alexa Simplistic Application of 3-Methy-2-Benzothiazoline Hydrazone (MBTH),an Oxidative Coupling Chromogenic Reagent for Quantification ofMetaxalone and Dabigatran Etexilate Mesylate Bulk Drug and Their Dosage Forms | Open Access Journals
ISSN : 2153-2435
Pharmaceutica Analytica Acta
Like us on:
Make the best use of Scientific Research and information from our 700+ peer reviewed, Open Access Journals that operates with the help of 50,000+ Editorial Board Members and esteemed reviewers and 1000+ Scientific associations in Medical, Clinical, Pharmaceutical, Engineering, Technology and Management Fields.
Meet Inspiring Speakers and Experts at our 3000+ Global Conferenceseries Events with over 600+ Conferences, 1200+ Symposiums and 1200+ Workshops on
Medical, Pharma, Engineering, Science, Technology and Business

Simplistic Application of 3-Methy-2-Benzothiazoline Hydrazone (MBTH),an Oxidative Coupling Chromogenic Reagent for Quantification ofMetaxalone and Dabigatran Etexilate Mesylate Bulk Drug and Their Dosage Forms

Pani Kumar DA*, Archana G, Sunitha G, Rachel Paul K, Harika R and Sowndarya NSKR

Department of Pharmaceutical Analysis and Quality Assurance, Gokaraju Rangaraju College of Pharmacy, Osmania University, Hyderabad, India

*Corresponding Author:
Panikumar DA
Gokaraju Rangaraju College of Pharmacy
Department of Pharmaceutical Analysis
Hyderabad, Telangana State -500090, India
Tel: 040-32912927
E-mail: [email protected]

Received date: February 27, 2015; Accepted date: April 13, 2015; Published date: April 20, 2015

Citation: Pani Kumar DA, Archana G, Sunitha G, Rachel Paul K, Harika R, et al. (2015) Simplistic Application of 3-Methy-2-Benzothiazoline Hydrazone (MBTH), an Oxidative Coupling Chromogenic Reagent for Quantification of Metaxalone and Dabigatran Etexilate Mesylate Bulk Drug and Their Dosage Forms. Pharm Anal Acta 6:362. doi:10.4172/2153-2435.1000362

Copyright: © 2015 Pani Kumar DA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Visit for more related articles at Pharmaceutica Analytica Acta

Abstract

Purpose: A simple and sensitive spectrophotometric method in visible region has been developed and validated for quantification of metaxalone (MET) and dabigatran etexilate mesylate (DAB) in their bulk and pharmaceutical dosage forms. Methods: The method is based on the oxidative coupling reaction of 3-methyl-2-benzothiazoline hydrazone (MBTH) with MET and DAB in the presence of ferric chloride to form green coloured chromogen with absorption maximum at 666 nm and 632 nm respectively. Results: Beer’s law is obeyed in concentration range of 4-20 and 1-6 μg/mL for MET and DAB respectively with correlation coefficient of 0.999. Limit of detection and quantification were 0.46 μg/mL and 1.518 μg/mL for MET and 0.0578 μg/mL and 0.298 μg/mL for DAB. When marketed formulations were analyzed, the results obtained by the proposed method were in good agreement with labelled amounts. The developed method was validated statistically as per ICH guidelines. Conclusion: The developed method is simple, sensitive, specific and can be successfully employed in routine analysis of MET and DAB pharmaceutical dosage forms.

Keywords

MBTH; Metaxalone (MET); Dabigatran etexilate mesylate (DAB); Visible spectrophotometry

Introduction

Metaxalone (MET) which is chemically 5-(3, 5-dimethylphenoxymethyl)-1, 3-oxazolidin-2-one (Figure 1.A) is a muscle relaxant used to relieve pain caused by strain, sprains and other musculoskeletal conditions [1] Dabigatran etexilate(DAB) is chemically ethyl 3-(1-{2-[({4-[amino({[(hexyloxy)carbonyl]imino}) methyl]phenyl}amino)methyl]-1-methyl-1H-1,3-benzodiazol-5- yl}-N-(pyridin-2-yl)formamido)propanoate (Figure 1). DAB is oral anticoagulant. It is a pro-drug which is converted to its active form, by esterase-catalyzed hydrolysis in the plasma and liver. DAB is competitive and reversible direct inhibitor of thrombin.

pharmaceutica-analytica-acta-Structure-MET

Figure 1: Structure of MET (A) and DAB (B).

