Portable near infrared spectroscopy for the isomeric differentiation of new psychoactive substances.

Rapid and efficient identification of the precise isomeric form of new psychoactive substances (NPS) by forensic casework laboratories is a relevant challenge in the forensic field. Differences in legal status occur for ring-isomeric species of the same class, thus leading to different penalties and judicial control. Portable systems such as near-infrared (NIR) spectroscopy recently emerged as suitable techniques for the on-scene identification of common drugs of abuse such as cocaine, MDMA and amphetamine. This way, the overall forensic process becomes more efficient as relevant information on substance identity becomes available directly at the scene of crime. Currently, no NIR-based applications exist for the rapid, on-scene detection of NPS isomers. Herein, we present the differentiation of cathinone and phenethylamine-type NPS analogues based on their NIR spectrum recorded in 2 seconds on a portable 1350 - 2600 nm spectrometer. A prior developed data analysis model was found suitable for the identification of the methylmethcathinone (MMC) isomers 2-MMC, 3-MMC and 4-MMC. In 51 mixtures and 22 seized casework samples, the correct isomeric form was detected in all cases except for a few mixtures with an active ingredient content of 10 wt%. These results show the feasibility of on-site NPS detection as presumptive test performed directly at the scene of crime with a small size NIR-spectrometer. Additionally, in the illicit drug analysis laboratory the combination of NIR and GC-MS analysis might be suitable for robust identification of NPS isomers and analogues.

How to perform spectrum-based LC-HR-MS screening for more than 1,000 NPS with HighResNPS consensus fragment ions.

INTRODUCTION: The ever-changing market of new psychoactive substances (NPS) poses challenges for laboratories worldwide. Analytical toxicologists are constantly working to keep high-resolution mass spectrometry (HR-MS) screening libraries updated for NPS. This study sought to use the online crowd-sourced HighResNPS database for spectrum comparison screening, thereby broadening its utility to all HR-MS instruments. METHOD: HighResNPS allows formation of a set of consensus fragment ions for a NPS and prioritises among multiple entries of collision-induced fragment ions. A subset of 42 NPS samples was analysed in data-independent acquisition (DIA) and data-dependent acquisition (DDA) modes on two different instruments. HighResNPS-computed spectra were generated with either Absolute (all fragment ions set to 100%) or Fractional (50% intensity reduction of former fragment ion) intensity. The acquired NPS data were analysed using the consensus library with computed ion intensities and evaluated with vendor-neutral screening software. RESULTS: Overall, of the 42 samples, 100% were identified, with 88% identified as the top candidate. Three samples had the correct candidate proposed as the second highest ranking NPS. In all three of those samples, the top proposed candidate was a positional isomer or closely related compound. Absolute intensity assignment provided identical scoring between the top two proposed compounds in two samples with DIA. DDA had a slightly higher identification rate in the spectra comparison screening with fractional intensity assignment, but no major differences were observed. CONCLUSION: The fractional intensity assignment was slightly more advantageous than the absolute assignment. It was selective between proposed candidates, showed a high identification rate and had an overall higher fragmentation scoring. The candidates proposed by the HighResNPS library spectra comparison simplify the determination of NPS for researchers and toxicologists. The database provides free monthly updates of consensus spectra, thereby enabling laboratories to stay at the forefront of NPS screening by LC-HR-MS with spectra screening software.

The Lysosomotropic Activity of Hydrophobic Weak Base Drugs is Mediated via Their Intercalation into the Lysosomal Membrane.

