Journal of Chromatographic Science, 2016, Vol. 54, No. 4, 647–652 doi: 10.1093/chromsci/bmv184 Advance Access Publication Date: 12 January 2016 Article
Article
HPTLC Method for the Determination of Paracetamol, Pseudoephedrine and Loratidine in Tablets and Human Plasma Nehal Fayek Farid and Eglal A. Abdelaleem* Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Bani-Suef University, Alshaheed Shehata Ahmad Hegazy St., 62514 Beni-Suef, Egypt *Author to whom correspondence should be addressed. Email: [email protected] Received 30 August 2014; Revised 10 August 2015
Abstract A sensitive, accurate and selective high performance thin layer chromatography (HPTLC) method was developed and validated for the simultaneous determination of paracetamol (PAR), its toxic impurity 4-aminophenol (4-AP), pseudoephedrine HCl (PSH) and loratidine (LOR). The proposed chromatographic method has been developed using HPTLC aluminum plates precoated with silica gel 60 F254 using acetone–hexane–ammonia (4:5:0.1, by volume) as a developing system followed by densitometric measurement at 254 nm for PAR, 4-AP and LOR, while PSH was scanned at 208 nm. System suitability testing parameters were calculated to ascertain the quality performance of the developed chromatographic method. The method was validated with respect to USP guidelines regarding accuracy, precision and specificity. The method was successfully applied for the determination of PAR, PSH and LOR in ATSHI® tablets. The three drugs were also determined in plasma by applying the proposed method in the ranges of 0.5–6 µg/band, 1.6–12 µg/band and 0.4–2 µg/band for PAR, PSH and LOR, respectively. The results obtained by the proposed method were compared with those obtained by a reported HPLC method, and there was no significance difference between both methods regarding accuracy and precision.
Introduction Paracetamol (PAR) is acetamide, N-(4-hydroxy phenyl) (1, 2), which is widely used as a minor analgesic, and is used as an alternative to aspirin without the side effects of salicylate on gastric mucosa (3). 4-Aminophenol (4-AP) is considered to be PAR impurity and related substance (1, 2), which has nephrotoxic (4) and teratogenic (5) effects. Pseudoephedrine HCl (PSH) is (1S,2S)-2-(methylamino)-1phenylpropan-1-ol hydrochloride (2). It is a stereoisomer of ephedrine and has similar action. PSH and its salts are given by mouth for the symptomatic relief of nasal congestion and are commonly combined with other ingredients in preparations intended for the relief of cough and cold symptoms (6). Loratadine (LOR) (ethyl-4-(8-chloro-5,6-dihydro-11H-benzo[5,6] cyclohep-ta[1,2-b]pyridin-11-ylidine)-1-piperidine carboxylate) is a second-generation antihistamine. LOR is a selective and moderately strong antagonist of histamine H1 receptors (7). Reviewing the
literature in hand, different methods have been reported for determination of PAR, PSH and LOR. Two reported methods have been published for determination of PAR and LOR in their binary mixtures using the RP-HPLC method. Chromatographic separation was achieved isocratically using methanol–acetonitrile–water–THF (50:40:10:2.5%, by volume) as mobile phase at a flow rate of 0.8 mL/min (8) and the TLCdensitometric method (9). Different methods have been reported for determination of PSH and LOR using spectrophotometric techniques (10–13), HPLC method (12–15) and high performance thin layer chromatography (HPTLC). The HPTLC method employs a silica gel 60 F254 on Al foil and a mobile phase comprising n-hexane–dichloromethane–triethylamine in a ratio of (5.5 : 4.0 : 0.5, by volume). Detection was carried out at 235 nm (16). PAR and PSH were determined in the presence of cetirizine using the RP-HPLC method (17).
© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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For the ternary mixture of PAR, PSH and LOR, only two methods have been published. The first method used liquid chromatography-tandem mass spectrometry with a monolithic column. Separation was achieved using a gradient component of methanol–0.1% formic acid at a flow rate of 1.0 mL min−1 (18). The second method employed HPLC for separation of the three drugs using a CN column and mixture of water (0.05% triethylamine, adjusted to pH 3 with phosphoric acid): acetonitrile (45:55) as a mobile phase, and the flow rate was at 1.0 mL min−1 with UV detection at 216 nm (19). Reviewing the literature up to the present reveals that there were only two HPLC methods for the simultaneous determination of the three studied drugs. The developed HPTLC method is more selective than the reported HPLC method (19) as it can be used for the simultaneous determination of PAR, PSH and LOR together with the nephrotoxic, teratogenic 4-PA and can be considered as time and cost-effective in comparison to the published HPLC method; moreover, our study was also extended to the in vitro determination of the three drugs in spiked human plasma. No reported method, to the best of our knowledge, was found for the simultaneous determination of PAR, PSH and LOR together with the nephrotoxic, teratogenic 4-PA. This encouraged us to develop a simple, sensitive, selective HPTLC method for their determination.
