1Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar 522510, India, 2GITAM University Bengaluru, Karnataka 562163, India
Email: [email protected]
Received: 03 Oct 2018, Revised and Accepted: 19 Nov 2018
Objective: The present study was aimed at the development of a simple visible spectrophotometric method for the assay of mianserin, a drug used for the treatment of depression.
Methods: The method was developed using tropaeolin-ooo (TPooo) as an ion associative complex forming a chromophore. Developed the chromophore by sequential mixing of aqueous solutions of mianserin, hydrochloric acid, and TPooo. Chromophore was extracted into an organic solvent (chloroform) and absorbance values of organic layers were measured. As per the existing guidelines of an international conference on harmonization (ICH), various parameters of the method were tested for validation.
Results: At the optimized reaction conditions, the formed chromophore (λ max 524 nm) was stable and sensitive. Regression analysis (r>0.9999) shows that the plotted calibration curve exhibits good linearity in the studied range of concentration (4–24 µg/ml). Accuracy of the method was evident from the % recovery values (99.50–99.87 range). Satisfactory precision (both intra and inter day) for the proposed method was clear as ranges of percentage of relative standard deviation (%RSD) values were 1.382-1.781 and 1.128-1.765 respectively. Since RSD is less than 2 %, this method was reproducible and accurate.
Conclusion: Due to lack of pre-treatment process for this method, it was simple. All the tested parameters of the method were validated as per ICH guidelines.
Keywords: Mianserin, Tropaeolin-ooo, Ion associative complex, Assay, Method Development, Validation
© 2019 The Authors.Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Mianserin is an atypical antidepressant. It also works on brain nerve cells. It is useful to relive from depression. In liver, an enzyme cytochrome P450 2D6 metabolizes it via N-oxidation, aromatic hydroxylation, and N-demethylation. C20H20N2 is its molecular formula and is a tetracyclic piperazinoazepine (fig. 1) [1-2]. In spite of its activity in relieving from dyskinesia and PD psychosis, its prospective clinical usage is limited by hindering the action of L-DOPA antiparkinsonian . In stressed animals, mianserin exhibits a protective role on the amounts of cytokine and decreases the levels of IL-6 and TNFa . It exhibits antinociceptive effect along with antidepressant activity and hence it was suggested as a substitute to treat both mood disorders and neuropathic pain associated with diabetes . It is clear from the literature survey that reports were published for its quantitative determination using various analytical methods. Those methods include usage of both UV and visible spectrophotometric [6-9], HPLC [10-13], capillary gas chromatography and electrophoresis [14-17] and Gas chromatography [18-19]. In UV spectrophotometric method proposed by S fair et al. , linearity was tested in the range of 20.0-140.0 μg/ml and continued study by liquid chromatographic method in which an Ace C18 column was used along with the mobile phase comprising of methanol and KH2PO4 buffer (pH 7.0) in the ratio of 85:15 v/v. Farag et al.  developed an extractive colorimetric method using four dyes where pH has to be maintained carefully using buffer solutions. In the proposed methods for the determination of mianserin concentration in human plasma, lower limits of quantitation were good but poor recovery values were reported [10, 11, 14, 15]. Taking into consideration of the cost of the chromatographic/electrophoresis instruments and difficulty in the maintenance of reaction conditions for the above spectro-photometric methods, in the present study, TPooo was used as a chromogen to develop colour for its determination both in bulk drug and dosage forms.
Fig. 1: Chemical structure of mianserin
TECHOMP (UV 2310) double beam UV-Visible Spectrophotometer with HITACHI software version 2.0 was used to measure the absorbance. Quartz cuvetts (10 mm path length) were used for the analysis. Digital pH meter (Elico LI-120) and balance (Shimadzu AUX-220) were used to weigh the samples and to measure pH respectively. Spectroscopic measurements were conducted at room temperature (30±1 °C). All chemicals used in the present study were AR grade. In the entire process, used water was double distilled.
Preparation of reagents
Tropaeolin-ooo solution (0.2% w/v): 200 mg of tropaeolin-ooo (TPooo) was dissolved in 100 ml of distill water.
Preparation of standard drug solution: The standard mianserin (25 mg) was weighed accurately and transferred to 25 ml volumetric flask. It was dissolved properly and diluted up to the mark with methanol to obtain the final concentration of 1000 µg/ml (stock solution). 2.0 ml from the stock solution was further diluted to 10.0 ml to get a standard stock solution having 200 µg/ml of mianserin.
