol (PAP) and Theophylline (THEO) as potential impurities of both drugs respectively. First method is HPTLC which depends on separation and
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quantitation of the studied drugs on aluminium plates pre-coated with silica gel 60F254 as astationary phase using chloroform: methanol: ethyl acetate: glacial acetic acid (8:0.8:0.6:0.2, v/v/v/
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v) as mobile phase followed by densitometric measurement of the bands at 254 nm. Second method is RP-HPLC which comprises separation of the studied drugs on a Phenomenex C8 column by
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gradient elution using mobile phase consisting of sodium dihydrogen phosphate buffer (0.05M): methanol: acetonitrile (85:10:5, v/v/v) at a flow rate of 1 mL / min for first 7.5 minutes and at a flow rate of 1.5 mL / min for the next 5 minutes. The proposed methods
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(70:20:10, v/v/v)
were successfully applied for determination of the potential impurities of PCM and PAM after resolving them from the pure drugs. The developed methods have been validated and proved to
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meet the requirements delineated by ICH guidelines with respect to linearity, accuracy, precision, specificity and robustness. The validated methods were successfully applied for determination of
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the studied drugs in their pharmaceutical formulation. The results were statistically compared to those obtained by the reported RP-HPLC method where no significant difference was found;
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indicating the ability of proposed methods to be used for routine quality control analysis of these drugs.
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Keywords: HPTLC; RP-HPLC; Paracetamol; Pamabrom; P-aminophenol; Theophylline.
Introduction:
Paracetamol (PCM); N-(4-Hydroxy phenyl) acetamide [1], (Figure.1.a.), is one of the most popular over-the counter analgesic and antipyretic drugs. Pamabrom (PAM); 8-bromotheophylline compound with 2-amino-2-methyl-1-propanol (1:1) [1], (Figure.1.b.), is a weak diuretic that has been used with analgesics and antihistamines for symptomatic relief of the premenstrual syndrome [2]. PCM helps to reduce menstrual pains and PAM reduces associated bloating. P- aminophenol (PAP); 4-amino phenol, is an impurity and potential degradation product of PCM [3], (Figure.1.c.). Theophylline (THEO); 1,3-dimethyl-7H-purine-2,6-dione[1], is an impurity of a methylxanthine drug (Figure.1.d).
that
bears
structural
and
pharmacological
PAM. It is
similarity to caffeine.,
Literature survey revealed that few methods were reported for simultaneous
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determination of PCM and PAM by LC/MS [4], HPLC [5,6], Spectrophotometry [7,8], simultaneous determination of both drugs in presence of PAP by RP-LC and TLC [9]. The aim of this work is to develop sensitive, selective and accurate HPTLC and RP-HPLC methods for determination of Paracetamol (PCM) and Pamabrom (PAM) in presence of P-aminophenol (PAP)
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and Theophylline (THEO) as potential impurities of both drugs respectively.
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2.Experimental 2.1. Apparatus
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2.1.1. For HPTLC method
HPTLC aluminum plates (20 x 20 cm) coated with 0.25 mm silica gel 60 F254 (Merck, Germany),
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TLC scanner 3 densitometer (Camag, Muttenz, Switzerland), Linomat IV with 100 μL syringe (Camag, Muttenz, Switzerland), Sonix TV ss-series ultrasonicator (USA) and UV lamp with short wavelength 254 nm (Vilber Lourmat, Marne La Vallee, Cedex, France).
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2.1.2. For RP-HPLC method
HPLC (Shimadzu) instrument was equipped with a model series LC-10 ADVP pump, SCL-10 AVP
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controller, DGU-12 A degasser and SPD-10 AVP UV-VIS detector. Separation and quantitation were made at room temperature on a 250 mm × 4.6 mm (i.d.) RP C8 column (4.6 µm particle size).
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The detector was set at 277 nm.
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2.2. Materials and reagents 2.2.1 Pure standard
PCM and PAM were kindly supplied by SIGMA Pharmaceutical Industries, Cairo, Egypt. Purity was reported to be 99.4% and 99.84% for PCM and PAM respectively according to the company’s analysis certificate.
2.2.2 Pharmaceutical formulation
Menobrocond ® film coated tablets (Batch No.32801) are labeled to contain 500 mg of PCM and 25 mg of PAM per tablet; it is manufactured by SIGMA Pharmaceutical Industries. 2.2.3 Chemicals All chemicals and solvents used throughout this work were of analytical grade. PAP was purchased from Riedel-dehaen-AG- Germany; with certified purity of 99%. THEO was supplied from SigmaAldrich (St. Louis, MO, USA) with certified purity of ≥ 99% according to analysis certificate.
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Others include chloroform and methanol, HPLC grade (Sigma
Aldrich, Chemie GmbH,
Germany), Ethyl acetate and glacial acetic acid (El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt). 2.3 Preparation of standard solutions
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Stock standard solutions of PCM, PAM, PAP and THEO were prepared in methanol in the concentration of (1 mg/mL). Working standard solution of PCM, PAM, PAP and THEO were
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prepared in methanol in the concentration of (100 µg/mL). 2.4 Laboratory prepared mixtures
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Mixtures containing different ratios of PCM, PAM, PAP and THEO were prepared using their respective stock standard solutions in methanol. 2.5 Sample preparation
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Ten film coated tablets of Menobrocond® were finely powdered and mixed well. An amount equivalent to 25 and 500 mg of PAM and PCM, respectively, was accurately weighed, transferred
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into a 25mL volumetric flask, and dissolved in 20 mL methanol. The solution was ultrasonicated for 30 min, filtered and then completed to volume with methanol.
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Part of the solution was diluted to obtain (100μg/mL) working solution for PAM and another part
2.6 Method development
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was diluted to obtain (100μg /mL) working solution for PCM using methanol as a solvent.
Most of reported methods were for determination of PCM and PAM alone without taking into
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consideration their potential impurities and due to the nephrotoxic effect of PAP and the different pharmacological action of THEO from the parent drug PAM, Hence, it was necessary to develop and validate simple, sensitive and selective HPTLC and RP-HPLC methods for simultaneous determination of PCM, PAM in presence of their potential impurities in bulk material and in their pharmaceutical formulation.
2.6.1 Construction of calibration curves HPTLC method Into a set of 10-mL volumetric flasks, different aliquots equivalent to 0.6 – 2.4, 0.6 – 2, 0.4 – 2.8 and 0.4 – 1.8 mg of each of PCM, PAM, PAP and THEO respectively were accurately transferred from their standard stock solution (1 mg/mL), and the volume was completed with methanol.