Solutions Stock standard solution (1 mg/mL) of PAR, 4-AP,PSH and LOR were prepared in methanol. Pharmaceutical dosage form solution Ten tablets were crushed and triturated well in a mortar. The average tablet weight was determined, and a powder sample equivalent to 500 mg of PAR was transferred into a 100-mL volumetric flask. About 75-mL methanol was added, and the flask was sonicated for 15 min. The solution was filtered, and the volume was completed to the mark with methanol.
Apparatus The used apparatuses are as follows: CAMAG Linomat 5, autosampler (Switzerland); HPTLC aluminum plates, precoated with silica gel 60 F254 (20 × 20 cm), 0.2 mm thickness (Merck, Germany); CAMAG microsyringe, 100 µL (Switzerland); glass chamber (Macherey-Nagel, Germany); UV lamp-short wavelength 254 nm; and CAMAG HPTLC Densitometric Scanner 3S/N 130319 with WINCATS software (CAMAG, Muttenz, Switzerland).
Procedure
Experimental Samples Pure samples Pure samples were kindly supplied by Al Rowad Pharmaceutical Industries Co., with certified purities of 99.84, 98.50 and 100.07% for PAR, PSH and LOR, respectively. Pure standard 4-AP was purchased from Sigma-Aldrich Co., Cairo, Egypt, with a certified purity of 99.56%. Pharmaceutical samples ATSHI® tablets (batch no. 2234), manufactured by Al Rowad Pharmaceutical Industries Co., 10th of Ramadan City, Egypt, were labeled to contain 500 mg PAR, 120 mg PSH and 5 mg LOR. Chemicals and reagents Acetone, hexane, methanol and ammonia 33% were of analytical grade (El Nasr Pharmaceutical Chemicals Co., Abu-Zabaal, Cairo, Egypt).
Chromatographic conditions Samples were applied in the form of bands of 6 mm width with a 100-µL sample syringe on aluminum plates precoated with silica gel 60F254 (20 × 10 cm), using an autosampler. A constant application rate of 0.1 µL/s was used, and the space between bands was 8.9 mm. The slit dimension was 6.0 × 0.3 µm, and the scanning speed was 20 mm/s. The mobile phase consisted of acetone–hexane– ammonia (4:5:0.4, by volume). Linear ascending development was carried out in a glass chamber saturated with the mobile phase. Development of the plates was left till the mobile phase migrates 8 cm. Following the development, the plates were air dried, spots were visualized under a UV lamp at 254 nm and densitometric scanning was performed using a CAMAG TLC scanner in the reflectance–absorbance mode at 254 nm for PAR, 4-AP and LOR and at 208 nm for PSH and operated by WINCATS software. The radiation source was a deuterium lamp.
Table I. Regression and Analytical Parameters of the Proposed HPTLC-Densitometric Method for the Determination of PAR, 4-Amino Phenol, LOR and Pseudoephedrine Parameters
PAR
4-AP
LOR
Pseudoephedrine
Linearity Range (µg/band) Slope Intercept Correlation (r) Accuracy (mean ± % RSD) Robustness ±5 mL hexane in 100 mL (% RSD) ±0.05 mL NH3 in 100 mL (% RSD) Duration of saturation (±5 min) Precision (% RSD) Repeatability* Intermediate precision**
0.1–6
0.2–3.5
0.1–2
1.6–12
−678.59a, 8299.1b 976.14 0.9995 99.07 ± 2.198
−130.71a, 1469.4b 4372.5 0.9996 100.26 ± 1.413
−1568a, 7459.1b 189.85 0.9996 99.67 ± 0.867
244.78 204.9 0.9987 100.20 ± 2.309
1.682 2.174 No change in Rf
2.776 1.370 No change in Rf
1.526 2.820 No change in Rf
1.323 2.237 No change in Rf
1.517 1.843
1.601 1.956
2.128 2.260
1.330 1.764
Following a polynomial regression A ¼ aX2 þ bX þ c; where A is the peak area, X is the concentration in µg/band, a and b are coefficients of the slope and c is the intercept. *The intraday and **the interday (n = 9) average of three different concentrations (1, 2 and 4 µg/band for PAR; 0.6, 1 and 2 for para-amino phenol; 0.4, 0.6 and 2 µg/band for LOR and 1.6, 4 and 6 µg/band for pseudoephedrine).
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PAR, PSH and LOR in Tablets and Human Plasma Calibration Volumes equivalent to 0.1–6 mL for PAR, 0.2–3.5 mL for 4-AP, 1.6– 12 mL for PSH and 0.1–2 mL LOR from stock standard solution of each were accurately transferred into four series of 10-mL volumetric flasks, and the volumes were completed to the mark with methanol. Then, 10 µL from each flask was spotted in replicates on HPTLC plates. The procedure under chromatographic conditions was performed. The peak areas were plotted against concentrations to obtain the calibration graphs.
solution was applied to the HPTLC plate and the procedure was followed as described under calibration. The regression equations were calculated.