Lipophilic weak base therapeutic agents, termed lysosomotropic drugs (LDs), undergo marked sequestration and concentration within lysosomes, hence altering lysosomal functions. This lysosomal drug entrapment has been described as luminal drug compartmentalization. Consistent with our recent finding that LDs inflict a pH-dependent membrane fluidization, we herein demonstrate that LDs undergo intercalation and concentration within lysosomal membranes. The latter was revealed experimentally and computationally by (a) confocal microscopy of fluorescent compounds and drugs within lysosomal membranes, and (b) molecular dynamics modeling of the pH-dependent membrane insertion and accumulation of an assortment of LDs, including anticancer drugs. Based on the multiple functions of the lysosome as a central nutrient sensory hub and a degradation center, we discuss the molecular mechanisms underlying the alteration of morphology and impairment of lysosomal functions as consequences of LDs' intercalation into lysosomes. Our findings bear important implications for drug design, drug induced lysosomal damage, diseases and pertaining therapeutics.

Development of an Integrated Tissue Pretreatment Protocol for Enhanced MALDI MS Imaging of Drug Distribution in the Brain.

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) technique has attracted intense interest in the visualization of drug distribution in tissues. Its capability to spatially resolve individual molecules makes it a unique tool in drug development and research. However, low drug content and severe ion suppression in tissues hinder its broader application to resolve drug tissue distribution, especially small molecule drugs with a molecular weight below 500 Da. In this work, an integrated tissue pretreatment protocol was developed to enhance the detection of central nervous system drugs in the mouse brain using MALDI MSI. To evaluate the protocol, brain sections from mice dosed intraperitoneally with donepezil, tacrine, clozapine, haloperidol, and aripiprazole were used. The tissue sections were pretreated serially by washing with ammonium acetate solution, incubation with trifluoroacetic acid vapor, and n-hexane washing before MALDI MSI. Compared with the untreated sample, the signal intensities for the test drugs increased by 4.7- to 31.5-fold after pretreatment. Besides the enhancement of signal intensity, fine optimization of pretreatment time and washing solvents preserved the spatial distribution of target drug molecules. The utility of the developed protocol also provided tissue-specific distribution for five drugs which were well resolved when imaged by MALDI MS.

Oxidative removal of carbamazepine by peroxymonosulfate (PMS) combined to ionizing radiation: Degradation, mineralization and biological toxicity.

Carbamazepine is one of pharmaceutical and personal care products (PPCPs) and has been widely used to treat depression and seizures, and it cannot be effectively removed during the conventional wastewater treatment processes. In this study, three processes were used for the carbamazepine degradation, including single radiation, radiation in the presence of peroxymonosulfate (PMS) and radiation followed by PMS oxidation. The results show that radiation in the presence of PMS could enhance the degradation and mineralization of carbamazepine, decreasing the absorbed dose required for completely degrading carbamazepine from 800 Gy to 300 Gy, no matter what the molar ratio of PMS to carbamazepine was. The radiation followed by PMS oxidation significantly increased the mineralization, and the maximum mineralization achieved 46.5% at the dose of 600 Gy. Eight intermediates were tentatively identified. Compared to single radiation process, the radiation in the presence of PMS enhanced the transformation of intermediates and the release of ammonium ion. In real wastewater, the radiation in the presence of PMS could effectively remove carbamazepine and considerably decreased the biological toxicity of the wastewater containing carbamazepine.

Development and Validation of Stability Indicating High-Performance Thin-Layer Chromatographic (HPTLC) Method for Quantification of Asiaticoside from Centella asiatica L. and its Marketed Formulation.

Background: Ayurvedic medicines help in healing disease with fewer undesirable effects in comparison with an allopathic system of medicine to treat central nervous system (CNS) disorders, as the latter is more expensive. Centella asiatica L. is often used in Ayurvedic formulations for the treatment of CNS disorders. Objective: A stability test using an HPTLC method for the estimation of an important marker asiaticoside (ASI) from C. asiatica powder and marketed formulation was developed. Methods: The marker compound ASI from plant powders and marketed formulations were resolved using toluene-ethyl acetate-methanol-glacial acetic acid (2+7+3+1, v/v/v/v) as the mobile phase and then was derivatized. The plant powder and marketed formulation were also subjected to stability studies. Results: The Rf value of ASI was found in range of 0.43-0.47 for the standard ASI, plant powder, and marketed formulation. It was found that the plant powder and formulation exhibited first-order degradation kinetics. Conclusions: The contents of ASI in the formulation (Churna) and its flow characters reduced at the end of the 6 months during an accelerated stability study. The developed method can be used to quantify ASI in the presence of its degradation products. Highlights: The developed method helps in determining batch to batch variation in the content of ASI in herbal formulations.

