© 2019 IJRAR March 2019, Volume 6, Issue 1 www.ijrar.
org (E-ISSN 2348-1269, P- ISSN 2349-5138)
Development And Validation of HPTLC for
Simultaneous Estimation of Montelukast Sodium,
Levocetirizine Dihydrochloride And Ambroxol In
Bulk And In Three-Component Capsule
Formulation
2Manisaikumar D, 1Dr. U.Srinivasulu*,3Mahesh. M,
Post graduate student, Reader in Chemistry, Asst. Professor
1
Dept of Chemistry, 2&3Dept of Pharm. Analysis
Abstract
This method was analyzed in simple, quick, precise and accurate for both bulk and fixed-dose capsules by using densitometric
detection. Separation was performed by using mobile phase isethylacetate, methanol, toluene and ammonia (7:2.5:2.5:1v/v/v/v).The
Rf values were found at0.21, 0.59 and 0.79 for Levocetirizine Dihydrochloride,Montelukast Sodium and Ambroxol Hydrochloride at
224nm respectively. The correlation coefficient values were found to be above 0.999 indicated that the calibration graphs were
adequately linear at the evaluated concentration ranges. F-test for the quantification says the variances among the repeatabilities and
was within the limit (Fcrit). The pooled %RSD for repeatability of the amount (assay of theformulation) were found to be 1.6567,
0.4882 and 0.5487, for time different intermediate precision for intraday were 1.1657, 0.2379 and 0.2508 and for interday were
0.3168, 0.5234 and 0.3269 for Levocetirizine Dihydrochloride, Montelukast Sodium and Ambroxol Hydrochloride respectively. The
range of recoveries was 97.54-98.66 for Montelukast Sodium, 98.60-100 for Levocetirizine Dihydrochloride and 99.26-100.1 for
Ambroxol Hydrochloride (n=3). Most factors evaluated in the robustness test were found to have an insignificant effect on the
selected responses at 95% confidence level.
Keywords: Montelukast Sodium, Levocetirizine Dihydrochloride, Ambroxol Hydrochloride, HPTLC, Densitometric detection.
1. INTRODUCTION
Asthma, a worldwide health problem, may have negative impacts on life quality and in fact on life span. Asthma is the
chronic inflammatory disease of the airways characterized by variable and recurring symptoms, reversible airflow obstruction and
bronchospasm. The first line of treatment includes single drug therapy and second treatment is the multiple drug therapy. The drug
therapy was given through oral route in the form of aerosols and powdered inhalers. The multiple dosage forms were used previously
to treat asthma alone but now a days this combined dosage form especially was used to prevent breathing problems during exercise
and seasonal allergic rhinitis. Montelukast Sodium (MON) is a potent cysteinyl leukotriene receptor antagonist which is being used
very effectively in the treatment of chronic asthma and chemically it is 1-[[[(1R)-1-3-[1E)-2- (7-chloro-2-quinolinyl) ethenyl]
phenyl]-3- [2-(1- hydroxy-1-methylethyl) phenyl] propyl] thio] methyl] cyclopropane acetic acid sodium[1-3]. Levocetirizine
Dihydrochloride (LEVO) is the R-enatiomer of Cetirizine, a non sedating antihistamine and used for the treatment for symptomatic
relief of allergic conditions including rhinitis and chronic urticaria. Chemically it is (R)-2-[2-[4-[(4-chlorophenyl)
phenylmethyl]piperazine-1-yl]ethoxy]acetic acid dihydrochloride [1,4]. Ambroxol Hydrochloride (AMB) is a metabolic product of
Bromhexine and used as mucolytic expectorant and chemically it is trans-4-[(2-Amino-3,5-dibromobenzyl)amino]cyclohexanol
hydrochloride [1,5]. The combined dosage form especially was used to prevent breathing problems during exercise and seasonal
allergic rhinitis. Literature survey revealed that the LEVO is official in IP [6] and AMB is official in IP [6] and BP [7]. The molecular
structure of LEVO, MON and AMB are shown in fig. 1, 2 and 3.
