Potential of Borojoa patinoi Cuatrecasas water extract to inhibit nosocomial antibiotic resistant bacteria and cancer cell proliferation in vitro.

Borojó (Borojoa patinoi Cuatrec.) is a fruit used in Colombian traditional medicine with supposed antihypertensive, antitumoral, diuretic, healing, immunological, anti-inflammatory and aphrodisiac effects. To explore the relative merits in terms of biological activities of borojó aqueous extract (BAE), we investigated in vitro its antimicrobial activity on nosocomial pathogenic and multidrug resistant (MDR) strains of Pseudomonas aeruginosa (6), Staphylococcus aureus (1) and Candida species (6), as well as its cytotoxicity on human conjunctive Wong-Kilbourne derivative (WKD) cells and Caco-2 cells from heterogeneous human epithelial colorectal adenocarcinoma. The bacteriostatic activity was observed overall on P. aeruginosa strains, as evidenced by the increase of the lag phase (43 hours) and reduction of the maximum growth rate detected using 187.5 mg BAE per mL. The bactericidal activity, instead, was observed at 375 mg BAE per mL. On the other hand, BAE showed an anti-proliferative effect against the Caco-2 cell line and was shown to be toxic on the WKD cell line at concentrations ranging from 0.05 to 187.5 µg mL-1. The analysis of the phenolic fraction of the fruit aqueous extract (BAE) using UHPLC-MS/MS showed the presence of 26 compounds, with vanillic, syringic and o-coumaric acids as the most abundant. Among these molecules, 7.81 ng mL-1 luteolin and myricetin, singly tested, were able to reduce bacterial growth. To the best of our knowledge, we are unaware of any previous studies demonstrating the anti-bacterial activity of borojó aqueous extract against antibiotic resistant strains of P. aeruginosa, and its anti-proliferative effect against WKD and Caco-2 cell lines. The latter result offers a potential base for new interest and investigations in relation to colon carcinoma models and borojó fruit consumption, since in Colombia this fruit is consumed also for its supposed antitumoral effects.

Analysis of new psychoactive substances in oral fluids by means of microextraction by packed sorbent followed by ultra-high-performance liquid chromatography-tandem mass spectrometry.

In recent years, new drugs, commonly known as new psychoactive substances (NPS), appeared on the market, which include, among others, synthetic cannabinoids, cathinones, and tryptamine analogs of psilocin. The aim of this work was to develop and validate a new method for simultaneous screening and quantification of 31 NPS in oral fluid by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The chosen target analytes represented different chemical and toxicological NPS classes, such as synthetic cathinones, piperazines, phenethylamines, synthetic cannabinoids, and their metabolites. The procedure involved a rapid sample preparation based on protein precipitation followed by clean-up utilizing microextraction by packed sorbent (MEPS); the quantitative analysis was performed by UHPLC-MS/MS. The MEPS clean-up, regardless of non-quantitative recoveries for some analytes, provided an effective removal of interfering compounds, as demonstrated by reduced matrix effects found at different concentrations for all the analytes. The validation protocol, based on SWGTOX guidelines, demonstrated the suitability of the proposed method for quantitative analysis: linearity range ranged over 3 or 4 orders of magnitude; precision and accuracy tests gave RSD% values below 25%, and accuracy ranged from 85.9% to 107%, accomplishing SWGTOX requirements. Limits of detection (LODs) ranged between 0.005 ng/mL and 0.850 ng/mL and limits of quantification (LOQs) from 0.015 to 2.600 ng/mL.

Multi-class analysis of new psychoactive substances and metabolites in hair by pressurized liquid extraction coupled to HPLC-HRMS.

In this paper, an analytical method has been developed and validated for the analysis of new psychoactive substances (NPS) and metabolites in hair samples. The method was based on pressurized liquid extraction (PLE) followed by solid-phase extraction (SPE) clean-up and high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) analysis. To evaluate extraction efficiency and the applicability of the method, hair samples were fortified by soaking in order to obtain a good surrogate for drug users' hair; the amount of incorporated drugs related to their lipophilicity, similarly to in vivo drug incorporation. To the best of our knowledge, this is the first method that allowed for the analysis of both cathinones (5) and synthetic cannabinoids (7) in hair with a single extraction procedure and chromatographic run. A phenethylamine (2C-T-4), 4- fluorophenylpiperazine and methoxetamine were also included showing that PLE coupled to SPE clean-up was suitable for a multi-class analysis of NPS in hair. In addition, the use of PLE significantly reduced hair analysis time: decontamination, incubation, clean-up, and liquid chromatography-mass spectrometry (LC-MS) analysis were carried out in approximately 45 min. The method was fully validated according to Scientific Working Group for Forensic Toxicology (SWGTOX) and Society of Hair Testing (SoHT) guidelines. Limit of quantification (LOQ) values ranged from 8 to 50 pg mg-1 for cathinones, phenetylamines and piperazines, and from 9 to 40 pg mg-1 for synthetic cannabinoids (10 pg mg-1 for methoxetamine). Matrix effects were below 15% for all the analytes, demonstrating the effectiveness of the clean-up step. Inaccuracy was lower than 9% in terms of bias. Copyright © 2016 John Wiley & Sons, Ltd.

