The authors have declared that no competing interests exist.
Diphenhydramine HClis a weakly fluorescent drug having tertiary amine group forming ion pair complex with eosin Y in dichloromethane at pH 5 in disodium hydrogen phosphate-citric acid buffer solution. The complex formation was the basis for the development of new analytical method for determination of active diphenhydramine in pharmaceutical formulations. The stoichiometric ratio between diphenhydramine and eosin Y was studied by mole ratio method and found to be 2:1. The ion-pair complex showed maximum fluorescence emission intensity at 554 nm with excitation at 259 nm. The linear dynamic range was obtained in the concentration range of 2-22 µg mL-1 with a linear equation of FI = 0.361 + 13.675 C. The apparent Gibb’s free energy (ΔGº) was calculated and found to be -80.783 KJ mol-1, confirmed the feasibility of the reaction. The proposed method was successfully applied to the determination of diphenhydramine HCl in pharmaceutical formulations and in good agreement with the reference method.
Diphenhydramine hydrochloride is chemically known as 2-(Diphenylmethoxy)-N,N-dimethylethylamine hydrochloride (CAS: 147-24-0; M.W.: 291.82).
Fluorescence spectra and all measurements were done on Agilent’s Cary Eclipse FluorescenceSpectrophotometer (Thermo Scientific, Australia) equipped with a xenon 150 W arc lamp and 1-cm quartz cells. Excitation and emission wavelengths were set with slit widths of 10 nm.
Hanna pH meter (USA) was used for pH measurements. IR spectra were recorded on IR Affinity-1 spectrophotometer (Shimadzu, Kyoto, Japan) in the range of 4000-400 cm-1 using KBr pellet technique.
All reagents and solvents were AR grade. 0.02% eosin Y disodium salt (CAS: 17372-87-1, M.W.: 691.85, Fluka Chemie AG, Switzerland) solution was freshly prepared in distilled water. McIlvaine Na2HPO4-citric acid buffer solutions of different pH ranging from 2.2-6.6 were prepared
Pure diphenhydramine hydrochloride reference drug was gifted by National Pharmaceutical Industries Company, Oman. 0.02% of pure diphenhydramine hydrochloride solution was tested for linear dynamic range and other validation parameters.
Marketed products of diphenhydramine hydrochloride such as Stopkof syrup 100 mL (National Pharmaceutical Industries Company, Oman), Histalix syrup 150 mL (Wallace manufacturing chemists Ltd, UK) and Amydramine syrup 120 mL (Julphar, UAE) were investigated for active diphenhydramine HCl in the presence of pharmaceutical excipients.
Into a series of 10 mL volumetric flasks, varying aliquots (0.1-1.1 mL) of 0.02% diphenhydramine hydrochloride equivalent to 2-22 µg mL-1 were pipetted with 1.4 mL of 0.02% eosin Y and 2.0 mL of Na2HPO4-citric acid buffer of pH 5. The contents of the flask were diluted with distilled water at 25±1 ̊C and transferred into the separating funnel with 10 mL dichloromethane. The solution was shaken and mixed for 2 min for the separation of organic layer. The layer was separated and treated with anhydrous sodium sulphate to remove any traces of water. The fluorescence emission intensity of the associated ion pair complex in organic layer was recorded at 554 nm after excitation at 259 nm and the linear regression equation was developed for the estimation of active diphenhydramine in pharmaceutical formulations.
Into a series of 10 mL standard volumetric flask, different volumes (0.05, 0.1, 0.15, 0.2, and 0.25 mL) of 0.02% pure diphenhydramine HCl were taken and diluted up to the mark with distilled water at 25±1 ̊C. The fluorescence emission intensity at 286 nm was recorded after keeping excitation wavelength fixed at 207 nm against distilled water as a blank solution. The calibration graph was constructed by plotting fluorescence emission intensity versus the concentration of diphenhydramine HCl in μg mL-1. Alternatively, the linear regression equation was generated for finding out the concentration of active diphenhydramine HCl in pharmaceutical preparations.
3 commercially available bottled syrups i.e. Stopkof, Histalix and Amydramine were shaked and poured each into a 50 mL beaker. 7.4, 7.2 and 7.4 mL of the respective syrups equivalent to 20 mg active diphenhydramine HCl were transferred into a 100 mL volumetric flask and diluted up to the mark with distilled water. The recommended procedures were then followed for determination of active diphenhydramine HCl in commercial dosage forms.
