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Thin-layer chromatography method for the simultaneous quantification and stability testing of alprazolam and mebeverine in their combined pharmaceutical dosage form
Usmangani K. Chhalotiya *, Nishma M. Patel, Dimal A. Shah, Falgun A. Mehta, Kashyap K. Bhatt
Indukaka Ipcowala College of Pharmacy, Beyond GIDC, P.B. No. 53, Vitthal Udyognagar 388 121, Gujarat, India Received 25 March 2015; received in revised form 8 June 2015; accepted 10 June 2015
Abstract
A sensitive, selective and precise high-performance thin-layer chromatographic method was developed and validated for the simultaneous determination of alprazolam and mebeverine, both as bulk drugs and in formulations. The method employed HPTLC aluminium plates that had been pre-coated with silica gel 60F-254 as the stationary phase, while the solvent system was ace-tone:methanol:acetic acid (6:4:0.1, v/v/v). The Rf values of alprazolam and mebeverine were observed to be 0.80 ±0.08 and 0.60 ± 0.05, respectively. The densitometric analysis was carried out in absorbance mode at 225 nm. The linear regression analysis for the calibration plots showed a good linear relationship for alprazolam and mebeverine over concentration ranges of 600 to 3600ng/spot and 1000 to 6000ng/spot, respectively. The limit of detection and the limit of quantification for alprazolam (mebeverine) were determined to be 63.97 (11.35) ng/spot and 193.85 (34.40) ng/spot, respectively. Alprazolam and mebeverine stock solutions were subjected to acid and alkali hydrolysis, chemical oxidation, dry heat degradation and photo-degradation. Both drugs were found to be susceptible to acid and alkali hydrolysis, chemical oxidation and photo-degradation, whereas both were found to be stable towards dry heat. The degraded product peaks were well resolved from the pure drug peak and displayed a significant difference in their Rf values. Stressed samples were assayed using the developed HPTLC method. The proposed method was validated with respect to linearity, accuracy, precision and robustness. The method was successfully applied to the estimation of alprazolam and mebeverine in marketed formulations. Statistical analysis showed that the method is repeatable, selective, and precise. ©2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Taibah University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Alprazolam; Mebeverine; HPTLC; Stress degradation; Validation
* Corresponding author. Tel.: +91 9924712908. E-mail address: usmangani84@gmail.com (U.K. Chhalotiya). Peer review under responsibility of Taibah University.
1. Introduction
Alprazolam (ALP) is a white powder and is amorphous in nature; it is formally referred to as 8-chloro-1-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3,-a] [1,4]benzodiazepine [1] (Fig. 1A). It binds non-specifically to benzodiazepine receptors BNZ1, which mediates sleep, and BNZ2, which affects muscle
http://dx.doi.org/10.1016/j.jtusci.2015.06.012
1658-3655 © 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Taibah University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Fig. 1. Chemical structure of (A) alprazolam and (B) mebeverine.
relaxation, anticonvulsant activity, motor coordination, and memory [2]. Mebeverine (MEB) is a white powder; chemically, it is known as 3,4-dimethoxybenzoic acid 4-[ethyl[2-(4-methoxyphenyl)-1-methylethyl]amino]-butyl ester [3] (Fig. 1B). It is very soluble in water and alcohol [3]. Mebeverine acts on gut muscles at the cellular level to relieve painful muscle spasms without affecting the gut's normal motility; it is also an inhibitor of calcium depot replenishment. Therefore, it has a dual mode of action which normalizes the small motility. The combination of alprazolam with mebeverine is useful and safe for treatment of irritable bowel syndrome.
The International Conference on Harmonization (ICH) guideline titled 'stability testing of new drug substances and products' requires stress testing to be carried out to elucidate the stability characteristics of active substances [4]. Hydrolytic and photolytic stability, as well as a susceptibility to oxidation, are required tests. An ideal method to indicate stability is one that quantifies the drug and resolves its degradation products.
A survey of the literature revealed that alprazolam can be estimated by spectrophotometry [5] and liquid chromatographic methods [6-11], individually or in combination with other drugs, while mebeverine can be estimated by liquid chromatographic methods individually or concurrently with other drugs [12-16]. The reported methods are highly sophisticated, costly, and time-consuming, often requiring special sample preparation. In comparison with LC and other methods, HPTLC is considered a good alternative, and it should be widely explored as an important tool in routine drug analysis. A major advantage of HPTLC is its ability to simultaneously analyze multiple samples using only a small amount of mobile phase. This reduces time and cost of analysis, minimizes exposure risks, and significantly reduces disposal problems associated with toxic organic
solvents, thereby reducing the possibilities of environmental contamination. HPLC analysis requires more time compared with HPTLC; therefore, HPTLC is a preferred method of analysis. Finally, a method employing UV Spectroscopy has been reported for the simultaneous estimation of both alprazolam and mebeverine in combined dosage form [17].
