The authors have declared that no competing interests exist.
A spectrofluorimetric method has been developed for the determination of imipramine HCl in bulk and commercial dosage forms. The method was based on measuring the fluorescence emission intensity of imipramine-eosin Y ion pair complex (λem = 558 and λex = 319) in dichloroethane at buffer solution (sodium acetate and acetic acid) of pH 4.8. The stoichiometric ratio between imipramine and eosin Y was studied by Job's method of continuous variations and found to be 2:1. Formation constant (Kf) and Gibb’s free energy change (ΔG) were calculated and pointed towards the spontaneous nature of the reaction. A series of variables were studied to optimize the reaction conditions. The proposed method was validated as per ICH guidelines and successfully applied for the determination of active imipramine HCl in commercial dosage forms with high degree of accuracy and precision.
Imipramine hydrochloride is chemically known as 5-3-(dimethylamino)propyl-10,11-dihydro-5H-dibenzbf-azepine monohydrochloride (Chemical Abstract Service: 113-52-0; Molecular Weight: 316.88). The drug is a dibenzazepine derivative of tricyclic antidepressant, competitively blocks the reuptake of norepinephrine and serotonin in synapses in brain
Imipramine HCl is officially listed in British Pharmacopoeia
Fluorescence intensity and spectra were recorded on Thermo Scientific Agilent’s Cary Eclipse FluorescenceSpectrophotometer (Australia) equipped with a xenon 150 W arc lamp and 1-cm quartz cells. Excitation and emission wavelengths were set with slit widths of 5 nm. pH values were measured using Hanna pH meter (USA).
All reagents and solvents used were of analytical reagent grade. 0.03% eosin Y disodium salt (CAS: 17372-87-1, M.W.: 691.85, Fluka Chemie AG, Switzerland) solution was prepared by dissolving 0.03 g of eosin Y in 100 mL standard volumetric flask and diluted up to the mark with distilled water.
Walpole sodium acetate-acetic acid buffer solution of different pH (3.72-5.57) were prepared using different volumes of 0.2M sodium acetate and 0.2M acetic acid in a total volume of 10 mL
Imipramine hydrochloride was purchased from Sigma-Aldrich (USA). 0.02% imipramine HCl solution was prepared by dissolving 0.02 g drug in 100 mL distilled water. Imipramine 25 (Actavis, UK) and imipramine HCl 25 (SGH, Singapore) were procured from SQU hospital (Oman) and Ibn Sina Hospital (Oman), respectively.
0.15-1.0 mL of 0.02% imipramine HCl and 2 ml of sodium acetate-acetic acid buffer solution of pH 4.8 were added with 1.6 ml of 0.03% eosin Y into 10 mL standard volumetric flask. The contents of the flask were diluted up to the mark with distilled water at 25±1 ̊C and transferred into a 100 mL separating funnel. 10 mL of dichloroethane was poured in the separating funnel and the contents of the funnel were thoroughly mixed for 2 min. 2 layers were formed and the dichloroethane layer was enriched with fluorescent ion-pair complex. The dichloroethane layer was separated and treated with anhydrous sodium sulphate (4g). The said layer was now subjected for recording fluorescence emission intensity at 558 nm after keeping excitation wavelength at 319 nm. The calibration graph was plotted and the linear regression equation was developed for the estimation of imipramine HCl in commercial tablets.
Into a series of 10 mL standard volumetric flask, 0.2-0.5 mL of 0.02% imipramine HCl solution was taken and the solution was diluted up to the mark with distilled water at room temperature. The fluorescence emission intensity at 407 nm was measured after keeping excitation wavelength at 259 nm against distilled water as a blank. The calibration graph was constructed by plotting fluorescence emission intensity against the initial concentration of imipramine HCl. The linear regression equation was generated using Origin Pro6.1 software and utilized for quantitation of active imipramine in pharmaceutical formulations.
Ten tablets of Imipramine 25 (Actavis, UK) and imipramine HCl 25 (SGH, Singapore) were weighed and finely powdered using agate mortar and pestle. An amount of powdered tablets equivalent to 20 mg imipramine HCl was transferred in 100 mL beaker and dissolved in 50 mL of distilled water. The solution was passed through a filter funnel equipped with Whatmann No 42 filter paper in 100 mL standard volumetric flask. The residue on filter paper was washed with 4×10 mL portions of distilled water. The filtrate was diluted up to the mark with distilled water in 100 mL volumetric flask and finally cleaned using 0.45µm polyethersulfone membrane (Filter-Lab) using 5mL syringe. The recommended procedures were followed for the determination of active imipramine HCl in commercial dosage forms.
