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: S0731-7085(15)00276-9
DOI: http://dx.doi.org/doi:10.1016/j.jpba.2015.04.043
Reference: PBA 10079

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 5-3-2015

Revised date: 28-4-2015
Accepted date: 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

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 1 HPTLC and RP-HPLC Methods for Simultaneous Determination of
2 Paracetamol and Pamabrom in Presence of Their Potential Impurities
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4 Eglal A. Abdelaleem, Ibrahim A. Naguib, Eman S. Hassan *, and Nouruddin W. Ali.

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5 Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef

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6 University, Alshaheed Shehata Ahmad Hegazy St., 62514, Beni-Suef, Egypt.

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9 *Corresponding author information:

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Corresponding author: Eman Sherif Hassan Khairy

11 Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef

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12 University, Alshaheed Shehata Ahmad Hegazy St., Beni-Suef, 62514, Egypt.

13 Email address: dr.emansherif@yahoo.com

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14 Tel: +2/ 01009158403

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15 Fax: +082/2317950
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22 Abstract
23 Two chromatgraphic methods were developed for determination of Paracetamol (PCM) and
24 Pamabrom (PAM) in presence of P-aminophenol (PAP) and Theophylline (THEO) as potential
25 impurities of both drugs respectively. First method is HPTLC which depends on separation and
26 quantitation of the studied drugs on aluminium plates pre-coated with silica gel 60F254 as

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27

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astationary phase using chloroform: methanol: ethyl acetate: glacial acetic acid (8:0.8:0.6:0.2, v/v/v/
28 v) as mobile phase followed by densitometric measurement of the bands at 254 nm. Second method

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29 is RP-HPLC which comprises separation of the studied drugs on a Phenomenex C8 column by
30 gradient elution using mobile phase consisting of sodium dihydrogen phosphate buffer (0.05M):

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31 methanol: acetonitrile (85:10:5, v/v/v) at a flow rate of 1 mL / min for first 7.5 minutes and
32 (70:20:10, v/v/v) at a flow rate of 1.5 mL / min for the next 5 minutes. The proposed methods
33

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were successfully applied for determination of the potential impurities of PCM and PAM after
34 resolving them from the pure drugs. The developed methods have been validated and proved to
35 meet the requirements delineated by ICH guidelines with respect to linearity, accuracy, precision,
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36 specificity and robustness. The validated methods were successfully applied for determination of
37 the studied drugs in their pharmaceutical formulation. The results were statistically compared to
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38 those obtained by the reported RP-HPLC method where no significant difference was found;
39 indicating the ability of proposed methods to be used for routine quality control analysis of these
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40 drugs.
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Keywords: HPTLC; RP-HPLC; Paracetamol; Pamabrom; P-aminophenol; Theophylline.

42
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43 Introduction:
44 Paracetamol (PCM); N-(4-Hydroxy phenyl) acetamide [1], (Figure.1.a.), is one of the most popular
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45 over-the counter analgesic and antipyretic drugs. Pamabrom (PAM); 8-bromotheophylline

46 compound with 2-amino-2-methyl-1-propanol (1:1) [1], (Figure.1.b.), is a weak diuretic that has
47 been used with analgesics and antihistamines for symptomatic relief of the premenstrual syndrome
48 [2]. PCM helps to reduce menstrual pains and PAM reduces associated bloating. P- aminophenol
49 (PAP); 4-amino phenol, is an impurity and potential degradation product of PCM [3], (Figure.1.c.).
50 Theophylline (THEO); 1,3-dimethyl-7H-purine-2,6-dione[1], is an impurity of PAM. It is
51 a methylxanthine drug that bears structural and pharmacological similarity to caffeine.,
52 (Figure.1.d). Literature survey revealed that few methods were reported for simultaneous
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53 determination of PCM and PAM by LC/MS [4], HPLC [5,6], Spectrophotometry [7,8],
54 simultaneous determination of both drugs in presence of PAP by RP-LC and TLC [9]. The aim of
55 this work is to develop sensitive, selective and accurate HPTLC and RP-HPLC methods for
56 determination of Paracetamol (PCM) and Pamabrom (PAM) in presence of P-aminophenol (PAP)
57 and Theophylline (THEO) as potential impurities of both drugs respectively.