OH Cl O COOH
N
Br N
NH
H
. HCl . 2 HCl
NH2
Br
Fig no.: 1.Ambroxol Hydrochloride Fig no.: 2.Levocetirizine Dihydrochloride
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+
S COONa
Cl N
HO
H3C
CH3
Fig no.: 3. Montelukast Sodium
Several analytical methods have been reported for estimation of MON using UV spectroscopy [8], HPLC [9, 10] and by LC-MS [11],
AMB by UV spectroscopy [12], HPLC [13, 14] and by LC-MS [15,16] and for LEVO by HPLC [17,] and by LC-MS [18]. Methods reported in
the literature for the simultaneous estimation of MON and LEVO in formulations by UV spectrophotometry [19], HPLC [20,21] and
HPTLC [22], simultaneous estimation of LEVO and AMB in formulations by UV [23] spectrophotometry have been previously
reported. The purpose of this research reported here was to develop a simple, accurate, specific, and precise HPTLC method with
densitometric detection. The proposed method was optimized and validated as per ICH guidelines [24].
2. MATERIALS AND METHODS
2.1 Chemicals:Levocetirizine Dihydrochloride, Ambroxol Hydrochloride and Montelukast sodium were gifted by Madras
Pharmaceuticals Pvt. Ltd., India and Dr. Reddy’s Laboratories Pvt. Ltd., India. Commercially available RENEA, capsule of AMB
manufactured by Shield Healthcare Pvt. Ltd., Indiawere procured from the local market.
2.2Instrumentation:Spotting device: Linomat V sample spotter Camag (Switzerland), Syringe: 100 L Hamilton (Bonaduz,
Switzerland), TLC Chamber: Twin trough chamber (Camag, Muttenz, Switzerland), Densitometer: TLC scanner III with CATS
software (1.4.4 6337) Camag.
2.3Chromatography Condition:Stationary phase: Silica gel 60 F254 aluminum sheets, Mobile Phase:ethyl acetate-methanol-
toluene-ammonia (7:2.5:2.5:1), Scanning Wavelength: 224 nm, Applied Volume: 0.5–5 µL.
3. METHODS
3.1 Preparation of standards: 20 mg of MON, 20 mg of LEVO and 75 mg of AMB were weighed accurately by using a
Shimadzu AUX - 220 Digital balance and transferred in to 50 mL volumetric flasks separately, dissolved in methanol and made up to
the volume to 50 mL with methanol. The standard stock solutions contain 400 µg/ mL of Montelukast Sodium, 400 µg/ mL of
Levocetirizine Dihydrochloride and 1500 µg/ mL of Ambroxol Hydrochloride. 1 mL of each of the standard stock solutionwas
transferred in to a 10 mL volumetric flask and the solution was diluted to 10 mL with methanol. From the solution, 0.5 – 5 µL were
applied on silica gel 60 F254 aluminum sheets. Mobile phase components were mixed prior to use and the development chamber was
left to saturate with mobile phase vapour for 1h before each run. Then the plates were dried on a hot plate. Room temperature and
relative humidity were always maintained at 20°C ± 2 and 55 % ± 5, respectively. The separation conditions were based on the TLC
screening test for LEVO, MON and AMB, these conditions were transferred to the HPTLC system and the results were evaluated with
the aim of achieving an optimum separation between spots (Rs 1.5), and a migration of spots with Rf values at 0.21, 0.59 and 0.79 for
Levocetirizine Dihydrochloride, Montelukast Sodium and Ambroxol Hydrochloride, in order to ensure separation reproducibility was
shown in fig-4(b). The calibration graph was plotted using peak area vs concentration.