In the literature, it was reported that chlorpheniramine
The molar combining ratio between diphenhydramine HCl and eosin Y was investigated by mole ratio method
Eq.1
Where Fobs. and Fextp. are observed and extrapolated fluorescence emission intensities of the complex. CIM, CE and are initial concentration of imipramine, eosin Y and limiting concentration (=CE) in mol L-1, respectively. The Kf of the complex was found to be 1.437×1014. The apparent Gibbs free energy (ΔGº) was calculated using ΔGº = -2.303 RT log Kf and found to be -80.783 KJ mol-1, confirming the feasibility of the reaction.
The experimental variables such as reaction time, concentration of eosin Y, buffer solutions of different pH, solvents and shaking time for extraction of complex were optimized for proposed spectrofluorimetric method.
The effect of the reaction time on the fluorescence emission intensity of ion-pair complex was studied from 1 to 70 min and found to be stable for 1 h at 25 ± 1˚C.
The influence of the volumes of 2.89×10-4 M eosin Y was studied at 20.0 μg mL-1 diphenhydramine hydrochloride on the fluorescence emission intensity of ion pair complex. The maximum fluorescence emission intensity was achieved at 1.0 mL of 2.89×10-4 M eosin Y and above this volume up to 1.6 mL, the fluorescence emission intensity remained constant. 1.3 mL of 2.89×10-4 M eosin Y was used as optimum volume for the determination of diphenhydramine hydrochloride.
The influence of pH on the fluorescence emission intensity of diphenhydramine-eosin Y complex was investigated using 2.0 mL of Na2HPO4-citric acid buffer solution of different pH in the range of 3.0-6.6 with 20.0 μg mL-1 diphenhydramine and 1.3 mL of 2.89×10-4 M eosin Y. The fluorescence emission intensity was recorded for each determination and the maximum fluorescence intensity of ion-pair complex was achieved at pH 4.2 and constant up to pH 5.8. Above pH 5.8, the fluorescence emission intensity decreased. Therefore, 2.0 mL of Na2HPO4-citric acid buffer solution of pH 5.0 was selected as the optimum pH for determination of diphenhydramine hydrochloride.
The effect of the shaking time for the extraction of the ion pair complex was tested in the range of 0.5-3.0 min. The maximum fluorescence emission intensity of the complex was obtained at 1.5 min. After this time, the intensity remained constant up to 2.5 min. Therefore, the shaking time of 2.0 min was adequate for the extraction of ion-pair complex into dichloromethane.
The fluorescence emission intensity of the ion-pair complex was recorded in different solvents of benzene, hexane, chloroform, carbon tetrachloride, dichloromethane, dichloroethane, and ethyl acetate and found to be maximum in dichloromethane. There was no fluorescence emission intensity recoded in benzene and hexane. The fluorescence emission intensity in the blank solution was also recorded and found to be negligible in the solvent. Therefore, dichloromethane was selected as the best solvent for extraction of the complex.
For the proposed method, a series of nine different concentrations of diphenhydramine were prepared and the fluorescence emission intensity of the complex was recorded at emission wavelength of 554 nm. The calibration graph was constructed by plotting fluorescence emission intensity against initial concentration of diphenhydramine in µg mL-1. The linear relationship between the measured fluorescence emission intensity and initial concentration of diphenhydramine HCl was found to be in the concentration range 2-22 µg mL-1. The calibration data were utilized to get slope, intercept, coefficient of correlation, detection limit and quantitation limit, standard deviation of intercept and slope, variance, standard deviation of the calibration line of proposed and reference methods using OriginPro 6.1 software. The results are summarized in
t=a/Sa Eq. 2
Parameters | Proposed method | Reference method |
Absorption wavelength (nm) | 259 | 285 |
Maximum emission wavelength (nm) | 554 | 207 |
Linear dynamic range (µg mL-1) | 2-22 | 1-5 |
Linear regression equation | FI = 0.361 + 13.675 C | FI = 0.410 + 39.155 C |
Standard deviation of intercept, Sa | 0.329 | 0.384 |
Confidence limit of the intercept | 0.778 | 1.222 |
Standard deviation of slope, Sb | 0.022 | 0.116 |
Confidence limit of the slope | 0.052 | 0.369 |
Correlation coefficient (r) | 0.999 | 0.999 |
Standard deviation of calibration line (So) | 0.426 | 0.367 |
Variance (So2) | 0.181 | 0.135 |
Limit of detection, LOD (µg mL-1) | 0.103 | 0.031 |
Limit of quantification, LOQ (µg mL-1) | 0.310 | 0.094 |
The value of t was found to be 1.095 which is less than the tabulated t-value (2.365, =7) at 95% confidence level
The precision of the proposed method was evaluated in terms of intra-day and inter-day precisions. The intra-day precision was tested using test samples for five times per day at 3 concentration levels i.e. 4, 12 and 20 µg mL-1. Inter-day precision was evaluated by investigating the test samples for five times once daily for five consecutive days. The results are summarized in
Actual concentration(mg mL-1) | Intra day assay:Found concentration ± SD(μg mL-1); RSD (%)a | Inter day assay:Found concentration ± SD(μg mL-1); RSD (%)a |
4.0 | 3.979± 0.010; 0.28 | 4.026 ±0.033; 0.82 |
12.0 | 11.974±0.022; 0.19 | 12.004 ± 0.068; 0.57 |
20.0 | 20.014±0.013; 0.07 | 20.002 ± 0.048; 0.24 |
The statistics was applied to establish the confidence limit
Where and are average concentration (diphenhydramine) and fluorescence emission intensity values, respectively, for n standard solutions.
