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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 15
| Issue : 1 | Page : 76-82 |
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Development of a new validated stability-indicating high-performance thin-layer chromatography method for determination of dosulepin HCl in bulk and marketed formulation with characterization of its degradants by liquid chromatography–Mass spectrometry
Aishwarya Ramchandra Balap1, Ravina R Waghmare2
1 Department of Pharmaceutical Chemistry, Progressive Education Society's Modern College of Pharmacy, Pune, Maharashtra, India
2 Department of Quality Assurance Techniques, Progressive Education Society's Modern College of Pharmacy, Pune, Maharashtra, India
| Date of Submission | 03-Jan-2023 |
| Date of Decision | 28-Feb-2023 |
| Date of Acceptance | 02-Mar-2023 |
| Date of Web Publication | 31-Mar-2023 |
Correspondence Address:
Aishwarya Ramchandra Balap
Department of Pharmaceutical Chemistry, Progressive Education Society's Modern College of Pharmacy, Nigdi, Pune - 411 044, Maharashtra
India
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/ajprhc.ajprhc_2_23
Aim: Dosulepin hydrochloride is a tricyclic antidepressant. It acts by inhibition of the reuptake of biogenic amine and increasing available neurotransmitter levels at the synaptic cleft. The objective of this work is to develop a new validated stability-indicating high-performance thin-layer chromatography (HPTLC) method for the determination of dosulepin hydrochloric acid (HCl) in bulk and marketed formulation and characterization of the degradation products by liquid chromatography–mass spectrometry (LC–MS). Materials and Methods: The HPTLC method development of dosulepin HCl was performed on aluminum plates precoated with silica gel 60 F254 using dichloromethane: toulene: methanol: glacial acetic acid (GAA) (4:4:2:0.2 v/v) with densitometric detection at 220 nm. The method validation was done using linearity, precision, accuracy, and robustness parameter. Results: Linearity range for dosulepin HCl was found in between 150 and 900 ng/band correlation coefficient was 0.996. The % relative standard deviation for accuracy and precision was found <2%. The developed and validated HPTLC method is simple, accurate, precise, and specific. Conclusion: This study concludes that the dosulepin HCl undergoes degradation to different extent under different stress conditions. From the peak purity profile studies, it was confirmed that the peak of the degradation product was not interfering with the peak of drugs. Major acidic and oxidation degradation products were isolated and characterized by LC–MS and probable degradation pathway for dosulepin HCl was determined.
Keywords: Dosulepin HCl, high-performance thin-layer chromatography, liquid chromatography–mass spectrometry, stress degradation
How to cite this article:
Balap AR, Waghmare RR. Development of a new validated stability-indicating high-performance thin-layer chromatography method for determination of dosulepin HCl in bulk and marketed formulation with characterization of its degradants by liquid chromatography–Mass spectrometry. Asian J Pharm Res Health Care 2023;15:76-82 |
How to cite this URL:
Balap AR, Waghmare RR. Development of a new validated stability-indicating high-performance thin-layer chromatography method for determination of dosulepin HCl in bulk and marketed formulation with characterization of its degradants by liquid chromatography–Mass spectrometry. Asian J Pharm Res Health Care [serial online] 2023 [cited 2023 Jun 8];15:76-82. Available from: http://www.ajprhc.com/text.asp?2023/15/1/76/373372 |
| Introduction | |  |
Dosulepin hydrochloride or dothiepin hydrochloride (DOS) is a tricyclic antidepressant.[1] Chemically, it is 3-dibenzo (b, e) thiepin-11 (6H)-ylidene-N, N-dimethyl-1-propanamine hydrochloride [Figure 1].[2],[3] DOS act by inhibition of the reuptake of biogenic amine and increasing available neurotransmitter levels at the synaptic cleft. This helps to extend the mood-lightening effect of any released adrenaline and serotonin.[4],[5] DOS is prescribed in patients who do not respond to alternative antidepressant therapies. It is prescribed in the treatment of major depressive disorders, specifically for aged patients with heart disease, and is proven to be useful for early morning stiffness.[6]
As per literature, several analytical methods have been reported for the determination of DOS in pharmaceuticals which contains high-performance liquid chromatography (HPLC),[7],[8],[9],[10] colorimetry,[11] spectrofluorimetry,[12] ultraviolet (UV)/visible spectrophotometry,[13],[14],[15] and stability-indicating UV and HPLC methods.[16],[17] In a previous literature survey, not a single paper was found on the high-performance thin-layer chromatography (HPTLC) technique for DOS in a single composition or in combination. Hence, the present work was focused on the stress degradation study of DOS in bulk drug and its marketed formulation and its characterization of degradation products by liquid chromatography–mass spectrometry (LC–MS) method.
| Materials and Methods | | |
Materials
Drug sample of DOS was procured from Yarrow chemical Products, Mumbai. AR grade methanol, toluene, and dichloromethane were supplied by Merck Specialties Pvt. Ltd., Mumbai. During the entire study, double distilled water was used.
