Effect of chronic γ-hydroxybutyrate (GHB) administration on GHB toxicokinetics and GHB-induced respiratory depression.

BACKGROUND: γ-hydroxybutyrate (GHB) has a high potential for illicit use; overdose of this compound results in sedation, respiratory depression and death. Tolerance to the hypnotic/sedative and electroencephalogram effects of GHB occurs with chronic GHB administration; however, tolerance to respiratory depression has not been evaluated. GHB toxicodynamic effects are mediated predominantly by GABAB receptors. Chronic treatment may affect monocarboxylate transporters (MCTs) and alter the absorption, renal clearance and brain uptake of GHB. OBJECTIVES: To determine effects of chronic GHB dosing on GHB toxicokinetics, GHB-induced respiratory depression, and MCT expression. METHODS: Rats were administered GHB 600 mg/kg intravenously daily for 5 days. Plasma, urine and tissue samples and respiratory measurements were obtained on days 1 and 5. Plasma and urine were analyzed for GHB by LC/MS/MS and tissue samples for expression of MCT1, 2 and 4 and their accessory proteins by QRT-PCR. RESULTS: No differences in GHB pharmacokinetics or respiratory depression were observed between days 1 and 5. Opposing changes in MCT1 and MCT4 mRNA expression were observed in kidney samples on day 5 compared to GHB-naïve animals, and MCT4 expression was increased in the intestine. CONCLUSIONS: The lack of tolerance observed with GHB-induced respiratory depression, in contrast to the tolerance reported for the sedative/hypnotic and electroencephalogram effects, suggests that different GABAB receptor subtypes may be involved in different GABAB-mediated toxicodynamic effects of GHB. Chronic or binge users of GHB may be at no less risk for fatality from respiratory arrest with a GHB overdose than with a single dose of GHB.

[Progress on Determination and Analysis of Zopiclone in Biological Samples].

As a new hypnotic, zopiclone is widely used in clinical treatment. There are many methods for determination of zopiclone, including spectrophotometry, chromatography and chromatography mass spectrum, etc. Present paper reviews different kinds of biological samples associated with zopiclone, extraction and purification methods, and determination and analysis methods, which aims to provide references for the relevant research and practice.

Brain extracellular γ-hydroxybutyrate concentrations are decreased by L-lactate in rats: role in the treatment of overdoses.

PURPOSE: L-lactate represents a potential treatment for GHB overdose by inhibiting GHB renal reabsorption mediated by monocarboxylate transporters. Our objective was to assess the dose-dependence of L-lactate treatment, with and without D-mannitol, on GHB toxicokinetics/toxicodynamics (TK/TD). METHODS: Rats were administered GHB 600 mg/kg i.v. with L-lactate (low and high doses), D-mannitol, or L-lactate (low dose) with D-mannitol. GHB-induced sleep time and GHB plasma, urine and brain extracellular fluid (ECF) concentrations (by LC/MS/MS) were determined. The effect of L-lactate and D-mannitol on the uptake and efflux of GHB was assessed in rat brain endothelial RBE4 cells. RESULTS: L-lactate treatment increased GHB renal clearance from 1.4 ± 0.1 ml/min/kg (control) to 2.4 ± 0.2 and 4.7 ± 0.5 ml/min/kg after low and high doses, respectively, and reduced brain ECF AUC values to 65 and 25% of control. Sleep time was decreased from 137 ± 12 min (control) to 91 ± 16 and 55 ± 5 min (low and high L-lactate, respectively). D-mannitol did not alter GHB TK/TD and did not alter L-lactate's effects on GHB TK/TD. L-lactate, but not D-mannitol, inhibited GHB uptake, and increased GHB efflux from RBE4 cells. CONCLUSIONS: L-lactate decreases plasma and brain ECF concentrations of GHB, decreasing sedative/hypnotic effects.

Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC-MS, LC-MSn, and LC-HR-MSn.

