Accepted Manuscript Title: Development and Validation of HPTLC method for simultaneous estimation of Clonazepam and Paroxetine hydrochloride using DOE approach Author: Purvi Shah Jalpa Patel Kalpana Patel Tejal Gandhi PII: DOI: Reference:
S1658-3655(15)00179-X http://dx.doi.org/doi:10.1016/j.jtusci.2015.11.004 JTUSCI 259
To appear in: Received date: Revised date: Accepted date:
7-8-2015 30-10-2015 11-11-2015
Please cite this article as: P. Shah, J. Patel, K. Patel, T. Gandhi, Development and Validation of HPTLC method for simultaneous estimation of Clonazepam and Paroxetine hydrochloride using DOE approach, Journal of Taibah University for Science (2015), http://dx.doi.org/10.1016/j.jtusci.2015.11.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development and Validation of HPTLC method for simultaneous estimation of Clonazepam and Paroxetine hydrochloride using DOE approach Purvi Shah*, Jalpa Patel, Kalpana Patel, Tejal Gandhi Department of Quality Assurance, Anand Pharmacy College, Near Town Hall, Anand 388
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315, Gujarat, India.
Author of Correspondence
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Dr. Purvi Shah Professor
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Dept of Quality Assurance, Anand Pharmacy College, Anand
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Email:
[email protected]
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Abstract The present study deals with simultaneous multiple response optimization using the Derringer’s desirability function for the development of HPTLC method for the
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determination of Clonazepam and Paroxetine hydrochloride in pharmaceutical dosage form. CCD was used for the optimization of chromatographic conditions in HPTLC. The
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independent variables used for the optimization were n-butanol content in mobile phase, chamber saturation time and distance travel. HPTLC separation was performed on aluminium
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plates pre-coated with silica gel 60 F254 as the stationary phase using n-butanol: glacial acetic acid: water (9:2:0.5 % v/v/v) as mobile phase. Quantification was achieved by densitometric analysis of Clonazepam and Paroxetine hydrochloride over the concentration range of 40-
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240ng/band and 300-1800 ng/band respectively at 288 nm. The method gave compact and well resolved band at Rf of 0.77 ± 0.02 and 0.34 ± 0.02 respectively for Clonazepam and
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Paroxetine hydrochloride. The linear regression analysis for the calibration plots showed r2 = 0.9958 and r2 = 0.9989 for Clonazepam and Paroxetine hydrochloride respectively. The method was validated for precision, accuracy, robustness, specificity, limit of detection and
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limit of quantitation as per ICH guideline. The factors evaluated in the robustness test were
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found to have an insignificant effect on the selected responses. The results indicate that the
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method is suitable for the routine quality control testing of marketed tablet formulation.
Keywords
Clonazepam, Paroxetine hydrochloride, Central composite design, High performance thin layer chromatography, Validation
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1. Introduction Clonazepam (CLO) [5-(2-chlorophenyl)-7-nitro-2, 3-dihydro-1H-1, 4-benzodiazepin-2-one] is a benzodiazepine drug having anxiolytic, anticonvulsant, muscle relaxant, sedative, and
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hypnotic properties. It exerts its action through allosteric interactions between central benzodiazepine receptors and gamma-aminobutyric acid (GABA) receptors that potentiate the effects of GABA. This results in inhibition of synaptic transmission across the central
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nervous system [1-3]. Paroxetine hydrochloride (PH) [(-)-Trans-4R-(4'-fluorophenyl)-3S-[(3', 4’methylenedioxyphenoxy) methyl] piperidine hydrochloride], is a selective serotonin (5-
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hydroxy-tryptamine, 5-HT) reuptake inhibitor (SSRI) and potentiates 5-HT in the CNS. (Figure 1). PH is indicated for the treatment of major depressive disorder, social anxiety
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disorder, obsessive-compulsive disorder, panic disorder, generalized anxiety disorder, and posttraumatic stress disorder [1-4].
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Depression and anxiety disorders are distinct illnesses that often coexist. Patients with comorbid depression and anxiety are more debilitated than are patients with either condition alone. Mixed anxiety-depression is gaining recognition as a separate diagnosis and has been
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included in the International Classification of Diseases, 10th edition, and in the appendix of
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the Diagnostic and Statistical Manual of Mental Disorders, 4th edition. Nowadays, for the management of co-morbid depression and anxiety, fixed dose combination of an anti-
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depressant like PH and an anti-anxiety drug like CLO is an available treatment option [4, 5].