Instruments
The HPTLC system was used for the method development of the CAMAG system, which consisted Linomat 5 sample applicator, CAMAG twin trough chamber (TLC) scanner. Precoated aluminum plates (size 10 cm × 10 cm) coated with silica gel 60 F254 (63–200 μm) were used for application. Syringe of 100 μL capacity was used for injections. Degradation products were characterized by Agilent LC–MS (Model No. G65408).
Selection of analytical wavelength
DOS showed the maximum absorbance at 220 nm using dichloromethane: toluene: methanol: GAA (4:4:2:0.2 v/v) mobile phase as solvent.
Preparation of solutions
A stock solution of DOS was prepared by dissolving accurately about 75 mg of DOS with 100 ml methanol. Required dilutions were made with methanol to get working standard solution of DOS having a concentration of 750 μl/ml.
Analysis of tablet formulation
After weighing 20 tablets, the average weight was calculated. Tablets were triturated to fine powder. Tablet powder equivalent to about 75 mg DOS was dissolved and diluted up to 100 ml with methanol. From this solution, 10 ml was diluted up to 100 ml with methanol.
Linearity and range
A standard stock solution of DOS was applied to the HPTLC plate to deliver 150–900 ng of DOS per band. The plate was developed under optimized conditions. The plate was then scanned at 220 nm. The plot of peak area versus concentration was used for the standard calibration curve. Linear regression equation was used to calculate linearity, and the statistical data were calculated.[18]
Accuracy
Seventy-five milligrams tablet powder of DOS was weighed precisely. It was transferred individually in three different 100 ml volumetric flasks. To each of the flask, 60, 75, and 90 mg of DOS was added, respectively. Dilutions were made up with methanol and 15 min ultrasonication was performed. Filtration of the solutions was performed through Whatman filter paper No. 42. From the filtrate, further dilutions were made up with methanol.
Precision
Intraday precision
Analysis of tablet sample solutions at different times on the same day was used to determine the intraday precision. Under the mentioned circumstances, a sample solution of the tablet was made and examined.
Interday precision
Interday precision was calculated by analyzing tablet sample solutions on three separate days. Under the mentioned circumstances, a sample solution of the tablet was made and examined.
Limit of detection and limit of quantitation
The limit of detection (LOD) and limit of quantitation (LOQ) were calculated separately based on the calibration curve standard deviation (SD) values. The y-intercept SD and calibration curve slope were used to calculate the LOD and LOQ using the following formulae:
LOD = SD/Slope × 3.3 LOQ = SD/Slope × 10.
Robustness
For evaluation of the robustness for DOS, the small changes in the mobile-phase composition, mobile-phase volume, and duration of chamber saturation with mobile phase have been introduced for the proposed method, and then, the effects on the retention factor (RF) value of drugs were examined. Mobile-phase volume and duration of chamber saturation were varied at 10 ± 1 mL (9.2, 10, and 10.2 ml) and 10 ± 5 min (5, 10, and 15 min), respectively.
Forced degradation study
The stability of the method was checked by treatment of drug to different stressed conditions such as acidic, basic, oxidation, neutral, thermal, and photodegradation.
Hydrolysis in acidic and alkaline medium
Volume equivalent to 7.5 mg DOS was transferred in two subsequent 10 ml volumetric flask from the stock solution. A volume of 3 ml of 0.1N HCl and 0.1N NaOH each were added to these flasks for acidic and alkaline degradation study, respectively. These flasks were kept in a water bath at 80°C temperature for 4 h. These solutions were neutralized with 0.1N NaOH and 0.1N HCl, respectively, and diluted up to 10 ml. Final solutions were cooled at room temperature.
Oxidative degradation
To the volume equivalent to 7.5 mg from the standard stock solution, 3 ml of 3% (w/v) hydrogen peroxide was added. This flask was heated to 80°C temperature for 4 h. This solution was cooled to room temperature and diluted up to 10 ml.
Neutral degradation
Volume equivalent to 7.5 mg DOS was transferred in 10 ml volumetric flask from the stock solution. 3 ml of water was added to this flask for neutral degradation. This flask was kept in a water bath at 80°C temperature for 4 h. This solution was diluted up to 10 ml. The final solution was used at room temperature.
Thermally induced degradation
Volume equivalent to 7.5 mg from standard stock solution was diluted up to 10 ml. This solution was heated at 100°C for 45 min. The mixture was subsequently cooled to room temperature.
Photo degradation
Quantity equivalent to 7.5 mg from standard stock solution was diluted up to 10 ml. This solution was kept in UV radiation at 254 nm for 24 h inside the UV chamber.