b
c
a
Fig no.:4 chromatogram of standard drugs (a) LEVO (b) MON (c) AMB
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3.2 Preparation of samples: Twenty capsules (RENEA containing 10 mg of Montelukast Sodium, 5 mg of Levocetirizine
Dihydrochloride and 75 mg of Ambroxol Hydrochloride) were accurately weighed and average weight was found. Powdered the mixed
contents of the capsule powder, weighed accurately a quantity of the capsule powder equivalent to 75 mg of Ambroxol Hydrochloride
and transferred in to a 25 mL volumetric flask and extracted by using methanol. Finally the solution was further diluted to get
concentration of 20 ng/ L, 40 ng/ L and 300 ng/ L of LEVO, MON and AMB, respectively. For the intraday and interday analysis,
the analysis was done in three replicates daily and then repeated each replicate for three consecutive days. The repeatability, (S 2r), and
the time different intermediate precision, (S2I (t)), were then estimated at each concentration level from an ANOVA table and equation,
S2I (t) = S2r + S2between. Where S2between represents the between - days variance. Chromatogram shown in fig-4(c) and formulation alone fig
no.-5.
Fig no.:5 chromatogram of formulation (a) LEVO (b) MON (c) AMB
3.3 Preparation of 80%, 100% and 120% spiked samples for the recovery: The capsule powder was spiked with reference
standards MON, LEVO and AMB at 80%, 100% and 120% of the target concentrations of each compound. The obtained solutions
were applied on the HPTLC plates to form spots with 36, 40 and 44 ng/ spot of LEVO, 72, 80 and 88 ng/ spot of MON and 540, 600
and 660 ng/ spot of AMB. The analysis was repeated for three times for each concentration. The trueness was determined and
expressed as percentage recovery.
4. Results and discussion
4.1Linearity of the calibration line
Before performing regression, the homoscedasticity of the calibration standards was verified using a Cochran’s test. The
Ccalc values were 0.284, 0.276 and 0.327 for LEVO, MON and AMB, respectively. These test statistics were smaller than the critical
value, Ctab(α=0.05;k=5, n=9) = 0.439. Thus, the variances of the calibration standards were considered to be homoscedastic and
ordinary least squares could be used to estimate the regression lines. Regression analysis was performed using Microsoft Office Excel
XP. Equations of the calibrations lines for LEVO, MON and AMB were Area LEVO = 12.68CLEVO (ng/ spot) + (-6.45), AreaMON =
17.31CMON (ng/ spot) + 63.76, and AreaAMB = 4.17CAMB (ng/ spot) + 11.59, respectively. The corresponding values of the slopes and
intercepts with their 95% confidence limits were 12.68±1.14 and 6.45±0.73 for LEVO, 17.31±1.62 and 63.76±13.62 for MON, and
4.17±0.14 and 11.59±0.79 for AMB. The correlation coefficients were 0.9992, 0.9990 and 0.9993, for MON, LEVO and AMB,
respectively. Visual observation of the calibration curves gave the impression that they were linear. A student’s t-test was performed
to determine wether the experimental intercept (c) was not significantly different from the theoretical zero value. It concerns the
comparision of t = c/sc, where c is the intercept of the regression equation and sc is standard deviation of c, with tabulated data of the t-
distribution. As the calculated t-value (tMON=2.5632, tLEVO=0.7899 and tAMB=1.3491), does not exceed to (0.01%) 2.567, the intercept
of regression equation is not significantly differ from zero. (Table 1)
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Table 1Linear regression analysis data
PARAMETERS LEVO* MON* AMB*
Detection wavelength 224 nm 224 nm 224 nm
Linearity range (ng/l) 20 – 200 20 - 200 75 – 750
Correlation Coefficient 0.9990 0.9994 0.9993
(r)
Regression Equation Y = 12.6776x+(-6.4515) Y=17.4154x+22.9086 Y = 4.1748x+11.5865
(y=mx+c)
Slope (m) 12.677624 17.41543 4.174865
Intercept (c) -6.451515 22.90864 11.586515
LOD (ng/ L) 2.1260 1.0238 6.7883
LOQ (ng/ L) 6.4424 3.1025 20.5708
t-test 0.7899 2.5632 1.3491
Sa 8.1635 20.8897 8.5880
Sb 0.1242 0.2211 0.0460
Resolution --- 13.33 9.24
* Mean of six determinations, Sais standard deviation of intercept, Sb is standard deviation of slope
4.2 Precision
Precision of the method was determined by performing repeatability and intermediate precision. Repeatability of the method
was done by the repeated analysis of formulation for six times. The percentage purity of MON, LEVO and AMB in formulation
(RENEA) were found to be 99.97 ± 0.4881, 101.89 ± 1.6881 and 100.02 ± 0.5487 respectively. The F-value and variance are used for
comparing results that best fit into the data set and the data obtained are within limit compared to the critical values. Confidence
Interval (95 %) were found to be 99.45 – 100.48 for MON, 100.11 – 103.66 for LEVO and 98.39 – 101.64 for AMB, respectively.
