Learning from remote decentralised clinical trial experiences: A qualitative analysis of interviews with trial personnel, patient representatives and other stakeholders.

AIMS: The aim of the study was to identify actionable learning points from stakeholders in remote decentralised clinical trials (RDCTs) to inform their future design and conduct. METHODS: Semistructured interviews were carried out with a purposive sample of stakeholders, including senior managers, trial managers, technology experts, principal investigators, clinical investigators, research scientists, research nurses, vendors, patient representatives and project assistants. The interview data were coded using a thematic approach, identifying similarities, differences and clustering to generate descriptive themes. Further refinement of themes was guided by empirical phenomenology, grounding explanation in the meanings that interviewees gave to their experiences. RESULTS: Forty-eight stakeholders were interviewed. Actionable learning points were generated from the thematic analysis. Patient involvement and participant engagement were seen as critical to the success of RDCTs where in-person contact is minimal or nonexistent. Involving patients in identifying the research question, creating recruitment materials, apps and websites, and providing ongoing feedback to trial participants were regarded as facilitating recruitment and engagement. Building strong relationships early with trial partners was thought to support RDCT conduct. Multiple modes of capturing information, including patient-reported outcomes (PROs) and routinely collected data, were felt to contribute to data completeness. However, RDCTs may transfer trial activity burden onto participants and remote-working research staff, therefore additional support may be needed. CONCLUSION: RDCTs will continue to face challenges in implementing novel technologies. However, maximising patient and partner involvement, reducing participant and staff burden, and simplifying how participants and staff interact with the RDCT may facilitate their implementation.

A systematic review of methods used to conduct decentralised clinical trials.

AIMS: To evaluate, using quantitative and qualitative approaches, published data on the design and conduct of decentralised clinical trials (DCTs). METHODS: We searched MEDLINE, EMBASE, CENTRAL, PsycINFO, ProQuest Dissertations and Theses, ClinicalTrials.gov, OpenGrey and Google Scholar for publications reporting, discussing, or evaluating decentralised clinical research methods. Reports of randomised clinical trials using decentralised methods were included in a focused quantitative analysis with a primary outcome of number of randomised participants. All publications discussing or evaluating DCTs were included in a wider qualitative analysis to identify advantages, disadvantages, facilitators, barriers and stakeholder opinions of decentralised clinical trials. Quantitative data were summarised using descriptive statistics, and qualitative data analysed using a thematic approach. RESULTS: Initial searches identified 19 704 articles. After removal of duplicates, 18 553 were screened, resulting in 237 eligible for full-text assessment. Forty-five trials were included in the quantitative analysis; 117 documents were included in the qualitative analysis. Trials were widely heterogeneous in design and reporting, precluding meta-analysis of the effect of DCT methods on the primary recruitment outcome. Qualitative analysis formulated 4 broad themes: value, burden, safety and equity. Participant and stakeholder experiences of DCTs were incompletely represented. CONCLUSION: DCTs are developing rapidly. However, there is insufficient evidence to confirm which methods are most effective in trial recruitment, retention, or overall cost. The identified advantages, disadvantages, facilitators and barriers should inform the development of DCT methods. We recommend further research on how DCTs are experienced and perceived by participants and stakeholders to maximise potential benefits.

Postmortem redistribution of the heroin metabolites morphine and morphine-3-glucuronide in rabbits over 24 h.

The interpretation of postmortem drug levels is complicated by changes in drug blood levels in the postmortem period, a phenomena known as postmortem drug redistribution. We investigated the postmortem redistribution of the heroin metabolites morphine and morphine-3-glucuronide in a rabbit model. Heroin (1 mg/kg) was injected into anesthetised rabbit; after 1 h, an auricular vein blood sample was taken and the rabbit was euthanised. Following death rabbits were placed in a supine position at room temperature and divided into three groups namely (1) immediate autopsy, (2) autopsy after 30 minutes and (3) autopsy 24 h after death. Various samples which included femoral blood, cardiac blood, lung, liver, kidney, vitreous humour, subcutaneous and abdominal fat, liver, bone marrow and skeletal muscle were taken. The samples were analysed with a validated LC-MS/MS method. It was observed that within minutes there was a significant increase in free morphine postmortem femoral blood concentration compared to the antemortem sample (0.01 ± 0.01 to 0.05 ± 0.02 mg/L).Various other changes in free morphine and metabolite concentrations were observed during the course of the experiment in various tissues. Principal component analysis was used to investigate possible correlations between free morphine in the various samples. Some correlations were observed but gave poor predictions (>20 % error) when back calculating. The results suggest that rabbits are a good model for further studies of postmortem redistribution but that further study and understanding of the phenomena is required before accurate predictions of the blood concentration at the time of death are possible.