Isolation and identification of degradation products by liquid chromatography–mass spectrometry
Isolation of degradation products was done by applying degraded sample solution on TLC plates and this plate was developed under optimized chromatographic conditions. After drying the plate, it was observed under UV cabinet and degraded band was identified and marked. The band was then scrapped and soaked overnight in methanol. On the next day, the sample was filtered through Whatman filter paper Grade 1 (pore size 11 μm) and subjected to LC–MS. After the recording of LC–MS spectra, the identification of fragmentation patterns was made by observing LC/MS spectra.[19]
| Results and Discussion | | |
After different trials, the method development of DOS was performed on aluminum plates precoated with silica gel 60 F254 using dichloromethane: toulene: methanol: glacial acetic acid (4:4:2:0.2 v/v) with densitometric detection at 220 nm [Figure 2]. A summary of chromatographic conditions for the analysis of DOS is given in [Table 1]. The developed method was validated as per the international conference on harmonisation (ICH) Q2 R1 guidelines to evaluate the accuracy, precision, detection limit, quantitation limit, and robustness.[18] | Figure 2: Densitogram of DOS showing RF at 0.45. DOS: Dothiepin hydrochloride, RF: Rheumatoid factor
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 | Table 1: Chromatographic conditions for analysis of dothiepin hydrochloride
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Linearity and calibration curve
For linearity, the area of the peak and the concentration is directly proportional. The linearity curve was found to be linear and the regression coefficient was found to be 0.996 with equation y = 6.6339x + 379.86. The linearity curve is shown in [Figure 3].
Accuracy
The accuracy studies were performed to check the recovery of the pure drug from the tablet solution of the DOS. The study was carried out at three levels, i.e., 80%, 100%, and 120%. The results showed that the percent recovery was between the ranges of 102.36% and 104.2%, as the results shown in [Table 2].
Precision
Repeatability and intermediate precision for the HPTLC method were stated as the relative SD (RSD) of the peak area. As per the calculations, repeatability, and intra- and interday variation of the results were within the acceptable range. The coefficient of variation for the inter-day and intraday precision of the method was found to be below 1% [Table 3].
Limit of detection and limit of quantitation
- LOD of DOS = 0.2 μg/ml
- LOQ of DOS = 150 ng/mL.
Robustness
The robustness of the method evaluated by assessing the effect of variations in method parameters on peak areas showed low RSD values (<1.0%), indicating the robustness of the method [Table 4].
Analysis of bulk drug and marketed formulations
The % assay of the active substance in marketed formulations was calculated. The % RSD was found to be <2. The results of the analysis of the substance in the pharmaceutical formulation are shown in [Table 5].
Forced degradation study
The forced degradation behavior of DOS under acidic, alkaline, oxidative, photolytic, hydrolytic, and thermal conditions is presented in [Table 6] and the respective densitograms are presented in [Figure 4]. From densitograms of DOS under different conditions, it was observed that acidic and oxidative conditions give major degradation products. These degraded products were isolated further and subjected to LC–MS analysis. | Figure 4: (a) Densitogram of acid degradation of DOS in 0.1NHCl at room temperature after 45 min. (b) Densitogram of alkaline degradation of DOS in 0.05N Na OH at room temperature after 45 min. (c) Densitogram of oxidative degradation of DOS in 3% H2O2 at room temperature after 45 min. (d) Densitogram of photolytic degradation of DOS after 24 h exposure to UV light. (e) Densitogram of Hydrolytic degradation of DOS in distilled water at room temperature after 45 min. (f) Densitogram of Thermal degradation of DOS in distilled water at room temperature after 45 min. DOS: Dothiepin hydrochloride, UV: Ultraviolet
Click here to view |
Liquid chromatography–mass spectrometry of degradation products
The standard solutions of DOS were analyzed using LC–MS system using a direct injection probe with electrospray ionization (ESI). The LC–MS study of DOS was performed, and spectra are shown in [Figure 5] and [Figure 6]. In acidic degradation, the fragmentation pattern of DOS is exhibited degradation production at mass-to-charge ratio (m/z) (M-44) 251.20, further degradation of it at m/z (M-49) 202.18 and m/z (M-46) 178.12. In oxidative degradation, the LC–MS exhibited molecular degradation product at m/z (M-44) 251.018 m/z, further degradation of it at (M-22) 224.127 and (M-46) 178.122 m/z. The (M-1) peak was obtained at 294.13 m/z, which confirms the molecular weight of DOS.[20],[21],[22] After the interpretation of LC–MS of the degradation product and reported MS data, probable degradation pathway of the DOS is given in [Figure 7]. | Figure 5: LC–MS of acidic degradation product of DOS. DOS: Dothiepin hydrochloride, LC–MS: Liquid chromatography–mass spectrometry
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 | Figure 6: LC–MS of oxidation degradation product of DOS. DOS: Dothiepin hydrochloride, LC–MS: Liquid chromatography–mass spectrometry
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 | Figure 7: Proposed degradation pathway of DOS. DOS: Dothiepin hydrochloride
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| Conclusion | | |
From the above study, we can conclude that the developed HPTLC method is more sensitive, accurate, precise, and reproducible. The developed HPTLC method was selective for DOS in the presence of the degradation product obtained in the stability study. The peak purity profile confirms that the degradation products do not interfere with the peak of drugs. The developed method was validated as per ICH guidelines and can be applied for regular analysis of pharmaceutical formulations containing DOS. In the LC–MS data, degradation studies under acidic and oxidation degradation, drug undergoes protonation and deprotonation. The fragmentation pattern of degradant was interpreted and the probable degradation pathway of the DOS was predicted.
Author contributions
Aishwarya Balap – Concept, supervision, and writing, Ravina Waghmare – Investigation, data collection, and processing.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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