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Development and validation of a stability indicating RP-TLC/densitometric method for determination of Loratadine in bulk and in tablets
⁎Corresponding author. Tel.: +91 9823691502. atulshirkhedkar@rediffmail.com (Atul A. Shirkhedkar),
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
A new rapid, economical and environmentally friendly Reversed-Phase Thin-Layer chromatography (RP-TLC)/densitometry has been developed and validated for quantitative determination of Loratadine (LOR) in bulk and in tablets. RP-TLC separation was achieved on aluminium plates precoated with silica gel 60RP-18F 254S as the stationary phase using methanol:acetonitrile (90:10% v/v) as mobile phase. Quantitation was performed at 247 nm over the concentration range of 200–1200 ng/band. The method was found to give compact and well resolved band for LOR at retention factor (Rf) 0.58 ± 0.02. The linear regression analysis data for calibration graph showed good linear relationship with r2 = 0.998. The method was validated for recovery, precision, robustness, ruggedness and sensitivity as per International conference on Harmonisation (ICH) guidelines. The minimum detectable amount and limit of quantitation were found to be 19.40 ng and 61.51 ng, respectively. LOR was subjected to hydrolysis in acid, alkali, oxidation, photo-degradation and neutral condition. The drug demonstrated degradation under acid and alkali conditions. Statistical analysis proves that the method is sensitive, selective, precise and accurate for the estimation of LOR. The proposed developed RP-TLC/densitometry method can be applied for identification and quantitative determination of LOR in bulk drug and pharmaceutical dosage forms.
Keywords
Loratadine
RP-TLC/densitometry
Stability studies
Validation
1 Introduction
Loratadine (LOR), ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidine carboxylate (Budavari, 1996), is a second generation long-acting tricyclic antihistamine with selective peripheral histamine H1-receptor antagonistic activity (Ruperez et al., 2002; Pavalache et al., 2010; Nagappan et al., 2008; Chebrolu et al., 2011). LOR undergoes extensive first pass metabolism in the liver, forming an active metabolite, desloratadine (Sherbiny et al., 2007).
In the literature, several methods have been described for determination of LOR in pharmaceutical preparations including UV-Spectrophotometry (Mabrouk et al., 2003), spectrofluorometry (Gazy et al., 2002), capillary electrophoresis (Barbas et al., 2003), atomic absorption spectrometry, colorimetry, (El-Kousy and Bebawy, 1999), polarography (Squella et al., 1996) and HPLC (Salem et al., 2004; Yin et al., 2003; Paweł, 2001).
Most of these analytical methods are often time-consuming, expensive and cumbersome.
The advantage of RP-TLC is that a large number of samples can be simultaneously analysed in a shorter time period. Unlike HPLC, this method utilises less quantities of solvents, thus lowering the cost of analysis. Mobile phase having pH 8 and above can be employed. Suspensions, dirty or turbid samples can be directly applied. It facilitates automated application and scanning in situ. HPTLC facilitates repeated detection (scanning) of the chromatogram with the same or different parameters. Simultaneous assay of several components in a multicomponent formulation is possible (Ansari et al., 2005).
In our present research endeavour an effort has been made to establish stability-indicating RP-TLC/densitometry estimation of LOR in bulk and in tablets. Further the validation of developed method as per the International Conference on Harmonisation guidelines (ICH, 2003).
2 Experimental
2.1 Materials and reagents
LOR was supplied as a gift sample from Care-well pharmaceutical Ltd., Ahmedabad, India. All chemicals and reagents used were of Analytical grade and were purchased from Merck Chemicals, India.