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10 µL of each solution was applied to HPTLC plates as bands of 6 mm width using Camag Linomat IV applicator. The bands were spaced 5mm from each other and 10 mm apart from the bottom edge of the plate. Linear ascending development was performed in a chromatographic chamber previously saturated with chloroform, methanol, ethyl acetate and glacial acetic acid solution
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(8:0.8:0.6:0.2, v/v/v/v) as a developing system for 1 h at room temperature to a distance of 8 cm. The integrated peak areas were recorded using scanning wavelength at 254 nm under the specified
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instrumental conditions. The calibration curves were constructed by plotting the integrated peak area/104 versus the corresponding concentrations of each component and regression equations were
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computed. RP-HPLC method
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Accurate aliquots equivalent to 60 – 520, 40 – 400, 50 – 240, 20 – 240 µg were separately transferred from their respective working standard solutions (100 µg/mL) into four separate series of 10-mL
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volumetric flasks and the volume was completed by the mobile phase. Triplicate injections were made for each concentration; the peak area/103 was used to construct the calibration curve for each
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component from which its regression equation was computed. Chromatographic separation was carried out by gradient elution using sodium dihydrogen phosphate buffer: methanol: acetonitrile
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(85:10:5, v/v/v, pH=4) as a mobile phase delivered at a flow rate of 1 mL/min for first 7.5 minutes and (70:20:10, v/v/v) at a flow rate of 1.5 mL/min for the next 5 minutes. Injection volume was 20 µL and scanning was carried out at 277 nm at room temperature and the run time was 12.5 min.
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2.6.2 Application to pharmaceutical formulation Different concentrations (within the linearity range of each drug) were prepared from the previously prepared pharmaceutical formulation working solution (100 µg/mL) and the procedure mentioned under linearity and construction of calibration curves was followed. Concentrations of PCM and PAM were calculated from their respective regression equations and the percentage recoveries were then calculated.
3. Results and Discussion HPTLC and RP-HPLC are useful techniques for resolution and determination of drug mixtures. These techniques offer a simple way to quantify studied drugs in presence of their potential
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impurities. As reported in USP [1] PAP and THEO are considered as the potential impurities for PCM and PAM , and most of the methods reported in the literature determined PCM and PAM in their binary mixtures without taking into consideration the importance of determination of their potential impurities. Hence the presented work aims to develop and validate a highly selective
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analytical methods for simultaneous determination of PCM and PAM in presence of their potential impurities. These methods offer high sensitivity and selectivity for analysis of PCM and PAM in
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presence of PAP and THEO which are the potential impurities of both drugs respectively.
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3.1 Method optimization – HPTLC method
In order to optimize the developed HPTLC method, it was necessary to investigate the effect of
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different factors to get the desired chromatographic resolution. a- Mobile phase
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Different developing systems of different composition and ratios were tried for separation, e.g., chloroform-methanol-glacial acetic acid (8:1:0.2, v/v/v) showed good separation between PCM and
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PAP but separation between PAM and THEO wasn’t satisfying, chloroform-methanol-ethyl acetate (7.5:0.5: 0.5, v/v/v) , ethyl acetate enhanced separation between PAM and THEO but wasn’t
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suitable for separation of PCM and PAM, and chloroform-ethyl acetate-glacial acetic acid (7.5:2.5:0.5, v/v/v) in which PAP didn’t move from baseline which showed importance of methanol for seperation. The best mobile phase was chloroform-methanol-ethyl acetate-glacial acetic acid
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(8:0.8:0:6:0.2, v/v/v/v).This selected mobile phase allows good separation between the quaternary mixtures with good Rf values (3.21, 7.11, 8.26, 11.49 min.) for PCM, PAP, PAM and THEO respectively without tailing of the separated bands as shown in Figure 2. b- Scanning wavelength
Different scanning wavelengths were tried (220, 254 and 278 nm) in order to obtain good sensitivity of PCM, PAM in presence of PAP and THEO with minimum noise. The wavelength 254 nm was found to be the best wavelength regarding sensitivity of all components. Peaks were sharp and symmetrical with minimum noise, as shown in Figure 2. c- Band dimensions The slight spread of the developed bands due to ordinary diffusion should be taken into consideration, so the band width and interspaces between bands should be chosen carefully to avoid
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spread of bands outside the scanning tracks and interference between adjacent bands. Different band dimensions were tried to obtain sharp and symmetrical peaks. The optimum band width chosen was 6 mm and the inter-space between bands was 5 mm. d- Slit dimensions of scanning light beam
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The slit dimensions of the scanning light beam should ensure complete coverage of band dimensions on the scanned track without interference of adjacent bands. Different slit dimensions
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were tried where 6 mm×0.3 mm proved to be the slit dimensions of choice that provided highest
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sensitivity. –RP-HPLC
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To optimize the proposed RP-HPLC method, it was necessary to test the effects of different parameters that affect the sensitivity, selectivity and the efficiency of the chromatographic separation.
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a- Mobile phase
Different mobile phases with different compositions and polarities were tested to achieve the
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chromatographic separation e.g: water –methanol, water–acetonitrile, sodium dihydrogen phosphate buffer–methanol, sodium dihydrogen phosphate buffer–acetonitrile. Complete
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separation between studied components was achieved by gradient elution using sodium dihydrogen phosphate buffer (0.05M)–methanol–acetonitrile. Additionaly the effect of sodium dihydrogen
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phosphate buffer–methanol–acetonitrile ratio was studied to improve resolution. Maximum resolution was obtained using sodium dihydrogen phosphate buffer (0.05M)–methanol–acetonitrile in the ratio (85:10:5, v/v/v) for first 7.5 min. and (70:20:10, v/v/v) for the next 5 min, separation was obtained at retention times (tR) (3.21, 7.11, 8.26, 11.49 min.) for PCM, PAP, PAM and THEO respectively as shown in Figure3. b- pH effect
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Different pH values (3 – 6) were tested where pH = 4 gave the best chromatographic resolution
203 204
between PCM, PAM and their potential impurities. c- Scanning wavelength
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Different scanning wavelengths were tried (254, 270, 277 nm) in order to enhance the sensitivity of the method where scanning at 277 nm gave good sensitivity for all the studied components as shown in Figure 3. d- Flow rate
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Different flow rates were also tested to provide the best separation within acceptable run time. The
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best flow rate was obtained at 1 mL / min for the first 7.5 min and 1.5 mL / min for the next 5 min.