Intermediate precision was confirmed by performing the analysis of formulation for three times on the same day and one
time on three consecutive days. The %RSD for intraday and interday analysis were found to be 0.2379 and 0.5234 for MON, 1.1657
and 0.3168 for LEVO and 0.2508 and 0.3269 for AMB, respectively. The precision was confirmed by low %RSD values for Intraday
and Interday analysis. The results are shown in table 2 and 3.
Table 2 Results of analysis of the formulation
Drug Label claim Amount Drug SD Variance F value
(mg/ tablet) found content (%) RSD (%)
(mg) n=6
n=6
MON 10 9.997 99.97 0.4881 0.4882 0.5734 0.3328
LEVO 5 5.0945 101.89 1.6881 1.6567 3.1178 0.1589
AMB 75 75.015 100.02 0.5487 0.5486 0.4519 0.0001
SD is Standard deviation, RSD is Relative Standard Deviation, Ftab (α=0.05;df1=2, df2=17)= 3.6823
Table 3 Intraday and Interday results for the formulation
Intraday Interday
Drug
Percentage Variance %RSD F value Percentage Variance %RSD F value
99.79 0.3090 99.77 0.3200
MON* 99.34 0.0543 0.2379 0.1892 100.38 0.1860 0.5234 0.4243
99.67 0.0508 99.34 0.0882
101.08 2.2087 101.76 0.3612
LEVO* 99.50 0.6224 1.1657 0.1485 102.00 0.1045 0.3168 0.0499
100.26 0.7527 102.40 0.3345
100.05 0.0212 100.05 0.0280
AMB* 100.13 0.0632 0.2508 0.1591 100.08 0.0886 0.3269 0.0197
100.52 0.1669 99.50 0.3366
* Mean of three observations; Ftab (α=0.05;df1=2, df2=6)=5.1432
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4.3 Trueness
The mean percentage recovery for each compound was calculated at each concentration level and reported with its standard
deviation. The results obtained for LEVO at 80 %, 100 % and 120% concentration levels were 100 ± 0.6854, 98.60 ± 1.9744 and
99.68 ± 0.7633, respectively. The range of % recovery values was 96.34 – 100.73 %, while the mean recovery for all the
concentration levels was 99.42 ± 0.99.
For MON the % recoveries at the same concentrations levels were 97.54 ± 0.9086, 98.54 ± 1.3291 and 98.66 ± 0.7494,
respectively. The range of % recovery values was 97.14 - 100 %. The mean value covering all concentration levels was 98.25 ± 1.01.
The % recovery values for AMB were 99.88 ± 0.4650, 99.26 ± 0.9586 and 100.10 ± 0.3517 respectively. The range was
98.17 – 100.35 % and the overall mean was found to be 99.75 ± 0.59. The % bias values for the recovery shows the results obtained
were in good agreement with standard values. In conclusion, the method was considered to have an acceptable recovery and trueness.
The results were tabulated in table 4.