In the literature, it was reported that doxepin HCl
The selectivity offered by fluorescence measurements is invaluable because of distinct excitation and fluorescence spectra and wavelengths available for each fluorophore
The extraction equilibria can be represented as follows:
2HIM+ + E-2
(HIM2+ E-2)
where HIM+ and E-2 are protonated imipramine and di-anionic eosin Y, respectively, and the subscript o refers to the dichloroethane layer. The reaction sequence is shown in
The stoichiometric ratio between imipramine and eosin Y for the ion pair complex was established by Job's method of continuous variations
Where Fobs. and Fextp. are observed and extrapolated fluorescence emission intensities of the complex. CIM, CE and
For the development and optimization of analytical method, certain validation parameters and experimental variables were investigated separately.
The experimental variables such as reaction time, concentration of eosin Y, sodium acetate-acetic acid buffer solutions of different pH, volume of buffer solution at particular pH, extracting solvents and shaking time for extraction of complex were optimized with 20.0 mg mL-1 imipramine hydrochloride.
The effect of the reaction time was investigated. The maximum fluorescence emission intensity of the complex was achieved immediately and stable up to 1 h at 25 ± 1˚C. Therefore, the analysis can be performed within 1 h.
The influence of the volumes of 4.34 × 10-4 M eosin Y (0.03%) was studied. The maximum fluorescence emission intensity was obtained with 1.2 mL of 4.34 × 10-4 M eosin Y and remained constant up to 1.6 mL of 4.34 × 10-4 M eosin Y (
The effect of the pH of the aqueous phase on fluorescent ion-pair extraction was studied using sodium acetate-acetic acid buffer solutions over the pH range 3.72-5.57. The fluorescence intensity of dichloroethane extract was maximum and constant in the pH range 4.63-4.99. Above pH 4.99, the fluorescence intensity was decreased (
The effect of volume of sodium acetate-acetic acid buffer solution of pH 4.8 was examined in the range of 0.5-2.5 mL. The highest fluorescence intensity was obtained with 1.5 mL, later on remained constant (
The effect of the extracting solvent such as dichloroethane, dichloromethane, chloroform, hexane, ethyl acetate, and benzene was tested. The polarity of a solvent affects both the extraction efficiency and fluorescence intensity. There was no fluorescence intensity recoded in ethyl acetate, benzene and hexane. The fluorescence emission intensity of the blank solution was also recorded and found negligible in the solvent. The maximum fluorescence emission intensity was obtained using dichloroethane as extracting solvent (
The effect of the shaking time from 0.5 to 3 min was investigated. The maximum fluorescence intensity of the complex was obtained at 1.5 min. The time acquired above did not produce any increase in fluorescence intensity. Therefore, the shaking time of 2.0 min was sufficient for the extraction of ion-pair complex into dichloroethane.
The fluorescence emission intensity of proposed method (or reference method) at 558 nm (or 407 nm) after keeping excitation wavelength constant at 319 nm (or 259 nm) of drug-eosin complex (or drug solution) was recorded and the linear regression equation was generated using OriginPro 6.1 software. The linear dynamic range of the proposed and reference methods were established and found to be in the range of 3.0 to 20.0 μg mL-1 (proposed method, n =8) and 4 to 10 μg mL-1 (reference method, n=7). The calibration data was treated with OriginPro 6.1 software to generate slope, intercept, standard deviation of intercept and slope, correlation coefficient, standard deviation of calibration line (So). Optical characteristics, linear regression equation and statistical data along with detection limit and quantitation limit are summarized in
t=a/sa Eq. 2
The value of t- were found to be 1.81 and 1.78 for proposed and reference methods, which were less than the tabulated t-value (2.447,
Parameters | Analytical methods | ||
Proposed spectrofluorimetric method | Reference spectrofluorimetric method | ||
Maximum wavelength (nm) | λem=558; λex=319 | λem=407; λex=259 | |
Linear dynamic rang (µg mL-1) | 3-20 | 4-10 | |
No. of concentration levels | 8 | 7 | |
Linear regression equation | A= 0.36 + 11.13C | A= 0.29 + 4.39C | |