(a)
(b)
Figure 1 Chemical structure of (a) Clonazepam; (b) Paroxetine hydrochloride Literature survey states that CLO and PH are official in IP, USP and BP individually [1, 6-7] however, combination of CLO and PH is not official in any Pharmacopoeia. Various analytical methods like Spectrophotometry [8-12], Spectrofluorimetry [13, 14], HPLC [1525] and HPTLC [26-29] methods were reported in literature for the determination of CLO
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and PH alone and in combination with other drugs in pharmaceutical dosage forms. Spectrophotometric methods [30, 31] and stability-indicating HPLC method [32, 33] were reported for the simultaneous estimation of CLO and PH in combined pharmaceutical formulations. However, development of a high performance thin layer chromatographic
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(HPTLC) method for simultaneous estimation of CLO and PH in combined dosage form has not been reported till date.
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Hence, the present manuscript is the first one that describes the development and validation of HPTLC method as per ICH guidelines ICH Q2 (R1) for simultaneous estimation of CLO
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and PH. A multivariate approach using experimental design is used to study the simultaneous variation effect of the factors on the responses. The best experimental design approach for the purpose of modelling and optimization is the response surface design. In the present study,
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central composite design (CCD) was used for optimization of chromatographic conditions of HPTLC method. CCD is chosen due to its flexibility and can be applied to optimize
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chromatographic conditions by gaining better understanding of factor’s main and interaction effects [34]. Viewing the simplicity, economic, less time-consuming and few processing parameters, the objective of this research work was therefore to develop a simple, rapid,
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precise and accurate HPTLC method using DOE approach for quantitative analysis of CLO
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and PH and to validate the method in accordance with ICH guidelines.
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2. Materials and Methods 2.1 Materials
Analytically pure CLO (Vital Formulation, India) and PH (Torrent Pharmaceutical, India) were obtained as gift samples respectively. Marketed tablet formulation used was Pari-CR Plus, IPCA Laboratories, India (Label claim 0.5 mg of CLO and 12.5 mg of PH) procured from the local market. All solvents and chemicals used were of analytical grade, purchased from Merck Specialities Pvt. Ltd., India.
2.2 Instrumentation Hamilton microlitre syringe (Linomat syringe 659.0014, Hamilton-Bonaduz Schweiz,
Camag, Switzerland), precoated silica gel aluminium plate 60 F254, (10 × 10 cm, 100
m
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thickness; E. Merck, Darmstadt, Germany), Linomat 5 sample applicator (Camag, Switzerland), Twin trough chamber (20 × 10 cm; Camag, Switzerland), UV chamber (Camag, Switzerland), TLC scanner 4 (Camag, Switzerland), and operated by winCATS version 1.4.6 software (Camag, Switzerland) were used in the study. All drugs and chemicals
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were weighed on an elctronic balance (AUW 220, Shimadzu Corp., Japan).
2.3 Preparation of standard solutions
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10 mg of standard CLO and PH were accurately weighed, transferred to two separate 10 ml volumetric flasks, dissolved in methanol and then volumes were made up to the mark with
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methanol, to obtain solution containing 1000 µg/ml. Aliquots of the stock solutions were appropriately diluted with methanol to obtain working standards of 40 µg/ml of CLO and 300
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µg/ml of PH.
2.4 Chromatographic procedure
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The standard solutions of different concentrations were spotted in the form of bands having a band width of 6 mm with a micro syringe on pre-coated silica gel aluminium Plate 60F254, using a Camag Linomat 5 sample applicator. Linear ascending development was carried out
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in a twin trough glass chamber. The mobile phase consisted of n-butanol: glacial acetic acid:
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water (9:2:0.5, %v/v/v). The optimized chamber saturation time before chromatographic ◦
development was 35 min at room temperature (25 ± 2 C). The length of chromatographic run
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was 8 cm. Subsequent to the development; HPTLC plates were dried in a current of air with the help of an air dryer. Densitometric scanning was performed using a Camag TLC scanner 4 with winCATS software. All measurements were made in the reflectance–absorbance mode at 288 nm, slit dimension (6.00 x 0.30 mm, micro), scanning speed 20 mm/s, data resolution 100 µm/step. The source of radiation was deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm. Concentration of both drugs was determined from the intensities of diffusely reflected lights and evaluation was done by ordinary linear regression analysis of peak areas.