Application to human plasma Into a series of 10 mL volumetric flasks, 1 mL of drug-free human plasma sample was spiked with different concentrations of PAR, PSH and LOR from their stock solutions (1 mg/mL). The volumes were completed to the mark with methanol to provide final concentrations of 0.5–6, 1.6–12 and 0.4–2 mg/mL for PAR, PSH and LOR, respectively. The flasks were shaken vigorously and then centrifugated at 3,000 rpm for 15 min. Then, 1 mL of the protein-free supernatant was transferred into four series of 10-mL volumetric flasks, and the volume was completed with methanol. Furthermore, 10 µL of each
Application to pharmaceutical dosage form The procedure under calibration was followed using pharmaceutical dosage form solution.
The freeze–thaw stability Aliquots equivalent to 2, 4 and 0.8 µg/band for PAR, PSH and LOR, respectively, were prepared. These samples were subjected to three cycles of freeze–thaw operations in three consecutive days (at −25°C).
Results The HPTLC method offers a simple way to quantify directly on a HPTLC plate by measuring the optical density of the separated bands. The amounts of compounds are determined by comparing to a standard curve from reference materials chromatographed simultaneously under the same condition (20). The HPTLC densitometric method has advantages of low operating costs and high sample output, and the need for minimal sample preparation and mobile phase having pH 8 or more can be used (21). Although the proportion of the three studied drugs in their pharmaceutical formulation is complex (500:120:5, for PAR, PSH and LOR, respectively), the proposed method has offered a solution to this problem as HPTLC is a separation method. The calibration curves were constructed by plotting the peak areas versus the corresponding concentrations, and the regression equations were calculated for the three drugs and 4-AP. The results are summarized in Table I.
Discussion Method optimization
Figure 1. HPTLC chromatogram of a mixture of 2 µg/band PAR, 0.5 µg/band 4-AP, 0.5 µg/band LOR and 2 µg/band pseudoephedrine at 208 nm. This figure is available in black and white in print and in color at JCS online.
Several developing systems were tried to reach the optimum resolution of the four components. Trails were done using chloroform–methanol– acetone (9.5, 0.5, 0.2, by volume), chloroform–methanol–ammonia (9, 1, 0.2, by volume), chloroform–methanol–glacial acetic acid (9.5, 0.5, 0.25, by volume), hexane–ethyl acetate–triethylamine (6, 4, 0.1, by volume) and hexane–ethyl acetate–water (6, 5, 1, by volume); however, all these systems failed to resolve the four components
Table II. Determination of PAR, LOR and Pseudoephedrine in ATSHI® Tablets by the Proposed HPTLC-Densitometric Method and Results of the Standard Addition Technique Dosage form
ATSHI® tablets claimed to contain 500 µg PAR, 5 µg LOR and 120 µg pseudoephedrine per tablet B. no. 2234
a
Average of six determinations. Average of three determinations.
b
Founda ± % RSD
Compound taken (µg/band)
Taken µg/band
PAR
2
LOR
0.4
103.05 ± 0.755
Pseudoephedrine
4.8
100.82 ± 2.80
98.19 ± 0.755
Standard addition technique Pure added µg/spot
% Foundb µg/spot
1 2 3 0.2 0.4 0.6 2 4 8
97.56 98.10 99.79 99.58 101.06 102.36 99.89 102.38 97.58
Mean ± % RSD 98.48 ± 1.181
101.00 ± 1.377
99.95 ± 2.402
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from each other. Finally, a developing system consisting of hexane–acetone–ammonia (4, 5, 0.1, by volume) was used, which resulted in well sharp well-resolved peaks. Different scanning wavelengths were tried. On using 254 nm for PAR, 4-AP and LOR and 208 nm for PSH, sharper and symmetrical peaks with minimum noise were obtained. The Rf values were 0.18, 0.34, 0.69 and 0.76 for PAR, 4-AP, LOR and PSH, respectively. Typical chromatograms are shown in Figure 1.
Table III. System Suitability Parameters of the Proposed HPTLC-Densitometric Method for PAR, LOR and Pseudoephedrine Parameter
PAR
4-AP
LOR
Pseudoephedrine
Rf Resolution (Rs) Capacity factor (K′) Selectivity (α) Tailing factor
0.18 2 0.82 1.89 1
0.34 5.5 0.66 3.83 1
0.69 2 0.31 1.10 1
0.76 0.24 0.9
Method validation Validation was performed according to USP (1).
Linearity Under optimum chromatographic conditions, linearity of the method was evaluated by measuring the peak area of different concentrations each of PAR, 4-AP, PSH and LOR and then plotting calibration curves relating the peak area against the corresponding concentrations from which the regression equations were constructed. The calibration curve showed good relationship over the concentration ranges of 0.1–6 µg/band for PAR, 0.2–3.5 µg/band for 4-AP, 0.1–2 µg/band for LOR and 1.6–12 µg/band for PSH. Regression equation parameters are listed in Table I.