A detailed literature survey revealed that few analytical methods have been reported for the determination of MET by UV Spectrophotometry, RP–HPLC [2-5], HPTLC [6], hydrotropic solubilisation technique using UV spectroscopy [7] and HPLC [8,9], Where as normal UV spectroscopic method [10] for the determination of DAB. It concluded that there is no visible spectrophotometric method available for quantification of MET and DAB. Even the reported spectrophotometric methods are not so sensitive; require high concentrations of organic solvents and extraction procedures. Therefore according to the need, this work presents a simple, specific and sensitive visible spectrophotometric method for the quantification of MET and DAB based on its oxidative coupling interaction with 3-methyl-2-benzothiazoline hydrazone (MBTH) [11,12]. The proposed method was validated as per ICH guidelines.

Methodology

Materials

MET standard gift sample was provided by Sun Pharma and marketed tablet dosage form Flexura 400 was acquired from local pharmacies. DAB pure drug was a gift sample provided by Neuland laboratories limited and commercial hard gelatin capsule dosage form Pradaxa -110 mg was acquired from local pharmacies.

Chemicals and reagents: All chemicals and reagents used were of analytical grade and were freshly prepared in distilled water.

Preparation of 0.9%w/v of MBTH reagent: The reagent 900 mg was accurately weighed and dissolved in few mL of distilled water, further diluted upto 100 mL with distilled water.

Preparation of 3%w/v of ferric chloride solution: Ferric chloride 3 g was accurately weighed and dissolved in few ml of 0.1N HCl, further diluted upto 100 mL with 0.1N HCl.

Instrument: Double–beam Shimadzu UV–Visible Spectrophotometer 1800, with spectral bandwidth of 0.1 nm, wavelength accuracy ± 0.1 nm and a pair of 1 cm path length matched quartz cells were used to measure absorbance of the resulting solution.

Preparation of standard stock solutions

Preparation of MET standard stock solution: Accurately weighed 10 mg of MET (bulk drug) was dissolved in few mL of methanol in 10 mL volumetric flask and volume was made upto the mark with methanol to get 1000 μg/mL. From the above stock solution,1 mL was pipetted out into 10 mL volumetric flask and the volume was made upto the mark with methanol water(50:50) to obtain final concentration of 100 μg/mL.

Preparation of DAB standard stock solution: 10 mg of DAB was weighed accurately and dissolved in few mL of methanol in 10 mL volumetric flask and volume was made upto the mark with methanol to obtain 1000 μg/mL solution. Further dilutions were done to obtain 100 μg/mL solutions.

Procedure for determination of MET and DAB: Aliquots of standard drug solutions of MET ranging from 0.4-2.0 mL and DAB ranging from 0.1-0.6 mL were taken into a series of 10 mL volumetric flasks and aqueous solution of 0.9% w/v MBTH (2 mL), 3 %w/v ferric chloride in 0.1 N HCl (2 mL) were added. The solutions were finally made up to the mark with water and were kept aside for 15 minutes and 60 min for MET and DAB respectively. The absorbance of the green coloured chromogen was measured at 666 and 632 nm for MET and DAB respectively against the corresponding reagent blank. The linearity of calibration curve (absorbance vs concentration) in pure solution was checked over concentration ranges of about 4-20 μg/mL for MET and 1-6 μg/mL for DAB. The mean ± standard deviation for the slope, intercept and correlation coefficient of standard curves (n=6) were calculated.

Assay of MET and DAB in their respective dosage forms: The proposed method was applied to commercially available marketed formulations of MET tablets (Flexura 400) and DAB capsules (Pradaxa 110). Accurate quantity equivalent to 10 mg of active ingredient was dissolved in methanol and the volume was made upto 10 mL with methanol. Subsequent dilutions of this solution were made to obtain the required concentration within the linearity range and similar procedure was followed as that of the standard. The absorbance of the green coloured chromogen was measured at selected wave length against the corresponding reagent blank and analyzed for drug content and their results were statistically validated. There are no official standard methods available to show a real amount of MET and DAB in the dosage form till now, so that self-punched tablets of DAB and MET were made with the common excipients (lactose, starch, microcrystalline cellulose, magnesium stearate, titanium dioxide and talc) of the pharmaceutical formulation. (as prescribed under hand book of excipients) and evaluated by the proposed methods in order to check if any component of the formulation could generate a response (absorbance) similar to the drugs.

Method Validation

The method was validated according to the ICH guidelines to evaluate the overall performance of qualitative analytical method with respect to certain parameters such as linearity, precision, accuracy, and sensitivity [13].

