Development and Validation of Stability indicating RP-HPLC Assay Method for Trospium Chloride in Tablet Dosage Form
Vikas D. Kedar*, Pritam N. Dube, Sachin N. Kapse, Yogita B. Thombare
Research Scholar Matoshri College of Pharmacy Ekalahare, Nashik. 422105 Maharashtra.
*Corresponding Author E-mail: kedarvikas10@gmail.com
ABSTRACT:
A simple, rapid, accurate and sensitive method was developed for quantitative analysis of Trospium Chloride in tablet dosage form using Reverse Phase high performance liquid chromatography (RP-HPLC) with DAD detection. The chromatography separation was achieved Xterra RP18, (250 x 4.6mm, 5µParticle Size)), at column temperature 40°C±2, in the isocratic mobile phase mode using Acetonitrile (ACN): Buffer (65:35; v/v) at a flow rate of 1.0mL/min. The determination was performed using Buffer HPLC with DAD detector set at 215nm. Samples were prepared with diluent [Buffer: ACN (200:100; v/v)], and the volume injected was 50µL. The retention time for Trospium Chloride was 3.275 minute. The analytical curve was linear (r2 0.9993) over a wide concentration range (50.0-150.0 µg/mL). The presence of components of the tablet did not interfere in the results of the analysis. The method showed adequate precision with a relative standard deviation (RSD) less than 2%.
KEYWORDS: Trospium Chloride, Assay, RP-HPLC, Method validation.
INTRODUCTION:
The scope of developing and validating analytical method is to ensure a suitable method for a particular analyte more specific, accurate and precise the main objective for that is to improve the condition and parameter, which should be followed in the development and validation1,2. Trospium Chloride is an antispasmodic, antimuscarinic agent. It is used in the treatment of overactive bladder with urge incontinence. It is also used as anticholinergic compound. It acts as a direct antagonist at muscarinic acetylcholine receptors in cholinergically innervated organs3 The scope of developing and validating analytical method is to ensure a suitable method for a particular analyte more specific, accurate and precise the main objective for that is to improve the condition and parameter, which should be followed in the development and validation1,2. Trospium Chloride is an antispasmodic, antimuscarinic agent. It is used in the treatment of overactive bladder with urge incontinence.
It is also used as anticholinergic compound. It acts as a direct antagonist at muscarinic acetylcholine receptors in cholinergically innervated organs3 Analytical quality by design is the new emerging field for the analysis of drugs in the industries. Conventionally, anticholinergic drugs are those which block actions of ACh on autonomic effect tors and in the CNS exerted through muscarinic receptors1. The selective action of atropine can easily be demonstrated on a piece of guinea pig ileum where ACh induced contractions are blocked without affecting those evoked by histamines-HT or other spasmogens.2. The selectivity is, however, lost at very high doses. All anticholinergics are Trospium chloride is a muscarinic antagonist.3.Trospium chloride antagonizes the effect of acetylcholine on muscarinic receptors in cholinergic ally innervated organs including the bladder4. Its Para sympatholytic action reduces the tonus of smooth muscle in the bladder Competitiveantagonists5
Figure: 1 Trospium Chloride
Organoleptic properties of drug:
Colorless to slightly yellow, fine crystalline solid Powder.
Melting point determination:
Identification of Trospium Chloride was done by checking its melting point and it was found in the range of 255-258°C Standard
Solubility analysis:
1gm/2ml in water It is soluble in water. Freely soluble in methanol, ractically insoluble in methylene chloride.
Fourier Transform Infra-red Spectroscopy (FTIR):
1mg of Trospium Chloride API was mixed properly with 200mg of dried KBr, and then carefully triturated in a mortar pestle. At last this mixture was kept on a die and IR spectrum was taken using the Diffused Attachment reflectance mode.