HPTLC possesses the advantage that a large number of samples can be analyzed in a short period of time. Additionally, a new stationary plate can be used for each analysis, which prevents the problem of analyte carryover that can be found with HPLC. So, for routine quality control analysis, HPTLC is a more preferable method of analysis than HPLC. However, there has been no HPTLC method reported for the combined estimation of ALP and MEB.
2. Experimental
2.1. Instruments
The HPTLC instrument consists of a CAMAG (Mut-tenz, Switzerland) Linomat V sample applicator with 100 |xL syringe (Hamilton, Bonadauz, Switzerland). Chromatography was performed on 10 cm x 10 cm aluminium TLC plates pre-coated with silica gel 60-F254 (E. Merck, Darmstadt, Germany; supplied by Anchrom Technologists, Mumbai, India). A CAMAG TLC scanner 4 was used for the densitometric scanning of the developed chromatogram. All drugs and chemicals were weighed on a Mettler Toledo electronic balance (ME204/A04, METTLER TOLEDO Group).
2.2. Chemicals and reagents
Analytically pure ALP (99.5%) and MEB (99.5%) were obtained as samples from Sun Pharmaceutical Pvt. Ltd., Baroda, India. Methanol was purchased from E. Merck Ltd. (Mumbai, India). Acetone was obtained from Suvidhinath Laboratory (Baroda, India), and acetic acid was obtained from Chemdyes Corporation (Baroda, India).
2.3. Chromatographic system
2.3.1. Sample application
Standards and synthetic mixture samples of ALP and MEB were applied to the HPTLC plates in the form of narrow bands that were 6 mm in length. The bands were applied to the plate at a location that was 10 mm from the
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bottom and 15 mm from left edge. Samples were applied under a continuous drying stream of nitrogen.
2.3.2. Mobile phase and development
Plates were developed using a mobile phase consisting of acetone:methanol:acetic acid (6:4:0.1 v/v/v). Linear ascending development was carried out in a twin-trough glass chamber equilibrated with the mobile phase vapours for 25 min. Ten millilitres of mobile phase (5 mL in the trough containing the plate and 5 mL in the other trough) was used for each development and was allowed to travel 80 mm up the plate. After development, the HPTLC plates were completely dried.
2.3.3. Densitometric analysis
Densitometric scanning was performed in absorbance mode under control by win CATS planar chromatog-raphy software (CAMAG, Muttenz, Switzerland). The source of radiation was a deuterium lamp, and the bands were scanned using 225 nm. The slit dimensions were 5 mm in length and 0.45 mm in width, with a scanning rate of 20 mm/s. The concentrations of the compounds were determined from the intensity of diffusely reflected light and evaluated as a function of peak area versus concentration using a linear regression equation.
2.4. Preparation of standard stock solution
Ten milligrams of standard ALP and MEB were accurately weighed, transferred to two separate 10-ml volumetric flasks and dissolved in a few ml of methanol. The flasks were filled to the mark with methanol to yield solutions containing 1000 ^gml-1 of ALP and MEB. Aliquots from the stock solutions of ALP and MEB were appropriately diluted with methanol to obtain working standards of 120 ^gml-1 of ALP and 200 ^gml-1 of MEB.
2.5. Validation
Validation of the developed HPTLC method was carried out according to the International Conference on Harmonization (ICH) guidelines Q2 (R1) [18].
2.5.1. Linearity of calibration curves
Linearity of the method was evaluated by constructing calibration curves at six concentration levels over a range of 600-3600ng band-1 and 1000-6000ng band-1for ALP and MEB, respectively. The calibration curves were developed by plotting peak area versus concentration (n = 5). The linearity was assessed linear regression.
2.5.2. Accuracy
The accuracy of the method was determined by calculating the recoveries of ALP and MEB by the method of standard additions. Known amounts of ALP (0, 600, 1200, and 1800 ng band-1) and MEB (0, 1000, 2000, and 3000 ng band-1) were taken from the working standard solutions (120 ^g ml- 1of ALP and 200 ^g mL- 1of MEB). These known amounts were then added to pre-quantified sample solutions that contained tablet formulations, from which concentrations of 120 (ig ml-1of ALP and 200 ^g m-1 of MEB were prepared. From these solutions 10 ^l were applied to TLC plates and yielded final concentrations of 1200 ng band-1 ALP and 2000 ng band-1 MEB. The amounts of ALP and MEB were estimated by measuring the areas and fitting the values to the straight-line equations of the calibration curves.