Accuracy Accuracy was checked by determining nine different concentrations of each of PAR, LOR and PSH in the calibration range. It was further assured by performing recovery studies at three levels (80, 100 and
Figure 2. HPTLC chromatogram of (A) blank plasma at 254 nm, (B) blank plasma at 208 nm, (C) a mixture of 4 µg/band PAR, 0.8 µg/band LOR and 2 µg/band pseudoephedrine in spiked human plasma at 254 nm and (D) a mixture of 4 µg/band PAR, 0.8 µg/band LOR and 2 µg/band pseudoephedrine in spiked human plasma at 208 nm. This figure is available in black and white in print and in color at JCS online.
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PAR, PSH and LOR in Tablets and Human Plasma Table IV. Results of Freeze–Thaw Stability (Three Freeze–Thaw Cycles) (at −25°C for PAR, LOR and Pseudoephedrine) % Remaining after freeze–thaw cyclesa
PAR 2 µg/band
LOR 0.8 µg/band
Pseudoephedrine 4 µg/band
1 2 3
99.83 ± 1.422 99.66 ± 0.976 98.13 ± 2.378
100.55 ± 1.458 98.59 ± 1.574 96.84 ± 0.965
99.52 ± 2.117 98.92 ± 2.772 95.16 ± 1.237
a Calculated as the percentage of the initial concentration and expressed as mean ± SD.
Table V. Statistical Comparison of the Proposed HPTLC Method with the Reported HPLC Method Parameters
PAR
PSH
LOR
Degree of freedom F-test Student’s t-test
12 2.916 (4.283)a 0.928 (2.178)a
12 2.417 (4.283)a 1.1665 (2.178)a
12 3.283 (4.283)a 2.118 (2.178)a
a
The values in parenthesis are the corresponding theoretical values at P = 0.05.
120%), and the average percent recovery was then calculated. Good percentage recoveries were obtained and are given in Table I. Precision The precision of the method was verified by testing repeatability and intermediate precision. Three concentrations of (1, 2, 4 µg/band for PAR, 0.6, 1, 2 µg/band for 4-AP, 0.4, 0.6, 2 µg/band for LOR and 1.6, 4, 6 µg/band for PSH) were analyzed three times intra-daily by the proposed HPTLC method. The percentage recoveries and the relative standard deviation (RSD) were calculated (see Table I). The intermediate precision of the method was checked by repeating the previous procedure inter-daily seven times on four different days. Good results and acceptable RSD% (Table I) were obtained. Specificity Specificity of the method was tested by how accurately and specifically the analytes of interest are determined in the presence of other components including impurities, degradates or excipients (22). The selectivity of the method was achieved by the analysis of laboratory prepared mixtures of the three drugs together with 4-AP (Figure 1). Furthermore, good results obtained on applying the proposed HPTLC method on ATSHI® tablets (Table II), prove that tablet additives do not interfere with any of the three separated components. Robustness The method was demonstrated to be robust over an acceptable working range of its HPTLC co-operational conditions. Any small deliberate variation in the mobile phase and saturation time showed no dramatic change in Rt value, peak height, area or symmetry of the peaks (see Table I). System suitability System suitability parameters including resolution (Rs), peak symmetry, selectivity and capacity factor (K) were calculated to prove that the overall system performed well. The obtained values were in the acceptable ranges as shown in Table III.
Application of the method The proposed method has been successfully applied for the determination of PAR, PSH and LOR in ATSHI® tablets (Figure 2). The accuracy of the method was further assessed by applying the standard addition technique, where good results were obtained and are shown in Table II.
Application to human plasma No significant interference at the Rf of the three proposed drugs was observed in the plasma blank chromatogram at 254 and 208 nm as shown in Figures 2A and B. The proposed HPTLC method was applied for the determination of studied drugs in spiked human plasma in the concentration ranges of 0.5–6, 1.6–12 and 0.4–2 mg/mL for PAR, PSH and LOR with accuracy of the mean percentage recovery of 99.21, 101.02 and 101.20 for PAR, LOR and PSH, respectively (Figure 2C and D).
The freeze–thaw stability The results are shown in Table IV. Table V shows statistical comparison of the results obtained by the proposed method and the reported HPLC method (19) by applying on the dosage form (ATSHI® tablets). The calculated t and F values are less than the theoretical ones, indicating that there is no significant difference between the two methods with respect to accuracy and precision.