Linearity

Calibration curves were plotted for both MET and DAB. The linearity (absorbance vs concentration) in pure solution was checked over concentration ranges of about 4-20 μg/mL for MET and 1-6 μg/ mL for DAB. The mean ± standard deviation for the slope, intercept and correlation coefficient of standard curves (n=6) were calculated.

Sensitivity

The sensitivity of the method was determined with respect to LOD and LOQ. LOD is the lowest amount of an analyte in a sample that can be detected and LOQ is the lowest amount of an analyte in a sample that can be quantified with acceptable precision and accuracy under the stated experimental conditions. The LOD and LOQ levels shall be predicted based on the standard deviation and slope of the constructed calibration curve.

Precision

Precision is the degree of agreement among the individual test results when the procedure is applied repeatedly to multiple portions of a homogeneous sample. Precision of the method was determined by intra-day and inter-day precision as per ICH guidelines. For both intra-day and inter-day precision of the samples containing 8, 12, 16 μg/mL for MET and 2, 4, 6 μg/mL for DAB were analyzed six times on the same day (intra-day precision)and for three consecutive days (inter-day precision). The % RSD was calculated.

Accuracy

The accuracy of the method was determined by calculating recoveries of both the drugs by standard addition method. Three different levels (80%, 100% and 120%) of standards were spiked to pre quantified samples in triplicate. The mean of percentage recoveries and % RSD were statistically calculated.

Results and Discussion

Principle

The proposed colorimetric method is based on the oxidative coupling reaction between MBTH and drug molecule in the presence of oxidizing agent such as ferric chloride. Ferric chloride oxidizes MBTH to form an electrophilic intermediate by losing two electrons and one proton. The electrophilic intermediate forms green colored chromogen by coupling with the most nucleophilic site of MET and DAB as shown in Schemes 1 and 2. The absorbance of the green colored chromogen is measured at visible wavelength of 666 nm and 632 nm, Figures 2 and 3. The oxidative coupling reactions of DAB and MET with MBTH were time dependent, shown in Figure 4. The absorbance of DAB and MET were reached maximum at 60 min and 15 min respectively and stable upto 30 min.

pharmaceutica-analytica-acta-Spectrum-MET

Figure 2: Spectrum of MET at 666 nm with MBTH.

pharmaceutica-analytica-acta-Spectrum-DAB

Figure 3: Spectrum of DAB at 632 nm with MBTH.

pharmaceutica-analytica-acta-time-reaction

Figure 4: Effect of time on reaction between MBTH with DAB (A) and MET (B).

pharmaceutica-analytica-acta-reaction-mechanism

Scheme 1: Coupling reaction mechanism between MBTH and MET.

pharmaceutica-analytica-acta-Coupling-reaction

Scheme 2: Coupling reaction mechanism between MBTH and DAB.

Method validation

The method was validated according to the ICH guidelines.

Linearity

The developed method follows the beer’s law in the concentration range of 4-20 μg/mL with regression equation Y=0.035x + 0.246 and correlation value 0.999 for MET and concentration in the linearity range of 1-6 μg/mL with regression equation Y=0.1037x + 0.0083 and correlation value 0.9995 for DAB. The concentration vs absorbance plot is shown in Figures 5 and 6. The analytical data of the calibration curves including standard deviations for the slope and intercept and system suitability parameters are summarized in Table 1. This data indicates the linearity of the calibration graphs.

pharmaceutica-analytica-acta-curve-MET

Figure 5: Calibration curve of MET at 666 nm with MBTH.

pharmaceutica-analytica-acta-Calibration-curve

Figure 6: Calibration curve of DAB at 632 nm with MBTH.

Parameters MET DAB
Absorption Wavelength(nm) 666 632
Beers law range (µg/mL) 4-20 1-6
Molar absorptivity (L mol/cm) 0.00486 0.00075
Sandellsensitivitya, (µg/cm) 0.1744 0.0083
Limit of Detection (µg/mL) 0.46 0.0478
Limit of Quantification(µg/mL) 1.158 0.248
Correlation coefficient(r2) 0.999 0.9995
Slope(m) 0.035 0.1037
Intercept(c) 0.246 0.0083
Regression equation(yb) Y = 0.035x + 0.246 Y=0.1037x+0.0083

Table 1: System Suitability Parameters of MET and DAB.

Sensitivity

The sensitivity of the method was determined with respect to LOD and LOQ. These were separately determined based on standard calibration curve using 3.3 σ/s and 10 σ/s, formulae respectively, where s is the slope of the calibration curve and σ is the standard deviation of y-intercept of the regression equation. Results are shown in Table 1.