Impact of Post Manufacturing Handling of Protein-Based Biologic Drugs on Product Quality and User Centricity.

This article evaluates the current gaps around the impact of post-manufacturing processes on the product qualities of protein-based biologics, with a focus on user centricity. It includes the evaluation of the regulatory guidance available, describes a collection of scientific literature and case studies to showcase the impact of post-manufacturing stresses on product and dosing solution quality. It also outlines the complexity of clinical handling and the need for communication, and alignment between drug providers, healthcare professionals, users, and patients. Regulatory agencies provide clear expectations for drug manufacturing processes, however, guidance supporting post-product manufacturing handling is less defined and often misaligned. This is problematic as the pharmaceutical products experience numerous stresses and processes which can potentially impact drug quality, safety and efficacy. This article aims to stimulate discussion amongst pharmaceutical developers, health care providers, device manufacturers, and public researchers to improve these processes. Patients and caregivers' awareness can be achieved by providing relevant educational material on pharmaceutical product handling.

Examination of the Protein Drug Supply Chain in a Swedish University Hospital: Focus on Handling Risks and Mitigation Measures.

Protein drugs, such as monoclonal antibodies, have proved successful in treating cancer and immune system diseases. The structural complexity of these molecules requires careful handling to ensure integrity and stability of the drug. In this study, a failure mode and effects analysis was performed based on a Gemba Walk method in a Swedish University Hospital. The Gemba Walk is focused on pharmacists observing the actual supply process steps from distributor, pharmacy cleanroom to patient administration. Relevant protein drugs are chosen based on sales statistics within the hospital and the corresponding wards were observed. Further is the Double Diamond design method used to identify major risks and deliver mitigation strategies. The study identified potential stress factors such as temperature, shock by impact, shaking, vibration and light exposure. There were also risks associated with porters' and healthcare professionals' lack of awareness and access to information. These risk factors may cause loss of efficacy and quality of the protein drug, potentially leading to patient safety concerns. In this study, a simulation is also performed to list measures that theoretically should be in place to ensure the quality of the protein drug, for example validated and protocol-based compounding in cleanroom, training and validated transports.

Managing antibody stability: Effects of stressors on Ipilimumab from the commercial formulation to diluted solutions.

The stability of the monoclonal antibody Ipilimumab, the active ingredient of Yervoy®, used for the treatment of different types of cancer, has been investigated. Shaking/temperature, light exposure and dilution, protein drug renowned stressors, were applied on a 30-45-day series of experiments to observe the physicochemical and biological behavior of the molecule. Ipilimumab demonstrated stability under shaking and heat up to 45 days, without any unfolding during the induced combined stressors. Under artificial sunlight, the mAb showed to be sensitive even under the minimum dose tested (720 kJ/m2) with formation of aggregates, particularly when diluted in glucose solution. The light-induced soluble aggregates were higher in the case of diluted samples irradiated with much higher light doses (10460 kJ/m2). The aggregation of Ipilimumab took place also by irradiating the non-diluted formulation, indicating that the excipients did not protect completely the drug from photodegradation. Amino acid oxidation and deamidation were found. Anyway, after irradiation with both light doses, soluble Ipilimumab maintained its typical ß-sheets structure, and the tertiary structure was nearly maintained compared to the dark. As an additional stressor test, the effect of dilution on the formulation was monitored by using a saline solution (1 mg/mL Ipilimumab) applied during hospital infusion. After two days from dilution, the protein exhibited aggregation and chemical modifications including oxidation and deamidation. When stability conditions were compromised, the viability of human cell lines treated with the stressed formulation slight decreased suggesting low potential biological toxicity of the modified mAb. As this study has demonstrated the susceptibility of Ipilimumab to light, specific solutions, and excipients as well as the use of safe light in manufacturing, handling, and storage of this drug should be promoted. Moreover, the use of proper primary and secondary packaging should be indicated to avoid the detrimental effect of light on the mAb structure and efficacy. A detailed understanding of Ipilimumab physicochemical properties, integrity, and stability could assure the best storage and manipulation conditions for its safe and successful application in cancer therapy.

