Rossella Gottardo

Implementation and Performance Evaluation of a Database of Chemical Formulas for the Screening of Pharmaco/Toxicologically Relevant Compounds in Biological Samples Using Electrospray Ionization-Time-of-Flight Mass Spectrometry

Electrospray ionization (ESI)-time-of-flight (TOF) MS enables searching a wide number of pharmaco/toxicologically relevant compounds (PTRC) in biosamples. However, the number of identifiable PTRC depends on extension of reference database of chemical formulas/compound names. Previous approaches proposed in-house or commercial databases with limitations either in PTRC number or content (e.g., few metabolites, presence of non-PTRC). In the frame of development of a ESI-TOF PTRC screening procedure, a subset of PubChem Compound as reference database is proposed. Features of this database (approximately 50,500 compounds) are illustrated, and its performance evaluated through analysis by capillary electrophoresis (CE)-ESI-TOF of hair/blood/urine collected from subjects under treatment with known drugs or by comparison with reference standards. The database is rich in parent compounds of pharmaceutical and illicit drugs, pesticides, and poisons and contains many metabolites (including about 6000 phase I metabolites and 180 glucuronides) and related substances (e.g., impurities, esters). The average number of hits with identical chemical formula is 1.82 +/- 2.27 (median = 1, range 1-39). Minor deficiencies, redundancies, and errors have been detected that do not limit the potential of the database in identifying unknown PTRC. The database allows a much broader search for PTRC than other commercial/in-house databases of chemical formulas/compound names previously proposed. However, the probability that a search retrieves different PTRC having identical chemical formula is higher than with smaller databases, and additional information (anamnestic/circumstantial data, concomitant presence of parent drug and metabolite, selective sample preparation, liquid chromatographic retention, and CE migration behavior) must be used in order to focus the search more tightly.

Screening for synthetic cannabinoids in hair by using LC-QTOF MS: a new and powerful approach to study the penetration of these new psychoactive substances in the population

The current analytical technology for the determination of New Psychoactive Substances in biological samples is still largely inadequate, because the immunoassays are unsuitable for the detection of most of these compounds and the use of traditional gas chromatography–mass spectrometry techniques is hampered by the lack of chromatographic standards and mass fragmentation patterns. Taking advantage of the molecular recognition capability of high-resolution mass spectrometry, the present work aimed to apply liquid chromatography-quadrupole-time of flight mass spectrometry for the rapid identification of New Psychoactive Substances in the hair, a peculiar tissue which “keeps memory” of the recent history of drug intake of the subject. All the samples were screened for the presence of 50 different New Psychoactive Substances (synthetic cannabinoids, cathinones and phenethylamines), substances that had been reported officially by the National Early Warning System in the period 2009–2011. Among the 435 samples analyzed, 8 were found “positive” for the following compounds: JWH-018, JWH-073, JWH-081, JWH-250, JWH-122, in a broad range of concentrations (0.010–1.28 ng/mg). Results strongly support the use of hair analysis to monitor the diffusion of new psychoactive drugs in the community.

Capillary zone electrophoresis (CZE) coupled to time-of-flight mass spectrometry (TOF-MS) applied to the analysis of illicit and controlled drugs in blood.

A new method for the determination of illicit and abused drugs in blood by capillary zone electrophoresis–electrospray ionization–time‐of‐flight mass spectrometry is proposed, in view of its application in clinical and forensic toxicology. The analytes (methamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, methadone, cocaine, morphine, codeine, 6‐acethylmorphine, benzoylecgonine) were separated with capillary zone electrophoresis by applying 15 kV within 25 min, in an uncoated fused‐silica capillary (75 μm × 100 cm) using a 25 mM ammonium formate electrolyte solution (pH 9.5). The capillary electropherograph was coupled to time‐of‐flight mass spectrometry through an orthogonal electrospray ionization source, with a coaxial sheath liquid interface. The sheath liquid was composed of isopropanol‐water (1:1 v/v) containing 0.5% formic acid delivered at 4 μL/min. Forensic drugs were identified by exact mass determination (mass accuracy typically ≤5 ppm) and by matching of the isotopic pattern.

Analysis of synthetic cannabinoids in herbal blends by means of nano-liquid chromatography

In this study, a rapid and simultaneous separation of 12 synthetic cannabinoids and Δ(9)-tetrahydrocannabinol (Δ(9)-THC) in herbal blends was obtained by means of nano-liquid chromatography (nano-LC). The nano-LC experiments were performed in a 100μm i.d. capillary column packed with Cogent(®) bidentate C(18) silica particles for 25.0cm. All compounds were resolved using an isocratic elution mode in less than 30min. A mobile phase containing ACN/MeOH/H(2)O/formic acid 69/5/25/1 (v/v/v/v) was employed for the chromatographic separation. The developed analytical method was validated in terms of precision, linearity, sensitivity and accuracy. Under optimal nano-LC-UV conditions, the resulting RSD percentages for intra-day and inter-day repeatability, related to retention time and peak area, were below 2.98 and 6.40%, respectively. Limits of detection and quantification were 0.2 and 0.5μg/mL, respectively, for all the studied compounds. Linearity was assessed in the concentration range of interest for all analytes with determination coefficients r(2)≥0.9975. The method was then applied to the determination of synthetic cannabinoids in herbal blends. Quantitative analyses of the cannabimimetic compounds in six products showed that there was a wide difference in the concentration of the studied compounds among different products. Further, the nano-LC system was coupled with a mass spectrometer measuring the MS and MS-MS spectra to unequivocally identify the cannabinoids present in smoking mixtures.

