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TRANSCRIPT
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DEVELOPMENT AND VALIDATION OF HIGH PERFORMANCE
THIN LAYER CHROMATOGRAPHIC METHOD FOR
SIMULTANEOUS ESTIMATION OF LEVOSULPIRIDE AND
ESOMEPRAZOLE IN COMBINED PHARMACEUTICAL
FORMULATION
Rachana Patel*, Jagdish Kakadiya, Pinak Patel, Nehal Shah
Department of Quality Assurance, Indubhai Patel College of Pharmacy and
Research Centre, Dharmaj, Gujarat-388430, India
ABSTRACT
Current research work describes rapid high performance thin layer
chromatographic determination of Levosulpiride and Esomeprazole
from its combined pharmaceutical formulation. The mention drugs
were spotted on silica gel F254 TLC plates under pure nitrogen stream
by Linomat TLC spotter. Separation was carried out by using Toluene:
Methanol: Ethyl acetate: Ammonia as mobile phase in ratio of
4:3:3:0.2, v/v/v/v. Developed TLC plates were scanned by CAMAG
TLC scanner and detection was carried out at 301nm. Rf value of
separated drugs was found to be 0.34 ± 0.00288 and 0.66 ± 0.00577
for LEVO and ESO, respectively. The developed method was
validated as per ICH guidelines by studying various validation
parameters like Accuracy, Precision, Robustness, LOD, LOQ and solvent stability. The
developed and validated method was successfully applied for determination of LEVO and
ESO from its combined pharmaceutical formulation.
Keywords: Levosulpiride (LEVO), Esomeprazole magnesium trihydrate (ESO), High
performance Thin Layer chromatography, Analytical method validation.
INTRODUCTION
Levosulpiride (LEVO), is chemically N-[(1-ethylpyrrolidin-2-yl) methyl]-2-methoxy-5-
sulfamoylbenzamide. It levorotatory enantiomer of sulpiride, a substituted benzamide
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Article Received on 10 March 2014, Revised on 29 March 2014, Accepted on 26 April 2014
*Correspondence for Author
Dr. Jagdish Kakadiya
Department of Quality
Assurance, Indubhai Patel
College of Pharmacy and
Research Centre, Dharmaj,
Gujarat-388430, India
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Patel et al. World Journal of Pharmacy and Pharmaceutical Sciences
indicated as an antipsychotic, antidepressant drug[1-3](Figure 1a). LEVO is not official in
Pharmacopoeia, but it is listed. It consists of blocking the D2 dopaminergic receptors,
preferentially located on the presynaptic membranes in the dopaminergic pathways of the
brain, this means that sulpiride is a selective autoreceptor blocker. Literature survey revealed
that various, UV spectroscopy[4-14], Chromatographic[15-23] methods and LC/MS/MS
method[24] have been reported for quantitative estimation of LEVO in pharmaceutical
formulation and biological fluids individually or in combination with other drugs.
Esomeprazole magnesium trihydrate (ESO) is chemically, bis(5-methoxy-2-[(S)-[(4-
methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole-1-yl) magnesium
trihydrate[25-27] (Figure 1b). ESO is class of “proton pump inhibitor” that inhibits gastric acid
secretion through inhibition of K+/H+ ATPase in gastric parietal cells. It is S- Enantiomer
of Omeprazole . It is used for short term treatment of erosion and ulceration of the
esophagus caused by gastro-esophageal reflux disease (GORD), peptic ulcer, NSAID-
associated ulceration and zollinger-Ellision syndrome[27]. ESO is official in IP (2010)[28]. The
review of literature revealed that various analytical methods involving Spectrophotometry[11-
14, 29-31], HPLC[22, 23, 32-44]and HPTLC[45-46] have been reported for ESO in pharmaceutical
formulation and biological fluids individually or in combination with other drugs.
To the best of our knowledge, there is no published HPTLC method for this combination. So,
the present paper describes a simple, accurate and precise method for simultaneous
estimation of LEVO and ESO in combined Pharmaceutical formulation by HPTLC method.
The developed method was validated in accordance with ICH Guidelines[47] and successfully
employed for the assay of LEVO and ESO in their combined pharmaceutical formulation.