Precision

Repeatability (intra-day) and intermediate precision (inter-day) were assessed using three concentrations and three replicates of each concentration. The results for precision studied are summarized in Table 2. The relative standard deviations were found to be very small (<2%) indicating reasonable repeatability and intermediate precision of the proposed method.

Concentration (µg/mL) Intra-day precision Inter-day precision
Concentration estimated (µg/mL) A.M ± SDa % RSDb Concentration estimated (µg/mL)A.M ± SDa % RSDb
MET
8 8.04 ± 0.01 0.166 8.06+0.023 0.3833
12 12.12 ± 0.03 0.5 11.82+0.017 0.2833
16 15.86 ± 0.05 0.833 15.88+0.092 1.533
DAB
2 2.02 ± 0.002 0.990 2.01 ± 0.004 0.199
4 3.98 ± 0.02 0.502 4.02 ± 0.011 0.273
6 6.00 ± 0.014 0.230 6.01 ± 0.012 0.199

Table 2: Precision for MET and DAB.

Accuracy

The accuracy of the method was determined by calculating recoveries of both the drugs by standard addition method. The % recovery values for the method are shown in Table 3. The satisfactory recovery percentage results for MET and DAB indicate the accuracy of the method.

Brand name Recovery level(%) Theoretical content(mg) Amount found(mg) ± SD* %amountrecovered RSD (%)
FLEXURA 400(MET) 80 18 18.2+0.045 101.1 0.9
100 20 20.22+0.078 100.6 1.56
120 22 22.2+0.068 101.4 1.36
PRADAXA 110 mg(DAB)(DAB) 80 9 8.91 ± 0.0702 99 0.78
100 10 9.99 ± 0.014 99.9 0.14
120 11 10.9 ± 0.0115 99.9 0.10

Table 3: Accuracy (Recovery studies) for MET and DAB.

Application of the method to the marketed formulations

The proposed method was applied to standard drugs, self-punched and commercially available marketed formulations of MET (Flexura 400) and DAB (Pradaxa). The amount of drug was found to be 99.05 and 100.9 % for marketed formulations of MET and DAB respectively, as shown in Table 4. The sample recoveries in the formulation were in good agreement with their respective label claim and compared the assay values with standard drugs and self-punched tablets recoveries, which are reported in Table 5, revealed that there was no significant difference observed between them, which suggested no interference of formulation excipients in the estimation of the active ingredient.

Formulation Active ingredient Label claim (mg) Amount found (mg) A.M ± SDa %Assay % RSDb
Flexura- 400 MET 400 396.2+0.097 99.05 0.024
Pradaxa-110mg DAB 110 111 ± 0.11 100.9 0.099
aMean of three determinations
bRelative standard deviation (%)

Table 4: Assay of MET and DAB using MBTH.

Formulation DAB (110 mg) MET (400 mg)
AM ± SD % Assay AM ± SD % Assay
Bulk drug 110.5 ± 0.043 100.45 400.2 ± 0.027 100.05
Marketed Formulation 111.0 ± 0.11 100.9 396.2 ± 0.097 99.05
Self -punched tablets 110.9 ± 0.08 100.81 399.6 ± 0.105 99.90

Table 5: Comparison of assay values of marketed formulations with bulk drugs and self punched tablets.

Conclusion

It is concluded that the proposed method was found to be simple, sensitive, accurate and precise for the quantification of MET and DAB in pharmaceutical dosage forms. The assay values were in good agreement with their respective label claim. This spectrophotometric method has been found to be better because of its specificity, sensitivity, no extraction procedures and low concentration of solvent. These advantages encourage that the proposed method can be routinely employed in quality control for analysis of MET and DAB in the pharmaceutical dosage forms.

Acknowledgements

The authors are thankful to the management and Prof. C.V.S. Subramanyam, Principal, Gokaraju Rangaraju College of Pharmacy for supporting this work.

References

Select your language of interest to view the total content in your interested language
Post your comment

Share This Article

Recommended Conferences

Article Usage

  • Total views: 11676
  • [From(publication date):
    May-2015 - Aug 20, 2017]
  • Breakdown by view type
  • HTML page views : 7894
  • PDF downloads :3782
 

Post your comment

captcha   Reload  Can't read the image? click here to refresh

Peer Reviewed Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
International Conferences 2017-18
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings

Contact Us

 
© 2008-2017 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version
adwords