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58

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59 2.Experimental

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60 2.1. Apparatus
61 2.1.1. For HPTLC method

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62 HPTLC aluminum plates (20 x 20 cm) coated with 0.25 mm silica gel 60 F254 (Merck, Germany),
63 TLC scanner 3 densitometer (Camag, Muttenz, Switzerland), Linomat IV with 100 L syringe
64

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(Camag, Muttenz, Switzerland), Sonix TV ss-series ultrasonicator (USA) and UV lamp with short
65 wavelength 254 nm (Vilber Lourmat, Marne La Vallee, Cedex, France).
66 2.1.2. For RP-HPLC method
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67 HPLC (Shimadzu) instrument was equipped with a model series LC-10 ADVP pump, SCL-10 AVP
68 controller, DGU-12 A degasser and SPD-10 AVP UV-VIS detector. Separation and quantitation
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69 were made at room temperature on a 250 mm 4.6 mm (i.d.) RP C8 column (4.6 m particle size).
70 The detector was set at 277 nm.
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71
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72 2.2. Materials and reagents

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2.2.1 Pure standard

74 PCM and PAM were kindly supplied by SIGMA Pharmaceutical Industries, Cairo, Egypt. Purity
75 was reported to be 99.4% and 99.84% for PCM and PAM respectively according to the companys
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76 analysis certificate.
77 2.2.2 Pharmaceutical formulation
78 Menobrocond film coated tablets (Batch No.32801) are labeled to contain 500 mg of PCM and 25
79 mg of PAM per tablet; it is manufactured by SIGMA Pharmaceutical Industries.
80 2.2.3 Chemicals
81 All chemicals and solvents used throughout this work were of analytical grade. PAP was purchased
82 from Riedel-dehaen-AG- Germany; with certified purity of 99%. THEO was supplied from Sigma-
83 Aldrich (St. Louis, MO, USA) with certified purity of 99% according to analysis certificate.
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 84 Others include chloroform and methanol, HPLC grade (Sigma Aldrich, Chemie GmbH,
85 Germany), Ethyl acetate and glacial acetic acid (El-Nasr Pharmaceutical Chemicals Co., Cairo,
86 Egypt).
87 2.3 Preparation of standard solutions
88 Stock standard solutions of PCM, PAM, PAP and THEO were prepared in methanol in the

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89 concentration of (1 mg/mL). Working standard solution of PCM, PAM, PAP and THEO were

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90 prepared in methanol in the concentration of (100 g/mL).

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91 2.4 Laboratory prepared mixtures
92 Mixtures containing different ratios of PCM, PAM, PAP and THEO were prepared using their

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93 respective stock standard solutions in methanol.
94 2.5 Sample preparation
95 Ten film coated tablets of Menobrocond were finely powdered and mixed well. An amount
96
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equivalent to 25 and 500 mg of PAM and PCM, respectively, was accurately weighed, transferred
into a 25mL volumetric flask, and dissolved in 20 mL methanol. The solution was ultrasonicated
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98 for 30 min, filtered and then completed to volume with methanol.
99 Part of the solution was diluted to obtain (100g/mL) working solution for PAM and another part
100
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was diluted to obtain (100g /mL) working solution for PCM using methanol as a solvent.
101
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102 2.6 Method development

103 Most of reported methods were for determination of PCM and PAM alone without taking into
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104 consideration their potential impurities and due to the nephrotoxic effect of PAP and the different
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105 pharmacological action of THEO from the parent drug PAM, Hence, it was necessary to develop
106 and validate simple, sensitive and selective HPTLC and RP-HPLC methods for simultaneous
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107 determination of PCM, PAM in presence of their potential impurities in bulk material and in their
108 pharmaceutical formulation.
109
110 2.6.1 Construction of calibration curves
111 HPTLC method
112 Into a set of 10-mL volumetric flasks, different aliquots equivalent to 0.6 2.4, 0.6 2, 0.4 2.8
113 and 0.4 1.8 mg of each of PCM, PAM, PAP and THEO respectively were accurately transferred
114 from their standard stock solution (1 mg/mL), and the volume was completed with methanol.