Toward Higher Sensitivity in Quantitative MALDI Imaging Mass Spectrometry of CNS Drugs Using a Nonpolar Matrix.

Tissue-specific ion suppression is an unavoidable matrix effect in MALDI mass spectrometry imaging (MALDI-MSI), the negative impact of which on precision and accuracy in quantitative MALDI-MSI can be reduced to some extent by applying isotope internal standards for normalization and matrix-matched calibration routines. The detection sensitivity still suffers, however, often resulting in significant loss of signal for the investigated analytes. An MSI application considerably affected by this phenomenon is the quantitative spatial analysis of central nervous system (CNS) drugs. Most of these drugs are low molecular weight, lipophilic compounds, which exhibit inefficient desorption and ionization during MALDI using conventional polar acidic matrices (CHCA, DHB). Here, we present the application of the (2-[(2 E)-3-(4- tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile) matrix for high sensitivity imaging of CNS drugs in mouse brain sections. Since DCTB is usually described as an electron-transfer matrix, we provide a rationale (i.e., computational calculations of gas-phase proton affinity and ionization energy) for an additional proton-transfer ionization mechanism with this matrix. Furthermore, we compare the extent of signal suppression for five different CNS drugs when employing DCTB versus CHCA matrices. The results showed that the signal suppression was not only several times lower with DCTB than with CHCA but also depended on the specific tissue investigated. Finally, we present the application of DCTB and ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry to quantitative MALDI imaging of the anesthetic drug xylazine in mouse brain sections based on a linear matrix-matched calibration curve. DCTB afforded up to 100-fold signal intensity improvement over CHCA when comparing representative single MSI pixels and >440-fold improvement for the averaged mass spectrum of the adjacent tissue sections.

Hair analysis in toxicological investigation of drug-facilitated crimes in Denmark over a 8-year period.

Hair can serve as a specimen for identifying past drug exposure. Segmental hair analysis may differentiate a single exposure from chronic use. Consequently, segmental hair analysis is useful for disclosing a single drug ingestion, as well as for determining repeated exposures in drug-facilitated crimes (DFCs). This paper presents an overview of toxicological investigations that have used hair analysis in DFC cases from 2009 to 2016 in Denmark. Hair concentrations were determined for 24 DFC-related drugs and metabolites, including benzodiazepines and other hypnotics, antihistamines, opioid analgesics, antipsychotics, barbiturates, and illicit drugs from DFC cases. Drug detection in hair in DFC cases following a single or few intakes of chlorprothixene, codeine, diphenhydramine, oxazepam, oxycodone, promethazine, and phenobarbital is reported for the first time in forensic toxicology. A literature review on concentrations in the published DFC-related hair cases and on concentrations in hair of these substances after single and multiple doses is included. These cases demonstrate the value of segmental hair analysis in DFCs and facilitate future interpretations of results.

Importance of P-gp PET Imaging in Pharmacology.