Conclusion The HPTLC method was developed for determination of PAR, PSH and LOR in human plasma without interference from biological matrices, which was found to be specific, economical and fast. The proposed method has the advantage over the reported HPLC method in that it offers determination of the three studied drugs in human plasma that can be applied in pharmacokinetic study. On the other hand, the HPTLC method could effectively separate the PAR from its degradation product and it can be employed as a stability indicating method for PAR. Statistical analysis proves that the method is reproducible and selective for the analysis of the studied drugs as bulk drugs and in pharmaceutical formulation
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Article
HPTLC Method for the Determination of Paracetamol, Pseudoephedrine and Loratidine in Tablets and Human Plasma Nehal Fayek Farid and Eglal A. Abdelaleem* Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Bani-Suef University, Alshaheed Shehata Ahmad Hegazy St., 62514 Beni-Suef, Egypt *Author to whom correspondence should be addressed. Email: [email protected] Received 30 August 2014; Revised 10 August 2015
Abstract A sensitive, accurate and selective high performance thin layer chromatography (HPTLC) method was developed and validated for the simultaneous determination of paracetamol (PAR), its toxic impurity 4-aminophenol (4-AP), pseudoephedrine HCl (PSH) and loratidine (LOR). The proposed chromatographic method has been developed using HPTLC aluminum plates precoated with silica gel 60 F254 using acetone–hexane–ammonia (4:5:0.1, by volume) as a developing system followed by densitometric measurement at 254 nm for PAR, 4-AP and LOR, while PSH was scanned at 208 nm. System suitability testing parameters were calculated to ascertain the quality performance of the developed chromatographic method. The method was validated with respect to USP guidelines regarding accuracy, precision and specificity. The method was successfully applied for the determination of PAR, PSH and LOR in ATSHI® tablets. The three drugs were also determined in plasma by applying the proposed method in the ranges of 0.5–6 µg/band, 1.6–12 µg/band and 0.4–2 µg/band for PAR, PSH and LOR, respectively. The results obtained by the proposed method were compared with those obtained by a reported HPLC method, and there was no significance difference between both methods regarding accuracy and precision.
Introduction Paracetamol (PAR) is acetamide, N-(4-hydroxy phenyl) (1, 2), which is widely used as a minor analgesic, and is used as an alternative to aspirin without the side effects of salicylate on gastric mucosa (3). 4-Aminophenol (4-AP) is considered to be PAR impurity and related substance (1, 2), which has nephrotoxic (4) and teratogenic (5) effects. Pseudoephedrine HCl (PSH) is (1S,2S)-2-(methylamino)-1phenylpropan-1-ol hydrochloride (2). It is a stereoisomer of ephedrine and has similar action. PSH and its salts are given by mouth for the symptomatic relief of nasal congestion and are commonly combined with other ingredients in preparations intended for the relief of cough and cold symptoms (6). Loratadine (LOR) (ethyl-4-(8-chloro-5,6-dihydro-11H-benzo[5,6] cyclohep-ta[1,2-b]pyridin-11-ylidine)-1-piperidine carboxylate) is a second-generation antihistamine. LOR is a selective and moderately strong antagonist of histamine H1 receptors (7). Reviewing the
literature in hand, different methods have been reported for determination of PAR, PSH and LOR. Two reported methods have been published for determination of PAR and LOR in their binary mixtures using the RP-HPLC method. Chromatographic separation was achieved isocratically using methanol–acetonitrile–water–THF (50:40:10:2.5%, by volume) as mobile phase at a flow rate of 0.8 mL/min (8) and the TLCdensitometric method (9). Different methods have been reported for determination of PSH and LOR using spectrophotometric techniques (10–13), HPLC method (12–15) and high performance thin layer chromatography (HPTLC). The HPTLC method employs a silica gel 60 F254 on Al foil and a mobile phase comprising n-hexane–dichloromethane–triethylamine in a ratio of (5.5 : 4.0 : 0.5, by volume). Detection was carried out at 235 nm (16). PAR and PSH were determined in the presence of cetirizine using the RP-HPLC method (17).
© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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For the ternary mixture of PAR, PSH and LOR, only two methods have been published. The first method used liquid chromatography-tandem mass spectrometry with a monolithic column. Separation was achieved using a gradient component of methanol–0.1% formic acid at a flow rate of 1.0 mL min−1 (18). The second method employed HPLC for separation of the three drugs using a CN column and mixture of water (0.05% triethylamine, adjusted to pH 3 with phosphoric acid): acetonitrile (45:55) as a mobile phase, and the flow rate was at 1.0 mL min−1 with UV detection at 216 nm (19). Reviewing the literature up to the present reveals that there were only two HPLC methods for the simultaneous determination of the three studied drugs. The developed HPTLC method is more selective than the reported HPLC method (19) as it can be used for the simultaneous determination of PAR, PSH and LOR together with the nephrotoxic, teratogenic 4-PA and can be considered as time and cost-effective in comparison to the published HPLC method; moreover, our study was also extended to the in vitro determination of the three drugs in spiked human plasma. No reported method, to the best of our knowledge, was found for the simultaneous determination of PAR, PSH and LOR together with the nephrotoxic, teratogenic 4-PA. This encouraged us to develop a simple, sensitive, selective HPTLC method for their determination.