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115 10 L of each solution was applied to HPTLC plates as bands of 6 mm width using Camag Linomat
116 IV applicator. The bands were spaced 5mm from each other and 10 mm apart from the bottom edge
117 of the plate. Linear ascending development was performed in a chromatographic chamber
118 previously saturated with chloroform, methanol, ethyl acetate and glacial acetic acid solution
119 (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.

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120 The integrated peak areas were recorded using scanning wavelength at 254 nm under the specified

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121 instrumental conditions. The calibration curves were constructed by plotting the integrated peak

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122 area/104 versus the corresponding concentrations of each component and regression equations were
123 computed.

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124
125 RP-HPLC method
126 Accurate aliquots equivalent to 60 520, 40 400, 50 240, 20 240 g were separately transferred
127
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from their respective working standard solutions (100 g/mL) into four separate series of 10-mL
volumetric flasks and the volume was completed by the mobile phase. Triplicate injections were made
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129 for each concentration; the peak area/103 was used to construct the calibration curve for each
130 component from which its regression equation was computed. Chromatographic separation was
131
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carried out by gradient elution using sodium dihydrogen phosphate buffer: methanol: acetonitrile
132 (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
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133 (70:20:10, v/v/v) at a flow rate of 1.5 mL/min for the next 5 minutes. Injection volume was 20 L and
134 scanning was carried out at 277 nm at room temperature and the run time was 12.5 min.
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136 2.6.2 Application to pharmaceutical formulation

137 Different concentrations (within the linearity range of each drug) were prepared from the
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138 previously prepared pharmaceutical formulation working solution (100 g/mL) and the procedure
139 mentioned under linearity and construction of calibration curves was followed. Concentrations of
140 PCM and PAM were calculated from their respective regression equations and the percentage
141 recoveries were then calculated.
142
143 3. Results and Discussion
144 HPTLC and RP-HPLC are useful techniques for resolution and determination of drug mixtures.
145 These techniques offer a simple way to quantify studied drugs in presence of their potential

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146 impurities. As reported in USP [1] PAP and THEO are considered as the potential impurities for
147 PCM and PAM , and most of the methods reported in the literature determined PCM and PAM in
148 their binary mixtures without taking into consideration the importance of determination of their
149 potential impurities. Hence the presented work aims to develop and validate a highly selective
150 analytical methods for simultaneous determination of PCM and PAM in presence of their potential

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151 impurities. These methods offer high sensitivity and selectivity for analysis of PCM and PAM in

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152 presence of PAP and THEO which are the potential impurities of both drugs respectively.

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153
154 3.1 Method optimization

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155 HPTLC method
156 In order to optimize the developed HPTLC method, it was necessary to investigate the effect of
157 different factors to get the desired chromatographic resolution.
158
159
a- Mobile phase

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Different developing systems of different composition and ratios were tried for separation, e.g.,
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160 chloroform-methanol-glacial acetic acid (8:1:0.2, v/v/v) showed good separation between PCM and
161 PAP but separation between PAM and THEO wasnt satisfying, chloroform-methanol-ethyl acetate
162
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(7.5:0.5: 0.5, v/v/v) , ethyl acetate enhanced separation between PAM and THEO but wasnt
163 suitable for separation of PCM and PAM, and chloroform-ethyl acetate-glacial acetic acid
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164 (7.5:2.5:0.5, v/v/v) in which PAP didnt move from baseline which showed importance of methanol
165 for seperation. The best mobile phase was chloroform-methanol-ethyl acetate-glacial acetic acid
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166 (8:0.8:0:6:0.2, v/v/v/v).This selected mobile phase allows good separation between the quaternary
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167 mixtures with good Rf values (3.21, 7.11, 8.26, 11.49 min.) for PCM, PAP, PAM and THEO
168 respectively without tailing of the separated bands as shown in Figure 2.
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169 b- Scanning wavelength