2.2 HPTLC instrumentation
Chromatography was performed on aluminium plate pre-coated silica gel 60RP-18F254 S
(20 × 10 cm with 200 μm thickness, E. Merck). Before use, the plates were pre-washed and dried in oven at 100 °C for 5 min. Ten microlitre samples were spotted 6 mm from the edge of the plates by means of CAMAG Linomat 5 applicator. The slit dimension was kept at 6.00 × 0.45 mm (micro) and 20 mm/s scanning speed was employed. The linear ascending development of plates was performed to a distance of 8 cm in twin-trough chamber (20 × 10 cm) previously saturated for 25 min with the mobile phase methanol:acetonitrile (90:10% v/v). Subsequent to development; the RP-TLC plates were dried in a current of air with the help of an air dryer. Densitometry scanning was performed at 247 nm on a Camag TLC scanner 3 operated by winCATS software version 1.3.0.
2.3 Preparation of standard solution and linearity study
A stock solution containing 1 mg/mL of LOR was prepared by dissolving an accurately weighed 10 mg portion of the drug in 10 mL methanol. Different volumes of stock solution, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mL were taken and transferred to six different 10 mL volumetric flasks and the volume was made up to mark with methanol. From each flask 10 μL of solution was spotted on RP-TLC plate to obtain concentration of 200, 400, 600, 800, 1000 and 1200 ng/band of LOR, respectively. The data of peak area versus drug concentration were treated by linear least square regression.
3 Method validation
3.1 Precision
Precision can be performed at two different levels – repeatability and intermediate precision. Repeatability is an indication of how easy it is for an operator in a laboratory to obtain the same results for the same batch of material using the same method at different times using the same equipment and reagent. Repeatability of sample application and measurement of peak area were carried out using six replicates of the same band (800 ng/band of LOR).
The intermediate precision results from the variations such as different days, analysts and equipment. The intra-day variation studies were performed by using three different concentrations over the linearity range within the same day. The inter-day variations in the methods were assessed by studying three different concentrations for three different days over a period of week. The intra and inter-day variation for the determination of LOR was carried out at three different concentration levels of 400, 800, and 1000 ng/band.
3.2 Limit of detection (LOD) and limit of quantification (LOQ)
In order to determine the limit of detection and limit of quantification, LOR concentrations in the lower part of the linear range of the calibration curve were used. LOR solutions of 200, 240, 280, 320 360 and 400 ng/band were prepared and applied on RP-TLC plate. The LOD and LOQ were calculated using equation LOD = 3.3 × N/B and LOQ = 10 × N/B, where, N is the standard deviation of the peak areas of the drugs (n = 3), taken as a measure of noise, and ‘B’ is the slope of the corresponding calibration curve.
3.3 Specificity
The specificity of the method was ascertained by analysing the drug standard and sample. The spot for LOR in sample was confirmed by comparing the Rf values and spectra of the bandwidth with those of the standard. The peak purity of LOR 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.
3.4 Ruggedness
Ruggedness of the method was performed by spotting 800 ng/band of LOR by two different analysts keeping same experimental and environmental conditions.
3.5 Accuracy
The analysed samples were spotted with extra 80%, 100% and 120% of the standard LOR and the mixture was analysed by the proposed method. This was done to check the recovery of the drug at different levels in the formulations.
3.6 Robustness
Robustness measures the capacity of an analytical method to remain unaffected by small but deliberate variations in method parameters. By introducing small changes in the mobile phase composition, the effects on the results were examined. Mobile phases having a different composition of methanol:acetonitrile (90:10% v/v) were tried and chromatograms were run. The amount of the mobile phase, temperature and relative humidity was varied in the range of ± 5%. The plates were prewashed with methanol and activated at 100 ± 5 °C for 2, 5 and 7 min prior to chromatography. Time from spotting to chromatography and from chromatography to scanning was varied from 20, 30 and 40 min.
3.7 Application of proposed method to tablet formulation
To determine the concentration of LOR in tablets (Label claim: 10 mg/tablet), the contents of 20 tablets were weighed, their mean weight determined and they were finely powdered. The powder equivalent to 10 mg of LOR was weighed and transferred into a 50 mL volumetric flask containing 30 mL of methanol. To ensure complete extraction of the drug, it was sonicated for 30 min and the volume was made up to 50 mL. The resulting solution was filtered using 0.45 μm filter (Millfilter, Milford, MA). From it, 2.5 mL was diluted to 10 mL with methanol. A volume of 10 μL was applied on RP-TLC plate followed by development and scanning as described above.