Table 4 Recovery data analysis table
% Drug Amount Amount Amount % SD % RSD %Bias
added present added recovered Recovered*
(ng/ L) (ng/ L) (ng/ L)*
MON
80 72.0646 32 31.2144 97.54 0.9086 0.9115 2.56
100 80.2625 40 39.4102 98.54 1.3291 1.3417 1.56
120 88.5447 48 47.3591 98.66 0.7494 0.7595 1.44
LEVO
80 36.8344 16 16.0008 100.00 0.6854 0.6872 0.00
100 40.5545 20 19.7209 98.60 1.9744 2.0024 1.4
120 44.7638 24 23.9301 99.68 0.7633 0.7657 0.32
AMB
80 544.9804 240 239.7218 99.88 0.4650 0.4663 0.12
100 604.3981 300 299.4728 99.26 0.9586 0.9657 0.74
120 666.6271 360 360.0369 100.1 0.3517 0.3613 0.10
*Mean of three determinations
4.4 Specificity
The chromatogram of the solution of the non-spiked capsule matrix did not show any spots. On the other hand, the
chromatogram of the capsule matrix spiked with three compounds showed clear, compact and well-separated peaks of LEVO, MON
and AMB (Fig. no-6.). Moreover, no other peaks eluted besides the three active compounds. Therefore, the method was considered
specific.
Fig no.:6 blank chromatogram
4.5 Ruggedness
Ruggedness of the method was confirmed by the analysis of formulation was done with different analysts. The %RSD for
analyst 1 and analyst 2 were found to be 0.6224 and 0.4666 for Montelukast Sodium, 1.7359 and 1.8957 for Levocetirizine
Dihydrochloride and 1.1735 and 0.4660 for Ambroxol Hydrochloride respectively (Table 5).
4.6Robustness
HPTLC method robustness was determined during the method development. Here four factors were screened, i.e. the
developing distance, amount of toluene in the mobile phase, drying conditions applied to the HPTLC plate after development and spot
band size. Factor selection was based on observations during method development and own experience. The %RSD for the factors
screened were found to be 0.8753 for the developing distance, 0.7683 for amount of toluene in mobile phase, 1.2301 for drying
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conditions applied to the HPTLC plate after development and 1.1087 for spot band size, respectively. The above low %RSD values
indicated that the robustness of the method was confirmed (Table 5).
Table 5 Ruggedness and Robustness study for method validation
Ruggedness study
Different Analyst* MON (%RSD) LEVO(%RSD) AMB(%RSD)
Analyst I 0.6224 1.7359 1.1735
Analyst II 0.4666 1.8957 0.4660
Robustness study
Levels
Factor Nominal %RSD
(-) (+)
(0)
(A)Developing distance(cm) 6 7 8 0.7683
(B) Toluene content in the total mobile phase (mL) 3.5 4 4.5 0.8753
(c) drying conditions applied on the plate after development Air Hot plate oven 1.2301
(D) spot band size (mm) 6 7 8 1.1087
* Mean of six determinations.
4.7 Limits of detection and quantification
The limits of detection (LOD) and quantification (LOQ) were calculated from the calibration graphs. The method based on
the determination of slope of linearity plot and standard deviation of y intercept of linearity was used for the determination of LOD and
LOQ and found to be 1.02 ng/ L and 3.10 ng/ L for MON, 2.12 ng/ L and 6.44 ng/ Lfor LEVO and 6.79 ng/ L and 20.57 ng/ L
for AMB, respectively (Table 1).
5. SUMMARY CONCLUSION
A quick, precise and accurate method has been developed for the simultaneous routine analysis of MON, LEVO and AMB in
bulk and in fixed-dose combination capsules. The method was successfully validated for linearity, precision, trueness, specificity,
ruggedness and robustness. It has the advantage over HPLC methods in general. It consumes less than 25 mL of mobile phase per run
(18 samples per plate), whereas HPLC methods would consume not less than 100 mL per runs of the similar number of samples. If we
consider the time from sample preparation to densitometric evolution for one plate, the new method takes an average of 1h, whereas
HPLC methods would generally take more than 2h for the same number of samples. It is cheap, quick and does not use chloroform,
therefore suitable for the simultaneous routine quality control analysis of MON, LEVO and AMB in bulk and in fixed dose capsule
formulation.
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