Capillary endothelial cells in the brain express P-glycoprotein (P-gp), which works as a functional blood-brain barrier (BBB). P-gp pumps out multiple types of molecules from the brain parenchyma into the blood. Therefore, altered P-gp function at the BBB will change the concentrations of therapeutic drugs in the central nervous system (CNS) and hence impact the toxicity and efficacy of CNS drugs. Positron emission tomography (PET) is the only way to non-invasively measure P-gp function in the living human brain. PET imaging of P-gp function was first demonstrated in 1998 with the substrate tracer racemic [11C]verapamil. Since then, several drug interaction studies and proof-of-concept studies regarding drug resistance have been performed with P-gp PET imaging. Although preclinical findings have been very positive regarding the possibilities and importance of P-gp PET imaging, very few studies have shown the clinical relevance of P-gp PET imaging in different disorders of the brain. This review summarizes the pharmacological studies with PET using substrate tracers and emphasizes the importance of PET imaging to understand the mechanism of action of CNS drugs.

The use of a sulfonated capillary on chiral capillary electrophoresis/mass spectrometry of amphetamine-type stimulants for methamphetamine impurity profiling.

Chiral capillary electrophoresis/tandem mass spectrometry (CE/MS/MS) using a chemically modified capillary containing sulfonated groups was developed for the following 8 amphetamine-type stimulants (ATS): amphetamine, methamphetamine (MA), norephedrine, norpseudoephedrine, ephedrine (EP), pseudoephedrine (pEP), dimethylamphetamine and methylephedrine. The running buffer was 10 mM formic acid containing 20 mM highly sulfated γ-cyclodextrin (pH 2.5) as the chiral selector. All 16 enantiomers were well resolved within 60 min, and precisely identified due to their characteristic mass spectra. Further, the RSDs of the migration times of the analytes were no more than 0.3% without any standardization. (1R,2S)-(-)-EP and (1S,2S)-(+)-pEP, which are important ATS impurities originating in the precursors, were added to a highly concentrated MA solution (1 mg/mL) and analyzed as mock samples for MA impurity analysis. Acceptable repeatability of the migration times of (-)-EP and (+)-pEP (ca. 0.3% RSDs) was still observed without interference from the large amount of MA. The limits of detection (LOD) of (-)-EP and (+)-pEP were approximately 2 µg/mL, therefore, their LOD as the impurity concentrations were calculated at about 0.2%. Seized MA samples were dissolved in water at a high concentration (1 mg/mL) and analyzed by this method. (-)-EP and (+)-pEP were clearly detected as impurities. Although these compounds had similar migration times and mass spectral patterns, the fine repeatability allowed easy identification of the impurities by a simple comparison of the absolute migration times of the specimens and those of authentic standards. This study is the first to report the use of a chemically modified capillary for the impurity profiling on CE/MS/MS.

Comparing translational population-PBPK modelling of brain microdialysis with bottom-up prediction of brain-to-plasma distribution in rat and human.

The prediction of brain extracellular fluid (ECF) concentrations in human is a potentially valuable asset during drug development as it can provide the pharmacokinetic input for pharmacokinetic-pharmacodynamic models. This study aimed to compare two translational modelling approaches that can be applied at the preclinical stage of development in order to simulate human brain ECF concentrations. A population-PBPK model of the central nervous system was developed based on brain microdialysis data, and the model parameters were translated to their corresponding human values to simulate ECF and brain tissue concentration profiles. In parallel, the PBPK modelling software Simcyp was used to simulate human brain tissue concentrations, via the bottom-up prediction of brain tissue distribution using two different sets of mechanistic tissue composition-based equations. The population-PBPK and bottom-up approaches gave similar predictions of total brain concentrations in both rat and human, while only the population-PBPK model was capable of accurately simulating the rat ECF concentrations. The choice of PBPK model must therefore depend on the purpose of the modelling exercise, the in vitro and in vivo data available and knowledge of the mechanisms governing the membrane permeability and distribution of the drug.

In-stream attenuation of neuro-active pharmaceuticals and their metabolites.