Method validation
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ICH guidelines [10] for analytical method validation were followed. -Linearity
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Under optimum chromatographic conditions, linear relationships were obtained between the integrated peak area/104 versus the corresponding concentrations for HPTLC and integrated peak area/103 versus the corresponding concentrations for RP-HPLC; results were calculated and
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presented in Table 1. -Accuracy
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Accuracy was calculated as the percentage recoveries of blind pure PCM, PAM, PAP and THEO, it was further assured by performing standard addition technique at three levels and the average -Precision
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percentage recovery was then calculated and presented in Table 2.
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Both repeatability and intermediate precision were studied. Repeatability was calculated by the analysis of three different concentrations of pure components in triplicates on the same day. The experiment was repeated on the same concentrations seven times on four consecutive days to determine the intermediate precision. Good results and acceptable RSD% were obtained, Table 1. -Specificity
The specificity of the method was confirmed by how accurately and specifically the analytes of interest are determined in presence of other components (impurities, degradates or excipients) [11]. Specificity was confirmed as shown in the HPTLC and RP-HPLC chromatograms, Figures 2 & 3. Furthermore, good results were obtained on applying the method on Menobrocond® film coated tablets; Table 2 proves that tablet additives do not interfere with any of the separated components. -Limits of detection and quantitation (LOD and LOQ)
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LOD and LOQ were calculated using the following equations [11]. The low values of LOD and
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4. Conclusion
LOQ indicate the high sensitivity of proposed methods, Table 1. LOD = 3.3 x SD / slope.
LOQ = 10 x SD / slope.
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-Robustness
Deliberate small changes in HPTLC method parameters (e.g. changing in % chloroform in the
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mobile phase± 1% and change in scanning wavelength ±1nm) and in RP-HPLC method parameters (e.g. changing in % methanol in the mobile phase± 1%, changing in flow rate ± 0.05 mL/ min and values respectively, peak height or symmetry of the peaks. -System suitability
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change in scanning wavelength ± 1 nm) didn't lead to significant changes in changes in Rf and tr
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System suitability parameters were carried out to prove that the overall system performed well, it was checked by calculating different parameters and the obtained values were in the acceptable
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ranges according to USP as shown in Table 3.
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Results obtained by the suggested HPTLC and RP-HPLC methods for determination of PCM and PAM in their pharmaceutical formulation were statistically compared to those obtained by applying
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the reported HPLC method [6]. For HPTLC, the calculated t- values were (0.055, 0.166) and Fvalues were (1.027, 2.645) for PCM and PAM respectively. For RP-HPLC, calculated t- values
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were (0.612, 0.074) and F-values were (4.303, 3.719) for PCM and PAM respectively. The obtained values were found to be less than the theoretical ones, confirming accuracy and precision at 95% confidence level.
The present work is concerned with the development and validation of HPTLC and RP-HPLC methods for simultaneous determination of PCM, PAM, PAP and THEO without sample pretreatment and without interference from pharmaceutical formulation excipients. The developed methods have advantage over any reported method in being able to determine the studied drugs along with their potential impurities with high sensitivity, selectivity and short analysis time using one simple mobile phase for all components. Moreover, these methods were successfully applied for determination of PCM and PAM in Menobrocond® film coated tablets without interference
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from pharmaceutical formulation excipients. The developed methods can be easily applied for
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6. References:
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quality control of the studied drugs.
1. The United States Pharmacopeia, 32 Ed., National Formulary 27, United States Pharmacopeial
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convention INC, USA (2009).
2. Martindale. The complete drug reference “The Extra Pharmacopoeia, 31st Edition
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Pharmaceutical press London, 2007.
3. "The British Pharmacopoeia", The Stationary Office, London (2009).
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4. H. Xue, J. Liu, H. Liu, G. WANG, W. LU , Simultaneous determination of paracetamol and caffeine in human plasma by HPLC, Chin. J. Hosp. Pharm. (2005). 25(8), 708. 5. L.J. Zhou, L.H. Gu, Y.J. Wang, J.Y. Liang, Determination of two constituents in compound
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acetaminophen and pamabrom tablets in human plasma by high performance liquid chromatography, Chin. J. New Drugs Clin. Remedies. 2007; 3:008.
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6. D. Shah, B. Patel, A. Bhavsar, Development and Validation of Simultaneous Estimation of
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Paracetamol and Pamabrom in Bulk and Combined Pharmaceutical Dosage Form by RP-HPLC. Research & Reviews: JPPS. 2014; 3(2):16-23.
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7. S. Bambhrolia, S.J. Rajput, Simultaneous estimation of Paracetamol and Pamabrom in bulk drugs and in pharmaceutical formulation by spectrophotometry, Int. J. ChemTech. Res. 2013; 5(4):18027.
8. P. Hardik, M. Bhati, V. Sanjaysinh, P. Hemant, Development
and Validation of UV
Spectrophotometric methods for estimation of Paracetamol and Pamabrom in bulk and synthetic mixture by Simultaneous equation method. Inventi Rapid: Pharm. Anal. Q. A. 2012. 9. O.M. El-Houssini, RP-LC and TLC Densitometric Determination of Paracetamol and Pamabrom in presence of Hazardous Impurity of Paracetamol and Application to pharmaceuticals, Anal. Chem. Insights. 2013; 8:73. 10. ICH, Q2 (R1) Validation of Analytical Procedures, Proceedings of International Conference on Harmonization, Geneva, 2005. 11.USFDA guidance. “Analytical Procedures and Methods Validation”. “Food and Drug Administration”. Rockville. 2000.
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GRAPHICAL ABSTRACT “HPTLC and RP-HPLC methods for Simultaneous Determination of
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Paracetamol and Pamabrom in Presence of Their Potential Impurities”
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3D HPTLC chromatogram of resolved mixture of P-aminophenol (Rf = 0.11), Paracetamol (Rf=0.33), Theophylline (Rf = 0.48) and Pamabrom (Rf=0.65) using (chloroform: methanol: ethyl acetate: glacial acetic acid) (8: 0.8: 0.6: 0.2, by volume) as a developing system and scanning at 254 nm.
