Determination of diphenhydramine by HPLC method in biological liquids

Abstract

The aim of the study was to develop an algorithm for directed analysis of diphenhydramine in biological extracts from urine and blood using a unified method of HPLC research. Materials and methods. The extraction of diphenhydramine was carried out with chloroform at pH 9.0. The extracts were purified from impurities by a combination of TLC and extraction with hexane. TLC purification and identification of diphenhydramine were carried out under optimal conditions: organic solvents systems – chloroform-methanol (90:10); methanol; methanol-25 % solution of ammonium hydroxide (100:1.5) and chromatographic plates – Sorbfil PTLC-AF-A, Sorbfil PTLC-P-B-UV. For the detection of diphenhydramine, the most sensitive location reagents were used – UV light (λ=254 nm) and Dragendorff reagent in the modification of Mounier. HPLC analysis was carried out on a microcolumn liquid chromatograph "Milichrome A-02" in conditions: reversed-phase variant, column with non-polar sorbent Prontosil 120-5 C18 AQ, 5 μm; mobile phase in the mode of linear gradient – from eluent А (5 % acetonitrile and 95 % buffer solution) to eluent B (100 % acetonitrile) as during 40 min. The flow rate of the mobile phase has been formed 100 μl/min, injection volume – 4 μl. Multichannel detection of the substance was carried out using a UV spectrophotometer at 210, 220, 230, 240, 250, 260, 280 and 300 nm; the optimal value of column temperature – 37–40 °С and pressure of pump – 2.8–3.2 MPa. Results and its discussion. Extraction, purification, identification and quantitative determination of diphenhydramine were carried out according to the developed methods. It is established that when isolating diphenhydramine from blood according to the developed methods it is possible to allocate 34.2–38.4 % of substance 5.69 %, RSDx 2.04 % ε = ±3.91%, RSDx = 1.40 %. (ε = ± =) and from urine – 55.8–60.5 % of substance Conclusions. An algorithm has been developed for directed analysis of diphenhydramine in biological extracts from urine and blood using a unified HPLC method. Statistical processing of the experimental results indicates the reliability and reproducibility of the technique.