A secondary qualitative analysis of stakeholder views about participant recruitment, retention, and adherence in decentralised clinical trials (DCTs).

BACKGROUND: Decentralised clinical trials (DCTs) are clinical trials where all or most trial activities occur in or near participants' homes instead of hospitals or research sites. While more convenient for participants, DCTs may offer limited opportunities to build trust with investigators and trial teams. This qualitative analysis explored DCT stakeholder views to inform strategies for maximising participant recruitment, retention, and adherence. METHODS: A secondary analysis of original interview transcripts focused on participant engagement: recruitment, retention, and adherence. Semi-structured interviews were conducted with a purposive sample of stakeholders, including trial managers and administrators, investigators, nurses, vendors, and patient representatives. Interview data were coded using a thematic approach to generate descriptive themes. RESULTS: Forty-eight stakeholders were interviewed. Three components of participant engagement in DCTs were identified: identifying and attracting potential participants, retaining participants and encouraging adherence, and involvement of patients and the public. Interviewees believed that a potential participant's beliefs about research value and their trust in the research team strongly influenced the likelihood of taking part in a DCT. Early involvement of patients was identified as one way to gauge participant priorities. However, perceived burden was seen as a barrier to recruitment. Factors influencing retention and adherence were related to the same underlying motivators that drove recruitment: personal values, circumstances, and burden. Being part of a DCT should not conflict with the original motivations to participate. CONCLUSION: Recruitment, retention, and adherence in DCTs are driven by factors that have previously been found to affect conventional clinical trials. Increasing patient and public involvement can address many of these factors. In contrast to conventional trials, DCTs are perceived as requiring greater emphasis on communication, and contact, to engender trust between participants and researchers despite a relative lack of in-person interaction.

From Inception to ConcePTION: Genesis of a Network to Support Better Monitoring and Communication of Medication Safety During Pregnancy and Breastfeeding.

In 2019, the Innovative Medicines Initiative (IMI) funded the ConcePTION project-Building an ecosystem for better monitoring and communicating safety of medicines use in pregnancy and breastfeeding: validated and regulatory endorsed workflows for fast, optimised evidence generation-with the vision that there is a societal obligation to rapidly reduce uncertainty about the safety of medication use in pregnancy and breastfeeding. The present paper introduces the set of concepts used to describe the European data sources involved in the ConcePTION project and illustrates the ConcePTION Common Data Model (CDM), which serves as the keystone of the federated ConcePTION network. Based on data availability and content analysis of 21 European data sources, the ConcePTION CDM has been structured with six tables designed to capture data from routine healthcare, three tables for data from public health surveillance activities, three curated tables for derived data on population (e.g., observation time and mother-child linkage), plus four metadata tables. By its first anniversary, the ConcePTION CDM has enabled 13 data sources to run common scripts to contribute to major European projects, demonstrating its capacity to facilitate effective and transparent deployment of distributed analytics, and its potential to address questions about utilization, effectiveness, and safety of medicines in special populations, including during pregnancy and breastfeeding, and, more broadly, in the general population.

Application of Healthcare 'Big Data' in CNS Drug Research: The Example of the Neurological and mental health Global Epidemiology Network (NeuroGEN).

Neurological and psychiatric (mental health) disorders have a large impact on health burden globally. Cognitive disorders (including dementia) and stroke are leading causes of disability. Mental health disorders, including depression, contribute up to one-third of total years lived with disability. The Neurological and mental health Global Epidemiology Network (NeuroGEN) is an international multi-database network that harnesses administrative and electronic medical records from Australia, Asia, Europe and North America. Using these databases NeuroGEN will investigate medication use and health outcomes in neurological and mental health disorders. A key objective of NeuroGEN is to facilitate high-quality observational studies to address evidence-practice gaps where randomized controlled trials do not provide sufficient information on medication benefits and risks that is specific to vulnerable population groups. International multi-database research facilitates comparisons across geographical areas and jurisdictions, increases statistical power to investigate small subpopulations or rare outcomes, permits early post-approval assessment of safety and effectiveness, and increases generalisability of results. Through bringing together international researchers in pharmacoepidemiology, NeuroGEN has the potential to be paradigm-changing for observational research to inform evidence-based prescribing. The first focus of NeuroGEN will be to address evidence-gaps in the treatment of chronic comorbidities in people with dementia.