2.5.3. Precision
Precision was evaluated in terms of intra-day and inter-day precisions. Intra-day precision was determined by analysing sample solutions of ALP (600, 1800, and 3000 ng band-1) and MEB (1000, 3000, and 5000ng band-1), at three levels covering low, medium, and high concentrations on the calibration curve, three times on the same day (n = 3). Inter-day precision was determined by analysing sample solutions of ALP (600, 1800, 3000 ng band-1) and MEB (1000, 3000, 5000ng band-1) that covered the same three concentration levels over a period of 3 days (n = 3). The obtained peak areas were used to calculate the mean and the relative standard deviation (% RSD).
Repeatability of the measurement of peak area was determined by analysing ALP (600, 1800, 3000 ng band-1) and MEB (1000, 3000, 5000ng band-1) six times without changing the position of plates.
2.5.4. Specificity
A specificity study was carried out by performing the forced degradation study [19].
2.5.4.1. Forced degradation study.
2.5.4.1.1. Alkali hydrolysis. To perform the alkali degradation study, appropriate aliquots of stock solutions of ALP and MEB were added to two different 10-ml volumetric flasks and 2 ml of 0.1 N NaOH was added to each. Similarly, appropriate aliquots of stock solutions of ALP and MEB were added together in a third 10ml volumetric flask, to which 2 ml of 0.1 N NaOH was added. All of the mixtures were heated in a water bath at 80 °C for 1 h and allowed to cool to room temperature. Solutions were neutralized with 0.1 N HCl and diluted up to the mark with methanol. This produced a final
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concentration of 1000 ^g mL-1 of ALP and MEB. The final concentration of all of the solutions applied to the TLC plates was 1000ng/band, and the chromatograms were recorded.
2.5.4.1.2. Acid hydrolysis. To perform the acid degradation study, appropriate aliquots of stock solutions of ALP and MEB were added to two different 10-ml volumetric flasks and 2 ml of 0.1 N HCl was added to each. Similarly, appropriate aliquots of stock solutions of ALP and MEB were added together in a third 10-ml volumetric flask, to which 2 ml of 0.1 N HCl was added. All of the mixtures were heated in a water bath at 80 °C for 1 h and allowed to cool to room temperature. Solutions were neutralized with 0.1 N NaOH and diluted up to the mark methanol. This produced a final concentration of 1000 ^gmL-1 of ALP and MEB. The final concentration of all of the solutions applied to the TLC plates was 1000 ng/band, and the chromatograms were recorded.
2.5.4.1.3. Oxidative stress degradation. To perform the oxidative stress degradation, appropriate aliquots of stock solutions of ALP and MEB were added to two different 10-ml volumetric flasks and 2 ml of 3% hydrogen peroxide was added to each. Similarly, appropriate aliquots of stock solutions of ALP and MEB were added together in a third 10-ml volumetric flask and 2 ml 3% hydrogen peroxide was added. All of the mixtures were heated in a water bath at 80 °C for 1 h, allowed to cool to room temperature and diluted up to the mark with methanol. This produced a final concentration of 1000 ^gmL-1 of ALP and MEB. The final concentration of all of the solutions applied on the TLC plates was 1000 ng/band, and the chromatograms were recorded.
2.5.4.1.4. Dry heat degradation. Analytically pure samples of ALP and MEB were placed in an oven at 80 °C for2h. The solids were allowed to cool, and 10 mg each of ALP and MEB were weighed, transferred to two separate volumetric flasks (10 ml) and dissolved in a few ml of methanol. The volumes were diluted up to the mark with methanol. Aliquots from the stock solutions of ALP and MEB were appropriately diluted with methanol. This produced a final concentration of 1000 ^g mL-1 of ALP and MEB. The final concentration of all of the solutions applied on the TLC plates was 1000 ng/band, and the chromatograms were recorded.
