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ABOUT AUTHORS:
Shoumik Roy*, Samil D. Desai, Bhavna A. Patel, Shraddha J. Parmar
Post Graduation Department of Pharmaceutical Sciences,
Sardar Patel University, Vallabh Vidyanagar, Gujarat, India.
roy.shoumik@gmail.com
ABSTRACT
The main thrust of the paper was to develop and validate a simple, precise high performance thin-layer chromatographic (HPTLC) method for estimation of tapentadol hydrochloride in tablet dosage form. Chromatography was performed on silica gel 60 F254 plates with Chloroform: Acetone: Ammonia (2.5: 2.4: 0.1 v/v/v) as mobile phase. This mobile phase system gave a good resolution for tapentadol hydrochloride (Rf value of 0.49 ± 0.02). Detection and quantification were carried out at 272 nm. The linear regression data for the calibration plot showed a good relationship with r=0.999. The limits of detection and quantification were 62.68 and 189.94ng/spot for tapentadol hydrochloride. The amounts of the drugs in the marketed formulation were 99.98%.
INTRODUCTION
Tapentadol hydrochloride(TAP),3-[(1R,2R)-3-(dimethylamino)-1-ethyl-2-methylpropyl]phenol hydrochloride (FIG.1) is a centrally acting analgesic agent that affects the brain and body primarily by activating opioid receptors in the brain, spinal cord and gastrointestinal tract. In addition, Tapentadol inhibits the reuptake of the brain chemical nor epinephrine which possibly has an analgesic effect[1,2]. The drug has dual mode of action in single molecule as an agonist at µ-opioid receptor and as a nor epinephrine reuptake inhibitor. With this mode of action Tapentadol provides analgesia at comparable levels of more potent narcotic analgesics such as morphine, oxycodone and hydrocodone[3,4].
Literature survey reveals that Tapentadol was analyzed by HPLC methods and UV methods in bulk and in formulations.[5,6,7,8] Till now no HPTLC method is developed for quantitative analysis of tapentadol present in literature survey. Hence it was endeavored to develop & validate a simple & precise HPTLC method for estimation of tapentadol.
Experimental:
Materials:
Methanol (AR grade), Ethyl Acetate(AR grade),chloroform(AR grade), Acetone (AR grade), Toluene(AR grade), Ethyl Acetate(AR grade), Acetic Acid (AR grade), Sodium Hydroxide(AR grade) were supplied by Sisco Research Lab Pvt Ltd, Andheri, Mumbai and Hydrochloric Acid(AR grade) was supplied by S.d. fine-Chem Ltd, Mumbai.
HPTLC instrumentation:
A CAMAG HPTLC system equipped with Linomat 5 Auto-sampler, TLC scanner 4, and winCATS v1.4.7 software (CAMAG, Muttens, Switzerland) was used. The slit dimension was kept at 6.00 mm × 0.1 mm, and 20 mm/sec scanning speed was employed. Chromatography was performed on pre-coated silica gel 60 F254 TLC plates (20×20cm2, catalogue no.:1.05554.0007 cm, (E.Merck, Germany) using Chloroform : Acetone : Ammonia ( 2.5 : 2.4 : 0.1 v/v/v) as mobile phase. The band length 6 mm and distance between bands 10 mm were kept constant throughout the study. Each concentration was spotted five times on TLC plate. The application speed was 150 nL/sec. Ascending development to a distance of 75 mm was performed on (10x10cm2, 20x10cm2, and 20x20cm2) cm twin through chamber (CAMAG). Chromatograms were evaluated via peak area after scanning in absorbance mode at 272 nm.
Preparation of standard TAP solution:
Accurately weighed 10 mg of TAP was transferred into 10 ml volumetric flask and dissolved in water and diluted up to the mark with water to get a stock solution containing 1 mg/ml of TAP (1000 μg/ml TAP).
Preparation of working standard solution:
1 ml of TAP standard stock solution was diluted to 10 ml with water to get TAP working standard solution containing 100 μg/ml of TAP.
Preparation of sample solution of marketed formulation:
Twenty tablets were weighed and finely powdered. The powdered equivalent to 50 mg of TAP were weighed and transferred to 50 ml volumetric flask. To this, about 20 ml of water was added and sonicated for 15 min. The volume was made up to the mark with water and mixed well. The solution was filtered through Whatman filter paper no. 41 and 1 ml of filtrate was diluted to 10 ml with water to obtain a solution containing 100μg/ml TAP.
Method Validation:
This method was validated for the parameters listed below as per ICH guideline Q2 (R1).
Linearity:
Various dilutions of combined working standard solution were spotted on the TLC plates which cover the range of 700-1400 ng/spot to determine the linearity. Each concentration was spotted five times on TLC plate.
Accuracy (percent of recovery):
80%, 100%, and 120% level of concentration of drug were prepared using reference standard of drug and Placebo. The recovery was found to be 99.56- 100.6% for TAP. The closeness of the result nearly to 100 % assured the accuracy of the developed method for the purpose.