Xylazine is used in veterinary medicine for sedation, anesthesia, and analgesia. It has also been reported to be misused as a horse doping agent, a drug of abuse, a drug for attempted sexual assault, and as source of accidental or intended poisonings. So far, no data concerning human metabolism have been described. Such data are necessary for the development of toxicological detection methods for monitoring drug abuse, as in most cases the metabolites are the analytical targets. Therefore, the metabolism of xylazine was investigated in rat and human urine after several sample workup procedures. The metabolites were identified using gas chromatography (GC)-mass spectrometry (MS) and liquid chromatography (LC) coupled with linear ion trap high-resolution multistage MS (MS(n)). Xylazine was N-dealkylated and S-dealkylated, oxidized, and/or hydroxylated to 12 phase I metabolites. The phenolic metabolites were partly excreted as glucuronides or sulfates. All phase I and phase II metabolites identified in rat urine were also detected in human urine. In rat urine after a low dose as well as in human urine after an overdose, mainly the hydroxy metabolites were detected using the authors' standard urine screening approaches by GC-MS and LC-MS(n). Thus, it should be possible to monitor application of xylazine assuming similar toxicokinetics in humans.

Electrokinetic supercharging in CE for the separation and preconcentration of barbiturate drugs in urine samples.

Three barbiturate drugs, barbital, phenobarbital, and secobarbital were separated and analyzed by electrokinetic supercharging. The influence of different parameters on electrokinetic supercharging performance was evaluated using both univariated and multivariated optimization processes. The parameters studied were sample pH, concentration, and length of the leading and terminating electrolytes, electrokinetic injection of the sample and composition and hydrodynamic injection of the solvent plug. The leading electrolyte (50 mM NaCl) was hydrodynamically injected (50 mbar × 120 s) prior to the sample that was adjusted to pH 9.6 and electrokinetically injected at -8.5 kV for 300 s. The terminating electrolyte (100 mM of 2-(cyclohexylamino) ethanesulphonic acid) was then hydrodynamically injected (50 mbar × 140 s). The results showed that this strategy enhanced detection sensitivity around 1050-fold compared with normal hydrodynamic injection, providing detection limits ranging between 1.5 and 2.1 ng/mL for standard samples with good repeatability in terms of peak area (values of relative standard deviation, %RSD < 3). The applicability of the optimized method was demonstrated by the analysis of human urine samples spiked with the studied compounds at different concentration levels and further liquid-liquid extraction step. The estimated detection limits obtained in the urine samples extract ranged between 8 and 15 ng/mL.

A Bayesian approach for estimating detection times in horses: exploring the pharmacokinetics of a urinary acepromazine metabolite.

We describe the population pharmacokinetics of an acepromazine (ACP) metabolite (2-(1-hydroxyethyl)promazine) (HEPS) in horses for the estimation of likely detection times in plasma and urine. ACP (30 mg) was administered to 12 horses, and blood and urine samples were taken at frequent intervals for chemical analysis. A bayesian hierarchical model was fitted to describe concentration-time data and cumulative urine amounts for HEPS. The metabolite HEPS was modelled separately from the parent ACP as the half-life of the parent was considerably less than that of the metabolite. The clearance (Cl/F(PM)) and volume of distribution (V/F(PM)), scaled by the fraction of parent converted to metabolite, were estimated as 769 L/h and 6874 L, respectively. For a typical horse in the study, after receiving 30 mg of ACP, the upper limit of the detection time was 35 h in plasma and 100 h in urine, assuming an arbitrary limit of detection of 1 lg/L and a small (≈0.01) probability of detection. The model derived allowed the probability of detection to be estimated at the population level. This analysis was conducted on data collected from only 12 horses, but we assume that this is representative of the wider population.

Liquid chromatographic high-throughput analysis of the new ultra-short acting hypnotic 'HIE-124' and its metabolite in mice serum using a monolithic silica column.