2.5.4.1.5. Photo-degradation. Analytically pure samples of ALP and MEB were exposed to UV light for 6h. The solids were allowed to cool, and 10 mg each of ALP and MEB were weighed, transferred to two separate volumetric flasks (10 ml) and dissolved in a few ml of methanol. The volumes were diluted up to the mark with methanol. Aliquots from the stock solutions of ALP and MEB were appropriately diluted
with methanol. This produced a final concentration of 1000 ^gmL-1 of ALP and MEB. The final concentration of all of the solutions applied on the TLC plates was 2000 ng/band, and the chromatograms were recorded.
2.5.5. Sensitivity
The limit of detection (LOD) is defined as the lowest concentration of an analyte that can be reliably differentiated from background levels. The limit of quantification (LOQ) of an individual analytical procedure is the lowest amount of analyte that can be quantitatively determined with suitable precision and accuracy.
LOD and LOQ were calculated using the following equations as per ICH guidelines:
LOD = 3.3 x -S
LOD =10 x -S
where s is the standard deviation of the y-intercepts of the regression lines and S is the slope of the calibration curve.
As the LOD and LOQ were computed using equations given in the ICH Q2(R1), the starting point of the range used in the method was at a somewhat higher value than the LOQ value. This was done to obtain accurate and precise results.
2.5.6. Robustness
Small changes in the chamber saturation time and solvent migration distance were introduced, and the effects of these changes on the results were examined. The robustness of the method was determined in triplicate at concentration levels of 600 ng band-1 and 1000 ng band-1 for ALP and MEB, respectively. The mean and % RSD values of the peak areas were calculated.
2.6. Analysis of marketed formulations
Six tablets were accurately weighed and finely powdered. Tablet powders equivalent to 0.25 mg of ALP and 135 mg of MEB were accurately weighed and transferred to a 10-mL volumetric flask. A few mL (5mL) of methanol was added to the above flask, and the flask was sonicated for 5 min. The solution was filtered using a Whatman filter paper No. 1, and the filtrate was added to another 10-mL volumetric flask; the flask was then diluted to the mark with methanol yielding a final concentration of 150 ^gmL-1 for ALP. Twelve microliters of each solution was applied to an HPTLC plate and analyzed for ALP. Then, 0.25 ml was taken from the stock solution, placed in a 10-ml volumetric flask and
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diluted to the mark with methanol; the obtained concentration of MEB was 2000 ^gmL-1. Lastly, 1 ml of this solution was placed in a 10-ml volumetric flask, which was diluted to the mark with methanol. This yielded a final concentration of 200 ^gmL-1 for MEB. The possibility of interference in the analysis from the other components of the tablet formulations were studied using the developed chromatogram spot area, and Rf values were determined.
Table 1
Regression analysis of calibration curve.
Parameters
Linearity range (ng/spot) Slope
Standard deviation of slope Intercept
Standard deviation of intercept Correlation coefficient
600-3600 6.822
0.136042273 3418.6 186.8697407 0.998
1000-6000 2.9548 0.03538 402.9 10.16612 0.999
3. Results and discussion
3.1. Optimization of the mobile phase
To develop the HPTLC routine method of analysis for ALP and MEB, the selection of the mobile phase was carried out on the basis of polarity. A mobile phase that would give a dense and compact band with an appropriate Rf value for ALP and MEB was desired. Various mobile phases such as methanol-toluene, hexane-methanol, acetone-ammonia, acetone-ammonia-acetic acid, were evaluated in different proportions. A mobile consisting of acetone:methanol:acetic acid (6:4:0.1, v/v/v) provided good separation from the matrix for ALP and MEB. It was also observed that the chamber saturation time and the solvent migration distance were crucial in the chromatographic separation. A chamber saturation time of less than 25 min and solvent migration distances greater than 80 mm resulted in diffusion of the analyteband. Therefore, the acetone:methanol:acetic acid (6:4:0.1, v/v/v) mobile phase with a chamber saturation time of 25 min at 25 °C and a solvent migration distance of 80 mm was used. These chromatographic conditions produced a well-defined, compact band of ALP and MEB with optimum migrations at Rf 0.80 and 0.60, respectively (Fig. 2).
3.2. Selection of detection wavelength
The sensitivity of an HPTLC method that uses ultraviolet (UV) detection depends upon proper selection of the detection wavelength. An ideal wavelength is one that gives good responses for the drugs that are to be detected. In the present study, a mixture of solutions, with concentration ranges of 600-3600 ng band-1 for ALP and 1000-6000 ng band-1 for MEB, were prepared in methanol. A syringe was filled with a mixed solution and applied to a single plate in the form of band under a nitrogen stream. The plate was then developed using acetone:methanol:acetic acid (6:4:0.1, v/v/v) at ambient conditions and dried in air. The developed plate was subjected to densitometric measurements in scanning mode
over the UV region of 200-400 nm, and the overlaid spectrum was recorded using CAMAG TLC Scanner 4. The overlaid spectra showed that all drugs absorb appreciably at 244 nm. Therefore, this value was selected as the detection wavelength (Fig. 3).