309 310 311
RP-HPLC chromatogram of separated peaks of P-aminophenol (Rt = 3.21 min), Paracetamol (Rt=7.11 min), Theophylline (Rt = 8.26 min)
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and Pamabrom (Rt=11.49 min) by gradient elution using phosphate buffer: methanol: acetonitrile (85:10:5 by volume) as a mobile phase delivered at a flow rate of 1 mL / min for first 7.5 minutes and (70:20:10) at a flow rate of 1.5 mL / min for the next 5 minutes and scanning at 277 nm.
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Highlights:
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HPTLC & RP-HPLC methods for determination of PCM and PAM were developed. The two developed methods are validated and met ICH requirements. Developed methods are able to determine PCM & PAM in presence of their impurities.
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Figures
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Figure 1.a. Chemical structure of Paracetamol (PCM)
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C8H9NO2
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327 328 329
Paracetamol (PCM), acetaminophen
Pamabrom (PAM)
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Paminophenol (PAP),
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Figure 1.b. Chemical structure of Pamabrom (PAM)
C6H7NO Figure 1.c. Chemical structure of P-aminophenol (PAP)
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C11H18BrN5O3 Mol. Wt. 348.20
Theophylline (THEO) C7H8N4O2 Mol. Wt. 180.17
334 335 336
Figure 1.d. Chemical structure of Theophylline (THEO)
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Figure 2. HPTLC chromatogram of resolved mixture of PAP (Rf = 0.11), PCM (Rf=0.33), THEO (Rf = 0.48) and PAM (Rf=0.65) using (chloroform: methanol: ethyl acetate: glacial acetic acid) (8: 0.8: 0.6: 0.2, by volume) as a developing system and scanning at 254 nm.
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Figure 3. RP-HPLC chromatogram of separated peaks of PAP (tR = 3.21 min), PCM (tR=7.11 min), THEO (tR = 8.26 min) and PAM (tR=11.49 min) by gradient elution using phosphate buffer: methanol: acetonitrile (85:10:5 by volume) as a mobile phase delivered at a flow rate of 1 mL / min for first 7.5 minutes and (70:20:10) at a flow rate of 1.5 mL / min for the next 5 minutes and scanning at 277 nm.
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347 348 349 350 351 352 353 354
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344 345 346
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Table1: Regression and analytical parameters of the proposed HPTLC & RP-HPLC methods for determination of PCM and PAM in presence of PAP and THEO as potential impurities of PCM and PAM respectively. HPTLC RP-HPLC
Parameters
PCM
PAM
PAP
THEO
PCM
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354 355 356
PAM
Calibration range 0.6-2.4 μg/band Slope 0.472 Intercept 0.404 Correlation coefficient 1
0.6-2 μg/band 0.191 0.258 0.9999
0.4-2.8 μg/band 0. 251 0.345 0.9999
Accuracy% (mean ± SD) Repeatability (RSD%)
100.00±0.870
100.31± 0.671
100.30± 0.513
0.676
0.579
1.046
0.720
0.489
0.9
Intermediadte Precision (RSD%)
1.458
0.969
1.166
1.006
0.738
1.1
0.143 μg/band
LOQ
0.503 μg/band
0.435 μg/band
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0.166 μg/band
0.131 μg/band
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LOD
0.41.8μg/band 0.469 0.196 0.9999 100.02± 0.718
0.397 μg/band
6-52 μg/mL 0.018 0.006 1
4-40 μ 0.0 0.0 1
100.21±0.425 100.36
0.080 μg/band
1.560 μg/mL
0.815
0.243 μg/band
4.728 μg/mL
2.468
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357*(RSD%)a* and (RSD%)b*; the intra- and inter-day relative standard deviation of concentrations (0.8, 1.6, 2 µg/ band) for 358PCM, (0.8, 1.6, 2 µg/ band) for PAM, (1.6, 2, 2.4 µg /band) for PAP and (0.8, 1.6, 1.8 µg/ band) for THEO determined by 359HPTLC and (12, 20, 24 µg/ mL) for PCM, (10,16 , 20µg /mL) for PAM, (14,16 , 24 µg/ mL) for PAP and (12, 16 , 20 µg / 360mL) for THEO determined by RP-HPLC.
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361 Table 2: Determination of PCM and PAM in pharmaceutical formulation by the proposed methods 362 and results of standard addition technique. 363 *Average of 6 determinations HPTLC PAM
1 μg/band
0.8 μg/band
100.00 ± 1.448
101.05 ± 1.090
/
mean± SD
Method Parameters
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Pure added (μg/band )
1 1.2 1.4
102.75 101.71 100.94
0.8 1 1.2
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101.80 ± 0.906
99.99 ± 1.004
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Recovery %
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364 365 366
Pure added (μg/band )
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Standard addition technique
1
10 μg/mL
100
Recovery % Pure added Recovery % (μg/mL) 101.96 100.24 102.30 10 99.80 100.17 12 100.09 14
Pure add (μg/mL ) 10 12 14
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mg
Found % * ± SD
PCM
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PCM Taken
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RP-HPLC
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od
101.48± 1.142
99.
100.04 ± 0.224
Table 3: Parameters of system suitability of the developed HPTLC & RP-HPLC methods for the determination of PCM, PAM, PAP and THEO. HPTLC RP PCM
PAP
PAM
THEO
Refrence values
PCM
PAP
6.4
1-10
4.92
1.68
0.88
~1
1.17
1.12
Capacity factor (K')
4
Symmetry factor
1
1
1
8 1
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5.4
2.9
Selectivity (α)
3
1.35
3.69
1.45
Rs > 1.5
3.62
α >1
2.21
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Resolution (Rs)
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Column efficiency (N)
164.9
0.0198
0.15
us
HETP(cm/plate)
1262.03
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S0731-7085(15)00276-9 http://dx.doi.org/doi:10.1016/j.jpba.2015.04.043 PBA 10079
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
5-3-2015 28-4-2015 30-4-2015
Please cite this article as: E.A. Abdelaleem, I.A. Naguib, E.S. Hassan, N.W. Ali., HPTLC and RP-HPLC Methods for Simultaneous Determination of Paracetamol and Pamabrom in Presence of Their Potential Impurities, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.04.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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HPTLC and RP-HPLC Methods for Simultaneous Determination of
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Paracetamol and Pamabrom in Presence of Their Potential Impurities
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Eglal A. Abdelaleem, Ibrahim A. Naguib, Eman S. Hassan *, and Nouruddin W. Ali. Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef
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University, Alshaheed Shehata Ahmad Hegazy St., 62514, Beni-Suef, Egypt.