2.5 Software aided method optimization Central composite design (CCD) was used to optimize the compositional parameters and to evaluate main effect, interaction effects and quadratic effects of the factors on the retardation factor (Rf) of both drug. CCD is useful in response surface methodology, for exploring
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quadratic response surfaces and constructing second order polynomial models without need to use a complete three-level factorial experiment [34, 35]. The selection of critical factors and ranges examined for optimization was based on
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preliminary univariate studies of method development and chromatographic intuition. The composition of the mobile phase is the volume of n-butanol content with respect to total volume of mobile phase. Total fifteen experiments with five centre points were conducted by
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selection of three factors, n-butanol content in mobile phase (A), chamber saturation time (B), distance travel (C) and Rf of CLO and PH were the responses selected for both drugs depicted
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in Table 1. The nominal value for these all three factors, A, B and C were 8 ml, 30 min, and 8cm respectively. In context to this, n-butanol content (A) was kept between 6.59 and 9.41.
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Similarly, minimum and maximum values of chamber saturation time (B) were fixed as 22.93 min and 37.07 min respectively. Likewise, minimum and maximum values for distance travel(C) were fixed as 6.59 and 9.41 respectively. The coded value of α is 1.41. The data
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generated were analyzed using Design Expert (Version 9.0.0.1, Stat-Ease Inc., Minneapolis, MN, USA) trial version statistical software. The significance of the relevant factors was calculated using Fisher’s statistical test for Analysis of Variance (ANOVA) model. All
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experiments were conducted in a randomized order to minimize the bias effects of
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experimental error.
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uncontrolled variables. Replicates (n=5) of the centre points were performed to estimate the
2.6. Method validation
The HPTLC method was validated for accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), specificity, robustness, and ruggedness, in accordance with ICH Q2 (R1) guideline [36].
2.6.1 Linearity
Different volumes (1 to 6 μl) of standard solutions of both drugs were applied on the HPTLC plate to obtain a concentration range of 40–240 ng/band of CLO and 300-1800 ng/band for PH, in five replicate measurements. The measured peak areas versus corresponding concentration of both drugs were evaluated by ordinary linear regression analysis. The homoscedasticity of the variances along the regression line of each drug was verified using the Bartlett’s test [37].
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2.6.2 Sensitivity Limit of detection (LOD) and Limit of quantitation (LOQ) of the developed method were calculated from the standard deviation of the response and slope of the calibration curve of drugs using the formula as per ICH guideline,
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Limit of detection=3.3 × σ/S Limit of quantitation=10 × σ/S
Where, “σ” is standard deviation of y intercepts of regression lines, “S” is Slope of calibration
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curve
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2.6.3 Precision
The precision of the developed method was evaluated by performing Intra-day and Inter-day
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precision studies. Intra-day precision was carried out by performing three replicates of three different concentration (80, 160 and 240 ng/band for CLO; 600, 1200 and 1800 ng/band for PH) on same day and peak area measured was expressed in terms of percent relative standard
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deviation (% RSD). The inter-day precision study was performed on three different days using
2.6.4 Accuracy
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mentioned concentrations of both drugs in triplicate.
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The accuracy of method was ascertained in triplicates, at three concentration level of 50%, 100% and 150 %, by spiking known amount of CLO and PH standard to the pre-quantified
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samples and calculating the recovery and % RSD for both the drugs. Recovery studies were carried out by spiking three different amount of CLO standard (20, 40, 60 ng/band) to the dosage form (40 ng/band) and PH standard (300, 600 and 900 ng/band) to the dosage form (600 ng/band) by standard addition method [38].
2.6.5 Specificity
The specificity of the method was ascertained by comparing the samples of tablet formulation with standard drugs. The band for CLO and PH in sample was confirmed by comparing the Rf and overlaying peak purity spectra with that of standard. The peak purity of CLO and PH was assessed by comparing the spectra at three different levels, i.e., peak start (S), peak apex (M) and peak end (E) positions of the band.
