Ivo Moreno

Determination of piperazine-type stimulants in human urine by means of microextraction in packed sorbent and high performance liquid chromatography-diode array detection

A method using microextraction by packed sorbent (MEPS) and high performance liquid chromatography-diode array detection (HPLC-DAD) is described for the determination of piperazine-type stimulants in human urine. The studied compounds were 1-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl) piperazine (TFMPP), 1-(3-chlorophenyl) piperazine (mCPP) and 1-(4-methoxyphenyl) piperazine (MeOPP); 1-(2-chlorophenyl)-piperazine (oCPP) was used as internal standard (IS). The factors which might influence the extraction were screened previously using the fractional factorial design approach, and none of them influenced significantly the process. The procedure was linear for concentrations ranging from 0.1 (lower limit of quantitation--LLOQ) to 5 μg/mL, with determination coefficients (R(2)) higher than 0.99 for all analytes in all runs. The limits of detection were 0.1 μg/mL for BZP and TFMPP, while for MeOPP and mCPP 0.05 μg/mL was obtained. Intra- and interday precision ranged from 1 to 14%, and accuracy was within a ± 15% interval for all analytes, fulfilling the criteria normally accepted in bioanalytical method validation. Under the optimized conditions, extraction efficiency was higher than 80% for all analytes, except BZP (50%). MEPS showed to be a rapid (<2 min) and simple procedure for the determination of piperazine-type stimulants in human urine, allowing reducing the handling time and costs usually associated to this type of analysis. Furthermore, the fact that only 0.1 mL of sample is required make this method a valuable and powerful tool for drug monitoring in human urine in situations where those compounds are involved, for instance in forensic scenarios.

Rapid determination of piperazine-type stimulants in human urine by microextraction in packed sorbent after method optimization using a multivariate approach

This paper describes the analysis of piperazine-type stimulants [1-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP), 1-(3-chlorophenyl)piperazine (mCPP) and 1-(4-methoxyphenyl)piperazine (MeOPP)] in low volume urine samples (0.1 mL) by microextraction in packed sorbent and liquid chromatography-diode array detection. Analyte extraction has been comprehensively optimized, and the influencing factors were screened by means of the fractional factorial design approach. Several parameters susceptible of influencing the process were studied, and these included extraction sorbent type (C(8) and C(18)), sample dilution (1:2 and 1:4), number of aspirations through the device (2 and 8) and the amount of methanol on both the washing (0 and 10%) and eluting solvents (10 and 100%). The method was linear from 0.5 (lower limit of quantitation) to 5 μgmL(-1), with determination coefficients higher than 0.99 for all compounds. Intra- and interday precision ranged from 1 to 9%, trueness was within a ± 11% interval for all analytes, and analyte recoveries were of about 70% for mCPP and TFMPP, and of about 10% for MeOPP and BZP. The method has shown to be selective, as no interferences from endogenous substances were detected by analysis of blank samples, and the analytes were stable in the samples for short periods at room temperature, after three freeze/thaw cycles and in processed samples. Due to its simplicity and speed, this method can be successfully applied in the screening and quantitation of these compounds in urine samples, and is suitable for application in forensic toxicology routine analysis.

Analytical approach to determine biogenic amines in urine using microextraction in packed syringe and liquid chromatography coupled to electrochemical detection.

The goal of this work was to develop and validate an analytical method for the detection and quantification of the biogenic amines serotonin (5-HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC-ED) in urine. The method was validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mL for 5-HT and between 5 and 1000 ng/mL for DA and NE, with determination coefficients (R(2)) >0.99 for all compounds. The limits of quantification and detection were respectively 50 and 20 ng/mL for 5-HT, and 5 and 2 ng/mL for DA and NE. Within- and between-run precision ranged from 0.84 to 9.41%, while accuracy ranged from 0.79 to 12.76% for all compounds. The intermediate precision and accuracy were 1.50-8.36 and 0.54-13.51%, respectively. The method was found suitable for clinical routine analysis of the studied compounds, using a sample volume of 0.5 mL. This is the first study employing a commercially available MEPS column for the simultaneous detection and quantification of 5-HT, DA and NE in urine by coulometric detection.