In-stream attenuation was determined for 14 neuro-active pharmaceuticals and associated metabolites. Lagrangian sampling, which follows a parcel of water as it moves downstream, was used to link hydrological and chemical transformation processes. Wastewater loading of neuro-active compounds varied considerably over a span of several hours, and thus a sampling regime was used to verify that the Lagrangian parcel was being sampled and a mechanism was developed to correct measured concentrations if it was not. In-stream attenuation over the 5.4-km evaluated reach could be modeled as pseudo-first-order decay for 11 of the 14 evaluated neuro-active pharmaceutical compounds, illustrating the capacity of streams to reduce conveyance of neuro-active compounds downstream. Fluoxetine and N-desmethyl citalopram were the most rapidly attenuated compounds (t1/2 = 3.6 ± 0.3 h, 4.0 ± 0.2 h, respectively). Lamotrigine, 10,11,-dihydro-10,11,-dihydroxy-carbamazepine, and carbamazepine were the most persistent (t1/2 = 12 ± 2.0 h, 12 ± 2.6 h, 21 ± 4.5 h, respectively). Parent compounds (e.g., buproprion, carbamazepine, lamotrigine) generally were more persistent relative to their metabolites. Several compounds (citalopram, venlafaxine, O-desmethyl-venlafaxine) were not attenuated. It was postulated that the primary mechanism of removal for these compounds was interaction with bed sediments and stream biofilms, based on measured concentrations in stream biofilms and a column experiment using stream sediments.

Widespread occurrence of neuro-active pharmaceuticals and metabolites in 24 Minnesota rivers and wastewaters.

Concentrations of 17 neuro-active pharmaceuticals and their major metabolites (bupropion, hydroxy-bupropion, erythro-hydrobupropion, threo-hydrobupropion, carbamazepine, 10,11,-dihydro-10,11,-dihydroxycarbamazepine, 10-hydroxy-carbamazepine, citalopram, N-desmethyl-citalopram, fluoxetine, norfluoxetine, gabapentin, lamotrigine, 2-N-glucuronide-lamotrigine, oxcarbazepine, venlafaxine and O-desmethyl-venlafaxine), were measured in treated wastewater and receiving surface waters from 24 locations across Minnesota, USA. The analysis of upstream and downstream sampling sites indicated that the wastewater treatment plants were the major source of the neuro-active pharmaceuticals and associated metabolites in surface waters of Minnesota. Concentrations of parent compound and the associated metabolite varied substantially between treatment plants (concentrations±standard deviation of the parent compound relative to its major metabolite) as illustrated by the following examples; bupropion and hydrobupropion 700±1000 ng L(-1), 2100±1700 ng L(-1), carbamazepine and 10-hydroxy-carbamazepine 480±380 ng L(-1), 360±400 ng L(-1), venlafaxine and O-desmethyl-venlafaxine 1400±1300 ng L(-1), 1800±2300 ng L(-1). Metabolites of the neuro-active compounds were commonly found at higher or comparable concentrations to the parent compounds in wastewater effluent and the receiving surface water. Neuro-active pharmaceuticals and associated metabolites were detected only sporadically in samples upstream from the effluent outfall. Metabolite to parent ratios were used to evaluate transformation, and we determined that ratios in wastewater were much lower than those reported in urine, indicating that the metabolites are relatively more labile than the parent compounds in the treatment plants and in receiving waters. The widespread occurrence of neuro-active pharmaceuticals and metabolites in Minnesota effluents and surface waters indicate that this is likely a global environmental issue, and further understanding of the environmental fate and impacts of these compounds is warranted.

Fluorogenic and chromogenic probe for rapid detection of a nerve agent simulant DCP.

A fluorogenic and visual probe was devised to detect diethyl chlorophosphate (DCP), a nerve agent simulant. The probe, N-(rhodamine B)-lactam-2-aminoethanol (RB-AE), undergoes oxazoline formation following phosphorylation in the presence of DCP, which gives rapid and clear fluorescence and color change in the assay solutions.

Simultaneous determination of ginsenosides and lignans in Sheng-mai injection by ultra-performance liquid chromatography with diode array detection.