170 Different scanning wavelengths were tried (220, 254 and 278 nm) in order to obtain good
171 sensitivity of PCM, PAM in presence of PAP and THEO with minimum noise. The wavelength 254
172 nm was found to be the best wavelength regarding sensitivity of all components. Peaks were sharp
173 and symmetrical with minimum noise, as shown in Figure 2.
174 c- Band dimensions
175 The slight spread of the developed bands due to ordinary diffusion should be taken into
176 consideration, so the band width and interspaces between bands should be chosen carefully to avoid

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177 spread of bands outside the scanning tracks and interference between adjacent bands.
178 Different band dimensions were tried to obtain sharp and symmetrical peaks. The optimum band
179 width chosen was 6 mm and the inter-space between bands was 5 mm.
180 d- Slit dimensions of scanning light beam
181 The slit dimensions of the scanning light beam should ensure complete coverage of band

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182 dimensions on the scanned track without interference of adjacent bands. Different slit dimensions

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183 were tried where 6 mm0.3 mm proved to be the slit dimensions of choice that provided highest

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184 sensitivity.
185

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186 RP-HPLC
187 To optimize the proposed RP-HPLC method, it was necessary to test the effects of different
188

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parameters that affect the sensitivity, selectivity and the efficiency of the chromatographic
189 separation.
190 a- Mobile phase
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191 Different mobile phases with different compositions and polarities were tested to achieve the
192 chromatographic separation e.g: water methanol, wateracetonitrile, sodium dihydrogen
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193 phosphate buffermethanol, sodium dihydrogen phosphate bufferacetonitrile. Complete

194 separation between studied components was achieved by gradient elution using sodium dihydrogen
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195 phosphate buffer (0.05M)methanolacetonitrile. Additionaly the effect of sodium dihydrogen

196
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phosphate buffermethanolacetonitrile ratio was studied to improve resolution. Maximum

197 resolution was obtained using sodium dihydrogen phosphate buffer (0.05M)methanolacetonitrile
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198 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
199 was obtained at retention times (tR) (3.21, 7.11, 8.26, 11.49 min.) for PCM, PAP, PAM and THEO
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200 respectively as shown in Figure3.

201 b- pH effect

202 Different pH values (3 6) were tested where pH = 4 gave the best chromatographic resolution

203 between PCM, PAM and their potential impurities.

204 c- Scanning wavelength
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205 Different scanning wavelengths were tried (254, 270, 277 nm) in order to enhance the sensitivity
206 of the method where scanning at 277 nm gave good sensitivity for all the studied components as
207 shown in Figure 3.
208 d- Flow rate
209 Different flow rates were also tested to provide the best separation within acceptable run time. The

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210 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.

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211

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212 Method validation
213 ICH guidelines [10] for analytical method validation were followed.