Solutions Stock standard solution (1 mg/mL) of PAR, 4-AP,PSH and LOR were prepared in methanol. Pharmaceutical dosage form solution Ten tablets were crushed and triturated well in a mortar. The average tablet weight was determined, and a powder sample equivalent to 500 mg of PAR was transferred into a 100-mL volumetric flask. About 75-mL methanol was added, and the flask was sonicated for 15 min. The solution was filtered, and the volume was completed to the mark with methanol.
Apparatus The used apparatuses are as follows: CAMAG Linomat 5, autosampler (Switzerland); HPTLC aluminum plates, precoated with silica gel 60 F254 (20 × 20 cm), 0.2 mm thickness (Merck, Germany); CAMAG microsyringe, 100 µL (Switzerland); glass chamber (Macherey-Nagel, Germany); UV lamp-short wavelength 254 nm; and CAMAG HPTLC Densitometric Scanner 3S/N 130319 with WINCATS software (CAMAG, Muttenz, Switzerland).
Procedure
Experimental Samples Pure samples Pure samples were kindly supplied by Al Rowad Pharmaceutical Industries Co., with certified purities of 99.84, 98.50 and 100.07% for PAR, PSH and LOR, respectively. Pure standard 4-AP was purchased from Sigma-Aldrich Co., Cairo, Egypt, with a certified purity of 99.56%. Pharmaceutical samples ATSHI® tablets (batch no. 2234), manufactured by Al Rowad Pharmaceutical Industries Co., 10th of Ramadan City, Egypt, were labeled to contain 500 mg PAR, 120 mg PSH and 5 mg LOR. Chemicals and reagents Acetone, hexane, methanol and ammonia 33% were of analytical grade (El Nasr Pharmaceutical Chemicals Co., Abu-Zabaal, Cairo, Egypt).
Chromatographic conditions Samples were applied in the form of bands of 6 mm width with a 100-µL sample syringe on aluminum plates precoated with silica gel 60F254 (20 × 10 cm), using an autosampler. A constant application rate of 0.1 µL/s was used, and the space between bands was 8.9 mm. The slit dimension was 6.0 × 0.3 µm, and the scanning speed was 20 mm/s. The mobile phase consisted of acetone–hexane– ammonia (4:5:0.4, by volume). Linear ascending development was carried out in a glass chamber saturated with the mobile phase. Development of the plates was left till the mobile phase migrates 8 cm. Following the development, the plates were air dried, spots were visualized under a UV lamp at 254 nm and densitometric scanning was performed using a CAMAG TLC scanner in the reflectance–absorbance mode at 254 nm for PAR, 4-AP and LOR and at 208 nm for PSH and operated by WINCATS software. The radiation source was a deuterium lamp.
Table I. Regression and Analytical Parameters of the Proposed HPTLC-Densitometric Method for the Determination of PAR, 4-Amino Phenol, LOR and Pseudoephedrine Parameters
PAR
4-AP
LOR
Pseudoephedrine
Linearity Range (µg/band) Slope Intercept Correlation (r) Accuracy (mean ± % RSD) Robustness ±5 mL hexane in 100 mL (% RSD) ±0.05 mL NH3 in 100 mL (% RSD) Duration of saturation (±5 min) Precision (% RSD) Repeatability* Intermediate precision**
0.1–6
0.2–3.5
0.1–2
1.6–12
−678.59a, 8299.1b 976.14 0.9995 99.07 ± 2.198
−130.71a, 1469.4b 4372.5 0.9996 100.26 ± 1.413
−1568a, 7459.1b 189.85 0.9996 99.67 ± 0.867
244.78 204.9 0.9987 100.20 ± 2.309
1.682 2.174 No change in Rf
2.776 1.370 No change in Rf
1.526 2.820 No change in Rf
1.323 2.237 No change in Rf
1.517 1.843
1.601 1.956
2.128 2.260
1.330 1.764
Following a polynomial regression A ¼ aX2 þ bX þ c; where A is the peak area, X is the concentration in µg/band, a and b are coefficients of the slope and c is the intercept. *The intraday and **the interday (n = 9) average of three different concentrations (1, 2 and 4 µg/band for PAR; 0.6, 1 and 2 for para-amino phenol; 0.4, 0.6 and 2 µg/band for LOR and 1.6, 4 and 6 µg/band for pseudoephedrine).
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PAR, PSH and LOR in Tablets and Human Plasma Calibration Volumes equivalent to 0.1–6 mL for PAR, 0.2–3.5 mL for 4-AP, 1.6– 12 mL for PSH and 0.1–2 mL LOR from stock standard solution of each were accurately transferred into four series of 10-mL volumetric flasks, and the volumes were completed to the mark with methanol. Then, 10 µL from each flask was spotted in replicates on HPTLC plates. The procedure under chromatographic conditions was performed. The peak areas were plotted against concentrations to obtain the calibration graphs.
solution was applied to the HPTLC plate and the procedure was followed as described under calibration. The regression equations were calculated.