Among the various existing techniques for quantitative estimation of pharmaceutical drugs, the prevalent approach is an establishment of the coloured complex involving ion-association. This method can be extended to those drugs consisting of nitrogen, which accepts a proton in an acid medium (i.e., undergoes protonation) to form a cation. Most of the anion dyes develop a complex with the above-formed cation. Visible spectrophotometry is used to measure the absorbances of those complexes after extraction into organic solvents . An added advantage of ion-association extract method is its application to the determination of the exact compound in spite of its presence among different constituents of formulations. Impelled by these advantages, present study describes the establishment of a procedure centered on creation of an ion-association complex with the help of chromogenic dye like tropaeolin-ooo (TPooo). An absorption maximum was observed at 524 nm for the developed chormophore using TPooo in the determination of mianserin by visible spectrophotometry (fig. 2).
Fig. 2: Visible spectrum of mianserin-TPooo complex
Optimization of reactions conditions
At ambient temperature (30±1 °C), performed the optimization of reaction conditions. Coloured solution exhibited maximum absorbance using HCl (0.1 N; 5 ml) (fig. 3a). Optimized conditions for TPooo regarding volume and concentration were 2.0 ml and 0.2% (w/v) respectively (fig. 3b). Instant formation of colour was noticed by mixing of reactants and observed the maintenance of stable colour intensity for two hours. Out of the tested solvents (C6H6, C6H5NH2, C6H5NO2, CH2Cl2 and CHCl3), chloroform was identified as the best solvent for extraction (fig. 4). 10 ml of organic solvent (CHCl3) addition to the aqueous layer (15 ml) resulted in highest and constant absorbance. Therefore, two minutes of contact time was fixed for the organic and aqueous phase (2:3 v/v). Effective sequential addition was mianserin, HCl, and TPooo. As per the Job’s continuation method (Job, 1928), confirmed the formation of 1:1 ion-association complex between protonated mianserin and TPooo anion. Scheme-1 shows the formation of coloured ion association pair between TPooo anion (TP–) and mianserin-cation (MH+).
Scheme-1: Formation of coloured ion association pair
Optimized method procedure
Suitable aliquots of mianserin standard solution (200 µg/ml) were taken into a sequence of separating funnels (125 ml volume). It was followed by successive addition of 5.0 ml of hydrochloric acid (0.1 N) and 2.0 ml of TPooo solution (0.2%). Addition of distilled water made the total volume of the aqueous layer to 15 ml. Then shaken the contents for two minutes after the addition of chloroform (10 ml). Absorbance values of organic layers were measured after their separation from aqueous layers.
Fig. 3: Effect of volumes of (a) acid and (b) TPooo solution
Fig. 4: Effect of extraction solvent
Chromophore formation and chemistry
In acidic medium, sodium sulphonate of TPooo undergoes hydrolysis to give TPooo anion. A stoichiometry of 1:1 for ion-association complex indicates that out of the two nitrogen atoms present on mianserin, only one was protonated. Probably, the lone pair of electrons present on the other nitrogen (of azepine) were involved in resonance with the adjacent benzene ring. Hence, a lone pair of electrons on this nitrogen was least available for protonation. Therefore, the second nitrogen gets protonated. Fig. 5 shows the chemical reactions involved in the formation of coloured ion pair complex.
Fig. 5: Reaction of mianserin with TPooo
Validation of method
Linearity and range
By following the above-developed method, the colour was developed by taking different concentrations of mianserin in the range 4–24 µg/ml. For each concentration of mianserin, mean of three measurements for absorbance was taken (table 1). A linear calibration curve was obtained by plotting mean absorbance against mianserin concentration (fig. 6). y = 0.0493x-0.0103 was the equation obtained by linear regression of the data. The correlation coefficient was found to be greater than 0.9999. It indicates the successful testing of linearity of the suggested analytical method.
Table 2 represents key parameters of method development and validation.
Table 1: Calibration curve values
|Concentration (µg/ml)||Absorbance* (mean±SD)|
*Average of three determinations
Fig. 6: Calibration graph of mianserin
Table 2: Key parameters of method development and validation
|1.||Apparent molar absorptivity||1.27×104 l/mol/cm|
|2.||Sandell’s sensitivity||0.0207 µg/cm2/A|
|3.||Regression coefficient (r)||0.9999|
|1.||λ max||524 nm|
|2.||Linearity (Beer’s Law Limit)||4–24 µg/ml|
|3.||Limit of detection||0.30 µg/ml|
|4.||Limit of quantitation||1.00 µg/ml|
|5||Stability period||18 h|
In order to test the accuracy of the method, % recovery studies were carried out and values were given in table 3. In this regard, different amounts of the drug sample in the range of 50 to 150% were added to fixed amounts of mianserin so that total concentration lies within the range of linearity study. The observed percent recoveries of were in between 99.50 to 99.87. As both SD and % RSD values were<1%, the suggested method was accurate.
