The identification and quantitative determination of amitriptyline by the HPLC-MWD method

Aim. To develop the sensitive and specific HPLC-MWD method for the determination of amitriptyline suitable for use in bioanalytical studies in clinical and forensic toxicology. Materials and methods. The analysis was performed on a “MiLiChrome A-02” microcolumn liquid chromatograph attached to a dual-beam multi-wave UV spectrophotometer under the following conditions: 2×75 mm column ProntoSIL 120-5-C18 AQ, 5 μm; the gradient elution mode with a linear gradient, eluent A (5 % acetonitrile and 95 % perchlorate buffer) to eluent B (100 % acetonitrile) for 40 min, then 100 % eluent B for 3 min; the mobile phase flow rate – 100 μL/min; the thermostat temperature – 40°C; detection was carried out at 8 wavelengths (210, 220, 230, 240, 250, 260, 280, 300 nm). Results. The retention time of amitriptyline was 22.80 min; the absorbance ratios Sλ/S210 were also set as additional option to increase the reliability of identification. The calibration curve was represented by the dependence of a peak area (S, mm2) against concentration (c, μg/μL) and described by the regression equation у = 0.00514 × x. The method showed linearity in the range of 1.0–100.0 μg/mL. The correlation coefficient was lower than 0.99. The LOD and LOQ values were calculated from the parameters of the calibration curve, they were 0.3 μg/mlL and 0.9 μg/mL, respectively. The accuracy and precision of the method developed were determined at three concentration levels (low, middle, high) within one day (intraday); RSD did not exceed 1.0 % across the full concentration range studied when analyzing model solutions. Conclusions. The sensitive and specific method for the quantitative determination of amitriptyline using the HPLC-MWD method has been developed. The method meets the requirements set to techniques recommended for use in clinical and forensic toxicology, and it has been confirmed by a number of the validation parameters. The HPLC-MWD method developed can be recommended for implementation into the practice of regional bureaus of forensic medical examination and clinical and diagnostic laboratories of regional drug addiction centers.