In order to assure the applicability of the proposed method, a systematic quantitative study was performed in order to find out the tolerated amount of common excipients (ammonium chloride, sodium citrate, glucose, fructose, lactose, sodium benzoate, starch, povidone, methyl cellulose and micro crystalline cellulose) in drug formulations. The tolerated amount of excipients at 20 mg mL-1 diphenhydramine HCl was calculated using the following expression:
Mass/Volume(mg/L)=C×MW×1000 Eq.4
where C and MW are concentration and molecular weight of excipients, respectively.
The results are summarized in
Excipients | Tolerance amount (mg mL -1) |
Glucose | 5.504 |
Fructose | 5.504 |
Sodium benzoate | 1.502 |
NH4Cl | 1.002 |
Sodium citrate | 1.502 |
Lactose | 9.557 |
Starch | 0. 152 |
Povidone | 0. 152 |
The accuracy of the proposed method was investigated by standard addition technique. In this method, the known amount of pure diphenhydramine was spiked with constant amount of Stopkof syrup solution and the fluorescence emission intensity of the complex was recorded. Standard addition plot was constructed using fluorescence emission intensity at y-axis and initial concentration of drug at x-axis (
The value of SxE was found to be 0.04 µg mL-1. The confidence limit for the concentration of diphenhydramine in syrup was calculated using
The applicability of the proposed method for the determination of active diphenhydramine in Stopkof, Histalix and Amydramine has been tested. The results of the proposed method were statistically compared with those of the reference method using point and interval hypothesis tests. t- and F-values at 95% confidence level were calculated using point hypothesis test. The results are summarized in
Formulations | Proposed method | Reference method | Paired t-value |
F-value |
qL |
qU |
||
Recovery | RSD |
Recovery | RSD |
|||||
(%) | (%) | (%) | (%) | |||||
Stopkof |
99.88 | 0.371 | 100.10 | 0.291 | 1.564 | 2.83 | 0.996 | 1.008 |
Histalix |
99.81 | 0.311 | 100.05 | 0.235 | 1.366 | 1.75 | 0.995 | 1.007 |
Amydramine |
99.42 | 0.449 | 99.92 | 0.206 | 1.357 | 4.67 | 0.998 | 1.011 |
Stopkof, Histalix and Amydramine excipients are NH4Cl, sodium citrate and menthol.
Mean for 5 independent analyses.
Theoretical
A bias, based on recovery experiments, of ± 2% is acceptable.
Interval hypothesis test was also utilized to calculate bias within the acceptable range of ±2%
Lower limit (θL) and upper limit (θU) were calculated and found to be in the range of 0.98-1.02 indicating the compliance of regulatory guidelines
The proposed method based on spectrofluorimery is very useful because many of the excipients are non-fluorescent in nature. The method was successfully applied for the determination of active diphenhydramine in stopkof, histalix and amydramine in the presence of excipients. The proposed method is easy to operate, utilizing less analysis time and economical with commonly available solvent. The proposed method can be utilized as an alternate method for routine quality control analysis of active diphenhydramine in research laboratories, hospitals and pharmaceutical industries.
The authors are grateful to Heads of Applied Sciences and Chemistry Section, Higher College of Technology, Muscat, Oman for providing research facilities. The authors are thankful to the higher-up of the Ministry of ManPower (Higher College of Technology) Muscat, Sultanate of Oman for facilities. The authors wish to express their gratitude to M/s National Pharmaceutical Industries Company, Oman for providing the gift sample of pure diphenhydramine HCl.