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*Corresponding author information: Corresponding author: Eman Sherif Hassan Khairy
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Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef
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Email address: [email protected]
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Tel: +2/ 01009158403
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Fax: +082/2317950
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University, Alshaheed Shehata Ahmad Hegazy St., Beni-Suef, 62514, Egypt.
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Abstract Two chromatgraphic methods were developed for determination of Paracetamol (PCM) and Pamabrom (PAM) in presence of P-aminophenol (PAP) and Theophylline (THEO) as potential impurities of both drugs respectively. First method is HPTLC which depends on separation and
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quantitation of the studied drugs on aluminium plates pre-coated with silica gel 60F254 as astationary phase using chloroform: methanol: ethyl acetate: glacial acetic acid (8:0.8:0.6:0.2, v/v/v/
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v) as mobile phase followed by densitometric measurement of the bands at 254 nm. Second method is RP-HPLC which comprises separation of the studied drugs on a Phenomenex C8 column by
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gradient elution using mobile phase consisting of sodium dihydrogen phosphate buffer (0.05M): methanol: acetonitrile (85:10:5, v/v/v) at a flow rate of 1 mL / min for first 7.5 minutes and at a flow rate of 1.5 mL / min for the next 5 minutes. The proposed methods
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(70:20:10, v/v/v)
were successfully applied for determination of the potential impurities of PCM and PAM after resolving them from the pure drugs. The developed methods have been validated and proved to
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meet the requirements delineated by ICH guidelines with respect to linearity, accuracy, precision, specificity and robustness. The validated methods were successfully applied for determination of
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the studied drugs in their pharmaceutical formulation. The results were statistically compared to those obtained by the reported RP-HPLC method where no significant difference was found;
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indicating the ability of proposed methods to be used for routine quality control analysis of these drugs.
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Keywords: HPTLC; RP-HPLC; Paracetamol; Pamabrom; P-aminophenol; Theophylline.
Introduction:
Paracetamol (PCM); N-(4-Hydroxy phenyl) acetamide [1], (Figure.1.a.), is one of the most popular over-the counter analgesic and antipyretic drugs. Pamabrom (PAM); 8-bromotheophylline compound with 2-amino-2-methyl-1-propanol (1:1) [1], (Figure.1.b.), is a weak diuretic that has been used with analgesics and antihistamines for symptomatic relief of the premenstrual syndrome [2]. PCM helps to reduce menstrual pains and PAM reduces associated bloating. P- aminophenol (PAP); 4-amino phenol, is an impurity and potential degradation product of PCM [3], (Figure.1.c.). Theophylline (THEO); 1,3-dimethyl-7H-purine-2,6-dione[1], is an impurity of a methylxanthine drug (Figure.1.d).
that
bears
structural
and
pharmacological
PAM. It is
similarity to caffeine.,
Literature survey revealed that few methods were reported for simultaneous
2 Page 2 of 19
determination of PCM and PAM by LC/MS [4], HPLC [5,6], Spectrophotometry [7,8], simultaneous determination of both drugs in presence of PAP by RP-LC and TLC [9]. The aim of this work is to develop sensitive, selective and accurate HPTLC and RP-HPLC methods for determination of Paracetamol (PCM) and Pamabrom (PAM) in presence of P-aminophenol (PAP)
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and Theophylline (THEO) as potential impurities of both drugs respectively.
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2.Experimental 2.1. Apparatus
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2.1.1. For HPTLC method
HPTLC aluminum plates (20 x 20 cm) coated with 0.25 mm silica gel 60 F254 (Merck, Germany),
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TLC scanner 3 densitometer (Camag, Muttenz, Switzerland), Linomat IV with 100 μL syringe (Camag, Muttenz, Switzerland), Sonix TV ss-series ultrasonicator (USA) and UV lamp with short wavelength 254 nm (Vilber Lourmat, Marne La Vallee, Cedex, France).
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2.1.2. For RP-HPLC method
HPLC (Shimadzu) instrument was equipped with a model series LC-10 ADVP pump, SCL-10 AVP
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controller, DGU-12 A degasser and SPD-10 AVP UV-VIS detector. Separation and quantitation were made at room temperature on a 250 mm × 4.6 mm (i.d.) RP C8 column (4.6 µm particle size).
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The detector was set at 277 nm.
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53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83
2.2. Materials and reagents 2.2.1 Pure standard
PCM and PAM were kindly supplied by SIGMA Pharmaceutical Industries, Cairo, Egypt. Purity was reported to be 99.4% and 99.84% for PCM and PAM respectively according to the company’s analysis certificate.
2.2.2 Pharmaceutical formulation
Menobrocond ® film coated tablets (Batch No.32801) are labeled to contain 500 mg of PCM and 25 mg of PAM per tablet; it is manufactured by SIGMA Pharmaceutical Industries. 2.2.3 Chemicals All chemicals and solvents used throughout this work were of analytical grade. PAP was purchased from Riedel-dehaen-AG- Germany; with certified purity of 99%. THEO was supplied from SigmaAldrich (St. Louis, MO, USA) with certified purity of ≥ 99% according to analysis certificate.
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Others include chloroform and methanol, HPLC grade (Sigma
Aldrich, Chemie GmbH,
Germany), Ethyl acetate and glacial acetic acid (El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt). 2.3 Preparation of standard solutions
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Stock standard solutions of PCM, PAM, PAP and THEO were prepared in methanol in the concentration of (1 mg/mL). Working standard solution of PCM, PAM, PAP and THEO were
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prepared in methanol in the concentration of (100 µg/mL). 2.4 Laboratory prepared mixtures
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Mixtures containing different ratios of PCM, PAM, PAP and THEO were prepared using their respective stock standard solutions in methanol. 2.5 Sample preparation
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Ten film coated tablets of Menobrocond® were finely powdered and mixed well. An amount equivalent to 25 and 500 mg of PAM and PCM, respectively, was accurately weighed, transferred
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into a 25mL volumetric flask, and dissolved in 20 mL methanol. The solution was ultrasonicated for 30 min, filtered and then completed to volume with methanol.
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Part of the solution was diluted to obtain (100μg/mL) working solution for PAM and another part
2.6 Method development
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was diluted to obtain (100μg /mL) working solution for PCM using methanol as a solvent.