2.6.6 Robustness
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The effect of small and deliberate variations on method parameters like change in volume of n-butanol content in mobile phase composition, saturation time, distance travel and
wavelength was evaluated. The effect of these changes on both the
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2.7 Analysis of marketed formulation
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areas was examined by calculating the % RSD for each parameter.
values and peak
To determine CLO and PH content of tablet dosage form, twenty tablets were accurately weighed; their mean weight was determined and finely powdered in a glass mortar. A powder
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equivalent to 0.4 mg of CLO and 10 mg PH was accurately weighed and transferred into the 10 ml volumetric flask with 5 ml methanol. The mixture was diluted to volume with
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methanol and sonicated for 15 min and then filtered through Whatman no. 42 filter paper wetted with methanol. The solutions were diluted to obtain the sample stock solution of 40µg/ml of CLO and 1000 µg/ml of PH. One microlitre of the filtered solution (40 ng/band
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of CLO and 1000 ng/band of PH) was applied on the HPTLC plate followed by development
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3. Results and discussion
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and scanning. The analysis was repeated in triplicate.
3.1 Selection of wavelength
The sensitivity of HPTLC method with ultraviolet detection depends on an appropriate wavelength. The developed plate was subjected to densitometric measurements in scanning mode in the UV-Visible region of 200–700 nm, and the overlain spectrum was recorded on a CAMAG TLC Scanner 4. Both drugs absorbed appreciably at 288 nm, and selected as the detection wavelength (Figure 2).
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Figure 2 Overlain absorption spectra of Clonazepam and Paroxetine hydrochloride at
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288 nm
3.2 Method optimization
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The optimizations of chromatographic conditions were done with a view to develop HPTLC method for simultaneous determination of CLO and PH in bulk and in pharmaceutical dosage
3.2.1 Preliminary study
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form.
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From literature review, it is revealed that HPTLC method for CLO and PH alone or with other drug combination had been reported, where selected mobile phase comprised of n-butanol,
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glacial acetic acid and water [27]. Hence, various combinations of such components in different proportions such as n-butanol: acetic acid (6:4, v/v); n-butanol: glacial acetic acid: water (6:4:0.5, 7: 3: 0.5, 8: 1: 1, 9: 1: 1, 8: 2: 0.5, v/v/v) were tried at fixed 30 min chamber saturation time and 80 mm solvent migration distance. However, satisfactory resolution of the drugs was not achieved with acceptable Rf value. Generally, chamber saturation time and solvent migration distance were crucial to HPTLC chromatographic separation. Here, chamber saturation time of less than 25 min and solvent migration distances greater than 80 mm resulted in diffusion of the analyte band. n-butanol: glacial acetic acid: water (8: 2: 0.5, v/v/v) was found to be a satisfactory mobile phase, giving good separation of CLO and PH. But, Rf value of CLO was found to near 0.8 and was also affected by chamber saturation time. Therefore, further chromatographic conditions were optimized to obtain well-defined, compact bands of CLO and PH with acceptable Rf value (< 0.8) of both drugs using CCD. 3.2.2 Optimization of chromatographic conditions using CCD
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CCD is chosen due to its flexibility and can be applied to optimize HPTLC separation by gaining better understanding of factor’s main and interaction effects. A three-factorial, rotatable Central Composite statistical experimental design was performed using 15 experimental run including five centre points. The independent variables such as n-butanol
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content in mobile phase (A), chamber saturation time (B) and distance travel (C) and the responses for all 15 optimized trial experimental runs are summarized in Table 1. During model selection, it was observed that the best-fitted model for Rf of CLO and PH was linear
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and quadratic model respectively based on lowest PRESS value and adjusted R2 value nearer
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to 1.