CEC-ESI ion trap MS of multiple drugs of abuse.

This article describes a method for the separation and determination of nine drugs of abuse in human urine, including amphetamines, cocaine, codeine, heroin and morphine. This method was based on SPE on a strong cation exchange cartridge followed by CEC‐MS. The CEC experiments were performed in fused silica capillaries (100 μm×30 cm) packed with a 3 μm cyano derivatized silica stationary phase. A laboratory‐made liquid junction interface was used for CEC‐MS coupling. The outlet capillary column was connected with an emitter tip that was positioned in front of the MS orifice. A stable electrospray was produced at nanoliter per minute flow rates applying a hydrostatic pressure (few kPa) to the interface. The coupling of packed CEC columns with mass spectrometer as detector, using a liquid junction interface, provided several advantages such as better sensitivity, low dead volume and independent control of the conditions used for CEC separation and ESI analysis. For this purpose, preliminary experiments were carried out in CEC‐UV to optimize the proper mobile phase for CEC analysis. Good separation efficiency was achieved for almost all compounds, using a mixture containing ACN and 25 mM ammonium formate buffer at pH 3 (30:70, v/v), as mobile phase and applying a voltage of 12 kV. ESI ion‐trap MS detection was performed in the positive ionization mode. A spray liquid, composed by methanol–water (80:20, v/v) and 1% formic acid, was delivered at a nano‐flow rate of ∼200 nL/min. Under optimized CEC‐ESI‐MS conditions, separation of the investigated drugs was performed within 13 min. CEC‐MS and CEC‐MS2 spectra were obtained by providing the unambiguous confirmation of these drugs in urine samples. Method precision was determined with RSDs values ≤3.3% for retention times and ≤16.3% for peak areas in both intra‐day and day‐to‐day experiments. LODs were established between 0.78 and 3.12 ng/mL for all compounds. Linearity was satisfactory in the concentration range of interest for all compounds (r2≥0.995). The developed CEC‐MS method was then applied to the analysis of drugs of abuse in spiked urine samples, obtaining recovery data in the range 80–95%.

Rapid and direct determination of creatinine in urine using capillary zone electrophoresis

BACKGROUND In clinical medicine creatinine determination is used for the diagnosis of renal diseases and muscular dysfunctions. Also, in forensic toxicology creatinine concentration is used as a standardization tool for the quantitative measurement of therapeutic or illicit drugs and xenobiotics in urine. The present work was aimed at developing a robust and reliable CE determination of creatinine in urine, meeting the needs of simplicity, rapidity and low cost required by routine toxicological screening. METHODS The optimized buffer electrolyte was composed of 200 mM phosphate and 200 mM acetic acid (pH 3.8). The separation capillary (50 microm x 10 cm of effective length) was made of naked fused silica. Separations were carried out under 25 kV potential. Urine samples were diluted 20 fold with water and directly injected. Direct UV absorption detection at 200 nm was employed. RESULTS Linearity was assessed in the range 0.2-32 mM. Precision tests resulted in CVs % below 0.56% for migration times and below 3.78% for peak area ratios (analyte/I.S.). CONCLUSIONS The described CE creatinine assay meets the strict requirements of forensic analysis and looks particularly useful to test the possible adulteration or dilution of urine samples undergoing toxicological screening.

Screening for new psychoactive substances in hair by ultrahigh performance liquid chromatography-electrospray ionization tandem mass spectrometry

In the latest years, many new psychoactive substances (NPS) from several drug classes have appeared in the illicit drug market. Their rapid, sensitive and specific identification in biological fluids is hence of great concern for clinical and forensic toxicologists. Here is described a multi-analyte method for the determination of NPS, pertaining to different chemical classes (synthetic cannabinoids, synthetic cathinones, ketamine, piperazines and amphetamine-type substances-ATS) in human hair using ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) in electrospray ionization mode. We focused on a sample preparation able to extract the different classes of NPS. About 30mg of hair was decontaminated and incubated overnight under sonication in different conditions depending on the type of analytes to be extracted: (a) with 300μL of HCOOH 0.1% for cathinones, piperazines and ATS; (b) with 300μL of MeOH for synthetic cannabinoids. Ten microliter of the extracts were then injected in UHPLC-ESI-MS/MS in MRM mode. The LODs varied from 2pg/mg to 20pg/mg. The method was linear in the range from the LOQ to 500pg/mg and showed acceptable precision (%RSD<15) and accuracy (%E<15) for all the analytes. The method was finally applied on 50 samples from real forensic cases (driving license re-granting, postmortem toxicological analyses, workplace drug testing). In three samples we detected synthetic cannabinoids, in four samples cathinones or ephedrines, in two samples ketamine.