For the first time, a fast, high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of the new ultra-short hypnotic HIE-124 and its metabolite in mice serum. Each compound, together with carbamazepine (internal standard) was extracted from the serum matrix using liquid-liquid extraction (LLE). Chromatographic resolution of the analytes was performed on a Chromolith Speed Rod monolithic silica column (100 mm × 4.6 mm i.d.) under isocratic conditions using a mobile phase of 65:35 (v/v), 20 mM phosphate buffer (pH 7.0 adjusted with phosphoric acid)-acetonitrile. The elution of the analytes were monitored at 240 nm and conducted at ambient temperature. Because of high column efficiency the mobile phase was pumped at a flow rate of 2.5 mL min(-1). The total run time of the assay was 2 min. The method was validated over the range of 60-2000 ng mL(-1) for HIE-124 and 200-1600 ng mL(-1) for the metabolite (r(2) = 0.99). The limit of detection (LOD) for HIE-124 and its metabolite were 20 ng mL(-1) and 65 ng mL(-1), respectively. The proposed method was validated in compliance with ICH guidelines, in terms of accuracy, precision, limits of detection and quantitation and other aspects of analytical validation. The developed method could be used for the trace analyses of HIE-124 and its metabolite in serum and was finally used for the pharmacokinetic study investigation of HIE-124 in mice serum.

Pharmacokinetic considerations for moderate and deep sedation.

Moderate and deep sedation can be provided using several routes of drug administration including oral (PO), inhalation, and parental injection. The safety and efficacy of these various techniques is largely dependent on pharmacokinetic principles. This continuing education article will highlight essential principles of absorption, distribution, and elimination of commonly used sedative agents.

Rapid urine drug screens: diphenhydramine and methadone cross-reactivity.

BACKGROUND: Rapid urine screens to detect drugs of abuse are often used in pediatric emergency departments (PEDs). A positive result may lead to further clinical testing, social evaluation, and increased stress/inconvenience. A PED patient with suspected diphenhydramine (DPH) ingestion had a positive methadone result on the rapid urine drug screen, One Step Multi-Drug, Multi-Line Screen Test Device (ACON Laboratories, San Diego, Calif). There was no history of methadone exposure so the patient was admitted while confirmatory testing was performed. Gas chromatography/mass spectroscopy testing of the urine failed to confirm the presence of methadone. We present this unreported false-positive methadone result and evaluation of the kit for cross-reactivity of DPH and methadone. METHODS: The same One Step urine drug screen was tested at an independent laboratory for cross-reactivity between methadone and DPH including the DPH metabolites. Drug-free urine was fortified with DPH, nordiphenhydramine, or dinordiphenhydramine at 0, 10, 25, 50, and 100 µg/mL for each analyte. One hundred microliters of the solutions were added to each of the 4 wells on test cassettes. Urine was allowed to migrate according to manufacturer instructions. Each cassette was interpreted by 2 analysts to ensure consistent interpretation and accurate data recording. RESULTS: In vitro laboratory testing results showed cross-reactivity between methadone and DPH but not for nordiphenhydramine or dinordiphenhydramine. CONCLUSIONS: Rapid urine drug screens using immunoassays based on the principle of competitive binding may show false-positive methadone results for patients who have ingested DPH. Product information for urine drug screens may not include all cross-reacting agents and should be used with caution when interpreting drug screen results in PED patients.

Proof of Concept: First Pediatric [14 C]microtracer Study to Create Metabolite Profiles of Midazolam.

Growth and development affect drug-metabolizing enzyme activity thus could alter the metabolic profile of a drug. Traditional studies to create metabolite profiles and study the routes of excretion are unethical in children due to the high radioactive burden. To overcome this challenge, we aimed to show the feasibility of an absorption, distribution, metabolism, and excretion (ADME) study using a [14 C]midazolam microtracer as proof of concept in children. Twelve stable, critically ill children received an oral [14 C]midazolam microtracer (20 ng/kg; 60 Bq/kg) while receiving intravenous therapeutic midazolam. Blood was sampled up to 24 hours after dosing. A time-averaged plasma pool per patient was prepared reflecting the mean area under the curve plasma level, and subsequently one pool for each age group (0-1 month, 1-6 months, 0.5-2 years, and 2-6 years). For each pool [14 C]levels were quantified by accelerator mass spectrometry, and metabolites identified by high resolution mass spectrometry. Urine and feces (n = 4) were collected up to 72 hours. The approach resulted in sufficient sensitivity to quantify individual metabolites in chromatograms. [14 C]1-OH-midazolam-glucuronide was most abundant in all but one age group, followed by unchanged [14 C]midazolam and [14 C]1-OH-midazolam. The small proportion of unspecified metabolites most probably includes [14 C]midazolam-glucuronide and [14 C]4-OH-midazolam. Excretion was mainly in urine; the total recovery in urine and feces was 77-94%. This first pediatric pilot study makes clear that using a [14 C]midazolam microtracer is feasible and safe to generate metabolite profiles and study recovery in children. This approach is promising for first-in-child studies to delineate age-related variation in drug metabolite profiles.