Figure: 2 FT-IR spectrum of Trospium Chloride
UV Spectrophotometric method:
Optimization of Detection Wavelength:
Initially the Ultra violet (U.V) spectrum of Trospium Chloride was produced using appropriate U.V spectrophotometer, so as to determine the absorbance maxima or Lambda max (λ max).6 This is essential since HPLC detection is basically UV based, thus a 25ppm solution of Trospium Chloride in acetonitrile was used to get the following spectra.221nm7
Preparation of standard solution of Trospium Chloride:
Weigh accurately about 50mg of Trospium Chlorideworking standard and transfer it into 50mL volumetric flask.8 Add 30mL of Diluent sonicate to dissolve. Make up to the mark with diluent. Further dilute 5 mL of the above solution to 50mL with Diluent 9
Determination of Max wavelength:
The scanning of wavelength 400nm to 200nm. From the spectrum, 221nm were recorded for estimation of drug10
Fig. 3 UV Spectrum of Trospium Chloride.
Development of HPLC method for Trospium Chloride:
High performance liquid chromatographic method was developed and validated for determination of Trospium Chloride in bulk form. Mobile phase consists of BUFFER: ACN (65:35) % Chromatogram obtained was shows the maximum wavelength where the drug shows maximum response was 215nm.11
Graph No.4: Typical chromatogram of Trospium Chloride
Table No. 1: System Suitability Parameters
|
Sr. No. |
Weight of standard (mg) Trospium Chloride |
Area of standard Trospium Chloride |
|
1 |
25.00 |
1552764 |
|
2 |
1554281 |
|
|
3 |
1553724 |
|
|
4 |
1552926 |
|
|
5 |
1554680 |
|
|
6 |
1558235 |
|
|
Mean |
1554435 |
|
|
SD |
2004.9231 |
|
|
%RSD |
0.13 |
|
Graph no. 5: Chromatogram of system suitability
Table No.2: Data of calibration curve of Trospium Chloride
|
% Conc. of sample |
Conc. (PPM) |
Mean Response (Area) |
Statistical analysis |
|
|
50 |
50 |
177647 |
Correlation |
0.9993 |
|
80 |
80 |
283337 |
||
|
90 |
90 |
321520 |
Intercept |
6758.5493 |
|
100 |
100 |
357126 |
||
|
110 |
110 |
397189 |
Slope |
19817.6487 |
|
120 |
426974 |
|||
|
150 |
150 |
533956 |
||
Graph No.6: Calibration curve for TROS
Graph No. 7: Chromatogram of Linearity
Graph no. 8: Chromatogram obtained from assay
Graph no. 9: Chromatogram obtained from assay
Graph no.10: Chromatogram obtained from assay
Graph no.11: Chromatogram obtained from assay
Force Degradation of Different Wavelength:
(a)
(b)
(c)
(d)
Table 3: Observations of Forced degradation
|
Condition |
% Assay |
% Degradation |
Purity angle |
Purity threshold |
|
Control |
100.6 |
- |
- |
- |
|
Acid Degradation |
102.9 |
-2.3 |
0.027 |
0.212 |
|
Base Degradation |
94.3 |
6.3 |
0.028 |
0.211 |
|
Peroxide Degradation |
99.9 |
0.7 |
- |
- |
|
Photolytic Degradation |
92.9 |
8.8 |
0.59 |
0.212 |
|
Heat Degradation |
98.6 |
3.1 |
0.032 |
0.215 |
CONCLUSION:
Purity angle of Trospium Chloride was found to be less than Purity Threshold in all degradation condition.12 The peak is pure and thus the method is stability indicating with respect to forced degradation studies’ simple, sensitive, isocratic and accurate stability indicating reverse phase HPLC method was described for determination of Trospium Chloride in tablet dosage form.13 The developed method was validated by testing Linearity, Precision, Accuracy, Specificity, Robustness, Ruggedness, Solution stability and forced degradation study.14The method is good enough to separate out API (active ingredient) from the degradation products (produced during forced degradation studies)[15]It is also clear from the chromatogram that the active ingredient peaks in all the stress conditions were free from any sort of degradation impurities. All these convinced us to conclude that the method can be successfully used for any sort of stability and validation studies.
ACKNOWLEDGEMENTS:
The author would like to thank management, Matoshri College of Pharmacy Ekalahare, Nashik. For providing the facilities to carry out the research work.
CONFLICTS OF INTEREST:
There are no conflicts of interest.
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Received on 26.06.2023 Modified on 01.08.2023
Accepted on 11.10.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(4):1595-1598.
DOI: 10.52711/0974-360X.2024.00252