Figure 1: Chemical structure of (a) LEVO and (b) ESO
MATERIALS AND METHODS
Materials
LEVO reference standard was procured from Intas Pharmaceuticals Ltd. Ahmedabad,
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Gujarat, India, as gift sample for research purpose and ESO reference standard was procured
from Torrent pharmaceuticals Ltd, Capsules containing LEVO 75 mg and ESO 40 mg was
procured from local market (NEXPRO L). Silica Gel F254 plates was purchased from E
Merck India Pvt. Ltd. Mumbai. methanol, toluene, ethyl acetate, and glacial acetic acid was
purchased from S.D. fine Chemicals.
Instrument and Experimental Conditions
HPTLC analysis was carried out on silica gel 60 F254 HPTLC plates (10 × 10 cm) by means
of a Linomat V automatic spotter equipped with a 100 µL syringe and operated with settings
of band length, 6 mm; distance between bands, 5 mm; distance from the plate edge, 10 mm;
and distance from the bottom of the plate, 10 mm. The plate was developed in a twin‐trough
chamber previously saturated for 20 min with the mobile phase for a distance of 7 cm. For
densitometry analysis, the spots on the air dried plate were scanned with the Scanner III at
301nm using the deuterium source. Photograph of developed plates were taken by
REPROSTAR camera at 254 nm.
Preparation of working solutions
Based upon trial and error at laboratory scale finally it was decided to prepare stock solution
of 750 µg/ml and 400 µg/ml of LEVO and ESO respectively.
Final mobile phase consisting of Toluene: Methanol: Ethyl Acetate: Ammonia
(4:3:3:0.2,v/v/v/v) was placed in CAMAG TLC chamber and saturation was performed for 30
min. Final Separation was achieved using above mobile phase.
Analytical Method validation:
Preparation of calibration curves/ Linearity and range
For preparation of linearity and range, Synthetic mixture containing 750 µg/ml and 400µg/ml
of LEVO and ESO was prepared and 100 µl Hamilton syringe was filed and aliquots of 1µl,
1.5µl, 2µl, 2.5µl, 3µl, 3.5µl was applied under pure nitrogen stream to give rise to spots
containing LEVO in range of 750-2625 ng/spot and ESO 400-1400 ng/spot. Spotted plates
were developed under stated condition and dried plates were scanned at 301 nm. Procedure
was repeated for further 5 times (Total n=6). Finally mean area was plotted against
concentration (ng/spot) with help of WINCATS software.
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Accuracy studies (Recovery)
Accuracy studies were performed by spiking test solution with standard solution. Accuracy
studies were performed at spiking level of 50, 100 and 150% of target concentration. Here
stock solution containing 750 µg/ml of LEVO and 400 µg/ml of ESO was prepared from
capsule formulation. Resulting solution was filtered and 1 ml of solution was transferred to
each four 10ml volumetric flask. Now from standard stock solution of 1000 µg/ml of LEVO
and 500 µg/ml of ESO various aliquots was transferred to each 10 ml volumetric flask.
Volume was made upto mark with methanol and 6 µl of solution was applied from each
volumetric flask on to plate. Procedure was repeated for further 2 times and mean recovery
for each level was calculated (n=3).
Method Precision (Repeatability)
For repeatability studies the linearity studies was repeated for 6 times without changing the
syringe and position of plates. Data are collected from each set and Mean area, standard
deviation and Coefficient of variance was calculated. Intermediate Precision (Reproducibility)
Precisions of the proposed HPTLC methods were determined by analyzing mixed standard
solution of LEVO and ESO at 3 different concentrations (750, 1500, 2625 ng/spot for LEVO
and 400, 800, 1400 ng/spot for ESO) 3 times on the same day (intraday precision) and on 3
different days (Interday Precision). The results are reported in terms of relative standard
deviation (RSD).
Limit of Detection and Limit of Quantitation (LOD and LOQ)
The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived
by using the following equations as per International Conference on Harmonization (ICH)
guidelines which is based on the calibration curve.
LOD = 3.3 × σ /S
LOQ = 10 × σ /S
Where σ = the standard deviation of y-intercepts of regression lines
S = Slope of calibration curve. Specificity Studies
The excipients such as starch, lactose were spiked into a reweighed quantity of drugs to
assess the specificity of the methods. The peak area was measured to determine the quantity
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of the drugs.
Robustness
Robustness was performed by changing various method parameters like Composition of
mobile phase, Size of TLC Chamber, Saturation time and plate pretreatment. Finally effect of
this changes was observed for change in Rf value and change in peak area.