Table 1 Central composite rotatable design arrangement and responses Factors A:
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Type
n-Butanol B: Chamber C:
content
saturation
Distance travel
(ml)
time (min) 0.00
Responses Rf
of Rf
CLO
PH
d
Run
(cm) 0.00
0.81
0.43
0.00
-1.41
0.84
0.4
0.00
0.00
0.81
0.37
Axial
-1.41
2
Axial
0.00
3
Centre
0.00
Axial
0.00
-1.41
0.00
0.81
0.38
Axial
0.00
+1.41
0.00
0.79
0.26
Centre
0.00
0.00
0.00
0.8
0.39
Fact
-1.00
+1.00
+1.00
0.77
0.35
Fact
+1.00
+1.00
-1.00
0.84
0.27
Centre
0.00
0.00
0.00
0.82
0.41
10
Fact
-1.00
-1.00
-1.00
0.84
0.45
11
Axial
0.00
0.00
+1.41
0.73
0.35
12
Fact
+1.00
-1.00
+1.00
0.78
0.25
13
Centre
0.00
0.00
0.00
0.81
0.41
14
Axial
+1.41
0.00
0.00
0.79
0.27
15
Centre
0.00
0.00
0.00
0.80
0.42
5 6 7 8 9
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4
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1
of
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n = 3 replicates, CLO = Clonazepam, PH = Paroxetine hydrochloride The model was also validated by analysis of variance (ANOVA) using Design Expert software and the results are as presented in Table 2. Significant effects had P value less than
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0.05. Adequate Precision, measure of the signal (response) to noise ratio, greater than 4 is desirable, and the obtained ratio for both drugs indicated an adequate signal [39]. The coefficient of variation (% CV) that measures the reproducibility of the model less than 10%
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and high adjusted R-square values indicated a good relationship between the experimental data and those of the fitted models. Here, the adjusted R2 were well within acceptable limits
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of R2 ≥ 0.80 which revealed the experimental data were a good fit to the polynomial equations [40, 41]. The final equation, in terms of actual components and factors, is as shown
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in the Table 2. A positive value represents an effect that favours the optimization, while a negative value indicates an inverse relationship between the factor and the response. Table 2 Predicted response models and statistical parameters obtained from ANOVA
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for CCD Response Type of model
Polynomial
(Rf )
equation
Adjusted Model %
model R2
Linear
0.81 + 5.214E-
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CLO
CV
precision
value
d
for Y
P-
Adequate
0.8889
0.0099 2.53 8.125
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003A -0.012B -
PH
Quadratic
0.028C
0.40 - 0.057A - 0.9766
0.0001 2.70 22.782
0.042B -0.018C + 7.322E-003AB
-
0.017AC
+
8.431E-003 BC 0.021A2 - 0.036B2 - 8.519E-003C2
Factors are in coded levels, CLO = Clonazepam, PH = Paroxetine hydrochloride Three-dimensional Response surface plots and perturbation plots were constructed to evaluate the effect of the factors on the retention factor of each drug. In Figure 3, perturbation plots were presented for predicted model in order to gain a better understanding of the
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investigated procedure. It gives the idea about how the response changes as each factor moves from its defined reference value, with all other factors held constant at a reference point, and steepest slope or curvature indicates sensitiveness to specific factor. Figure 3 (a) shows that distance travel (factor C) had the most significant effect on Rf value of CLO as
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compared to other factors. While in Figure 3 (b) n-butanol content (A) and chamber saturation time (B) had more significant effect on Rf value of PH followed by distance travel (factor C). Figure 4(a) represents a variation in Rf value of CLO as a function of chamber
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saturation time and distance travel while n-butanol is held constant and retention factor of CLO decreases as distance travel increases. Analysis of the perturbation plots and response
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plots of optimization model revealed that n-butanol content (A) and chamber saturation time
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(a)
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(B) had greater significant effect on responses as compared to factor C, i.e. distance travel.
(b)
Figure 3 Perturbation graph showing the effect of each factor A, B, and C on (a) Rf of
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Clonazepam and (b) Rf value of Paroxetine hydrochloride The optimum conditions of separation were estimated by Derringer’s desirability function [35]. During numerical optimization, firstly the target of individual factors and responses were fixed. Out of 15 different solutions of optimization provided by software two conditions were selected that have desirability near to 1. The response surface obtained for the maximum Derringer’s desirability function is presented in Figure 4 C. In order to investigate the predictability of the proposed model, the agreement between experimental and predicted responses for both the predicted optimums 1 and 2 are shown in Table 3. The Percentage of prediction error was calculated using formula, Predicted Error = ExperimentalPredicted/Predicted × 100.
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From the Table 3 and % predicted error, it is concluded that a set of coordinates producing high desirability value (D = 1) at optimum condition 1, hence proposed for selecting an optimum experimental condition for analyzing CLO and PH in combination. The optimized composition selected was n-butanol: glacial acetic acid: water (9:2:0.5 v/v/v), for the final CLO (40 ng/band) and 0.34 for PH (300 ng/band) is depicted in Figure 5.