Three different concentrations were selected in the linearity range (4–24µg/ml) to check the precision of the proposed method. A series of 6 independent analyses were done for each concentration on concurrent days (of 6 numbers) (table 4). The proposed method satisfied precision studies as % RSD values for intraday and interday were in between 1.382-1.781 and 1.128-1.765 respectively.
Table 3: Recovery of mianserin
|Level of recovery (%)||Amount of drug recovered (µg/ml) (practical)||Statistical evaluation||% Recovery = practical x 100/theoretical|
Nominal concentration used (a): 8 µg/ml, Amount of drug added (b): 4, 8 and 12 µg/ml respectively for 50%, 100% and 150% recovery levels, Theoretical amount: Total amount of drug (a+b) = 12, 16, 20 µg/ml respectively for 50%, 100% and 150% recovery levels
Table 4: Intraday and inter-day precision readings
|Concentration of mianserin (µg/ml)||Concentration*|
|Intraday (mean±SD) (µg/ml)||% RSD||Inter-day (mean±SD) (µg/ml)||% RSD|
*Average of six determinations
Table 5: Ruggedness data of mianserin
Test concentration of mianserin
|mean±SD (µg/ml)||% RSD|
*Average of six determinations
Assay of different amounts of mianserin (4, 16 and 24 µg/ml) was carried out by two different analysts on different days under the above-given method optimized conditions in order to appraise the ruggedness of the current developed method. Lack of significant difference in the values produced by different analysts indicates the evidence for reproducible results (table 5). Hence, ruggedness of this method was confirmed.
Limits of detection and quantification (LOD and LOQ)
As per the ICH guidelines (2005), LOD and LOQ were calculated to determine the sensitivity of the proposed method using formula (3.3 × σ/S) and (10 × σ/S) respectively taking into consideration of ratio between signal and noise [21-22], where S (calibration curve slope) and σ (SD of the response). The corresponding calculated values for mianserin determination were given below.
LOD = 0.30 μg ml-1 and
LOQ =1.00 μg ml-1
Analysis of pharmaceutical formulations
Mianserin tablet (Depnon®) extracts were treated with chromogen to develop the chromophore and measured the absorbances in order to determine the API amount in the formulation (tablet) taking into consideration of average weight as a basis (table 6). To determine the amount of mianserin present in the tablet formulations, the above-suggested method can be used because the recovery values of the API was good. It indicates the non-interference to the above method from common excipients. In developing countries, the most opted analytical technique is spectrophotometry to carry out the routine analysis in QC laboratories of industries [23-28].
Hence, the above method which comprises mianserin as a complexing agent can be applied to determine the quantity of Depnon present in pure and tablet formulations.
Table 6: Estimation of mianserin from its formulation
|Formulation||Labeled amount (mg)||Amount found* (mg)||% Drug recovered||%RSD|
*Average of three determinations
The method suggested using TPooo as an ion-pair forming agent was simple as there is no need to maintain complicated conditions (like an elaborate procedure for sample treatment, maintenance of critical optimum pH etc). Hence, no need of sophisticated or costly instruments. These benefits encourage the usage of the current method in quality control wings for mianserin routine analysis, both in the tablet dosage form and bulk drug.
All the authors have contributed equally.
Hamadjida A, Nuara SG, Gourdon JC, Huot P. The effect of mianserin on the severity of psychosis and dyskinesia in the parkinsonian marmoset. Prog Neuropsychopharmacol Biol Psychiatry 2018;81:367-71.
Yang M, Liu S, Hu L, Zhan J, Lei P, Wu M. Effects of the antidepressant mianserin on the early development of fish embryos at low environmentally relevant concentrations. Ecotoxicol Environ Saf 2018;150:144-51.
Hamadjida A, Nuara SG, Gourdon JC, Huot P. The effect of mianserin on the severity of psychosis and dyskinesia in the parkinsonian marmoset. Prog Neuro-Psychopharmacol Biol Psychiatry 2018;81:367-71.
Manikowska K, Mikołajczyk M, Mikołajczak PŁ, Bobkiewicz Kozłowska T. The influence of mianserin on TNF-α, IL-6 and IL-10 serum levels in rats under chronic mild stress. Pharmacol Rep 2014;66:22-7.
Ucel Uİ, Can OD, Ozkay UD, Ozturk Y. Antihyperalgesic and antiallodynic effects of mianserin on diabetic neuropathic pain: a study on mechanism of action. Eur J Pharmacol 2015;756:92-106.
Sfair LL, Graeff JS, Steppe M, Schapoval EES. Ultraviolet spectrophotometric method for analytical determination of mianserin hydrochloride in coated tablets and comparison with LC. Braz J Pharm 2015;51:833-7.