3.3. Validation
3.3.1. Linearity and calibration curves
The linearity of an analytical method is its ability, within a given range, to obtain test results that are directly, or through mathematical transformation, proportional to the concentration of the analyte. The method was found to be linear in a concentration range of 600-3600 ng/band (n =5) for ALP and 1000-6000 ng/band (n = 5) for MEB with respect to peak area. Fig. 4 displays a three-dimensional overlay of the HPTLC densitograms of the calibration bands for ALP and MEB at 225 nm. The regression data shown in Table 1 reveal a good linear relationship over the concentration range examined, which demonstrates the suitability of the method for analysis.
3.3.2. Accuracy
Accuracy of an analytical method is the closeness of test results to the true value (100%). This was determined by application of the analytical procedure to recovery studies, where a known amount of standard is spiked into pre-analyzed sample solutions. % Recoveries were found to be 99.42-101.67% and 99.93-100.93% for ALP and MEB, respectively (Table 4). These values demonstrate that the method is accurate.
3.3.3. Precision
Intra-day precision refers to the use of an analytical procedure within a laboratory over a short period of time by the same operator and with the same equipment, whereas inter-day precision involves the estimation of variations in analysis when a method is used within a laboratory on different days. The RSD values of the response were less than 5% for intra-day and inter-day precisions.
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Fig. 2. Densitogram of ALP (1800 ng/band) and MEB (3000 ng/band) using the mobile phase acetone:methanol:acetic acid (6:4:0.1, v/v/v).
3.3.4. Limit of detection and limit of quantification
Under the experimental conditions used, the lowest quantities of the drugs that could be detected (LOD) for ALP and MEB were found to be 63.97 and 11.35 ng band-1, respectively. The limits of quantification (LOQ) for ALP and MEB were found to be 193.85 and 34.40 ng band-1, respectively (Table 2). This indicates that the nanogram quantities of all drugs can be estimated accurately and precisely, which reflects the sensitivity of the method.
Table 2
Summary of validation parameter.
Parameters ALP MEB
Rf 0.80 ±0.01 0.60 ±0.01
Detection limit (ng/spot) 89.60624 11.3538
Quantitation limit (ng/spot) 271.53406 34.40544
Accuracy (%) 99.42-101.67 99.93-100.93
Intra-day (n = 3) (% RSD) 1.09-1.57 0.78-1.60
Inter-day (n = 3)(%RSD) 2.05-2.67 1.13-3.89
Repeatability study (n = 6) (% RSD) 1.17-2.32 1.32-2.53
Fig. 3. Three dimensional overlay of HPTLC densitograms of calibration bands of ALP (600-3600 ng/band) and MEB (1000-6000 ng/band).
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Fig. 4. Densitogram of 0.1 M NaOH-treated ALP (1000 ng/band) and MEB (1000 ng/band) under reflux for 2h at 80 °C.
Table 3
Robustness study of proposed method.
Parameters Amt of ALP Amt of ALP recovered ± SDa Amt of MEB Amt of MEB recovered ± SDa
Chamber saturation time: 20 min 600 607.33 ± 20.98 1000 994 ± 7.93
Chamber saturation time: 30 min 600 600.33 ± 9.07 1000 100.33 ± 8.50
Change in Wavelength: 223 nm 600 600 ± 9.53 1000 997.66 ± 14.50
Change in Wavelength: 227 nm 600 597.66 ± 7.09 1000 996.33 ± 6.02
Mobile phase composition changed
Methanol:Acetone:Acetic acid (6:5:0.2 v/v/v) 600 609.37 ± 28.91 1000 1004.95 ± 26.38
Methanol:Acetone:Acetic acid (5:4:0.3 v/v/v) 600 611.60 ± 26.202 1000 993.66 ± 13.50
a n = 3.
Table 4
Accuracy study of the proposed method.