Most of reported methods were for determination of PCM and PAM alone without taking into
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consideration their potential impurities and due to the nephrotoxic effect of PAP and the different pharmacological action of THEO from the parent drug PAM, Hence, it was necessary to develop and validate simple, sensitive and selective HPTLC and RP-HPLC methods for simultaneous determination of PCM, PAM in presence of their potential impurities in bulk material and in their pharmaceutical formulation.
2.6.1 Construction of calibration curves HPTLC method Into a set of 10-mL volumetric flasks, different aliquots equivalent to 0.6 – 2.4, 0.6 – 2, 0.4 – 2.8 and 0.4 – 1.8 mg of each of PCM, PAM, PAP and THEO respectively were accurately transferred from their standard stock solution (1 mg/mL), and the volume was completed with methanol.
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10 µL of each solution was applied to HPTLC plates as bands of 6 mm width using Camag Linomat IV applicator. The bands were spaced 5mm from each other and 10 mm apart from the bottom edge of the plate. Linear ascending development was performed in a chromatographic chamber previously saturated with chloroform, methanol, ethyl acetate and glacial acetic acid solution
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(8:0.8:0.6:0.2, v/v/v/v) as a developing system for 1 h at room temperature to a distance of 8 cm. The integrated peak areas were recorded using scanning wavelength at 254 nm under the specified
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instrumental conditions. The calibration curves were constructed by plotting the integrated peak area/104 versus the corresponding concentrations of each component and regression equations were
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computed. RP-HPLC method
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Accurate aliquots equivalent to 60 – 520, 40 – 400, 50 – 240, 20 – 240 µg were separately transferred from their respective working standard solutions (100 µg/mL) into four separate series of 10-mL
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volumetric flasks and the volume was completed by the mobile phase. Triplicate injections were made for each concentration; the peak area/103 was used to construct the calibration curve for each
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component from which its regression equation was computed. Chromatographic separation was carried out by gradient elution using sodium dihydrogen phosphate buffer: methanol: acetonitrile
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(85:10:5, v/v/v, pH=4) as a mobile phase delivered at a flow rate of 1 mL/min for first 7.5 minutes and (70:20:10, v/v/v) at a flow rate of 1.5 mL/min for the next 5 minutes. Injection volume was 20 µL and scanning was carried out at 277 nm at room temperature and the run time was 12.5 min.
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2.6.2 Application to pharmaceutical formulation Different concentrations (within the linearity range of each drug) were prepared from the previously prepared pharmaceutical formulation working solution (100 µg/mL) and the procedure mentioned under linearity and construction of calibration curves was followed. Concentrations of PCM and PAM were calculated from their respective regression equations and the percentage recoveries were then calculated.
3. Results and Discussion HPTLC and RP-HPLC are useful techniques for resolution and determination of drug mixtures. These techniques offer a simple way to quantify studied drugs in presence of their potential
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impurities. As reported in USP [1] PAP and THEO are considered as the potential impurities for PCM and PAM , and most of the methods reported in the literature determined PCM and PAM in their binary mixtures without taking into consideration the importance of determination of their potential impurities. Hence the presented work aims to develop and validate a highly selective
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analytical methods for simultaneous determination of PCM and PAM in presence of their potential impurities. These methods offer high sensitivity and selectivity for analysis of PCM and PAM in
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presence of PAP and THEO which are the potential impurities of both drugs respectively.
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3.1 Method optimization – HPTLC method
In order to optimize the developed HPTLC method, it was necessary to investigate the effect of
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different factors to get the desired chromatographic resolution. a- Mobile phase
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Different developing systems of different composition and ratios were tried for separation, e.g., chloroform-methanol-glacial acetic acid (8:1:0.2, v/v/v) showed good separation between PCM and
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PAP but separation between PAM and THEO wasn’t satisfying, chloroform-methanol-ethyl acetate (7.5:0.5: 0.5, v/v/v) , ethyl acetate enhanced separation between PAM and THEO but wasn’t
te
suitable for separation of PCM and PAM, and chloroform-ethyl acetate-glacial acetic acid (7.5:2.5:0.5, v/v/v) in which PAP didn’t move from baseline which showed importance of methanol for seperation. The best mobile phase was chloroform-methanol-ethyl acetate-glacial acetic acid
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(8:0.8:0:6:0.2, v/v/v/v).This selected mobile phase allows good separation between the quaternary mixtures with good Rf values (3.21, 7.11, 8.26, 11.49 min.) for PCM, PAP, PAM and THEO respectively without tailing of the separated bands as shown in Figure 2. b- Scanning wavelength
Different scanning wavelengths were tried (220, 254 and 278 nm) in order to obtain good sensitivity of PCM, PAM in presence of PAP and THEO with minimum noise. The wavelength 254 nm was found to be the best wavelength regarding sensitivity of all components. Peaks were sharp and symmetrical with minimum noise, as shown in Figure 2. c- Band dimensions The slight spread of the developed bands due to ordinary diffusion should be taken into consideration, so the band width and interspaces between bands should be chosen carefully to avoid
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spread of bands outside the scanning tracks and interference between adjacent bands. Different band dimensions were tried to obtain sharp and symmetrical peaks. The optimum band width chosen was 6 mm and the inter-space between bands was 5 mm. d- Slit dimensions of scanning light beam
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The slit dimensions of the scanning light beam should ensure complete coverage of band dimensions on the scanned track without interference of adjacent bands. Different slit dimensions
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were tried where 6 mm×0.3 mm proved to be the slit dimensions of choice that provided highest
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sensitivity. –RP-HPLC
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To optimize the proposed RP-HPLC method, it was necessary to test the effects of different parameters that affect the sensitivity, selectivity and the efficiency of the chromatographic separation.
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a- Mobile phase
Different mobile phases with different compositions and polarities were tested to achieve the
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chromatographic separation e.g: water –methanol, water–acetonitrile, sodium dihydrogen phosphate buffer–methanol, sodium dihydrogen phosphate buffer–acetonitrile. Complete
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separation between studied components was achieved by gradient elution using sodium dihydrogen phosphate buffer (0.05M)–methanol–acetonitrile. Additionaly the effect of sodium dihydrogen
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177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201
phosphate buffer–methanol–acetonitrile ratio was studied to improve resolution. Maximum resolution was obtained using sodium dihydrogen phosphate buffer (0.05M)–methanol–acetonitrile in the ratio (85:10:5, v/v/v) for first 7.5 min. and (70:20:10, v/v/v) for the next 5 min, separation was obtained at retention times (tR) (3.21, 7.11, 8.26, 11.49 min.) for PCM, PAP, PAM and THEO respectively as shown in Figure3. b- pH effect
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Different pH values (3 – 6) were tested where pH = 4 gave the best chromatographic resolution
203 204
between PCM, PAM and their potential impurities. c- Scanning wavelength
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Different scanning wavelengths were tried (254, 270, 277 nm) in order to enhance the sensitivity of the method where scanning at 277 nm gave good sensitivity for all the studied components as shown in Figure 3. d- Flow rate
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Different flow rates were also tested to provide the best separation within acceptable run time. The
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best flow rate was obtained at 1 mL / min for the first 7.5 min and 1.5 mL / min for the next 5 min.