Precision:
The precision is the measure of either the degree of reproducibility or of repeatability of the analytical method under normal operating conditions. It provides an indication of random error; results should be expressed as relative standard deviation or co-efficient of variation.
a) Intraday Precision:
Variation of results within the same day is called Intraday precision. The intra-day precision (% RSD) was determined by analyzing samples at 3 different concentrations for three times on the same day.
Intraday precision was determined by analyzing standard solutions in the concentration range of 800, 1000, 1200 ng/spot of TAP for 3 times in same day.
b)Inter-Day Precision:
Variation of results within different days is called Inter-day precision. The inter-day precision (% RSD) was determined by analyzing samples at 3 different concentrations for 3 days.
The inter-day precision was determined by analyzing standard solutions in the concentration range of 800, 1000, 1200 ng/spot of TAP at the interval of 3 days.
Limit of Detection (LOD) and Limit of Quantification (LOQ):
The LOD and LOQ were estimated by using following equation.
LOD = 3.3 × (SD/Slope)
LOQ = 10 × (SD/Slope)
Where,
SD = Standard deviation of the Y- intercepts of the 5 calibration curves.
Slope = Mean slope of the 5 calibration curves.
Specificity and Selectivity:
To determine the selectivity of the method Rf value of TAP standard solution and sample solution were compared and peak shape was observed. The specificity was estimated from the purity of the peak obtained at specific Rf value of the analyte.
Procedure of assay:
Twenty tablets were weighed and finely powdered. The powdered equivalent to 50 mg of TAP were weighed and transferred to 50 ml volumetric flask. To this, about 20 ml of water was added and sonicated for 15 min. The volume was made up to the mark with water and mixed well. The solution was filtered through watmann filter paper no. 41 and 1 ml of filtrate was diluted to 10 ml with water to obtain a solution containing 100μg/ml TAP.
Result and discussion:
Selection of detection wavelength:
The sensitivity of HPTLC method that uses UV detection depends upon proper selection of detection wavelength. An ideal wavelength is the one that gives good response for the drugs that are to be detected. In the present study solution of 100 μg/ml of TAP was prepared in water. A solution was filled in the syringe and under nitrogen stream it was applied in form of band of having concentration of 1000 ng/spot of TAP on a single TLC plate at10mm from bottom edge using Camag Linomat V semiautomatic spotting device. Plate was developed in ascending mode in twin-trough chamber previously saturated for 20 min. using Chloroform: Acetone: Ammonia (2.5:2.4:0.1 v/v/v) upto75 mm at 25 ± 1ºC and dried in air. Developed plate was subjected to densitometric measurements in scanning mode in the UV region of 200 – 400 nm and the overlain spectrum was recorded using Camag TLC Scanner 5. Here, 272 nm is the wavelength for the drug which gives higher absorbance for the drug. Hence 272nm is selected as a detection wavelength.
Method validation:
Linearity:
The linearity for TAP found in the range of 700-1400 ng/spot was shown in chromatogram of TAP. Calibration data of TAP was presented in table. Correlation coefficient, linearity equations (slope and intercept) were presented in Table1.
Table:1 Result Of Linearity
|
Parameter for |
TAP |
|
Linearity range |
700-1400 ng/spot |
|
Linearity equation |
y = 2.0451x – 124.02 |
|
Correlation co-efficient |
0.9994 |
|
SD of Slope |
0.040 |
|
SD of Intercept |
38.84 |
Accuracy (% Recovery):
80%, 100%, and 120% level of concentration of drug were prepared using reference standard of drug and Placebo. The recovery was found to be 99.56- 100.6% for TAP. The closeness of the result nearly to 100 % assured the accuracy of the developed method for the purpose.
Table: 2 Accuracy data of TAP (n=3)
|
Drug |
Amt of std drug added (ng/spot) |
%Recovery |
%RSD |
|
TAP |
800 |
99.79 |
1.14 |
|
1000 |
100.60 |
0.97 |
|
|
1200 |
99.56 |
1.42 |
Precision:
Intraday precision:
The data for intraday precision of method were summarized in Table 3. The % RSD was found to be 1.17-1.64 % for TAP which is less than 2 indicated that the method was precise.
Table:3 Intraday precision
|
Sr No. |
Area of TAP |
Mean Peak Area ± SD |
% RSD |
|
1 |
800 |
1498.65 ± 23.85 |
1.59 |
|
2 |
1000 |
1962.34 ± 33.25 |
1.64 |
|
3 |
1200 |
2385.13 ± 38.86 |
1.17 |
Inter day precision:
The data for inter day precision of method were summarized in table4.The %RSD was found to be 1.45-1.78 % for TAP which is less than 2 indicated that the method was precise.