Pressurized liquid extraction for the determination of cannabinoids and metabolites in hair: Detection of cut-off values by high performance liquid chromatography-high resolution tandem mass spectrometry.

Hair analysis has become a routine procedure in most forensic laboratories since this alternative matrix presents clear advantages over classical matrices; particularly wider time window, non-invasive sampling and good stability of the analytes over time. There are, however, some major challenges for the analysis of cannabinoids in hair, mainly related to the low concentrations of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH), that is the major metabolite. In this study a fast, accurate and sensitive method for the determination of cannabinol, cannabidiol, THC and THC-COOH in hair has been developed. The extraction of analytes from hair (50mg) is based on an automated pressurized liquid extraction (PLE) using water modified with the surfactant sodium dodecyl sulphate as eluent phase. PLE extract is then cleaned up by SPE using polymeric reversed phase cartridges Strata XL before the injection in the HPLC-HRMS/MS system. Chromatographic conditions obtained with a fused-core column allowed a good separation of the analytes in less than 4min. The whole procedure has been validated according to SWGTOX guidelines. The LLOQs obtained for THC-COOH and the other analytes were respectively 0.1 and 2pg/mg. To the best of our knowledge, this is the first LC-MS/MS based method that allows the detection of THC-COOH in hair at values lower than the cut-off.

Determination of illicit drugs and metabolites in oral fluid by microextraction on packed sorbent coupled with LC-MS/MS.

Oral fluid (OF) has become a valuable biologic specimen for toxicological analysis, especially in driving under the influence of drugs (DUID) investigations, because of easy and non-invasive collection procedures. In OF testing, being the sample volume is limited, multi-analyte procedures are particularly advantageous since they save time and resources. In this work, a procedure for the simultaneous analysis of 20 illicit drugs, belonging to the classes of cocaine, amphetamines, natural and synthetic opioids and hallucinogens, is presented. The sample preparation is based on microextraction by packed sorbent (MEPS), a novel technique which is based on the miniaturization of solid-phase extraction (SPE). The presented method, which includes all the most diffused illicit drugs and their metabolites, has been fully validated according to the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines. LLOQs ranged from 0.5 to 30 ng mL(-1) (diacetylmorphine); the presented method allows the detection of all the selected drugs quite below the cutoff values recommended by Substance Abuse and Mental Health Services Administration (SAMHSA) for abuse identification.

A µ-SPE procedure for the determination of cannabinoids and their metabolites in urine by LC-MS/MS.

In this paper the development and validation of a method for the analysis of THC-COOH, THC, THC-OH, CBD and CBN in their total form in urine by LC-MS/MS is presented. Tandem hydrolysis, i.e. enzymatic and basic, has been found optimal for the simultaneous analysis of the selected analytes in urine: basic hydrolysis is more effective for the cleavage of THC-COOH glucuronide while enzymatic hydrolysis allows the cleavage of the conjugated cannabinoids possessing ether bonds (THC, THC-OH, CBD). The whole procedure requires a 2h enzymatic hydrolysis using only 90µL of urine by µ-SPE extraction technique with C18 tips. Clear advantages in terms of time and of enzyme reduction are obtained and the cost of the analysis can be dramatically reduced. Satisfactory recovery values and matrix effect are obtained, and the chromatographic run, performed with a fused-core column, allowed the complete analyte separation in only 3min (total run 5.8min) with a common HPLC system. Furthermore the whole procedure has been validated according to SWGTOX guidelines: LOQs are between 6 and 10ppb, quite lower than the requested cut-off for urine testing; intermediate reproducibility of the selected analytes is below 10% and accuracy is between 85% and 113%, except for CBD, included only for semi-quantitative determination.