Method validation
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ICH guidelines [10] for analytical method validation were followed. -Linearity
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Under optimum chromatographic conditions, linear relationships were obtained between the integrated peak area/104 versus the corresponding concentrations for HPTLC and integrated peak area/103 versus the corresponding concentrations for RP-HPLC; results were calculated and
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presented in Table 1. -Accuracy
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Accuracy was calculated as the percentage recoveries of blind pure PCM, PAM, PAP and THEO, it was further assured by performing standard addition technique at three levels and the average -Precision
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percentage recovery was then calculated and presented in Table 2.
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205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234
Both repeatability and intermediate precision were studied. Repeatability was calculated by the analysis of three different concentrations of pure components in triplicates on the same day. The experiment was repeated on the same concentrations seven times on four consecutive days to determine the intermediate precision. Good results and acceptable RSD% were obtained, Table 1. -Specificity
The specificity of the method was confirmed by how accurately and specifically the analytes of interest are determined in presence of other components (impurities, degradates or excipients) [11]. Specificity was confirmed as shown in the HPTLC and RP-HPLC chromatograms, Figures 2 & 3. Furthermore, good results were obtained on applying the method on Menobrocond® film coated tablets; Table 2 proves that tablet additives do not interfere with any of the separated components. -Limits of detection and quantitation (LOD and LOQ)
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235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257
LOD and LOQ were calculated using the following equations [11]. The low values of LOD and
258 259 260 261 262 263 264 265
4. Conclusion
LOQ indicate the high sensitivity of proposed methods, Table 1. LOD = 3.3 x SD / slope.
LOQ = 10 x SD / slope.
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-Robustness
Deliberate small changes in HPTLC method parameters (e.g. changing in % chloroform in the
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mobile phase± 1% and change in scanning wavelength ±1nm) and in RP-HPLC method parameters (e.g. changing in % methanol in the mobile phase± 1%, changing in flow rate ± 0.05 mL/ min and values respectively, peak height or symmetry of the peaks. -System suitability
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change in scanning wavelength ± 1 nm) didn't lead to significant changes in changes in Rf and tr
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System suitability parameters were carried out to prove that the overall system performed well, it was checked by calculating different parameters and the obtained values were in the acceptable
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ranges according to USP as shown in Table 3.
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Results obtained by the suggested HPTLC and RP-HPLC methods for determination of PCM and PAM in their pharmaceutical formulation were statistically compared to those obtained by applying
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the reported HPLC method [6]. For HPTLC, the calculated t- values were (0.055, 0.166) and Fvalues were (1.027, 2.645) for PCM and PAM respectively. For RP-HPLC, calculated t- values
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were (0.612, 0.074) and F-values were (4.303, 3.719) for PCM and PAM respectively. The obtained values were found to be less than the theoretical ones, confirming accuracy and precision at 95% confidence level.
The present work is concerned with the development and validation of HPTLC and RP-HPLC methods for simultaneous determination of PCM, PAM, PAP and THEO without sample pretreatment and without interference from pharmaceutical formulation excipients. The developed methods have advantage over any reported method in being able to determine the studied drugs along with their potential impurities with high sensitivity, selectivity and short analysis time using one simple mobile phase for all components. Moreover, these methods were successfully applied for determination of PCM and PAM in Menobrocond® film coated tablets without interference
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266 267 268 269
from pharmaceutical formulation excipients. The developed methods can be easily applied for
270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296
6. References:
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quality control of the studied drugs.
1. The United States Pharmacopeia, 32 Ed., National Formulary 27, United States Pharmacopeial
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convention INC, USA (2009).
2. Martindale. The complete drug reference “The Extra Pharmacopoeia, 31st Edition
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Pharmaceutical press London, 2007.
3. "The British Pharmacopoeia", The Stationary Office, London (2009).
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4. H. Xue, J. Liu, H. Liu, G. WANG, W. LU , Simultaneous determination of paracetamol and caffeine in human plasma by HPLC, Chin. J. Hosp. Pharm. (2005). 25(8), 708. 5. L.J. Zhou, L.H. Gu, Y.J. Wang, J.Y. Liang, Determination of two constituents in compound
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acetaminophen and pamabrom tablets in human plasma by high performance liquid chromatography, Chin. J. New Drugs Clin. Remedies. 2007; 3:008.
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6. D. Shah, B. Patel, A. Bhavsar, Development and Validation of Simultaneous Estimation of
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Paracetamol and Pamabrom in Bulk and Combined Pharmaceutical Dosage Form by RP-HPLC. Research & Reviews: JPPS. 2014; 3(2):16-23.
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7. S. Bambhrolia, S.J. Rajput, Simultaneous estimation of Paracetamol and Pamabrom in bulk drugs and in pharmaceutical formulation by spectrophotometry, Int. J. ChemTech. Res. 2013; 5(4):18027.
8. P. Hardik, M. Bhati, V. Sanjaysinh, P. Hemant, Development
and Validation of UV
Spectrophotometric methods for estimation of Paracetamol and Pamabrom in bulk and synthetic mixture by Simultaneous equation method. Inventi Rapid: Pharm. Anal. Q. A. 2012. 9. O.M. El-Houssini, RP-LC and TLC Densitometric Determination of Paracetamol and Pamabrom in presence of Hazardous Impurity of Paracetamol and Application to pharmaceuticals, Anal. Chem. Insights. 2013; 8:73. 10. ICH, Q2 (R1) Validation of Analytical Procedures, Proceedings of International Conference on Harmonization, Geneva, 2005. 11.USFDA guidance. “Analytical Procedures and Methods Validation”. “Food and Drug Administration”. Rockville. 2000.
