Drug screening in human plasma by cloud-point extraction and HPLC

Cloud-point extraction (CPE) with RP-HPLC/DAD detection was used to develop a screen for six model basic drugs (paracetamol, promazine, amitriptyline, nortriptyline, clomipramine and chlorpromazine) in human plasma. These drugs’ varied hydrophobicities entail different affinities for the micelle-rich phase and CPE extraction efficiencies. Extraction recovery (except paracetamol) was above 80% and reproducibility (RSD%) ranged from 2.88 to 10.26 intraday and from 3.12 to 12.33 interday. The limits of detection were: 0.125 μg mL -1 (promazine and chlorpromazine), 0.25 μg mL -1 (amitriptyline and nortriptyline) and 0.5 μg mL -1 (paracetamol and clomipramine). The method was linear over the ranges: 0.125 -1.0 μg mL -1 (promazine and chlorpromazine), 0.25-1.0 μg mL -1 (amitriptyline and nortriptyline), 0.5-1.0 μg mL -1 (clomipramine) and 0.5-10 μg mL -1 (paracetamol). The procedure is a good alternative to the SPE or LLE sample preparation usually used

Application of micelle-mediated extraction in preparation of body fluids for HPLC-DAD screening of acidic and neutral drugs

Experimental conditions of cloud-point extraction (CPE) for the selected five acidic and neutral medicaments (salicylic acid, opipramol, carbamazepine, lorazepam, and alprazolam) in human plasma were studied and optimized. Separation and detection of the tested drugs were performed by the high-performance liquid chromatography with diode array detection (HPLC-DAD) method in an appropriate gradient mode using a column Nucleosil C8. Under the optimized conditions, main validation parameters were determined for all the compounds. The extraction yields (%) ranged from 54.12 to 82.17 with intra- and interday repeatability (RSD, %) 5.70–9.92 and 5.79–10.19, respectively. The detection limit was 0.5 μg mL−1 for all the tested drugs with exception of salicylic acid (LOD = 2.5 μg mL−1). The linearity of the proposed method was examined for the four drugs: opipramol, carbamazepine, lorazepam, and alprazolam in the concentration range of 0.5–2.0 μg mL−1 (correlation coefficient r2 = 0.995–0.999) and for salicylic acid in the concentration range of 2.5–10.0 μg mL−1 (correlation coefficient r2 = 0.993). The analytical parameters for the medicaments tested in whole blood were unsatisfactory, especially in terms of extraction recovery and repeatability, and application of the developed procedure for this biological matrix requires further study

Determination of N-benzylpiperazine in human plasma using derivatization-free liquid–liquid extraction and gas chromatography mass spectrometry

A simple, fast and low-cost liquid–liquid extraction (LLE) method without a derivatization step for quantification of N-benzylpiperazine (BZP) in human plasma using a gas chromatography mass spectrometry (GC-MS) technique was developed. Among the tested extraction agents the best results were obtained with the use of ethyl acetate at pH 12. Optimization of the gas chromatography conditions was conducted including split ratio, temperature gradient of the analytical column and injection volume. The main validation parameters of the proposed method were determined. The extraction recovery was 68% with intra-day and inter-day precision (RSD%) below 5% (n = 3). The limit of detection (LOD) and quantification (LOQ) of BZP was 24.8 and 82.7 ng mL−1, respectively. A linear calibration curve in the range from LOQ to 1000 ng mL−1 was obtained with the use of the selected ion monitoring (SIM) mode with determination coefficient (r2) higher than 0.999. The optimized and validated LLE-GC-MS method was effectively applied for analysis of clinical plasma samples. The proposed sample preparation protocol was found to be a good alternative to the derivatization step and may be applicable in tests for proof of BZP intake in toxicological routine analysis

An in vitro study of the neurotoxic effects of N-benzylpiperazine : a designer drug of abuse

Recently, the number of new psychoactive substances has significantly increased. Despite the systematic introduction of prohibition in trade of medicinal products which mimic the effects of illegal drugs, the problem concerning this group of drugs is still important although knowledge about the mechanism of action of those types of substances is scarce. This study aimed to follow the neurotoxic effect of N-benzylpiperazine (BZP), the central nervous system psychostimulant, using the human cancer LN-18 cell model. The statistically significant elevation of LDH levels, increased mitochondrial membrane potential, decreased ATP and increased ROS production, increased levels of DNA damage marker (8-OHdG) and activation of caspases: -3 and -9 confirmed by Real-Time PCR imply the activation of mitochondrial proapoptotic pathways induced by BZP after 24 h incubation. This study is a novel, preliminary attempt to explain the toxicity of one of the most popular designer drug of abuse at the cellular level