Amount of sample (ng/spot) Set Amount drug spiked (ng/spot) Average amount recovered (ng/spot) % Recovery
ALP MEB ALP MEB ALP MEB ALP MEB
1200 2000 1 0 0 1198.83 2003.97 99.90 100.19
1200 2000 1 600 1000 1804.28 2998.89 100.35 99.93
2 600 1000
3 600 1000
1200 2000 1 1200 2000 2420.09 3990.49 101.67 100.78
2 1200 2000
3 1200 2000
1200 2000 1 1800 3000 2993.13 5004.43 99.42 100.93
2 1800 3000
3 1800 3000
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Fig. 5. Densitogram of 0.1M HCl-treated ALP (1000 ng/band) and MEB (1000 ng/band) under reflux for 2h at 80 °C.
Fig. 6. Densitogram of 3% H2O2-treated ALP (1000 ng/band) and MEB (1000 ng/band) under reflux for 2 h at 80 °C.
MEB dégradent I ALP degradent
2 il L a
daBni ' \ J ■
Fig. 7. Densitogram of UV light-treated ALP (2000 ng/band) and MEB (2000 ng/band) for 24 h.
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Table 5
Forced degradation study of ALP and MEB.
Conditions Time (h) % Amount of drug found Rf value of degradation products (min)
ALP MEB ALP MEB ALP MEB
Base 0.1 M NaOH 2 2 87.23 90.12 0.88 0.43
Acid 0.1 HCl 2 2 85.62 92.74 0.92 0.50
3% hydrogen peroxide 2 2 80.49 95.50 0.90 0.45
Dry heat 2 2 99.01 98.79 0.80 0.60
UV light degradation 24 24 82.47 85.90 0.94 0.24
3.3.5. Forced degradation study
The chromatogram of the base hydrolysis performed at 80 °C for 2 h under reflux showed degradation of ALP and MEB with degradation product peaks at retention factors (Rf) 0.88 and 0.43, respectively (Fig. 5). The chromatogram of the acid hydrolysis performed at 80 °C for 2 h under reflux exposed degradation of ALP and MEB with degradation product peaks at retention factors (Rf) 0.92 and 0.50, respectively (Fig. 6).
The chromatogram of oxidized ALP and MEB with 3% hydrogen peroxide at 80 °C for 2 h under reflux confirmed degradation of ALP and MEB with degradation product peaks at retention factors (Rf) 0.90 and 0.45 (Fig. 7). The chromatogram of ALP and MEB exposed to UV light for 24 h evidenced degradation of ALP and MEB with degradation product peaks at retention factors (Rf) 0.94 and 0.24, respectively (Fig. 8). ALP and MEB were revealed to be stable when exposed to dry heat at 80 °C for 2h.
The degradation study indicated that ALP was found to be stable to dry heat, while it was susceptible to base hydrolysis, acid hydrolysis, oxidation (3% hydrogen peroxide), and photo-degradation. The degradation peaks were well resolved from the drug peak, and no degradation products from the different stress conditions affected the determination of ALP, which suggests that the method is selective and specific. The degradation study indicated that MEB was also susceptible to base hydrolysis, acid hydrolysis, oxidation (3% hydrogen peroxide), and photo-degradation, while it was found to be stable to dry heat. The degradation peaks were well resolved from the drug peak, and no degradation products from the different stress conditions affected determination of MEB, which indicates that the method is selective and specific. The force degradation data are shown in Table 5.
3.3.6. Robustness
The % RSD values were obtained after introducing small, deliberate changes in parameters of the developed
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Fig. 8. Densitogram of dry heat-treated ALP (1000 ng/band) and MEB (1000 ng/band) under reflux for 2h at 80 °C.
HPTLC method. All values were less than 5%, which confirms this method's robustness (Table 3).
3.4. Analysis of marketed formulations
Marketed formulations were analyzed using the proposed method and produced percent recoveries of 98.95-100.14% for ALP and 98.42-100.40% for MEB. A well resolved band at Rf 0.80 and 0.60 was observed in the chromatogram of ALP and MEB, and no interference from the excipients present in the marketed tablet formulations was observed.
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4. Conclusion
Introducing an HPTLC method to pharmaceutical analysis represents a major step in quality assurance. A specific HPTLC analytical method was developed for the simultaneous estimation of alprazolam and mebev-erine as bulk and in pharmaceutical formulations. The method was validated and found to be sensitive, accurate and precise. Statistical analysis proved that the method was repeatable and selective for the analysis of alprazo-lam and mebeverine without any interference from the excipients. The above results indicate the suitability of the method for acid, base, oxidation, dry heat and pho-tolytic degradation studies. As the method separates the drugs from its degradation products, it can be applied to the analysis of samples obtained during accelerated stability experiments to predict expiration dates of pharmaceuticals.
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