Micellar electrokinetic chromatography: a new simple tool for the analysis of synthetic cannabinoids in herbal blends and for the rapid estimation of their logP values.

For the first time a capillary separation based on micellar electrokinetic chromatography (MEKC) with diode array detection (DAD) was developed and validated for the rapid determination of synthetic cannabinoids in herbal blends. Separations were carried out on a 30 μm(ID) × 40 cm uncoated fused silica capillaries. The optimized buffer electrolyte was composed of 25 mM sodium tetraborate pH 8.0, 30 mM SDS and n-propanol 20% (v/v). Separations were performed at 30 kV. Sample injection conditions were 0.5 psi, 10s. Diazepam and JWH-015 were used as internal standards. The determination of the analytes was based on the UV signal recorded at 220 nm, corresponding to the maximum wavelength of absorbance of the molecules, whereas peak identification and purity check were also performed on the basis of the acquisition of UV spectra between 200 and 400 nm wavelengths. Under the described conditions, the separation of the compounds was achieved in 25 min without any significant interference from the matrix. Linearity was assessed within a concentration range from 5 to 100 μg/mL. The intra-day and inter-day imprecision values were below 2.45% for relative migration times and below 10.75% for relative peak areas. The present method was successfully applied to the direct determination of synthetic cannabinoids in 15 different herbal blend samples requiring only sample dilution. In addition, the developed MEKC separation was also applied to estimate the octanol/water partition coefficients (logP) of these new and poorly known molecules.

Analysis of drugs of forensic interest with capillary zone electrophoresis/time-of-flight mass spectrometry based on the use of non-volatile buffers.

he present work is aimed at investigating the influence of the background electrolyte composition and concentration on the separation efficiency and resolution and mass spectrometric detection of illicit drugs in a capillary zone electrophoresis‐electrospray ionization‐time of flight mass spectrometry (CZE‐ESI‐TOF MS) system. The effect of phosphate, borate and Tris buffers on the separation and mass spectrometry response of a mixture of 3,4‐methylenedioxyamphetamine, 3,4‐methylenedioxymethamphetamine, methadone, cocaine, morphine, codeine and 6‐monoacetylmorphine was studied, in comparison with a reference ammonium formate separation buffer. Inorganic non‐volatile borate and Tris buffers proved hardly suitable for capillary electrophoresis‐mass spectrometry (CE‐MS) analysis, but quite unexpectedly ammonium phosphate buffers showed good separation and ionization performances for all the analytes tested. Applications of this method to real samples of hair from drug addicts are also provided.

BH4 (tetrahydrobiopterin)‐dependent activation, but not the expression, of inducible NOS (nitric oxide synthase)‐2 in proinflammatory cytokine‐stimulated, cultured normal human astrocytes is mediated by MEK–ERK kinases

Nitric oxide (NO) from astrocytes is one of the signalers used by the brains extensive glial‐neuronal‐vascular network, but its excessive production by pro‐inflammatory cytokine‐stimulated glial cells can be cytodestructive. Here, we show how three pro‐inflammatory cytokines (IL‐1β, TNF‐α, and IFN‐γ) together stimulated the activation, but not the prior expression, of NOS‐2 protein via a mechanism involving MEK–ERKs protein kinases in astrocytes from adult human cerebral temporal cortex. The cytokines triggered a transient burst of p38 MAPK activity and the production of NOS‐2 mRNA which were followed by bursts of MEK–ERK activities, synthesis of the NOS‐2 co‐factor tetrahydrobiopterin (BH4), a build‐up of NOS‐2 protein and from it active NOS‐2 enzyme. Selectively inhibiting MEK1/MEK2, but not the earlier burst of p38 MAPK activity, with a brief exposure to U0126 between 24 and 24.5 h after adding the cytokine triad affected neither NOS‐2 expression nor NOS‐2 protein accumulation but stopped BH4 synthesis and the assembly of the NOS‐2 protein into active NOS‐2 enzyme. The complete blockage of active NOS‐2 production by the brief exposure to U0126 was bypassed by simply adding BH4 to the culture medium. Therefore, this cytokine triad triggered two completely separable, tandem operating mechanisms in normal human astrocytes, the first being NOS‐2 gene expression and accumulation of NOS‐2 protein and the second being the synthesis of the BH4 factor needed to dimerize the NOS‐2 protein into active, NO‐making NOS‐2 enzyme.