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214 -Linearity
215 Under optimum chromatographic conditions, linear relationships were obtained between the
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integrated peak area/104 versus the corresponding concentrations for HPTLC and integrated peak
217 area/103 versus the corresponding concentrations for RP-HPLC; results were calculated and
218 presented in Table 1.
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219 -Accuracy
220 Accuracy was calculated as the percentage recoveries of blind pure PCM, PAM, PAP and THEO, it
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221 was further assured by performing standard addition technique at three levels and the average
222 percentage recovery was then calculated and presented in Table 2.
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223 -Precision
224
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Both repeatability and intermediate precision were studied. Repeatability was calculated by the
225 analysis of three different concentrations of pure components in triplicates on the same day. The
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226 experiment was repeated on the same concentrations seven times on four consecutive days to
227 determine the intermediate precision. Good results and acceptable RSD% were obtained, Table 1.
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228 -Specificity
229 The specificity of the method was confirmed by how accurately and specifically the analytes of
230 interest are determined in presence of other components (impurities, degradates or excipients) [11].
231 Specificity was confirmed as shown in the HPTLC and RP-HPLC chromatograms, Figures 2 & 3.
232 Furthermore, good results were obtained on applying the method on Menobrocond film coated
233 tablets; Table 2 proves that tablet additives do not interfere with any of the separated components.
234 -Limits of detection and quantitation (LOD and LOQ)

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235 LOD and LOQ were calculated using the following equations [11]. The low values of LOD and
236 LOQ indicate the high sensitivity of proposed methods, Table 1.
237 LOD = 3.3 x SD / slope. LOQ = 10 x SD / slope.
238
239 -Robustness

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240 Deliberate small changes in HPTLC method parameters (e.g. changing in % chloroform in the

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241 mobile phase 1% and change in scanning wavelength 1nm) and in RP-HPLC method parameters

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242 (e.g. changing in % methanol in the mobile phase 1%, changing in flow rate 0.05 mL/ min and
243 change in scanning wavelength 1 nm) didn't lead to significant changes in changes in Rf and tr

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244 values respectively, peak height or symmetry of the peaks.
245 -System suitability
246 System suitability parameters were carried out to prove that the overall system performed well, it
247
248 an
was checked by calculating different parameters and the obtained values were in the acceptable
ranges according to USP as shown in Table 3.
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249
250 Results obtained by the suggested HPTLC and RP-HPLC methods for determination of PCM and
251
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PAM in their pharmaceutical formulation were statistically compared to those obtained by applying
252 the reported HPLC method [6]. For HPTLC, the calculated t- values were (0.055, 0.166) and F-
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253 values were (1.027, 2.645) for PCM and PAM respectively. For RP-HPLC, calculated t- values
254 were (0.612, 0.074) and F-values were (4.303, 3.719) for PCM and PAM respectively. The
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255 obtained values were found to be less than the theoretical ones, confirming accuracy and precision
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256 at 95% confidence level.

257
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258 4. Conclusion
259 The present work is concerned with the development and validation of HPTLC and RP-HPLC
260 methods for simultaneous determination of PCM, PAM, PAP and THEO without sample pre-
261 treatment and without interference from pharmaceutical formulation excipients. The developed
262 methods have advantage over any reported method in being able to determine the studied drugs
263 along with their potential impurities with high sensitivity, selectivity and short analysis time using
264 one simple mobile phase for all components. Moreover, these methods were successfully applied
265 for determination of PCM and PAM in Menobrocond film coated tablets without interference
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266 from pharmaceutical formulation excipients. The developed methods can be easily applied for
267 quality control of the studied drugs.
268
269
270 6. References:

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271 1. The United States Pharmacopeia, 32 Ed., National Formulary 27, United States Pharmacopeial
272 convention INC, USA (2009).

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273 2. Martindale. The complete drug reference The Extra Pharmacopoeia, 31st Edition
274 Pharmaceutical press London, 2007.

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275 3. "The British Pharmacopoeia", The Stationary Office, London (2009).
276 4. H. Xue, J. Liu, H. Liu, G. WANG, W. LU , Simultaneous determination of paracetamol and

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277 caffeine in human plasma by HPLC, Chin. J. Hosp. Pharm. (2005). 25(8), 708.
278 5. L.J. Zhou, L.H. Gu, Y.J. Wang, J.Y. Liang, Determination of two constituents in compound
279 acetaminophen and pamabrom tablets in human plasma by high performance liquid
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280 chromatography, Chin. J. New Drugs Clin. Remedies. 2007; 3:008.
281 6. D. Shah, B. Patel, A. Bhavsar, Development and Validation of Simultaneous Estimation of
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282 Paracetamol and Pamabrom in Bulk and Combined Pharmaceutical Dosage Form by RP-HPLC.
283
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Research & Reviews: JPPS. 2014; 3(2):16-23.