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HPTLC analysis. HPTLC densitogram under optimized conditions showing Rf of 0.77 for
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Table 3 Comparison of experimental and predictive values of different experimental runs under optimum conditions
content
saturation
(ml)
(min)
(cm)
9.00
35.00
8.00
PH
0.770
0.340
0.773
0.352
0.390
3.824
0.760
0.350
Predictive
0.769
0.364
Predicted error %
1.139
4.000
Predictive 35.00
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Predicted error % 8.612
Rf of
CLO
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time Distance travel
Experimental
2
Rf of
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conditions 1
Chamber C:
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A: n- Butanol B:
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Optimum
8.00
Experimental
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CLO = Clonazepam, PH = Paroxetine hydrochloride
(a)
(b)
(c)
Figure 4 Three-dimensional plots of the RSM for both responses (a) Variation in Rf of Clonazepam (CLO) as function of B and C while fixed factor A; (b) Variation in Rf of
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Paroxetine hydrochloride (PH) as function of A and B while fixed factor C; (c)
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Graphical representation of the maximum derringer’s desirability function
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Figure 5 HPTLC densitogram under optimized conditions showing Rf value 0.77 for
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Clonazepam (40 ng/band) and 0.34 for Paroxetine hydrochloride (300 ng/band)
3.3 Method validation 3.3.1 Linearity
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The linearity of an analytical method is its ability, within a given range, to provide results that
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are directly, or through a mathematical transformation, proportional to the concentration of the analyte. The CLO and PH showed a good correlation coefficient (r2 = 0.9958 for CLO and
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r2 = 0.9989 for PH) in the proposed concentration range 40-240 ng/band for CLO and 3001800 ng/band for PH (Table 4). Homoscedasticity of variance was confirmed by Bartlett’s test and the response of peak area for both drugs showed homogenous variance that was exemplified by the χ2 value less than the tabulated value (Table 4). Figure 6 shows a threedimensional overlay of the HPTLC densitogram for CLO and PH, with calibration bands at 288 nm.
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Figure 6 Three-dimensional densitogram for linearity of Clonazepam and Paroxetine hydrochloride at 288 nm
Parameters
CLO
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Linearity
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Table 4 Analytical validation parameters for CLO and PH by HPTLC method PH
40 - 240
300 – 1800
Correlation coefficient (r2) a
0.9958
0.9989
Slope ± SD
22.03 ± 1.142
5.2423 ± 0.104
20.071 - 24.020
4.995 - 5.488
Intercept ± SD
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Confidence limit of slope
b
d
Linearity range(ng/band)
43.10 ± 57.865
400.760 ± 102.092
40.945 - 45.255
380.722 - 420.798
0.0118
0.0109
LOD (ng/band)
8.840
63.416
LOQ (ng/band)
26.789
195.260
r (S,M)
0.99994
0.99981
r (M,E)
0.99990
0.99977
Intra-day Precision
1.141-1.546
0.520-0.948
Inter-day Precision
1.538-1.788
0.753-1.203
50%
101.46 ± 0.316
100.87 ± 1.832
100%
101.54 ± 0.129
101.21 ± 1.689
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Confidence limit of interceptb Bartlett’s test c (χ2) Sensitivity
Specificity
Precisiond (%RSD)
Accuracye
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101.46 ± 0.259
150%
101.51 ± 1.339
SD = standard deviation, % RSD = relative standard deviation, CLO = Clonazepam, PH = Paroxetine hydrochloride Average of five determinations
b
Confidence interval at 95% confidence level and 5 degree of freedom (t=2.57)
c
Calculated value less than tabulated value, χ2critical value 9.488 at 95% confidence interval
d
Average of three determinations for each concentration
e
Average of three determinations at each level
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a
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3.3.2 LOD and LOQ
LOD and LOQ of developed method were found to be 8.840 and 26.789 ng/band respectively
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for CLO while 63.416 and 195.260 ng/band respectively for PH indicating the sensitivity of the proposed method (Table 4).
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3.3.3 Precision
The experiment was repeated three times in a day (Intra-day precision) and the average % RSD values of the results were calculated. Similarly, the experiment was repeated on three
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different days (Inter-day precision) and the average % RSD values for peak area of CLO and
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PH was calculated. Results of intra-day and inter-day precision expressed in terms of % RSD
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less than 2 confirm precision of the method (Table 4).
3.3.4 Accuracy
The proposed method when used for evaluation of recovery at three concentrations levels, 50%, 100% and 150% after spiking with standard, showed percentage recovery between 101.46 to 101.54 for CLO and 101.21 to 101.87 for PH which were within acceptable ranges of 100 ± 2 % [38].
3.3.5 Specificity The chromatogram of the pharmaceutical formulation using the developed method, showed only two peak at Rf of 0.77 and 0.34 for CLO and PH respectively, that was found to be at the same Rf for both standard drugs by comparison of chromatograms (Figure 7). The peak purity of both drugs in pharmaceutical dosage form was confirmed when evaluated by comparing the overlaid spectra at peak start, peak apex and peak end positions of the band. It was observed
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from results shown in Table 4 that purity was more than 0.999 for all peaks, indicating
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specificity of method in the presence of various excipients (Figure 7).