Standard deviation of intercept, Sa | 0.199 (v=6) | 0.163 (v=5) | |
Confidence limit of the intercept,±tSa | 0.486 | 0.418 | |
Standard deviation of slope, Sb | 0.0154 | 0.0118 | |
Confidence limit of the slope, ±tSb | 0.038 (v=6) | 0.030 (v=5) | |
Correlation coefficient (r) | 0.999 (n=8) | 0.999 (n=7) | |
Variance (So2) | 0.071 | 0.031 | |
Standard deviation of calibration line (So) | 0.267 | 0.176 | |
Limit of detection, LOD (µg mL-1) | 0.079 | 0.132 | |
Limit of quantification, LOQ (µg mL-1) | 0.240 | 0.400 |
Ideally the true relation between found and added concentration will provide straight line passing through the origin (intercept 0) with a slope equal to unity
Limits of detection (LOD) and quantitation (LOQ) were calculated according to the International conference on Harmonization guidelines
The statistical analysis of the calibration data was tested for calculating the error (Sc) in the determination of a given concentration of imipramine HCl using the following expression
Where
Excipients | Tolerating volume (mL) | Fluorescence intensity | Tolerance amount (mg mL-1) | ||
Sucrosea | 0.01 | - | 1.0 | 223 | 3.42 |
Povidone | - | 0.2 | 0.25 | 224 | 0.05 |
Methyl cellulose | - | 0.2 | 0.25 | 222 | 0.05 |
Starch | - | 0.1 | 0.25 | 221 | 0.025 |
Poly ethylene glycol | - | 0.1 | 0.25 | 223 | 0.025 |
The precision of the proposed method was tested in terms of intraday (over a single day, n=5) and interday (over 5 consecutive days in a week, n=5) precisions at 3 concentration levels i.e. 4, 10 and 18 mg mL-1 imipramine HCl. The results are summarized in
Actual concentration (µg mL-1) | Intraday assay and interday precisions | |||||
Measured concentration ±SD(µg mL-1) | RSD |
Recovery,% | ||||
Intraday | Interday | Intra-day | Inter-day | Intraday | Interday | |
4 | 3.95 ± 0.32 | 3.94 ± 0.26 | 0.73 | 0.59 | 98.75 | 98.5 |
10 | 10.02 ± 0.16 | 10.03 ± 0.11 | 0.14 | 0.10 | 100.2 | 100.3 |
18 | 17.96 ± 0.24 | 17.97 ± 0.31 | 0.12 | 0.15 | 99.78 | 99.83 |
5 independent analyses
The specificity and selectivity of the proposed method was investigated. The influence of foreign substances (sucrose, methyl cellulose, povidone, starch and polyethylene glycol) that can commonly accompany imipramine in pharmaceutical preparations was studied. Solution of imipramine (20 mg mL-1) and each said compound were mixed to obtain samples. The tolerance limit of each interfering substances was calculated as the maximum concentration yielding a relative error of ±2% at a concentration of imipramine HCl in the analytical signal. The tolerated amount of excipients at 20 mg mL-1 imipramine HCl was calculated using the following expressions:
Mass/Volume (g L-1 or mg mL-1) = Molar concentration × MW Eq. 4
where MW is the molecular weight of excipients.
Mass/Volume (g L-1 or mg mL-1) = Volume taken (mL) × % concentration Eq. 5
The results are summarized in
The robustness of the proposed method was tested by deliberately changing the reaction conditions and studying the effect on the fluorescence intensity. The effect of varying volumes (1.4 ± 0.2 mL) of 4.34 × 10-4 M eosin Y, buffer solution of pH 4.63, 4.8 and 4.99, volumes (2± 0.5 mL) of buffer solution of pH 4.8, shaking time (2± 0.5 min) showed that these changes did not affect the percentage recovery of the drug. Results of variation in the experimental parameters were acceptable at room temperature, hence proved that the proposed method is robust.
The accuracy of the proposed method was investigated by standard addition technique. A series of known amount of pure imipramine was spiked with constant amount of Tofranil solution and the fluorescence emission intensity of the associated complex was recorded. Standard addition plot was constructed using fluorescence emission intensity at y-axis and initial concentration of imipramine at x-axis (
The value of Sc was found to be 0.044 µg mL-1. The confidence limit for the concentration of imipramine in Tofranil tablet was calculated by Ci ± tSc
Dosage forms | Proposed method | Reference method | t-valueb | F-valueb | θL |
θU |
||
Recovery (%) | RSD |
Recovery (%) | RSD |
|||||
Imipramine HCl 25 (SGH) | 100.11 | 0.36 | 100.16 | 0.23 | 1.09 | 2.28 | 0.989 | 1.004 |
Imipramine HCl 25 (Actavis, UK) | 99.96 | 0.235 | 99.94 | 0.50 | 1.37 | 1.75 | 0.99 | 1.008 |
5 independent analyses.
Theoretical t (
A bias, based on recovery experiments, of ± 2% is acceptable.