284 7. S. Bambhrolia, S.J. Rajput, Simultaneous estimation of Paracetamol and Pamabrom in bulk drugs
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285 and in pharmaceutical formulation by spectrophotometry, Int. J. ChemTech. Res. 2013; 5(4):1802-
286
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7.
287 8. P. Hardik, M. Bhati, V. Sanjaysinh, P. Hemant, Development and Validation of UV
288 Spectrophotometric methods for estimation of Paracetamol and Pamabrom in bulk and synthetic
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289 mixture by Simultaneous equation method. Inventi Rapid: Pharm. Anal. Q. A. 2012.
290 9. O.M. El-Houssini, RP-LC and TLC Densitometric Determination of Paracetamol and Pamabrom
291 in presence of Hazardous Impurity of Paracetamol and Application to pharmaceuticals, Anal.
292 Chem. Insights. 2013; 8:73.
293 10. ICH, Q2 (R1) Validation of Analytical Procedures, Proceedings of International Conference on
294 Harmonization, Geneva, 2005.
295 11.USFDA guidance. Analytical Procedures and Methods Validation. Food and Drug
296 Administration. Rockville. 2000.
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297 GRAPHICAL ABSTRACT
298 HPTLC and RP-HPLC methods for Simultaneous Determination of
299 Paracetamol and Pamabrom in Presence of Their Potential
300 Impurities
301
302

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303
304 3D HPTLC chromatogram of resolved mixture of P-aminophenol (Rf =
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305 0.11), Paracetamol (Rf=0.33), Theophylline (Rf = 0.48) and Pamabrom

306 (Rf=0.65) using (chloroform: methanol: ethyl acetate: glacial acetic
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307 acid) (8: 0.8: 0.6: 0.2, by volume) as a developing system and scanning
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308 at 254 nm.

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309
310 RP-HPLC chromatogram of separated peaks of P-aminophenol (Rt =
311 3.21 min), Paracetamol (Rt=7.11 min), Theophylline (Rt = 8.26 min)

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312 and Pamabrom (Rt=11.49 min) by gradient elution using phosphate
313 buffer: methanol: acetonitrile (85:10:5 by volume) as a mobile phase
314 delivered at a flow rate of 1 mL / min for first 7.5 minutes and
315 (70:20:10) at a flow rate of 1.5 mL / min for the next 5 minutes and
316 scanning at 277 nm.
317

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317 Highlights:
318 HPTLC & RP-HPLC methods for determination of PCM and PAM
319 were developed.
320 The two developed methods are validated and met ICH
321 requirements.
322 Developed methods are able to determine PCM & PAM in presence

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323 of their impurities.

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324
325

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325 Figures
326

Paracetamol
(PCM),

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327

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328 acetaminophen
329 C8H9NO2 Figure 1.a. Chemical structure of Paracetamol (PCM)

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Pamabrom (PAM)
C11H18BrN5O3
Mol. Wt. 348.20

330
331 an
Figure 1.b. Chemical structure of Pamabrom (PAM)
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P-
aminophenol
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(PAP),
332 C6H7NO
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333 Figure 1.c. Chemical structure of P-aminophenol (PAP)

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Theophylline (THEO)
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C7H8N4O2
Mol. Wt. 180.17

334
335 Figure 1.d. Chemical structure of Theophylline (THEO)
336

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336
337

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338
339
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340
341 Figure 2. HPTLC chromatogram of resolved mixture of PAP (Rf = 0.11), PCM (Rf=0.33), THEO
342 (Rf = 0.48) and PAM (Rf=0.65) using (chloroform: methanol: ethyl acetate: glacial acetic acid)
343 (8: 0.8: 0.6: 0.2, by volume) as a developing system and scanning at 254 nm.
344

