Design, synthesis and in vitro evaluation of phenylbenzamidine derivatives as SSRIs

A series of phenylbenzamidine analogs were synthesized and tested for their biological activities of inhibiting the reuptake of 5- HT. All of them were new compounds, and their structures were confirmed by 1HNMR, MS and XRD.     

Development of a capillary gas chromatographic procedure

A simple and fast capillary gas chromatographic method with flame ionisation detection (FID) has been developed for the analysis of fluoxetine, fluvoxamine, citalopram, sertraline and paroxetine in their pharmaceutical preparations, using clomipramine as internal standard in order to achieve quantification. The reported method is the first screening one that allows the determination of the five selective serotonin reuptake inhibitors by GC, permitting also to avoid prederivatization for the first time and it is even a pioneering work including an extensive analytical validation and robustness treatment on placebo pharmaceutical formulations too. Optimal conditions for the quantitative gas capillary separation were investigated: column head pressure (100 kPa), injector and detector (FID) temperatures (210 and 260 °C), time and temperature for the splitless step (0.80 min and 80 °C, respectively), volume injected (2 μL) and oven temperature program, providing analysis times shorter than 7 min. Aspects such as stability of solutions, linearity, accuracy, precision, detection and quantitation limits are examined on a selected placebo in order to validate this method. Peak purity is assessed using mass-selective detection (DMS). The robustness of this method was evaluated using the Plackett-Burman fractional factorial experimental design with a matrix of 15 experiments and the statistical treatment proposed by Youden and Steinner. The scope of the validated method is proved in the analysis of almost existing pharmaceutical preparations, with recoveries between 98.5% and 102.4% with regard to their nominal contents. Finally, the proposed method could be also a very successfully option for the analysis of these SSRIs in real urine samples introducing a previous solid phase extraction-preconcentration step.     

Monitoring of progestins: Development of immunochemical methods for purification and detection of levonorgestrel

A polyclonal antibody (Ab) for the progestin levonorgestrel (LNG) was generated, and immunochemical assays for its detection, clean-up and concentration were developed. A highly specific microplate diagnostic assay for the detection of LNG was developed that used the enzyme linked immunosorbent assay (ELISA) method. The LNG ELISA developed was sensitive and reproducible; it exhibited I₅₀ and I₂₀ values of 3.3 ± 1.8 ng mL⁻¹ and 0.6 ± 0.4 ng mL⁻¹, respectively, and the Abs did not cross react with any of the tested steroid hormones. The above Abs were used to develop a sol-gel-based immunoaffinity purification (IAP) method for concentration and clean-up of LNG that is compatible with subsequent immunochemical or instrumental chemical analytical procedures, such as liquid chromatography followed by mass spectrometry (LC-MS/MS). Development of the columns included successful entrapment of Abs within a tetramethoxysilane (TMOS)-based SiO₂ polymer network. The Abs could bind the free analyte from solution, and the bound analyte could be easily eluted from the sol-gel matrix at high recoveries. The Ab selectivity towards the antigen was high, in both ELISA and the sol-gel columns, but the entrapped Abs cross-reacted with two other steroid hormones - ethynylestradiol (EE2) and nortestosterone (NT) - which share similar epitopes with LNG, despite the lack of cross reactivity in the ELISA. The validity of the method was investigated by LC-MS/MS and a good analytical correlation was obtained.     

新型5-HT重摄取抑制剂的设计、合成及活性评价

在对已知各种结构类型的5-HT重摄取抑制剂分子结构全面分析的基础上, 建立了SSRIs药效团模型. 基于该模型应用UNITY程序对NCI-3D和Maybridge-3D数据库进行三维结构的限制性查询, 在获得的命中结构的信息指导下, 设计合成了3种全新结构类型的化合物, 并完成了初步的药理活性评价. 这些化合物均显示出不同程度的5-HT重摄取抑制活性, 其中5个化合物显示高抑制活性. 哌嗪取代的二苯脒类化合物的结构新颖, 较好地符合5-HT重摄取抑制剂药效团模型, 与SSRIs类化合物三维定量构效关系研究得到的CoMFA模型有较好的适配性.     

A simple HPLC method for the simultaneous determination of two selective serotonin reuptake inhibitors and two serotonin-norepinephrine reuptake inhibitors in hair, nail clippings, and cerebrospinal fluid

A novel and simple high-performance liquid chromatography method has been developed for the simultaneous determination of two selective serotonin reuptake inhibitors (fluoxetine and paroxetine) and two serotonin-norepinephrine reuptake inhibitors (venlafaxine and duloxetine) in alternative samples of toxicological interest such as hair, nail clippings, and cerebrospinal fluid (CSF). The separation was achieved on a Hichrom Kromasil 100-5C(18) (250 × 4.6 mm) 5 μm column by using ammonium acetate (0.05 M)-acetonitrile (59:41% v/v) as the mobile phase, delivered isocratically at a flow rate of 1.3 mL/min, within ca. 10 min. Ultraviolet detection at 235 nm was used for monitoring the eluting analytes. Validation was performed in terms of linearity, selectivity, accuracy, precision, and stability. Correlation coefficients were greater than 0.9954. The limits of quantitation ranged between 0.3 and 2.1 ng/μL for all analytes in the liquid matrix (CSF), while the respective values were in the range of 0.3-3.6 ng/mg for solid matrices (hair and nail clippings), with an injection volume of 20 μL. Repeatability and intermediate precision (relative standard deviation, RSD%) were less than 16.6%. The method was successfully applied to actual hair and nail samples from a patient under fluoxetine treatment.