Development and Validation of an LC-MS-MS Method for the Detection of 40 Benzodiazepines and Three Z-Drugs in Blood and Urine by Solid-Phase Extraction.

An analytical method for the detection of 40 benzodiazepines, (±)-zopiclone, zaleplon and zolpidem in blood and urine by solid-phase extraction liquid chromatography-tandem mass spectrometry was developed and validated. Twenty-nine of 43 analytes were quantified in 0.5 mL whole blood for investigating postmortem, drug-facilitated sexual assault (DFSA) and driving under the influence of drugs cases (DUID). The four different dynamic ranges of the seven-point, linear, 1/x weighted calibration curves with lower limits of quantification of 2, 5, 10 and 20 µg/L across the analytes encompassed the majority of our casework encountered in postmortem, DFSA and DUID samples. Reference materials were available for all analytes except α-hydroxyflualprazolam, a hydroxylated metabolite of flualprazolam. The fragmentation of α-hydroxyflualprazolam was predicted from the fragmentation pattern of α-hydroxyalprazolam, and the appropriate transitions were added to the method to enable monitoring for this analyte. Urine samples were hydrolyzed at 55°C for 30 min with a genetically modified ß-glucuronidase enzyme, which resulted in >95% efficiency measured by oxazepam glucuronide. Extensive sample preparation included combining osmotic lysing and protein precipitation with methanol/acetonitrile mixture followed by freezing and centrifugation resulted in exceptionally high signal-to-noise ratios. Bias and between-and within-day imprecision for quality controls (QCs) were all within ±15%, except for clonazolam and etizolam that were within ±20%. All 29 of the 43 analytes tested for QC performance met quantitative reporting criteria within the dynamic ranges of the calibration curves, and 14 analytes, present only in the calibrator solution, were qualitatively reported. Twenty-five analytes met all quantitative reporting criteria including dilution integrity. The ability to analyze quantitative blood and qualitative urine samples in the same batch is one of the most useful elements of this procedure. This sensitive, specific and robust analytical method was routinely employed in the analysis of >300 samples in our laboratory over the last 6 months.

Analysis of multiple abused drugs and hypnotics in urine by sweeping CE.

Multiple drugs usage is very common in addicts (AD). However, some parent drugs were undetectable in urine, it was necessary to monitor their metabolites simultaneously. A sweeping CE was established for the determination of several kinds of abused drug and their metabolites in AD urine. This method was developed using chemometric experimental design to simplify the CE optimization. The capillary was filled by separation buffer (phosphate buffer (75 mM, pH 2.5) and methanol (70:30 v/v)) and then hydrodynamically injected large volume of samples into capillary (1 psi, 200 s). Following was using sweeping buffer (phosphate buffer (75 mM, pH 2.5) and methanol (90:10 v/v) containing 65 mM SDS) to sweep and stack analytes. The separation voltage was set at -15 kV (anode at detector end). During method validation, calibration plots were linear (r > or = 0.992) over a range of 0.1-3 microg/mL for codeine, ketamine, and methamphetamine, 0.15-3 microg/mL for morphine, 0.1-1 microg/mL for alprazolam and oxazepam, and 0.1-1.2 microg/mL for other other benzodiazepines and its metabolites. During intra- and inter-day analysis, relative standards and relative errors were less than 14%. The analytes could be simultaneously analyzed and have a detection limit down to 20-50 ng/mL (S/N=3). The results showed good coincidence with GC-MS or LC-ESI-MS. This method was feasible for application to detect trace levels of abused drugs in AD' urine.

Asylum-seeking children with severe loss of activities of daily living: clinical signs and course during rehabilitation.