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344
345
346

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347

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348
349 Figure 3. RP-HPLC chromatogram of separated peaks of PAP (tR = 3.21 min), PCM (tR=7.11
350 min), THEO (tR = 8.26 min) and PAM (tR=11.49 min) by gradient elution using phosphate
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351 buffer: methanol: acetonitrile (85:10:5 by volume) as a mobile phase delivered at a flow rate
352 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
353 minutes and scanning at 277 nm.
354
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 354 Table1: Regression and analytical parameters of the proposed HPTLC & RP-HPLC methods for
355 determination of PCM and PAM in presence of PAP and THEO as potential impurities of PCM
356 and PAM respectively.
HPTLC RP-HPLC

Parameters PCM PAM PAP THEO PCM PAM

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Calibration range 0.6-2.4 g/band 0.6-2 g/band 0.4-2.8 g/band 0.4- 6-52 g/mL 4-40

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Slope 0.472 0.191 0. 251 1.8g/band 0.018 0.0
Intercept 0.404 0.258 0.345 0.469 0.006 0.0
Correlation coefficient 1 0.9999 0.9999 0.196 1 1

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0.9999
Accuracy% (mean 100.000.870 100.31 0.671 100.30 0.513 100.02 0.718 100.210.425 100.36

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SD)
Repeatability (RSD%) 0.676 0.579 1.046 0.720 0.489 0.9
Intermediadte Precision 1.458 0.969 1.166 1.006 0.738 1.1

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(RSD%)
LOD 0.166 g/band 0.143 g/band 0.131 g/band 0.080 g/band 1.560 g/mL 0.815
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LOQ 0.503 g/band 0.435 g/band 0.397 g/band 0.243 g/band 4.728 g/mL 2.468

357*(RSD%)a* and (RSD%)b*; the intra- and inter-day relative standard deviation of concentrations (0.8, 1.6, 2 g/ band) for
358PCM, (0.8, 1.6, 2 g/ band) for PAM, (1.6, 2, 2.4 g /band) for PAP and (0.8, 1.6, 1.8 g/ band) for THEO determined by
359HPTLC and (12, 20, 24 g/ mL) for PCM, (10,16 , 20g /mL) for PAM, (14,16 , 24 g/ mL) for PAP and (12, 16 , 20 g /
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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

od HPTLC RP-HPLC

t
PCM PAM PCM

ip
cr
Taken 1 g/band 0.8 g/band 10 g/mL 1
nd
d

us
Found % * SD 100.00 1.448 101.05 1.090 99.99 1.004 100

an
Pure added Recovery Pure added Recovery % Pure added Recovery % Pure add
Standard addition (g/band ) % (g/band ) (g/mL) (g/mL )
mg technique 101.96 100.24
1 102.75 0.8 102.30 10 99.80 10
M
1.2 101.71 1 100.17 12 100.09 12
/ 1.4 100.94 1.2 14 14
d

mean SD 101.80 0.906 101.48 1.142 99.

te

100.04 0.224

364
p

365 Table 3: Parameters of system suitability of the developed HPTLC & RP-HPLC methods for the
366 determination of PCM, PAM, PAP and THEO.
ce

HPTLC RP
Method
Parameters PCM PAP PAM THEO Refrence PCM PAP
Ac

values

Capacity factor (K')

4 1 8 6.4 1-10 4.92 1.68

Symmetry factor 1 1 1 0.88 ~1 1.17 1.12

18
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 Resolution 5.4 2.9 3.69 Rs > 1.5 3.62
(Rs)

Selectivity () 3 1.35 1.45 >1 2.21

t
Column efficiency 1262.03 164.9

ip
(N)

cr
HETP(cm/plate) 0.0198 0.15

us
367

an
368 M
d
p te
ce
Ac

19
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