Farag RS, Afifi MS, Abd-Rabow MM. Extractive spectrophotometric determination of mianserin hydrochloride by acid-dye complexation method in pure and in pharmaceutical preparations. Int J Pharm Sci Res 2011;2:1197-203.
Han IU, Aman T, Kazi AA, Khan ZA. Spectrophotometric determination of mianserin in pure and pharmaceutical preparations. J Chem Soc Pakistan 2002;24:114-8.
Devani MB, Pandya SS, Shah SA. Spectrophotometric determinations of mianserin hydrochloride with 3-methyl-2-benzothiazolinonehydrozone. J Pharm Pharm 1990;52:123-4.
Xu P, Chen BM, Ma N, Yan M, Zhu YG. Determination of mianserin in human plasma by high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI/MS): application to a bioequivalence study in Chinese volunteers. J Pharm Biomed Anal 2008;47:994-9.
Łukaszkiewicz J, Piwowarska J, Skarzyńska E, Łojewska MS, Pachecka J. Development validation and application of the HPLC method for determination of mianserin in human serum. Acta Pol Pharm 2007;64:103-7.
Hefnawy MM, Aboul Enein HY. Fast high performance liquid chromatographic analysis of mianserin and its metabolites in human plasma using monolithic silica column and solid-phase extraction. Anal Chim Acta 2004;504:291-7.
Sun LL, Si TM, Shu LA, Zhang HY, Tian CH. HPLC determination of mianserin in human plasma. Zhongguo-Xinyao-Zazhi 2002;11:714-6.
Grodner B, Pachecka J. A simpler and faster capillary electrophoresis method for determination of mianserin enantiomers in human serum. Acta Pol Pharm 2006;63:9-14.
Martinez MA, Sanchez de la Torre C, Almarza E. A comparative solid-phase extraction study for the simultaneous determination of fluvoxamine, mianserin, doxepin, citalopram paroxetine, and etoperidone in whole blood by capillary gas-liquid chromatography with nitrogen-phosphorus detection. J Anal Toxicol 2004;28:174-80.
Andersen S, Halvorsen TG, Pedersen S, Rasmussen KE. Liquid-phase micro-extraction combined with capillary electrophoresis, a promising tool for the determination of chiral drugs in biological matrices. J Chromatogr 2002;963:303-12.
Wang F, Khaledi MG. Capillary electrophoresis chiral separation of basic pharmaceutical enantiomers with different charges using sulfated beta-cyclodextrin. J Microcolumn Sep 1999;11:11-21.
Ishii A, Kurihara R, Kojima T, San T, Mizuno Y, Yamakawa Y, et al. Sensitive determination of mianserin and setiptiline in body fluids by gas chromatography with surface ionization detection (GC-SID). Int J Legal Med 2000;2:115-8.
Lewis J, Cairncross KD. A simplified method for the estimation of mianserin in plasma. Br J Clin Pharmacol 1981;12:583-5.
Kiran Kumar K, Venkata Nadh R, Nagoji KEV. Extractive spectrophotometric determination of nicergoline through ion-pair complexation reaction. Oriental J Chem 2013;29:263-9.
Sethi PD. HPLC quantitative analysis of pharmaceutical formulations. CBS publications, India; 2001.
ICH guidelines, Validation of Analytical Procedures. Text and Methodology. Q2 (R1); 2015. p. 8-13.
Sudhir MS, Nadh RV. Diazo-coupling a facile mean for the spectrophotometric determination of rasagiline hemitartrate. Oriental J Chem 2014;29:1507-14.
Kumar KK, Nadh RV, Nagoji KE. Determination of bendamustine hydrochloride in pure and dosage forms by ion-associative complex formation. Oriental J Chem 2014;30:905-10.
Kulkarni Manasi B, Joshi Anagha M. An experimental design approach for optimization of the modified colorimetric first-order derivative method for estimation of serralysin in bulk and pharmaceutical formulation. Asian J Pharm Clin Res 2018;11:293-300.
Akram M, El-Didamony, Monuir Z, Saad Nora O, Saleem. Extractive-spectrophotometric determination of some antimuscarinic antagonist in tablet formulations using eriochrome cyanine R. Int J Pharm Pharm Sci 2018;10:2-28.
Sharma DK, Jasvir Singh, Pushap Raj. Spectrophotometric determination of propranolol hydrochloride and metoprolol tartrate in pharmaceutical dosage forms, spiked water, and biological fluids. Int J Pharm Pharm Sci 2018;10:107-15.
Godambe RD, Disouza JI, Jamkhandi CM, Kumbhar PS. Development of spectrophotometric and fluorometric methods for estimation of darunavir using Qbd approach. Int J Curr Pharm Res 2018;10:13-9.