Validation of quantitative determination methods of diphenhyramine tablets by high performance liquid chromatography

(HPLC), Diphenhydramine, Validation Studies, Assay. Aim. In order to quantify diphenhydramine tablets the methods based on reverse phase high-performance liquid chromatography with UV- spectrometry detection at 254 nm (HPLC-UV) have been used. Methods and results. The mobile phase used a mixture of a phosphate buffer: acetonitrile (80:20). The proposed method is applied to the two dosage forms of diphenhydramine – tablets to 0.1 and 0.05, produced by various Ukrainian pharmaceutical companies. The main characteristics are validated by Pharmacopoeia of Ukraine. Conclusion. According to the experimental data, the technique can be correctly reproduced, and is suitable for use in pharmaceutical and forensic analytic laboratories.

Purpose Purpose of this paper is to study and validate the requirements for Ukrainian pharmacopoeia to fi nd convenient, economical, highly sensitive methods of quantitative determination of diphenhyramine in dosage forms by high performance liquid chromatography.

Objects of research applied reagents and equipment
For this study, a sample Diphenhyramineum pharmacopoeia standard was used (SE «Ukrainian Research Center Pharmacopoeial quality of drugs» series 2) and medicines -pills «Diphenhydramine» 0.1 g (JSC «Kyiv Vitamin Factory», Ukraine) tablets «Diphenhydramine -Darnytsya «0.05 g (FF «Darnitsa», Ukraine), a series of 10 712 and EN31013 respectively. Solvents and reagents grade purity.
General method of quantitative determination of diphenhyramine hydrochloride Aliquot portion (0.0002 g) of diphenhyramine solution is used in the mobile phase and placed in a volumetric fl ask 10.00 mL, to bring the mobile phase to the mark and mix. Spend chromatography following conditions: column -reversed phase Hypersil Gold C18, Thermo Scientifi c TM, 4×150 mm, particle size sorbent -5 mm, mobile phase -a mixture of phosphate buffer: acetonitrile in the ratio of 80:20, the fl ow rate -1 ml/ min, detector -UV spectrophotometric wavelength 254 nm, semi-injector volume of samples is 20 ml.
Preparation of phosphate buffer In 1 liter of distilled water dissolve 3.0 g of potassium hydroxide, 12.0 g of 82% ortho-phosphoric acid and 3.0 g of diethylamine. The solution of said composition has a pH equal to 3. In the case of deviation from the specifi ed pH transmitting correction, add ortho-phosphoric acid or potassium hydroxide.
Preparation of solutions for the mobile phase Phosphate buffer should be mixed with acetonitrile in the ratio of 80:20, thoroughly mixed and fi ltered through a membrane fi lter with a pore size less than 20 microns.

Quantifi cation determination of diphenhyramine in tablets
The exact sample of thoroughly pounded tablet weight (0.005 g aliquots diphenhydramine) should be dissolved in 3 ml of mobile phase in a glass of 25 ml and fi ltered in a volumetric fl ask 25.00 ml which also carry on fi lter, washed with an additional 2 ml of mobile phase two, the resulting solution should be adjusted to the mark with the same solvent and mixed. 1.00 ml of this solution is transferred to a volumetric flask 10.00 ml and analyzed by the general procedure. Calculation of the content of the active substance is carried out according to the typical formula.
Results and discussion HPLC method is widely used in chemical analysis for quantitative studies of drugs of different pharmacological groups and chemical properties. Firstly, this is due to the wide versatility method to no signifi cant changes in terms of analysis to explore the different molecular structure of matter. Secondly, the relatively high selectivity of chromatographic performance is known in various substances.
The selected research method can be performed on equipment of broad spectrum. UV spectrometry detector combined with column allows you to explore a wide range of drugs. Also, this method does not use expensive and popular solvents -acetonitrile and phosphate buffer, and does not require long complex sample preparation. Therefore we can say that the chosen method best meets the intended purpose of our work.
At the preparatory stage of the study it was found that diphenhydramine is the main center in the molecule, so it should be well chromatographed on reverse phase column with a rather acidic mobile phase, which is the chosen mixture.
As a result chromatographic study, chromatograms containing one right diphenhyramine peak with retention time (t) 5.82 min, were obtained; this peak was absent in the chromatography blank sample (fi g. 1).

Вопросы фармации / Problems of pharmacy
The basic chromatographic parameters obtained by chromatogram were calculated, such as retention volume (V), the symmetry factor (As), number of theoretical plates (N), the signal / noise (S / N), the maximum allowable relative standard deviation (RSDmax) and the relative standard deviation (RSD%). The value calculated indicators show the high effi ciency of the selected chromatographic system (table 1).   Table 3 Determination of the convergence results of quantitative determination of drugs studied (n = 9, p = 0.95)

Validation of Analytical Procedures
According to the requirements of SPU validation procedure was carried out for the developed technique. Key validation specifi cations such as linearity, precision and accuracy were determined by a standardized procedure validation standard method [14].
The linear dependence. Linearity was determined within concentrations corresponding to 80-120% of nominal content of tablets.
Key fi gures linear relationship is shown in table 2, which shows that the technique confi rm linearity over the entire range of concentrations mentioned above.
Precision methods defi ned for each dosage form was determined at convergence. To do this, in each case parallel measurements were performed nine times (three sample three repetitions) and the results confi rmed the expected validated characteristics (table 3). It was found that in all cases the onesided confi dence interval Δh does not exceed the maximum uncertainty analysis, so this method is accurate as at the level of convergence.
Correctness in both cases was determined by supplementation. For this solution to the minimum sample three drugs were added in three different proportions of the sample solution standard pharmacopoeial diphenhyramine. Thus, three solutions of three concentrations of diphenhyramine were given. Next, the resulting solution was chromatographed by the method described above. The results can be considered accurate defi nitions, as systematic error is not statistically different from zero, that is the true value of the quantity does not exceed the specifi ed confi dence interval (table 4).

Conclusions
1. Studies of chromatography conditions of diphenhyramine were performed by HPLC. Accurate, economical and rapid chromatographic method of assay of diphenhyramine was chosen using mobile phase as a mixture of phosphate buffer: acetonitrile in the ratio of 80:20.
2. The calculated basic chromatographic parameters of diphenhyramine chromatogram were obtained with the selected method. They demonstrate the suitability of the chromatographic system according to the requirements of SPU quantitative analysis of diphenhyramine chosen method. 3. It had been proven that the method of quantifi cation of processed diphenhyramine applied to two studied dosage forms for such characteristics as linearity, precision, accuracy is valid and can be used in pharmaceutical and forensic analytical laboratories.