Postmortem tissue distribution of morphine and its metabolites in a series of heroin-related deaths.

The abuse of heroin (diamorphine) and heroin-related deaths are increasing around the world. The interpretation of the toxicological results from suspected heroin-related deaths is notoriously difficult, especially in cases where there may be limited samples. To help forensic practitioners with heroin interpretation, we determined the concentration of morphine (M), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) in blood (femoral and cardiac), brain (thalamus), liver (deep right lobe), bone marrow (sternum), skeletal muscle (psoas), and vitreous humor in 44 heroin-related deaths. The presence of 6-monoacetylmorphine (6-MAM) in any of the postmortem samples was used as confirmation of heroin use. Quantitation was carried out using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with solid-phase extraction. We also determined the presence of papaverine, noscapine and codeine in the samples, substances often found in illicit heroin and that may help determine illicit heroin use. The results of this study show that vitreous is the best sample to detect 6-MAM (100% of cases), and thus heroin use. The results of the M, M3G, and M6G quantitation in this study allow a degree of interpretation when samples are limited. However in some cases it may not be possible to determine heroin/morphine use as in four cases in muscle (three cases in bone marrow) no morphine, M3G, or M6G were detected, even though they were detected in other case samples. As always, postmortem cases of suspected morphine/heroin intoxication should be interpreted with care and with as much case knowledge as possible.

A rapid GC-MS determination of gamma-hydroxybutyrate in saliva.

GHB and its related compounds have been known for years in forensic toxicology because of their illicit use in drug-facilitated sexual assault, and to a lesser extent, as party drugs. A sensitive and specific method for the identification and quantification of gamma-hydroxybutyric acid (GHB) in saliva has been developed using gas chromatography-mass spectrometry with selective ion monitoring mode. One microliter of synthetic saliva was spiked with 1.0 microL of GHB-d(6) as the internal standard and 1.0 microL of 1,7-heptanediol as a surrogate spike to all samples. After a silyl-derivatization, the sample was injected at a split ratio of 10:1. The following ions were monitoring: 233 and 234 for GHB; 239, 240, and 241 for GHB-d(6); and 55, 73, and 97 for 1,7-heptanediol. The limit of quantitation was determined to be 0.5 mg/L with a linear dynamic range of 0.5-50.0 mg/L. Quality control samples (5.0, 20.0, and 30.0 mg/L) were prepared for the evaluation of precision. Analytical precision measured by coefficients of variation ranged from 2.1% to 12.50% in both intraday and day-to-day experiments. Surrogate recovery from saliva samples fell in the range of 94.6% to 100% with an average of 98.37% and a corresponding percent relative standard deviation of 1.2%.

Fully automated analysis of Carbohydrate-Deficient Transferrin (CDT) by using a multicapillary electrophoresis system.

BACKGROUND: The techniques universally believed as most reliable for determination of Carbohydrate-Deficient Transferrin (CDT) are HPLC and capillary electrophoresis (CE). Recently a reagent kit for CDT analysis to be used in a multicapillary electropherograph has been introduced. The present work was aimed at a validation of this new commercial system by application of the analytical chemistry parameters and by comparison with validated CE and HPLC methods. METHODS: One hundred forty serum samples were analyzed with multicapillary CE (Capillarys, Sebia, France) using kit reagents (CAPILLARYS CDT assay), with single capillary electropherograph (P/ACE MDQ, Beckman Coulter, USA) using an original method developed by our group and with HPLC, performed on a gradient HPLC (Shimadzu Europe, Germany), using column and reagents provided in kit (ClinRep, Recipe, Germany). RESULTS: The separation efficiency of the multicapillary system was about 15,000 theoretical plates/column. The resolution of the transferrin isoforms ranged from 1.23 to 1.67. The variation coefficient was < 10% in both intra-run and between run tests. The correlation of results of multicapillary CE with those obtained with single CE and HPLC was statistically significant. CONCLUSIONS: The multicapillary system shows high productivity with good analytical performances; however, a confirmation of positive results with an alternative method is required.

Broad-spectrum toxicological analysis of hair based on capillary zone electrophoresis-time-of-flight mass spectrometry.