Application to human plasma Into a series of 10 mL volumetric flasks, 1 mL of drug-free human plasma sample was spiked with different concentrations of PAR, PSH and LOR from their stock solutions (1 mg/mL). The volumes were completed to the mark with methanol to provide final concentrations of 0.5–6, 1.6–12 and 0.4–2 mg/mL for PAR, PSH and LOR, respectively. The flasks were shaken vigorously and then centrifugated at 3,000 rpm for 15 min. Then, 1 mL of the protein-free supernatant was transferred into four series of 10-mL volumetric flasks, and the volume was completed with methanol. Furthermore, 10 µL of each
Application to pharmaceutical dosage form The procedure under calibration was followed using pharmaceutical dosage form solution.
The freeze–thaw stability Aliquots equivalent to 2, 4 and 0.8 µg/band for PAR, PSH and LOR, respectively, were prepared. These samples were subjected to three cycles of freeze–thaw operations in three consecutive days (at −25°C).
Results The HPTLC method offers a simple way to quantify directly on a HPTLC plate by measuring the optical density of the separated bands. The amounts of compounds are determined by comparing to a standard curve from reference materials chromatographed simultaneously under the same condition (20). The HPTLC densitometric method has advantages of low operating costs and high sample output, and the need for minimal sample preparation and mobile phase having pH 8 or more can be used (21). Although the proportion of the three studied drugs in their pharmaceutical formulation is complex (500:120:5, for PAR, PSH and LOR, respectively), the proposed method has offered a solution to this problem as HPTLC is a separation method. The calibration curves were constructed by plotting the peak areas versus the corresponding concentrations, and the regression equations were calculated for the three drugs and 4-AP. The results are summarized in Table I.
Discussion Method optimization
Figure 1. HPTLC chromatogram of a mixture of 2 µg/band PAR, 0.5 µg/band 4-AP, 0.5 µg/band LOR and 2 µg/band pseudoephedrine at 208 nm. This figure is available in black and white in print and in color at JCS online.
Several developing systems were tried to reach the optimum resolution of the four components. Trails were done using chloroform–methanol– acetone (9.5, 0.5, 0.2, by volume), chloroform–methanol–ammonia (9, 1, 0.2, by volume), chloroform–methanol–glacial acetic acid (9.5, 0.5, 0.25, by volume), hexane–ethyl acetate–triethylamine (6, 4, 0.1, by volume) and hexane–ethyl acetate–water (6, 5, 1, by volume); however, all these systems failed to resolve the four components
Table II. Determination of PAR, LOR and Pseudoephedrine in ATSHI® Tablets by the Proposed HPTLC-Densitometric Method and Results of the Standard Addition Technique Dosage form
ATSHI® tablets claimed to contain 500 µg PAR, 5 µg LOR and 120 µg pseudoephedrine per tablet B. no. 2234
a
Average of six determinations. Average of three determinations.
b
Founda ± % RSD
Compound taken (µg/band)
Taken µg/band
PAR
2
LOR
0.4
103.05 ± 0.755
Pseudoephedrine
4.8
100.82 ± 2.80
98.19 ± 0.755
Standard addition technique Pure added µg/spot
% Foundb µg/spot
1 2 3 0.2 0.4 0.6 2 4 8
97.56 98.10 99.79 99.58 101.06 102.36 99.89 102.38 97.58
Mean ± % RSD 98.48 ± 1.181
101.00 ± 1.377
99.95 ± 2.402
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from each other. Finally, a developing system consisting of hexane–acetone–ammonia (4, 5, 0.1, by volume) was used, which resulted in well sharp well-resolved peaks. Different scanning wavelengths were tried. On using 254 nm for PAR, 4-AP and LOR and 208 nm for PSH, sharper and symmetrical peaks with minimum noise were obtained. The Rf values were 0.18, 0.34, 0.69 and 0.76 for PAR, 4-AP, LOR and PSH, respectively. Typical chromatograms are shown in Figure 1.
Table III. System Suitability Parameters of the Proposed HPTLC-Densitometric Method for PAR, LOR and Pseudoephedrine Parameter
PAR
4-AP
LOR
Pseudoephedrine
Rf Resolution (Rs) Capacity factor (K′) Selectivity (α) Tailing factor
0.18 2 0.82 1.89 1
0.34 5.5 0.66 3.83 1
0.69 2 0.31 1.10 1
0.76 0.24 0.9
Method validation Validation was performed according to USP (1).
Linearity Under optimum chromatographic conditions, linearity of the method was evaluated by measuring the peak area of different concentrations each of PAR, 4-AP, PSH and LOR and then plotting calibration curves relating the peak area against the corresponding concentrations from which the regression equations were constructed. The calibration curve showed good relationship over the concentration ranges of 0.1–6 µg/band for PAR, 0.2–3.5 µg/band for 4-AP, 0.1–2 µg/band for LOR and 1.6–12 µg/band for PSH. Regression equation parameters are listed in Table I.