Table:4 inter day precision
|
Sr No. |
Area of TAP |
Mean Peak Area ± SD |
% RSD |
|
1 |
800 |
1484.80 ± 21.53 |
1.45 |
|
2 |
1000 |
1935.75 ± 32.85 |
1.70 |
|
3 |
1200 |
2345.67 ± 41.86 |
1.78 |
Limit of detection and limit of quantification:
Table :5 LOD and LOQ
The LOD and LOQ values were 62.68 ng/spot and 189.94 ng/spot for TAP .The data shows that the method is sensitive for the determination of tapentadol.
|
DRUG |
LOD (ng/spot) |
LOQ (ng/spot) |
|
TAP |
62.68 |
189.94 |
Specificity and selectivity:
The specificity of the method was ascertained by the Rf values of the drug in standard and as well as in formulation.
The purity of TAP spot was ascertained by comparing the spectra of standard TAP with that of sample spectra at three levels (lower, middle, higher position of the spot). The confirmation of TAP spot was obtained by over laying the spectra of the spot of drug in standard track and corresponding spot in track of sample for spectral comparison.
The result shown in Table 6 indicated that the spot was of TAP, no other sample component was interfering to TAP.
Rf values of TAP
|
|
Standard |
Formulation |
|
Rf value of TAP |
0.48±0.02 |
0.49±0.02 |
Table :6 Data of Peak Purity
|
|
TAP |
|
|
r (s, m) |
r (m, e) |
|
|
Standard |
0.999968 |
0.999555 |
|
Formulation |
0.99972 |
0.999504 |
Application of validated method to pharmaceutical preparation:
Analysis of sample of marketed tablets was carried out by applying 10.0μl of prepared sample solution on HPTLC plate and the amount of TAP present in the sample solution was determined by fitting area values of peak corresponding to TAP into the calibration curves. The results obtained for TAP were compared with the corresponding labeled amounts and recorded in Table 7.
Table:7 Analysis of market formulation (n=3)
|
Formulation (Tablet) |
Label claimed (TAP) |
TAP % Assay*±% RSD |
|
VORTH-TP |
100 mg/tab |
99.98 ± 1.53 |
CONCLUSION
The HPTLC method for estimation of TAP from pharmaceutical dosage form was developed. The marketed pharmaceutical formulation containing TAP was subjected to quantitative analysis using the developed method, yielded nearly 100% assay result for TAP. Hence, it can be concluded that the developed method was sensitive, precise, repeatable and accurate as per ICH guideline and can be used for routine analysis.
REFERENCES:
1. Kneip C, R. Terlinden R, Beier H and Chen G. Investigations Into the Drug-Drug Interaction Potential of Tapentadol in Human Liver Microsomes and Fresh Human Hepatocytes. Drug Metabolism Letters, 2008; 2:67-75.
2. Tzschentke T, Christoph T, Babette K, Schiene K, Hennies H, W Englberger W, Haurand H. (–)-(1R,2R)-3-(3-Dimethylamino-1-ethyl-2-methyl-propyl)-phenol Hydrochloride (Tapentadol HCl): A Novel -Opioid Receptor Agonist/Norepinephrine Reuptake inhibitor with broad spectrum analgesic properties. The journal of pharmacology and experimental therapeutics, 2007; 32: 265-276.
3. Indian Pharmacopoeia. 6th ed. Ghaziabad: The Indian Pharmacopoeia Commission 2010.Volume- II, III, 1318-21, 1859-61.
4. United State Pharmacopoeia, 34th edition NF 29, Rockville: published by United State Pharmacopoeial Convention; 2011. Volume-II, III, 2784, 2786,1720, 1723.
5. Shaik A, Ramani DN, Agarwal NK. Method development and validation of Tapentadol Hydrochloride by RP-HPLC in Pure and Tablet Dosage Form. J Chem Pharma Sciences,2012; 5(4):178-80.
6. Ramanaiah G, Ramachandran D, Srinivas G, Jayapal G, Rao P, Srilakshmi V. Development and Validation of Stability Indicating RP-LC Method for Simultaneous Estimation of Tapentadol and Paracetamol in Bulk and its Pharmaceutical Formulations. Drug Inv Today, 2012:4(7):391-96.
7. Kanzariya RP, Patel TP, Kapuriya KG, Faldu SD. Method development and validation of Tapentadol hydrochloride by RP-HPLC method. Inventi Rapid:Pharm.Ana&Qual.Assur. 2012.
8. Babu BS, Pavan KK, Nataraj K, Ramakrishna K. Development and validation of UV-Visible spectrophotometric method for the determination of Tapentadol hydrochloride from tablet dosage form. Der Pharmacia Lettre, 2013; 5(2):377-382.
REFERENCE ID: PHARMATUTOR-ART-2256
|
PharmaTutor (ISSN: 2347 - 7881) Volume 2, Issue 10 Received On: 05/08/2014; Accepted On: 07/08/2014; Published On: 01/10/2014How to cite this article: S Roy, SD Desai, BA Patel, SJ Parmar; Development and Validation of HPTLC Method for Estimation of Tapentadol Hydrochloride; PharmaTutor; 2014; 2(10); 136-141 |
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