AIM: To investigate whether severe loss of activities of daily living (ADL) in asylum-seeking children is associated with physical disease or toxic influences and to describe the clinical course during the recovery process. METHODS: A total of 29 asylum-seeking children with severe loss of ADL were regularly assessed by physical examinations, laboratory tests and a structured evaluation of their ADL status during rehabilitation. RESULTS: A total of 12 children had previously recorded suicide attempts and 21 were recorded to have experienced traumatic events in their country of origin. The mean time from turning point to recovery was 6 months. Of the study participants, 22 needed enteral feeding and 18 gained weight during recovery. All children had a pulse rate and systolic blood pressure within the normal range. No sign of intoxication or physical disease was identified in laboratory tests or clinical examinations, with the exception of one case of epilepsy. CONCLUSION: Physical disease, pharmacological sedation or anorexia nervosa was not considered to be a probable cause of the loss of ADL in these children. The high rate of psychosocial risk factors and the stressful event of being in an asylum-seeking process call for further investigation of psychosomatic mechanisms.

The involvement of drugs and alcohol in drug-facilitated sexual assault: a systematic review of the evidence.

The rate of drug-facilitated sexual assault (DFSA; when an incapacitating drug is administered surreptitiously to facilitate sexual assault) is perceived to be increasing in the United Kingdom and elsewhere, causing international concern. This article examines evidence that quantifies the contribution of drugs in instances of alleged DFSA, identifies the substances involved, and discusses the implications of these findings. Of 389 studies examined, 11 were included in this review. The only study to consider covert drugging reported that 2% of alleged DFSA cases were attributable to surreptitious drug administration. Other studies failed to remove voluntary drug consumption from their cohort, biasing results. A study by the United Kingdom's National Forensic Services found no evidence to suggest that flunitrazepam (Rohypnol) had been used for DFSA during its 3-year investigation. In the United States, flunitrazepam is used recreationally, providing a likely explanation for its presence in samples of some alleged DFSA victims.

Zolpidem and Zolpidem Carboxylic Acid Results from Medication Monitoring.

Zolpidem (Ambien®) is one of the "Z" drugs often used to improve sleep in older patients and those suffering from insomnia. Schwope, D.M., DePriest, A., Black, D.L., Caplan, Y.H., Cone, E.J., Heltsley, R. (2014) Determing zolpidem compliance: urinary metabolite detection and prevalence in chronic pain patients . Journal of Analytical Toxicology, 38, 513-518 reported that zolpidem in urine is not very prevalent being present <23% of the time in patient urine while the major metabolite, zolpidem 4-phenyl carboxylic acid (ZCA), is much more prevalent in urine with positive rates as high as 50% of the patient samples reviewed. Results from patient testing over a year's time are in agreement with the reported zolpidem results. However, the data observed herein for ZCA are not consistent with the earlier report. These data suggest that monitoring ZCA may result in even higher levels of positivity. Further, while the Food and Drug Administration has pointed out that female dosing should be half that given to males, results of this population testing indicate that the majority of patients (83% male and 73% female) receive 10 mg/day or 12.5 mg/day for Ambien CR® with females demonstrating statistically significantly higher levels of ZCA albeit zolpidem levels are not statistically significantly different between men and women. Estimates of patient positivity are dependent upon the value of the limit of quantification (LOQ) as demonstrated by the zolpidem results herein (LOQ = 50 ng/mL vs. 4 ng/mL). However, even with a much higher LOQ of 50 ng/mL for ZCA in this work, the positivity from ZCA results is significantly higher (e.g., 64.8%) than reported earlier (50.3%). Nevertheless, these data support the addition of ZCA for monitoring zolpidem in urine.

A simple and rapid method for the identification of zolpidem carboxylic acid in urine.

Zolpidem is a non-benzodiazepine hypnotic that has been implicated in both drug-facilitated sexual assault and drink spiking. Detection of the drug in urine is extremely difficult because of its extensive metabolism. A method is presented for the detection and quantitation of zolpidem carboxylic acid (ZCA), the major urinary metabolite of zolpidem. The metabolite was extracted from urine at pH 4.5-5.0 with chloroform/isopropanol alkylated with ethyl iodide and identified by gas chromatography-mass spectrometry in the selected ion-monitoring mode. Following a single ingestion of 10 mg zopidem, ZCA is detected for up to 72 h. The limit of detection is 2 ng/mL, with an overall recovery of 80%. Using this procedure, zolpidem was identified in two cases of alleged drug-facilitated sexual assault.