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GRAPHICAL ABSTRACT “HPTLC and RP-HPLC methods for Simultaneous Determination of
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Paracetamol and Pamabrom in Presence of Their Potential Impurities”
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297 298 299 300 301 302
3D HPTLC chromatogram of resolved mixture of P-aminophenol (Rf = 0.11), Paracetamol (Rf=0.33), Theophylline (Rf = 0.48) and Pamabrom (Rf=0.65) using (chloroform: methanol: ethyl acetate: glacial acetic acid) (8: 0.8: 0.6: 0.2, by volume) as a developing system and scanning at 254 nm.
309 310 311
RP-HPLC chromatogram of separated peaks of P-aminophenol (Rt = 3.21 min), Paracetamol (Rt=7.11 min), Theophylline (Rt = 8.26 min)
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303 304 305 306 307 308
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and Pamabrom (Rt=11.49 min) by gradient elution using phosphate buffer: methanol: acetonitrile (85:10:5 by volume) as a mobile phase delivered at a flow rate of 1 mL / min for first 7.5 minutes and (70:20:10) at a flow rate of 1.5 mL / min for the next 5 minutes and scanning at 277 nm.
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Highlights:
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HPTLC & RP-HPLC methods for determination of PCM and PAM were developed. The two developed methods are validated and met ICH requirements. Developed methods are able to determine PCM & PAM in presence of their impurities.
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317 318 319 320 321 322 323 324 325
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Figures
325 326
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Figure 1.a. Chemical structure of Paracetamol (PCM)
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C8H9NO2
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327 328 329
Paracetamol (PCM), acetaminophen
Pamabrom (PAM)
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Paminophenol (PAP),
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332 333
Figure 1.b. Chemical structure of Pamabrom (PAM)
C6H7NO Figure 1.c. Chemical structure of P-aminophenol (PAP)
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330 331
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C11H18BrN5O3 Mol. Wt. 348.20
Theophylline (THEO) C7H8N4O2 Mol. Wt. 180.17
334 335 336
Figure 1.d. Chemical structure of Theophylline (THEO)
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338 339 340 341 342 343 344
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336 337
Figure 2. HPTLC chromatogram of resolved mixture of PAP (Rf = 0.11), PCM (Rf=0.33), THEO (Rf = 0.48) and PAM (Rf=0.65) using (chloroform: methanol: ethyl acetate: glacial acetic acid) (8: 0.8: 0.6: 0.2, by volume) as a developing system and scanning at 254 nm.
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Figure 3. RP-HPLC chromatogram of separated peaks of PAP (tR = 3.21 min), PCM (tR=7.11 min), THEO (tR = 8.26 min) and PAM (tR=11.49 min) by gradient elution using phosphate buffer: methanol: acetonitrile (85:10:5 by volume) as a mobile phase delivered at a flow rate of 1 mL / min for first 7.5 minutes and (70:20:10) at a flow rate of 1.5 mL / min for the next 5 minutes and scanning at 277 nm.
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347 348 349 350 351 352 353 354
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cr
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344 345 346
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Table1: Regression and analytical parameters of the proposed HPTLC & RP-HPLC methods for determination of PCM and PAM in presence of PAP and THEO as potential impurities of PCM and PAM respectively. HPTLC RP-HPLC
Parameters
PCM
PAM
PAP
THEO
PCM
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354 355 356
PAM
Calibration range 0.6-2.4 μg/band Slope 0.472 Intercept 0.404 Correlation coefficient 1
0.6-2 μg/band 0.191 0.258 0.9999
0.4-2.8 μg/band 0. 251 0.345 0.9999
Accuracy% (mean ± SD) Repeatability (RSD%)
100.00±0.870
100.31± 0.671
100.30± 0.513
0.676
0.579
1.046
0.720
0.489
0.9
Intermediadte Precision (RSD%)
1.458
0.969
1.166
1.006
0.738
1.1
0.143 μg/band
LOQ
0.503 μg/band
0.435 μg/band
cr
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0.166 μg/band
0.131 μg/band
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LOD
0.41.8μg/band 0.469 0.196 0.9999 100.02± 0.718
0.397 μg/band
6-52 μg/mL 0.018 0.006 1
4-40 μ 0.0 0.0 1
100.21±0.425 100.36
0.080 μg/band
1.560 μg/mL
0.815
0.243 μg/band
4.728 μg/mL
2.468
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te
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357*(RSD%)a* and (RSD%)b*; the intra- and inter-day relative standard deviation of concentrations (0.8, 1.6, 2 µg/ band) for 358PCM, (0.8, 1.6, 2 µg/ band) for PAM, (1.6, 2, 2.4 µg /band) for PAP and (0.8, 1.6, 1.8 µg/ band) for THEO determined by 359HPTLC and (12, 20, 24 µg/ mL) for PCM, (10,16 , 20µg /mL) for PAM, (14,16 , 24 µg/ mL) for PAP and (12, 16 , 20 µg / 360mL) for THEO determined by RP-HPLC.
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361 Table 2: Determination of PCM and PAM in pharmaceutical formulation by the proposed methods 362 and results of standard addition technique. 363 *Average of 6 determinations HPTLC PAM
1 μg/band
0.8 μg/band
100.00 ± 1.448
101.05 ± 1.090
/
mean± SD
Method Parameters
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Pure added (μg/band )
1 1.2 1.4
102.75 101.71 100.94
0.8 1 1.2
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101.80 ± 0.906
99.99 ± 1.004
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Recovery %
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364 365 366
Pure added (μg/band )
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Standard addition technique
1
10 μg/mL
100
Recovery % Pure added Recovery % (μg/mL) 101.96 100.24 102.30 10 99.80 100.17 12 100.09 14
Pure add (μg/mL ) 10 12 14
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mg
Found % * ± SD
PCM
cr
PCM Taken
nd d
RP-HPLC
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od
101.48± 1.142
99.
100.04 ± 0.224
Table 3: Parameters of system suitability of the developed HPTLC & RP-HPLC methods for the determination of PCM, PAM, PAP and THEO. HPTLC RP PCM
PAP
PAM
THEO
Refrence values
PCM
PAP
6.4
1-10
4.92
1.68
0.88
~1
1.17
1.12
Capacity factor (K')
4
Symmetry factor
1
1
1
8 1
18 Page 18 of 19
5.4
2.9
Selectivity (α)
3
1.35
3.69
1.45
Rs > 1.5
3.62
α >1
2.21
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Resolution (Rs)
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Column efficiency (N)
164.9
0.0198
0.15
us
HETP(cm/plate)
1262.03
Ac ce p
te
d
M
an
367 368
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