Analysis of Marketed formulation
A powder quantity equivalent to 75 mg LEVO and 40 mg ESO was accurately weighed and
transferred to volumetric flask of 100 ml capacity. 60 ml of methanol was transferred to this
volumetric flask and sonicated for 10 min. The flask was shaken and volume was made up to
the mark with methanol. The above solution was filtered through whattman filter paper
(0.45µ). From this solution 5 ml was transferred to volumetric flask of 50 ml capacity
(Containing 750 ng/µl LEVO and 400 ng/µl ESO). From above solution apply 6 µl of volume
on to pretreated Silica Gel F254 plates.
RESULT AND DISCUSSION
Method optimization
Several mobile phases were tried to accomplish good separation of LEVO and ESO and final
separation was achieved using Toluene: Methanol: Ethyl Acetate: Ammonia (4: 3: 3:0.2,
v/v/v/v) (Fig. 2, 3). The developed plate was analyzed by densitometry and densitogram was
recorded to check resolution (Fig.4). Rf value of LEVO and ESO was found to be 0.34 ±
0.00288 and 0.66 ± 0.00577 respectively. For quantitation spots were scanned at 301nm
(Fig. 7). Finally all chromatographic conditions were optimized (Table. 1).
Figure 2: Photograph of developed Plate in final Mobile phase
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Figure 3: Densitogram of LEVO and ESO 750 ng/spot and 400 ng/spot respectively using mobile phase Toluene: Methanol: Ethyl Acetate: Ammonia(4.0: 3.0: 3.0: 0.2, v/v/v/v)
Figure 4: Scanning spectra of LEVO and ESO in the range of 200-400 nm
Figure 5: Calibration curve of LEVO by HPTLC method
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Figure 6: Calibration curve of ESO by HPTLC method
Figure 7: Overlay views of all tracks of LEVO and ESO at
Table. 1: Optimized Chromatographic conditions
Parameter Condition Mobile phase Toluene : Methanol : Ethyl acetate:
Ammonia(4: 3: 3: 0.2) Diluent Methanol Stationary phase Silica gel G F254 Distance run 70 mm Chamber dimensions 10 x 10 cm Saturation time 20 minutes Temperature Ambient Detection wavelength 301 nm Rf value LEVO: 0.34 ± 0.0028
ESO:0.66 ± 0.0057
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Analytical Method Validation
Linearity and range
The method was found to be linear with concentration of 750 -2625 ng/spot of LEVO and
400-1400 ng/spot of ESO(Table. 2 and 3) as r2 value was found to be 0.997 for LEVO and
0.998 for ESO (Fig. 7 and 8). Overlain spectra in 3D view also showed a good linearity (Fig.
9).
Method Precision (Repeatability studies)
Method was found to be repeatable as value of coefficient of variance was found to be less
than 2. For LEVO and ESO at all concentration..
Intermediate Precision (Reproducibility)
Method was found to be reproducible as value of coefficient of variance was found to be less
than 2. For LEVO and ESO at all given concentration for both interday and intraday.
Accuracy Study
Accuracy was performed by spiking method at 50, 100,150% of target concentration.
Recovery was found in the range of 100.1 – 101.1 % for LEVO and 100.9 – 102.1% for
ESO (Table. 4).
Determination of LOD and LOQ
The LOD for LEVO and ESO was found to be 42.72 ng/spot and 49.021 ng/spot
respectively.
LOQ was found to be 129.4 ng/spot and 148.5 ng/spot of LEVO and ESO respectively
(Table.5).
Specificity Studies
The method was found to be specific as there was no interference from the commonly used
excipients. Analysis was performed with peak purity analysis and peak purity was found to
be greater than 0.999.
Robustness
Minor modification were made in method parameters and changes were observed in peak
area and Rf value. And it was found that the method was found to be robust as there was no
significant change in peak area and Rf value (Table 6).
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Assay of Marketed Formulation
The developed method applied for quantitation of LEVO and ESO from its combined dosage
form value was found to be 99.84 ± 0.167and 99.8 ± 0.115 respectively (Table 7)
Table 2: Linearity data for LEVO
Concentration of Drug
(ng/spot) Area
Mean ± S.D
C.V.
750 2978.9 ± 31.00 1.04 1125 3979.2 ± 32.93 0.82 1500 4926.0 ± 40.48 0.82 1875 5648.1 ± 46.09 0.81 2250 6426.7 ± 34.48 0.53 2625 7331.4 ± 49.98 0.68
Table 3: Linearity data for ESO
Concentration of
Drug (ng/spot)
Area Mean ± S.D
C.V.