The coupling of capillary electrophoresis-electrospray ionization and time-of-flight mass spectrometry, combining efficiency and speed of separation with high mass accuracy and fast scanning capability, was for the first time applied to the determination of drugs of abuse (amphetamine, methamphetamine, MDA, MDMA, ephedrine, cocaine, morphine, codeine) and their metabolites in hair (6-MAM, benzoylecgonine). Experimental conditions were as follows. Separation: voltage 15 kV, uncoated fused-silica capillary (75 microm ID, 100 cm total length), running electrolyte 25 mM ammonium formate, pH 9.5, field-amplified sample stacking injection. Forensic drugs could be identified by exact mass determination (mass accuracy typically < or = 5 ppm) and by match of the isotopic pattern. The method was fully validated, showing limit of detections (LODs) suitable for the determination of all the compounds below the cut-off usually adopted for hair analysis (0.1 ng/mg). Analytical precision in real matrices (tested at 0.1 and 1.0 ng/mg) was typically characterized by CV's < or = 24% in both intra-day and day-to-day experiments. Quantitative determination was also tested by using a single internal standard (folcodine). Results, although with a moderate accuracy, conceivably depending on the lack of deuterated internal standards, proved useful for diagnostic use of the results from hair analysis. A single liquid-liquid extraction procedure was applied for all analytes, allowing the detection of a broad spectrum of basic drugs and their major metabolites.

Hair analysis for illicit drugs by using capillary zone electrophoresis-electrospray ionization-ion trap mass spectrometry.

In forensic toxicology, hair analysis has become a well established analytical strategy to investigate retrospectively drug abuse histories. In this field, gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry are currently used, often after preliminary screening with immunoassays. However, on the basis of previous applications to pharmaceutical analysis, capillary zone electrophoresis coupled to ion trap mass spectrometry looks also highly promising. The purpose of the present work was the development of a simple and rapid CZE-MS method for sensitive and quantitative determination of the main drugs of abuse and their metabolites (namely, 6-monoacetylmorphine, morphine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethampthetamine (MDMA), benzoylecgonine, ephedrine and cocaine) in human hair. Hair samples (100 mg) were washed, cut and incubated overnight in 0.1 M HCl at 45 degrees C, then neutralized with NaOH and extracted by a liquid-liquid extraction method. CZE separations were carried out in a 100 cm x 75 microm (I.D.) uncoated fused silica capillary. The separation buffer was composed of 25 mM ammonium formate, pH 9.5; the separation voltage was 15 kV. Electrokinetic injections were performed at 7 kV for 30 s under field amplified sample stacking conditions. ESI-ion trap MS detection was performed in the ESI positive ionization mode using the following conditions: capillary voltage 4 kV, nebulizer gas (nitrogen) pressure 3psi, source temperature 150 degrees C and drying gas (nitrogen) flow rate 8l/min. A sheath liquid, composed of isopropanol-water (50:50, v/v) with 0.5% formic acid, was delivered at a flow rate of 4 microl/min. The ion trap MS operated in a selected ion monitoring mode (SIM) of positive molecular ions for each drug/metabolite. Collision induced fragmentation was also possible. Nalorphine was used as internal standard. Under the described conditions, the separation of all compounds, except amphetamine/methamphetamine, MDA/MDMA and morphine/6-MAM was achieved in 20 min, with limits of detection lower than the most severe cut-offs adopted in hair analysis (i.e. 0.1 ng/mg). Linearity was assessed within drug concentration ranges from 0.025 to 5 ng of each analyte/mg of hair. Analytical precision was fairly acceptable with RSD's < or = 3.06% for migration times and < or = 22.47% for areas in real samples, in both intra-day and day-to-day experiments. On these grounds, the described method can be proposed for rapid, selective and accurate toxicological hair analysis for both clinical and forensic purposes.

Carbohydrate-deficient transferrin (CDT) as a marker of alcohol abuse: a critical review of the literature 2001-2005.

The diagnosis of alcohol abuse based on objective data is a necessary requirement in both clinical and forensic environments. Among the different biomarkers of chronic alcohol abuse, carbohydrate-deficient transferrin (CDT) is world wide recognized as the most reliable indicator. However, several problems about the real meaning of CDT and the reliability of its use for the diagnosis of alcohol abuses are still open, as reported by numerous research articles and reviews. The present article presents a critical review of the literature on CDT appeared in the period from 2001 to 2005 (included). The article is organized in the following sections: (1) introduction, (2) definition and structure of human serum CDT, (3) pathomechanisms of the ethanol-induced CDT increase, (4) preanalysis, (5) analysis, (6) data interpretation, (7) review papers, (8) conclusions. As many as 127 papers appeared in the international literature and retrieved by the search engines PubMed and Scopus are quoted.