4 Stability studies of LOR
4.1 LOR stability in the mobile phase and Bench top-stability under normal light conditions for 24 h
To study the LOR stability in the mobile phase, sample solution was prepared in the mobile phase. Whereas, to study the bench top stability of LOR, sample solution was prepared in methanol and stored in a capped volumetric flask on a bench top under normal lighting conditions for 24 h.
Application, development and scanning of the RP-TLC plates were performed at 0 h. and after 24 h.
5 Forced degradation studies of LOR
In acid and base degradation studies, 10 mg of LOR was separately dissolved in 10 mL of methanolic solution of 1 N HCl and 1 N NaOH. These solutions were refluxed at 70 °C for 6 h. in the dark in order to exclude the possible degradative effect of light. 1 mL of above solutions was taken and neutralised, then diluted up to 10 mL with methanol. The resultant solution was applied on RP-TLC plate in triplicate (10 μL each, i.e. 1000 ng/band).
In oxidative degradation studies, 10 mg of LOR was dissolved in 10 mL of methanolic solution of hydrogen peroxide (10% v/v). The solution was refluxed at 70 °C for 6 h in the dark in order to exclude the possible degradative effect of light. The resultant solution was applied on RP-TLC plate in triplicate (1.0 μL each, i.e. 1000 ng/band).
In photo stability studies, LOR solution was exposed to direct sunlight for 24 h. (8 h/day). The resultant solution (1.0 μL, i.e.1000 ng/band) was applied on a RP-TLC plate.
In neutral condition, 10 mg of LOR was dissolved in 10 mL reverse osmosis water. The solution was refluxed at 70 °C for 6 h in the dark in order to exclude the possible degradative effect of light. The resultant solution was applied on RP-TLC plate in triplicate (1.0 μL each, i.e. 1000 ng/band).
The RP-TLC plates were developed and scanned as described above.
6 Results and discussion
6.1 Development of optimum mobile phase
Different compositions of the mobile phase for RP-TLC/densitometry analysis were tested in order to obtain high resolution and reproducible peaks. The desired objective was achieved using methanol:acetonitrile (90:10% v/v) as the mobile phase. The wavelength of 247 nm was found to be optimal for the highest sensitivity. Sharp and well defined peaks for the LOR were obtained at Rf 0.58 ± 0.02 when the chamber was saturated with the mobile phase for 25 min at room temperature.
6.2 Calibration curve
The acceptability of linearity data is often judged by examining the correlation coefficient and intercept of the linear regression line for the response versus concentration plot.
The linear regression data for the calibration curves showed good linear relationship over the concentration range of 200–1200 ng/band. Linear regression equation was found to be Y = 7.154 X + 1293.9. The regression coefficient (r2 = 0.998) is generally considered as evidence of acceptable fit. The results were shown in Figure 1.
Linearity studies of LOR in methanol:acetonitrile (90:10% v/v) as mobile phase.
6.3 Validation of method
6.3.1 Precision
The precision of the developed RP-TLC method was expressed in terms of %relative standard deviation (%RSD). The %RSD value for repeatability of sample application and amount of LOR was estimated. The %RSD value was found to be less than 2.
The results depicted revealed high precision of the method and results are shown in Table 1.
6.3.2 LOD and LOQ
Detection limit and quantification limit were calculated by the method as described above. The LOD and LOQ were found to be 19.40 and 61.51, respectively. This indicates adequate sensitivity of the method.
6.3.3 Specificity
The peak-purity of LOR was assessed by comparing the spectra at peak start, peak apex and peak end positions of the spot, i.e., r2 (S, M) = 0.996 and r2 (M, E) = 0.998. Good correlation (r2 = 0.99) was also obtained between standard and sample spectra of LOR (Figure 2).