400 3370.9 ± 55.27 1.63 600 4394.2 ± 78.38 1.78 800 5402 ± 72.53 1.34 1000 6293.3 ± 49.12 0.78 1200 7214.3 ± 62.86 0.87 1400 8007.4 ± 47.65 0.59
Table 4: Accuracy data for LEVO and ESO at 50, 100 and 150 % of target
concentration (n=3)
% Level of
Recovery
Amount of drug
in sample
(ng/spot)
Amount of standard
added (ng/spot)
Total amount of Drug (ng/spot)
Amount of drug
recovered ± SD
(ng/ml)
% Recovery ± SD
LEVO % LEVO Unspiked 750 -
50 % 750 375 1100 366.4 ± 11.4 100.1 ± 0.70 100 % 750 750 1500 767.9 ± 10.3 101.06 ±0.60 150 % 750 1125 1875 1144.2 ± 13.0 100.1 ± 0.45
ESO %ESO Unspiked 400 -
50 % 400 200 600 200.2 ± 25.3 100.6 ± 1.01 100 % 400 400 800 417.8 ± 19.8 101.1 ± 1.05 150 % 400 600 1000 628.8 ± 15.5 102.2 ± 0.83
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Table 5: Determination of LOD and LOQ
LEVO(ng/spot) ESO(ng/spot) LOD based on mathemathical equation
42.72 LOD based on mathemathical equation
49.02
LEVO ESO LOQ based on mathemathical equation
129.4 LOQ based on mathemathical equation
148.5
Table 6: Robustness studies for LEVO by HPTLC method
Parameter
Level of Change
Effect on Peak area
LEVO Area ± S.D. %Assay
Proportion of Mobile Phase
4: 3 : 3: 0.2 3052.4± 7.63 98.13 ± 0.106 7: 2.5 : 0.5 3075.9±7.02 99.26 ±0.255
Size of TLC chamber
20*20 cm 3073.7± 13.03 99.26 ± 0.115 10*10 cm 3108.8± 6.17 100.9 ± 0.458
Saturation time 20 min 3073.06 ±8.40 99.29 ±0.145 10 min 3054.63 ± 9.01 98.52 ±0.157
Plate Pretreatment
With Pretreatment
3113.28± 9.99 100.3 ±0.294
Without Pretreatment
3108.17 ± 4.09 100.6 ± 0.585
Table 7: Robustness studies for ESO by HPTLC method
Parameter
Level of Change
Effect on Peak area ESO
Area ± S.D. % Assay Concentration of Mobile Phase
4: 3 : 3: 0.2 3438.51 ± 59.0 99.21 ±1.00 7: 2.5 : 0.5 3441.95 ± 78.5 99.25 ± 0.7
Size of TLC chamber
20*20 cm 3449.3 ±53.4 99.8 ±0.66 10*10 cm 3423.3 ± 95.5 99.3 ± 1.02
Saturation time 20 min 3446.03 ± 58.7 99.07 ± 0.71 10 min 3457.9 ± 97.8 100.3 ± 0.43
Plate Pretreatment
With Pretreatment Without Pretreatment
3471.4± 66.15
3487.4 ± 56.8
100.1 ± 0.63
100.9 ±0.79
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Table 7: Assay of Marketed Formulation
Formulation
Drug Amount
Taken (ng/spot)
Amount Found
(ng/spot) (n = 3)
Labeled claim (mg)
Amount found per
Tablet (mg)
% Label claim ±
SD
Nexpro- L LEVO 750 747.5± 1.678 75 74.75 99.84 ±
0.167
ESO 400 399.5± 3.907 40 39.95 99.8 ±
0.115 Summery and Conclusion
The HPTLC method was successfully developed and validated as per ICH guidelines and was
successfully applied for rapid determination of LEVO and ESO from its combined
pharmaceutical formulation. The developed and validated HPTLC method for LEVO and
ESO was found to be simple specific and cost effective for analysis of LEVO and ESO in
their combined dosage form. The additives usually present in the pharmaceutical
formulations of the assayed analytes did not interfer with determination of LEVO and ESO.
The method can be used for the routine simultaneous analysis of LEVO and ESO in
pharmaceutical preparations.
ACKNOWLEDGEMENT
Authors are extremely grateful Intas Pharmaceutical Pvt Ltd and Torrent Pharmaceutical Ltd
for providing reference samples of LEVO and ESO respectively. The authors are also grateful
to SICART (Sophisticated instrumentation center for applied research and technology),
Vallabh Vidyanagar, Gujarat, India, for providing excellent facilities for carrying out this
research work.
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