Analysis of Salvinorin A in urine using microextraction in packed syringe and GC–MS/MS

BACKGROUND The aim of this work was to develop and validate a method for the determination of Salvinorin A in human urine using microextraction by packed sorbent (MEPS) and GC-MS/MS. RESULTS The technique uses a sample volume as low as 0.2 ml, and the analyte was extracted using a C18 sorbent. The method showed to be linear between 20 and 1000 ng/ml and presented a LOD of 5 ng/ml. Intra- and inter-day precision and accuracy were acceptable. Absolute recoveries ranged from 71 to 80%. CONCLUSION GC-MS/MS with MEPS demonstrated to be a fast and simple procedure for the quantification of Salvinorin A in urine. This is the first time that GC-MS/MS with MEPS was used for the determination of this compound in biological fluids. Furthermore, the device could be reused for up to 80 extractions, which accounted for a lower cost of analysis.

Effects of Hypericum perforatum extract and its main bioactive compounds on the cytotoxicity and expression of CYP1A2 and CYP2D6 in hepatic cells

AIMS Hypericum perforatum (H. perforatum) is one of the most used medicinal plants. However, it has been associated with relevant interactions with several drugs. This situation is probably mediated by cytochrome P450 enzymes (CYP450), namely the 1A2 (CYP1A2) and 2D6 (CYP2D6) isoforms This study aims to assess the cytotoxic and CYP1A2 and CYP2D6 inductive and/or inhibitory effects of a H. perforatum extract and its main bioactive components in hepatic cell lines. MAIN METHODS A MTT proliferation assay was performed in WRL-68, HepG2 and HepaRG cells after exposition to different concentrations of H. perforatum extract, hypericin and hyperforin for 24 and 72 h. Then, a real-time PCR analysis was accomplished after incubating the cells with these products evaluating the relative CYP1A2 and CYP2D6 expression. KEY FINDINGS These products have relevant cytotoxicity at a 10 μM concentration and it was also demonstrated for the first time that H. perforatum can lead to a significant CYP1A2 and CYP2D6 induction in all cell lines. Moreover, hypericin seems to induce CYP1A2 in HepG2 cells and to inhibit its expression in HepaRG cells while hyperforin induced CYP1A2 in HepG2 and in WRL-68 cells. Additionally, hypericin and hyperforin induce CYP2D6 in HepG2 cells but inhibits its expression in HepaRG and in WRL-68 cells. SIGNIFICANCE This study not only evidenced that H. perforatum extract and two of its bioactive components can have toxic effects in hepatic cell lines but also emphasized the potential risk of the consumption of H. perforatum with CYP1A2- and CYP2D6-metabolized drugs.

Effects of Hypericum perforatum hydroalcoholic extract, hypericin, and hyperforin on cytotoxicity and CYP3A4 mRNA expression in hepatic cell lines: a comparative study

Hypericum perforatum L. is one of the best-studied and consumed worldwide medicinal plants and several reports described various biological activities, including sedative, anti-inflammatory, anticancer, and antidepressant. H. perforatum, hypericin, and hyperforin can regulate the expression of CYP3A4 and, to our knowledge, studies are focused on in vivo and clinical evaluations and, there is no an in vitro study comparing CYP3A4 expression between hepatic cells. This study evaluated the effects of H. perforatum hydroalcoholic extract, hypericin, and hyperforin in cellular viability and CYP3A4 expression in HepG2, WRL-68, and HepaRG cells.It was found that, in general, the cytotoxicity promoted by H. perforatum is similar to that promoted by hyperforin alone, time- and dose dependent and the most sensitive cell line is WRL-68 followed by HepG2 and HepaRG cells. Hypericin did not significantly compromise the cellular viability of cells. Regarding their effects in CYP3A4 expression, hyperforin strongly induced CYP3A4 mRNA expression in WRL-68 and HepG2, while in HepaRG it remains unaltered. Hypericin increased CYP3A4 levels in WRL-68 and HepaRG and diminished it in HepG2 cells. H. perforatum induced or decreased CYP3A4 mRNA expression cell- and time-dependent. Computational docking studies showed a lower binding energy between hypericin or hyperforin and its four major metabolites and the catalytic center of the CYP3A4 protein in comparison with ketoconazole.Overall, depending on the cell line, concentration and time of incubation, H. perforatum, hypericin and hyperforin, can exert distinct effects both in cytotoxicity and in CYP3A4 mRNA expression. The results highlighted that at least the continuous and high-dose usage of H. perforatum should be carefully done and monitored, especially in combination with CYP3A4 metabolized drugs.