The applicability of proposed method for the determination of active imipramine HCl in Tofranil and Imipramine HCl tablets has been tested. Percentage recovery of active drug in tablets was estimated. The results of the proposed method were statistically compared with those obtained by the reference method and summarized in
Interval hypothesis test was also utilized to calculate bias and found to be within the acceptable range of ±2%
This quadratic equation in θ has two roots (θL and θU) provided θL and θU of 0.989 and 1.004, respectively in SGH imipramine tablets and 0.99 and 1.008, respectively in Actavis imipramine tablets. The results are acceptable and showed the compliance with regulatory guidelines
S.No. | Reagents/Mobile phase/Electrode | λmax (nm) | Linear range (μg mL-1) | Analysis time (min) | References |
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1 1 | Toluene: ethyl acetate: ethanol: diethanolamine (70: 15: 4: 1 v/v/v/v) | 288 | 5-9 | 30 |
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2 2 | Mobile phase: Phosphate buffer (pH 3.4)-acetonitrile (55:45)Flow rate: 1.0 mL/min | 250 | 12.5-125 | 2.2 |
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3 3 | Mobile phase: water (pH 6)-methanol-triethylamine (70:30:0.1 % v/v/v)Flow rate: 1.0 mL/min | 216 nm | 50-150 | 5.05 |
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4 4 | Amberlite-titanium dioxide nanoparticles modified glassy carbon paste electrode at pH 6 phosphate buffer (0.1M) | - | 0.0004-1.97 | Preprocessing time, approx. 24 h |
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5 5 | Carbon nanocomposite electrode designed by montmorillonite nanoclayinto a carbon ionic liquid electrode | - | 0.63-12.68 | Preprocessing time, approx. 3 h |
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6 6 | Graphite-polyurethane composite electrode | - | 0.10-0.73 | Preprocessing time, approx. 24 h |
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7 7 | Methyl orange in water-dichloroethane medium + universal buffer (pH) | 425 | 0.79-25.3 | Preprocessing time, approx. 3 h |
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8 | Drug was alkalinized with ammonia and extracted in chloroform. The drug solution was heated at 70°C using water bath for removing chloroform. The residue was dissolved in acetonitrile and reacted with 2,3-dichloro-5,6-dicyano-p-benzoquinone | 460 | 10-60 | Preprocessing time 1h |
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9 | Drug + ammonium metavanadate + 10M sulphuric acid | 620 | 0.6–40 | 30 min |
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10 | Drug + eriochrome cyanine R. Extracted in n-butanol | 520 | 10-80 | 5 min |
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11. (a) | Drug + ammonium peroxidisulfate + 10M phosphoric acid | 658 | 10-110 | 35 min |
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(b) | Drug + niobium(V) thiocyanate + 10M sulphuric acid. Extracted in n-butanol-chlorofrom (9:1) medium | 350 | 0.8–8 | 7 min |
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12. (a) | Drug + bromothymol blue + sodium acetate-HCl buffer solution of pH 2.8. Extracted in chloroform | 415 | 2.5-25 | 5 |
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(b) | Drug + bromophenol blue + sodium acetate-HCl buffer solution of pH 2.5. Extracted in chloroform | 415 | 3.0-25 | 5 |
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(c) | Drug + bromocresol green + sodium acetate-HCl buffer solution of pH 3.5. Extracted in chloroform | 415 | 2.5-25 | 5 |
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(d) | Drug + bromocresol purple + sodium acetate-HCl buffer solution of pH 2.5. Extracted in chloroform | 415 | 2.5-25 | 5 |
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(e) | Drug + I2, reacted in dichloroethane | 366 | 2.5-25 | 5 |
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(f) | Drug + KMnO4 + 0.45 M NaOH | 610 | 3.0-25 | 5 |
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13 | Erythrosine B in water-chloroform medium + acetate buffer (pH 5) | λex = 544λem= 560 | 0.12-2.8 | Preprocessing time, approx. 3 h |
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14 | Rhodamine B in ethylene glycol-distilled water medium | - | 0.1-20.0 | 10 |
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This work | Drug + Eosin Y + sodium acetate-acetic acid buffer solution of pH 4.8. Extracted in dichloroethane | λem = 558 and λex = 319) | 3.0-20 | 2 | Proposed method |
A comparison of the proposed method with those of published reported methods was presented in
The authors are thankful to Dean, Heads of Applied Sciences and Chemistry Section, Higher College of Technology, Muscat, Oman for the facilities. The authors are grateful to to the higher-up of the Ministry of ManPower (Higher College of Technology) Muscat, Sultanate of Oman for support to carry out this work.