DOI: https://doi.org/10.15587/2519-4852.2016.87257

THe quantitative determination of sildenafil in human plasma by high performance liquid chromatography - mass spectrometry (lc-ms/ms)

Игорь Эрнестович Кузнецов, Елена Алексеевна Науменко, Наталия Константиновна Резниченко, Андрей Юрьевич Костюк, Роман Прокопович Савяк, Дмитрий Сергеевич Олейников

Abstract


Purpose. For the study of pharmacokinetics of sildenafil oral spray “Tegrum”, developed by pharmaceutical company “Microkhim” (Rubezhnoe, Ukraine) with the aim to improve the pharmacokinetic characteristics of existing solid formulations for oral use it was worked out and validated a rapid, simple and specific method of quantitative determination of sildenafil concentration in human plasma using internal standard labeled with 8 deuterium atoms - sildenafil-d8.

Methods. Sildenafil was extracted from plasma by the liquid-liquid extraction, and quantitatively determined in supernatant by means of а high performance liquid chromatography - tandem mass spectrometry method (HPLC-MS/MS). Separation of components was performed using the chromatographic column Kinetex 2.6u C18, 50 x 4.6 mm, 2.6 μm (Phenomenex) at 45˚C at isocratic elution mode and mobile phase, that consisted of 2 mM aqueous ammonium acetate solution (pH 5,45 ± 0,05) and acetonitrile (10/90, vol./vol.); flow rate equaled to 0.4 ml/min. Ionization of sildenafil and sildenafil-d8 was performed in the positive electrospray mode (ESI, Positive). Multi reactions monitoring (MRM) mode was used for selected daughter and parent ions detection with the following settings of the mass spectrometer: m/z 475,20/283,20 and 483.20/283.20 for sildenafil and sildenafil-d8 respectively.

Results. The method described above was validated in the linear range 10,00-400,00 ng/mL of sildenafil with a correlation coefficient (r2) 0,99764 and demonstrated reliable accuracy and reproducibility of results.

Conclusions. Utilization of elaborated method of fast direct liquid - liquid sildenafil extraction from the blood plasma with simultaneous proteins precipitation, avoiding commonly used supernatant evaporation and subsequent restoration of dry residue at preanalytical stage allowed to achieve target sensitivity of determination and reduced the analysis runtime. Usage of the deuterated internal standard (sildenafil-d8) at analytical stage gave an opportunity to reduce the limit of quantification, to achieve high sensitivity and specificity, as well as suppress matrix effect. Rather high analytical characteristics of the method proposed allow its application in routine bioanalysis during pharmacokinetic studies.


Keywords


sildenafil; sildenafil-d8; oral spray; pharmacokinetics; blood plasma; quantification; HPLC-MS/MS; internal standard selection; matrix effect; analytical method validation

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Copyright (c) 2016 Игорь Эрнестович Кузнецов, Елена Алексеевна Науменко, Наталия Константиновна Резниченко, Андрей Юрьевич Костюк, Роман Прокопович Савяк, Дмитрий Сергеевич Олейников

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