Accuracy Accuracy was checked by determining nine different concentrations of each of PAR, LOR and PSH in the calibration range. It was further assured by performing recovery studies at three levels (80, 100 and
Figure 2. HPTLC chromatogram of (A) blank plasma at 254 nm, (B) blank plasma at 208 nm, (C) a mixture of 4 µg/band PAR, 0.8 µg/band LOR and 2 µg/band pseudoephedrine in spiked human plasma at 254 nm and (D) a mixture of 4 µg/band PAR, 0.8 µg/band LOR and 2 µg/band pseudoephedrine in spiked human plasma at 208 nm. This figure is available in black and white in print and in color at JCS online.
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PAR, PSH and LOR in Tablets and Human Plasma Table IV. Results of Freeze–Thaw Stability (Three Freeze–Thaw Cycles) (at −25°C for PAR, LOR and Pseudoephedrine) % Remaining after freeze–thaw cyclesa
PAR 2 µg/band
LOR 0.8 µg/band
Pseudoephedrine 4 µg/band
1 2 3
99.83 ± 1.422 99.66 ± 0.976 98.13 ± 2.378
100.55 ± 1.458 98.59 ± 1.574 96.84 ± 0.965
99.52 ± 2.117 98.92 ± 2.772 95.16 ± 1.237
a Calculated as the percentage of the initial concentration and expressed as mean ± SD.
Table V. Statistical Comparison of the Proposed HPTLC Method with the Reported HPLC Method Parameters
PAR
PSH
LOR
Degree of freedom F-test Student’s t-test
12 2.916 (4.283)a 0.928 (2.178)a
12 2.417 (4.283)a 1.1665 (2.178)a
12 3.283 (4.283)a 2.118 (2.178)a
a
The values in parenthesis are the corresponding theoretical values at P = 0.05.
120%), and the average percent recovery was then calculated. Good percentage recoveries were obtained and are given in Table I. Precision The precision of the method was verified by testing repeatability and intermediate precision. Three concentrations of (1, 2, 4 µg/band for PAR, 0.6, 1, 2 µg/band for 4-AP, 0.4, 0.6, 2 µg/band for LOR and 1.6, 4, 6 µg/band for PSH) were analyzed three times intra-daily by the proposed HPTLC method. The percentage recoveries and the relative standard deviation (RSD) were calculated (see Table I). The intermediate precision of the method was checked by repeating the previous procedure inter-daily seven times on four different days. Good results and acceptable RSD% (Table I) were obtained. Specificity Specificity of the method was tested by how accurately and specifically the analytes of interest are determined in the presence of other components including impurities, degradates or excipients (22). The selectivity of the method was achieved by the analysis of laboratory prepared mixtures of the three drugs together with 4-AP (Figure 1). Furthermore, good results obtained on applying the proposed HPTLC method on ATSHI® tablets (Table II), prove that tablet additives do not interfere with any of the three separated components. Robustness The method was demonstrated to be robust over an acceptable working range of its HPTLC co-operational conditions. Any small deliberate variation in the mobile phase and saturation time showed no dramatic change in Rt value, peak height, area or symmetry of the peaks (see Table I). System suitability System suitability parameters including resolution (Rs), peak symmetry, selectivity and capacity factor (K) were calculated to prove that the overall system performed well. The obtained values were in the acceptable ranges as shown in Table III.
Application of the method The proposed method has been successfully applied for the determination of PAR, PSH and LOR in ATSHI® tablets (Figure 2). The accuracy of the method was further assessed by applying the standard addition technique, where good results were obtained and are shown in Table II.
Application to human plasma No significant interference at the Rf of the three proposed drugs was observed in the plasma blank chromatogram at 254 and 208 nm as shown in Figures 2A and B. The proposed HPTLC method was applied for the determination of studied drugs in spiked human plasma in the concentration ranges of 0.5–6, 1.6–12 and 0.4–2 mg/mL for PAR, PSH and LOR with accuracy of the mean percentage recovery of 99.21, 101.02 and 101.20 for PAR, LOR and PSH, respectively (Figure 2C and D).
The freeze–thaw stability The results are shown in Table IV. Table V shows statistical comparison of the results obtained by the proposed method and the reported HPLC method (19) by applying on the dosage form (ATSHI® tablets). The calculated t and F values are less than the theoretical ones, indicating that there is no significant difference between the two methods with respect to accuracy and precision.
Conclusion The HPTLC method was developed for determination of PAR, PSH and LOR in human plasma without interference from biological matrices, which was found to be specific, economical and fast. The proposed method has the advantage over the reported HPLC method in that it offers determination of the three studied drugs in human plasma that can be applied in pharmacokinetic study. On the other hand, the HPTLC method could effectively separate the PAR from its degradation product and it can be employed as a stability indicating method for PAR. Statistical analysis proves that the method is reproducible and selective for the analysis of the studied drugs as bulk drugs and in pharmaceutical formulation
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