Spectra for comparison of LOR standard and LOR extracted from tablets.
6.3.4 Ruggedness
When the method was performed by two different analysts under the same experimental and environmental conditions, it was found to be rugged.
6.3.5 Recovery study
The accuracy of the method is used to check that other components in the pharmaceutical formulation do not interfere with the analytical method.
The proposed method when used for extraction and subsequent estimation of LOR from the pharmaceutical dosage form after over spotting with 80%, 100% and 120% of additional drug, afforded good recovery of LOR. The amounts of drug added and determined, the %recovery is listed in Table 2. The results obtained indicate that other components do not interfere with the analytical method.
6.3.6 Robustness of the method
The standard deviation of peak areas was calculated for each parameter and %RSD was found to be less than 2%. The low value of %RSD, Table 3 indicates the reliability of analytical method during normal usage.
Parameters
%RSD of peak area
Mobile phase volume (±2 mL)
0.94
Development distance (±0.5 cm)
0.79
Duration of saturation (±2 min)
0.81
Time from spotting to chromatography (±10 min)
0.43
Time from chromatography to scanning (±10 min)
0.82
6.3.7 Assay of tablets
A single spot at Rf 0.58 ± 0.02 was observed in the chromatogram of the LOR extracted from tablets. There was no interference from the excipients commonly present in the tablets. The drug content ± S.D. was found to be 99.85 ± 0.63. The amount of drug estimated was found to be in close agreement with label claim indicating the suitability of this method for routine analysis of LOR in pharmaceutical dosage forms.
6.4 Stability studies
6.4.1 LOR stability in the mobile phase and Bench top-stability under normal light conditions for 24 h
There was no significant change in concentration and spectra of LOR in ‘mobile phase stability’ and ‘bench top stability’ under normal light conditions for 24 h, indicating adequate stability of LOR under both these conditions (Figures 3 and 4).
Spectrum of stability studies in mobile phase after 24 h.
Spectrum of stability studies on lab bench top at normal light after 24 h.
6.5 Forced degradation studies
The bands of degradation products were well resolved from the drug bands. The peak of LOR was not significantly shifted in the presence of degradation peaks which show the stability indicating property of the method. The number of degradation products along with their Rf values under different stress conditions is given in Table 4. The chromatograms obtained from LOR contained additional peaks at Rf 0.35 in the acid-induced degradation and at Rf 0.35 and 0.46 in the base-induced degradation (Figures 5 and 6). The concentration of the drug was different from the initial concentration, indicating that LOR undergoes degradation under acidic and basic conditions, spectra of degraded sample shown in Figure 7. The chromatogram of LOR degraded with hydrogen peroxide, photochemical and neutral condition showed well separated spots of pure LOR. However there was no additional peak of degraded sample.
Sample exposure condition
No. of degradation product and Rf
% Amount recovereda
1.0N HCl, 6 h.,70 °C
1 (0.35)
85.11
1.0N NaOH, 6 h., 70 °C
2 (0.35, 0.46)
79.40
10% H2O2, 6 h., 70 °C
–
98.14
Photo, 24 h
–
99.67
Neutral condition 6 h.,70 °C
–
99.48
RP-TLC chromatogram of acid degraded LOR.
RP-TLC chromatogram of base degraded LOR.
Spectra of degraded sample at acidic and basic conditions.
7 Conclusion
This RP-TLC/densitometry method is precise, specific, sensitive, accurate, and stability-indicating. Statistical analysis proved that the method is reproducible and selective for analysis of LOR in the bulk drug and in tablet formulations. The method can be used to determine the purity of the commercially available drug by detecting the related impurities. Because the method could effectively separate and quantify the drugs from their degradation products, it can be regarded as specific and stability-indicating.
Acknowledgement
Authors are thankful to R.C. Patel Institute of Pharmaceutical Education and Research Shirpur Dist: Dhule(MS) India 425 405, for providing necessary research facilities.
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