Direct analysis of bromide in human serum by capillary electrophoresis.

The purpose of the present work was the development and validation of a simple, rapid and reliable method for direct bromide quantification in serum based on capillary electrophoresis (CE). The analysis was carried out with an automated capillary electropherograph. Analytical conditions were as follows. Capillary: uncoated fused silica, effective length 50 cm, internal diameter 50 microm; voltage: 20 kV in reverse polarity mode; temperature: 25 degrees C; running buffer: 90 mmol/L sodium tetraborate decahydrate and 10 mmol/L NaCl, pH 9.24; detection: direct UV absorption at 200 nm; sample treatment: dilution of serum 1:10 with the internal standard solution (2 mmol/L thiocyanate). Under the described conditions, bromide ions and internal standard were baseline separated in 7 min. No interferences from other serum components were observed. The analytical sensitivity was characterized by a LOD: 0.05 mmol/L and a LOQ of 0.1 mmol/L. Excellent linearity was verified in the range from 2.5 to 60 mmol/L [y = 0.0746x - 0.0372; R2 = 0.9995 (x = bromide concentration; y = bromide peak area/internal standard (I.S.) peak area)]. Quantitative imprecision in intra-day (n = 7) and day-to-day (n = 7) experiments was always within R.S.D. values <2%. Recovery was quantitative throughout the range of linearity of the method. Clinical cases of infants undergoing potassium bromide therapy for refractory epilepsy were analyzed with results in agreement with literature data. On the basis of these considerations, capillary electrophoresis can be proposed as the method of choice for bromide analysis in serum samples, especially for therapeutic drug monitoring purposes.

Nefopam Hydrochloride: A Fatal Overdose.

Nefopam is a non-opiate analgesic commonly used for the treatment of moderate to severe pain. A case of a 37-year-old male who was found dead in the morning is presented. An autopsy was performed and femoral venous blood, heart blood, urine, and vitreous humor were submitted for toxicological analysis. A general drug screen detected the presence of nefopam, caffeine, nicotine, citalopram, gabapentin, amitriptyline, diazepam and paracetamol in cardiac blood. Nefopam was quantitated by high-performance liquid chromatography with diode-array detection. Nefopam was found at the following concentrations: 13.6 mg/L in unpreserved femoral blood; 14.7 mg/L in preserved (fluoride-oxalate) femoral blood; 21.2 mg/L in unpreserved cardiac blood and 4.5 mg/L in preserved vitreous. Citalopram was present at a concentration of 0.7 mg/L (femoral blood) and 0.9 mg/L (cardiac blood). Ethanol analyzed by headspace gas chromatography (GC-FID) was detected in preserved (fluoride-oxalate) vitreous (14 mg/100 mL) and preserved (fluoride-oxalate) urine 50 mg/100 mL. Death was attributed to atherosclerotic coronary artery disease and therapeutic drug toxicity.

Rapid and direct analysis of gamma-hydroxybutyric acid in urine by capillary electrophoresis-electrospray ionization ion-trap mass spectrometry.

The present work was aimed at the development of a capillary electrophoretic analysis of gamma-hydroxybutyric acid (GHB) using electrospray ion trap mass spectrometry to achieve the direct and unequivocal detection of this analyte in human urine. Optimized capillary electrophoretic conditions were: injection, 20 s at 0.5 psi (1 psi = 6894.76 Pa); buffer electrolyte, 12.5 mM ammonium formate adjusted to pH 8.35 with diethylamine; fused silicacapillary: 100 cm x 50 microm i.d.; separation voltage, 25 kV (forward polarity) + 0.5 psi; room temperature. Electrospray and mass spectrometric conditions were: drying gas and nebulizing gas (nitrogen) at flow rate 3 l/min, temperature 250 degrees C, nebulizer pressure: 10 psi; sheath liquid solution: methanol-water (90:10) containing 0.1% ammonia delivered at 3 microl/min; spray voltage 3.5 kV. Mass spetrometric detection was carried out in the selected ion monitoring mode of negative molecular ions at 103 m/z for GHB and 115 m/z for maleic acid (I.S.). Under these conditions the baseline separation of GHB and the I.S. was obtained. The selectivity of the analysis allowed for direct injection of unextracted urine, previously diluted 1:4 with water. Linearity was assessed in the GHB concentration range from 80 to 1280 microg/ml in urine. Analytical sensitivity (as limit of detection) resulted about 5 microg/ml in water and 20 microg/ml in original urine. Analytical precision was fairly acceptable with R.S.D. values lower than 5% for migration times and 18% for quantitation in real samples, in both intra day and day-to-day experiments. On these grounds, the developed method can be adopted for rapid identification of acute intoxications from GHB in humans.