(a)
(b)
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Figure 7 Overlain peak purity spectra of (a) Clonazepam (CLO) and (b) Paroxetine hydrochloride (PH) with corresponding standard
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3.3.6 Robustness
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Deliberate change in different parameters like n-butanol content in mobile phase composition, chamber saturation time, distance travel and wavelength showed %Relative standard deviation
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of peak area less than 2%, indicating robustness of method (Table 5).
Table 5 Robustness study of developed HPTLC method
Change in Mobile phase ratio (n-butanol: Glacial acetic acid: Water, 9:2:0.5 ± 0.25 in n-butanol content)a
Drugs CLO PH
Ratio
Rf
Area ± SD (ng/band)
% RSD
8.75: 2:0.5
0.77 ± 0.01
1860.22 ± 10.161
0.546
9.25: 2:0.5
0.77 ± 0.01
1892.32 ± 32.859
1.736
8.75: 2:0.5
0.34 ± 0.01
3649.25 ± 18.616
0.510
9.25: 2:0.5
0.34 ± 0.01
3710.68 ± 24.821
Change in chamber saturation time (35 min ± 5) Drugs
Saturation
Rf
Area ± SD (ng/band)
0.668 a
% RSD
time (min)
Page 17 of 23
PH
30
0.77 ± 0.02
1895.16 ± 34.867
1.839
40
0.77 ± 0.02
1819.99 ± 18.285
1.004
30
0.34 ± 0.02
3599.61 ± 53.716
1.492
40
0.34 ± 0.02
3624.62 ± 43.103
1.192
Change in Distance travel (8 cm ± 1) Drugs
Distance
a
Rf
Area ± SD (ng/band)
7
0.77 ± 0.02
1812.95 ± 23.263
9
0.77 ± 0.02
1872.62 ± 18.929
7
0.34 ± 0.02
3622.95 ± 37.212
9
0.34 ± 0.02
3722.62 ± 33.264
PH
1.283 1.010 1.027
us
CLO
% RSD
cr
travel (cm)
ip t
CLO
0.893
Drugs
Wavelengt
an
Change in wavelength (288 nm ± 2) a Rf
Area ± SD (ng/band)
% RSD
286
0.76
1859.36 ± 9.411
0.506
290
0.76
1839.45 ± 4.525
0.246
286
0.35
3659.36 ± 11.467
0.313
290
0.35
a
3633.95 ± 29.434
0.809
te
PH
d
CLO
M
h (nm)
Average of three determinations of 40 ng/band for CLO and 300 ng/band for PH; SD =
Ac ce p
standard deviation, % RSD = relative standard deviation, CLO = Clonazepam, PH = Paroxetine hydrochloride
3.4 Analysis of marketed dosage form Analysis of tablet formulation containing 0.5 mg CLO and 12.5 mg PH showed good recovery where percentage amount for both drugs were 99.926 % for CLO and 100.980 % for PH indicating that the method can be applicable in routine quality control testing of the tablet dosage formulation. The %RSD value was found to be less than 2.
4. Conclusion CCD design and response surface methodology provides a better insight into essential information regarding the sensitivity of various chromatographic variables on Rf of CLO and PH. n-butanol content, chamber saturation time and distance travel were simultaneously
Page 18 of 23
optimized by applying useful experimental design tool: response surface design and Derringer’s desirability function. From the obtained results, it is concluded that the use of CCD design and multi-criteria decision making approach is a flexible procedure, able to reduce the number of the needed experiments for the development and optimization of
ip t
HPTLC method and it is an economic method that can be used to generate a maximum amount of information in a less time with small number of experiments. Methodological validation indicates that the established HPTLC method is simple, accurate, and reliable and
cr
suitable for rapid quantitative analysis of CLO and PH in routine tests. The proposed HPTLC method can be successfully utilized for the simultaneous estimation of CLO and PH in the
us
pharmaceutical dosage form without interference and any prior separation of individual
an
drugs.
Acknowledgement
We would like to express our gratitude to Vital Formulation, Viththal Udhyognagar, Anand
M
and Torrent Pharmaceutical, Ahmedabad for providing gift sample of standard Clonazepam
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