[Chemicotoxicological evaluation of doxylamine].

The conditions of doxilamine isolation from biological fluids are studied. The method of its extraction with a mixture of organic solvents in pH 9 is proposed. Identification of doxilamine with techniques of thin layer chromatography, UV-, IR-spectroscopy, chromato-mass-spectrometry, densitometry, gas-liquid chromatography is described. UV-spectrometry, gas chromatography and densitometry can be used for quantitation of doxilamine.

Metabolite characterization of a novel sedative drug, remimazolam in human plasma and urine using ultra high-performance liquid chromatography coupled with synapt high-definition mass spectrometry.

Remimazolam is a new chemical entity belonging to the benzodiazepine class of sedative drugs, which shows faster-acting onset and recovery than currently available short-acting sedatives. In the present study, ultra high performance liquid chromatography with synapt high-definition mass spectrometry method combined with MassLynx software was established to characterize metabolites of remimazolam in human plasma and urine. In total, 5 human metabolites were detected, including 3 phase I and 2 phase II metabolites. There was no novel human metabolite detected compared to that in rat. Hydrolysis, glucuronidation and oxidation were the major metabolic reactions. To our knowledge, this is the first report of the human metabolic profile of remimazolam.

Determination of Propofol by GC/MS and Fast GC/MS-TOF in Two Cases of Poisoning.

Two cases of suspected acute and lethal intoxication caused by propofol were delivered by the judicial authority to the Department of Sciences for Health Promotion and Mother-Child Care in Palermo, Sicily. In the first case a female nurse was found in a hotel room, where she lived with her mother; four 10 mg/mL vials and two 20 mg/mL vials of propofol were found near the decedent along with syringes and needles. In the second case a male nurse was found in the operating room of a hospital, along with a used syringe. In both cases a preliminary systematic and toxicological analysis indicated the presence of propofol in the blood and urine. As a result, a method for the quantitative determination of propofol in biological fluids was optimized and validated using a liquid-liquid extraction protocol followed by GC/MS and fast GC/MS-TOF. In the first case, the concentration of propofol in blood was determined to be 8.1 µg/mL while the concentration of propofol in the second case was calculated at 1.2 µg/mL. Additionally, the tissue distribution of propofol was determined for both cases. Brain and liver concentrations of propofol were, respectively, 31.1 and 52.2 µg/g in Case 1 and 4.7 and 49.1 µg/g in Case 2. Data emerging from the autopsy findings, histopathological exams as well as the toxicological results aided in establishing that the deaths were due to poisoning, however, the manner of death in each were different: homicide in Case 1 and suicide in Case 2.

Optimization and Validation of High-Resolution Mass Spectrometry Data Analysis Parameters.

High-resolution mass spectrometry (HRMS) has gained recognition as a valuable tool for comprehensive drug screening in a variety of biological matrices. HRMS instruments collect untargeted, accurate mass data, which permit identification of known and unknown compounds in a single analytical run. One of the most challenging aspects of implementing an HRMS drug screen is establishing appropriate data analysis parameters for identifying compounds. Unlike other types of mass spectrometry data, guidelines for HRMS data analysis and acceptability criteria have not been established. Although many laboratories have published on the utility of HRMS for drug screening, few have included details on how they determined allowable errors and set positivity criteria. Previously, we developed and validated a comprehensive 169-compound drug screen on a high-resolution quadrupole time of flight mass spectrometer. Here we report the detailed procedure that we used to determine appropriate positivity criteria for our screening procedure. Our approach was empirical; we collected data and analyzed it with commonly available software. We found that a combined scoring approach using a threshold of 70, with 70% weight given to library match and 10% weight given to each of mass error, retention time error and isotope pattern difference provided optimum drug identification efficiency of 99.2%. Our results demonstrate the importance of library matching in accurately identifying compounds, and underscore the utility of robust product ion spectra that contain information on the lineage, mass and relative abundance of fragments. The method we describe is easily adaptable to include alternative parameters that may be available in software associated with a variety of HRMS platforms. With careful selection of error limits and positivity criteria, HRMS instruments are capable of producing high-quality, high-confidence results that may reduce the need for confirmatory testing.