An ultra-performance liquid chromatography (UPLC) method with diode array detection was developed for simultaneous analysis of eight ginsenosides (ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3, Rd) and one lignan (schizandrin) in Sheng-mai injection, a traditional Chinese medicine prescription widely used for the treatment of cardiovascular diseases. The chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 column (1.8 microm, 100 mm x 2.1 mm i.d.) using a linear gradient elution over 28 min with a mixture of water and acetonitrile as the mobile phase. All calibration curves showed good linearity (r2 > 0.9998) within the test ranges. Validation proved the repeatability of the method was good and recovery was satisfactory. The validated method was successfully applied to 12 batches of Sheng-mai injection. The results showed that there was a great variation among different samples. Principal component analysis (PCA) further proved considerable variations among the samples from different factories and suggested that schizandrin, ginsenosides Rb1 and Rg1 might have the greatest influence on the variation of 12 samples. In conclusion, these results demonstrated that the UPLC method proposed was very useful for the analysis and quality evaluation of Sheng-mai injection.

Chemiluminescence and electrochemiluminescence detection of controlled drugs.

We review the determination of various controlled drugs (opioids, tranquilizers, stimulants, and hallucinogens) using flow-analysis methodologies (flow injection analysis, high performance liquid chromatography, capillary electrophoresis, and microfluidic devices) with chemiluminescence and electrochemiluminescence reagents such as luminol, diaryloxalates, tris(2,2'-bipyridine)ruthenium(II), permanganate, manganese(IV), and sulfite, for industrial, clinical, pharmaceutical, and forensic science applications.

[Application of liquid chromatography-high resolution mass spectrometry in toxicological screening].

Due to the diversity of toxicologically relevant substances, the uncertainty of target compounds and the specificity of samples, toxicological screening techniques have always been valued by the forensic toxicologists. Depending on its powerful separation ability, superhigh resolution and accurate mass measurement, combined with the two levels spectrum database matching and abundance ratio of isotope ion, the liquid chromatography-high resolution mass spectrometry (LC-HRMS) analyzers have increasingly advantage in screening and identification of chemical compound. This review focuses on the applications of LC-HRMS in screening and identification of drug-of-abuse, prescription drugs, pesticide and stimulant. The prospect of LC-HRMS in forensic toxicology analysis is also included.

Pupil function as an indicator for being under the influence of central nervous system-acting substances from a traffic-medicine perspective.

To evaluate an individual's ability to drive safely, objective measurement methods are required. The primary aim of this study was to assess the applicability and value of infrared pupillography as an objective measurement method to indicate possible influence of central nervous system-acting substances. We exposed healthy test subjects (n = 41), as well as persons who were under the influence of drugs and/or medication (n = 105), to different light stimuli and tested the pupillary light reflex using infrared technology. The synoptic examination of numerous parameters, and the possibility of examination under various conditions, made it possible to achieve highly significant differentiation between persons who were under the influence of drugs and/or medication and control persons. Infrared pupillography represents an objective method of measuring pupil function, providing a reliable indicator of previous consumption of drugs and/or medication. In order to increase legal certainty it would thus appear desirable to make infrared pupillography a routine part of police checks in the near future.

Impurity analysis of pharmaceuticals using capillary electromigration methods.

This review deals with the determination of impurities in pharmaceuticals by electromigration methods in the capillary format. These separation methods are either based on the different effective mobility of the charged analytes (as in zone electrophoresis and isotachophoresis) or include hybrid methods such as micellar electrokinetic chromatography, microemulsion electrokinetic chromatography and electrochromatography. The pharmaceutically active compounds under consideration belong to chemotherapeutic agents, central nervous system drugs, histamine receptor drugs, cardiovascular drugs, anticancer drugs, anti-inflammatory drugs and some other drugs. The review discusses about 150 publications from the period between 1980 and 2007 with special emphasis on the recent trends and gives details about the experimental conditions applied for analyses and the obtained analytical performance parameters.