Determination of gamma-hydroxybutyric acid in biological fluids by using capillary electrophoresis with indirect detection.

gamma-Hydroxybutyric acid (GHB) is a central nervous system (CNS) depressant and hypnotic which, in recent times, has shown an increasing abuse either as recreational drug (due to its euphoric effects and ability to reduce inhibitions) or as doping agent (enhancer of muscle growth). Analogues of GHB, namely gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD), share its biological activity and are rapidly converted in vivo into GHB. At present, GHB and analogues are placed in the Schedules of Controlled Substances. Numerous intoxications in GHB abusers have been reported with depressive effects, seizures, coma and possibly death. The purpose of the present work was the development of a rapid analytical method based on capillary zone electrophoresis for the direct determination of GHB in human urine and serum at potentially toxic concentrations. Analytical conditions were as follows. Capillary: length 40 cm (to detector), 75 microm i.d.; buffer: 5.0 mM Na(2)HPO(4), 15 mM sodium barbital adjusted to pH 12 with 1.0 M NaOH; voltage: 25 kV at 23 degrees C; indirect UV detection at 214 nm; injection by application of 0.5 psi for 5 s. alpha-Hydroxyisobutyric acid was used as internal standard (IS). Sample pretreatment was limited to 1:8 dilution. Under these conditions, the sensitivity was approximately 3.0 microg/ml (signal-to-noise ratio >3). Calibration curves prepared in water, urine and serum were linear over concentration ranges 25-500 microg/ml with R(2)>/=0.998. Analytical precision was fairly good with R.S.D.<0.60% (including intraday and day-to-day tests). Quantitative precision in both intraday and day-to-day experiments was also very satisfactory with R.S.D.

Preparation and analytical characterization of 1-(1-phenylcyclohexyl)piperidine (PCP) and 1-(1-phenylcyclohexyl)pyrrolidine (PCPy) analogues.

Classic examples of psychoactive arylcycloalkylamines include ketamine and 1-(1-phenylcyclohexyl)piperidine (PCP) and many others serve as important structural templates for neuropharmacological research. The recent emergence of PCP analogues that can be obtained from internet retailers requires the implementation of appropriate monitoring strategies for harm reduction purposes. Access to analytical data plays a key part when encountering these substances, especially if reference material is not available. The present study describes the synthesis of three substituted 1-(1-phenylcyclohexyl)piperidines, (3-MeO-, 4-MeO- and 3-Me-PCP) and three substituted 1-(1-phenylcyclohexyl)pyrrolidine analogues (3-MeO-, 4-MeO- and 3-Me-PCPy). Analytical characterizations of all six arylcyclohexylamines and their primary 1-phenylcyclohexanamine intermediates included gas chromatography ion trap electron- and chemical ionization and high resolution mass spectrometry, liquid chromatography electrospray hybrid triple-quadrupole linear ion trap tandem mass spectrometry, infrared, diode array detection and (1) H and (13) C nuclear magnetic resonance (NMR) spectroscopy. Solvent (CDCl3 vs. d6 -DMSO) and protonation effects (free bases vs hydrochloride salts) were studied in order to investigate the impact on shifts and splitting patterns, for example, when attempting to assign separate axial and equatorial proton chemical shifts of NMR spectra. Differentiation between the isomeric 3-MeO-/4-MeO-PCP and PCPy analogues was feasible under mass spectral conditions. Gas chromatography analysis appeared to induce notable degradation of the 4-MeO-substituted analytes, especially when dealing with the HCl salts which led to the detection of the substituted 1-phenylcyclohex-1-ene nucleus. This phenomenon was observed to be less pronounced with the 3-MeO isomers, possibly due to the resonance properties of the para-methoxy group followed by more facile elimination of the amine.