Development of an Analytical Method for Detection of Anesthetics and Sedatives in Fish.

BACKGROUND: Anesthetics and sedatives are frequently used to prevent abrasions caused by stress and to facilitate fish management. However, drug residues may persist and cause changes in fish conditions and induce side effects. In addition, drugs that are not permitted for use in edible fish are sometimes potentially used in fish. The drugs can also be found in wastewater and are likely to be detected in fish. OBJECTIVE: The purpose of this study was to establish a quantitative analytical method for 10 anesthetic and sedative (azaperone, chlorpromazine, diazepam, estazolam, haloperidol, nitrazepam, nordiazepam, oxazepam, perphenazine, and temazepam) residues in fish sold in Korean markets. METHOD: Shrimp, flounder, and eel samples were selected as matrices. Acetonitrile (ACN) containing 0.1% formic acid was selected as an extraction solvent for shrimp and 100% ACN for flounder and eel. The QuEChERS method with C18 and primary secondary amine (PSA) was used as the extraction procedure, and the analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Limit of quantitation, recovery, accuracy, and precision were validated, and satisfactory results were obtained for the drugs. All results applied to the real samples were negative. CONCLUSIONS: An optimal validation method was studied. Since the results for all samples were negative, it is considered that additional studies are needed by increasing the number of drugs. HIGHLIGHTS: The most effective QuEChERS pretreatment method and conditions of LC-MS/MS for the analysis of anesthetics and sedatives in fish were established.

Incorporation of five common hypnotics into hair after a single dose and application to a forensic case of drug facilitated crimes.

In order to obtain fundamental information on the disposition of hypnotics into hair after a single oral dose the quantitative hair analysis of triazolam (TZ), etizolam (EZ), flunitrazepam (FNZ), nitrazepam (NZ) and zolpidem (ZP) have been performed using a validated LC-MS/MS procedure. Hair specimens (straight, black) were collected from three subjects about one month and three months after a single 0.25 mg dose of TZ, 1 mg of EZ, 2 mg of FNZ, 5 mg of NZ and 10 mg of ZP tartrate. The subjects ingested just one out of five different hypnotics on each day, each of five days in turn. All ingested hypnotics have been detected in hair from each subject both one month and three months after intake, and their concentrations were in the range of 0.023-0.043 pg/hair strand (0.077-0.36 pg/mg) for TZ, 0.11-0.63 pg/hair strand (0.44-5.2 pg/mg) for EZ, 0.14-2.6 pg/hair strand (0.56-22 pg/mg) for FNZ, 0.33-1.7 pg/hair strand (1.3-17 pg/mg) for NZ and 20-40 pg/hair strand (120-270 pg/mg) for ZP. For FNZ and NZ, not only the parent drugs but also their metabolites, 7-amino-FNZ and 7-amino-NZ, were detected in the range of 2.3-9.2 pg/hair strand (9.2-82 pg/mg) and 2.4-9.1 pg/hair strand (8.0-55 pg/mg), respectively. The calculated incorporation ratios into hair against the dose were found to exhibit similarity between the four benzodiazepines. This finding suggests the ability to apply these quantitative data to approximately estimating the amounts of other benzodiazepines, which have similar chemical structures, in hair although it should be noted that the amounts of drugs in hair varies considerably depending on the hair color. On the other hand, the incorporation ratio of ZP showed 15-29 times higher than that of TZ, indicating that lipophilic ZP was more likely to incorporate into hair than benzodiazepines. In addition, the application of the present data to a drug-facilitated sexual assault was shown.

Evaluating the spectrum-effect profiling and pharmacokinetics of Tieshuang Anshen Prescription with better sedative-hypnotic effect based on Fe2+ than Hg2.

Although Zhusha Anshen Pill (ZSASP) is a commonly used traditional prescription for insomnia, the safety of cinnabar in the formula has always been controversial since its initial application in medical fields. Here, we developed a new prescription, Tieshuang Anshen Prescription (TSASP), by improving ZSASP with Fe2+ instead of Hg2+. Besides, TSASP was further optimized by establishing and testing the HPLC fingerprint and its sedative-hypnotic effect of formulas with different compatibility ratios and performing correlation spectrum analysis. The safety of TSASP was also evaluated by HE staining of liver and kidney. In addition, a validated and robust UHPLC-MS/MS method was established to demonstrate the pharmacokinetic characteristics of berberine, palmatine, jatrorrhizine, ligustilide, catalpol, loganin, liquiritin and liquiritigenin after oral administration of TSASP. Our study originally provides a new non-toxic prescription, TSASP, with better sedative-hypnotic effect in comparison with ZSASP, revealing that Fe2+ could replace Hg2+ to eliminate its toxicity and play a sedative role. Meanwhile, we believe that our pharmacokinetics results may contribute valuable reference to both TSASP's specific mechanism of action and its further clinical efficacy and effectiveness research.

All solid-state miniaturized potentiometric sensors for flunitrazepam determination in beverages.

Flunitrazepam is one of the frequently used hypnotic drugs to incapacitate victims for sexual assault. Appropriate diagnostic tools should be available to victims regarding the growing concern about "date-rape drugs" and their adverse impact on society. Miniaturized screen-printed potentiometric sensors offer crucial point-of-care devices that alleviate this serious problem. In this study, all solid-state screen-printed potentiometric flunitrazepam sensors have been designed. The paper device was printed with silver and carbon ink. Formation of an aqueous layer in the interface between carbon-conducting material and ion-sensing membrane nevertheless poses low reproducibility in the solid-contact electrodes. Accordingly, poly(3,4-ethylenedioxythiophene) (PEDT) nano-dispersion was applied as a conducting hydrophobic polymer on the electrode surface to curb water accumulation. Conditioning of ion-sensing membrane in the vicinity of reference membrane has been considered carefully using special protocol. Electrochemical characteristics of the proposed PEDT-based sensor were calculated and compared favorably to PEDT-free one. The miniaturized device was successfully used for the determination of flunitrazepam in carbonated soft drinks, energy drink, and malt beverage. Statistical comparison between the proposed sensor and official method revealed no significant difference. Nevertheless, the proposed sensor provides simple and user-friendly diagnostic tool with less equipment for on-site determination of flunitrazepam.

Comprehensive phytochemical analysis and sedative-hypnotic activity of two Acanthopanax species leaves.

Acanthopanax senticosus leaves (SCL) and Acanthopanax sessiliflorus leaves (SFL), which are usually made into functional teas, possess similar pharmacological activities. With the aim of revealing their chemical compositions and evaluating their sedative-hypnotic effects, comprehensive metabolite profiling analysis based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) and high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) as well as bioassay studies in mice were performed for the first time. Firstly, a total of 75 compounds (including 69 shared components) were identified or briefly characterized. Results indicated that the leaves of the two species were both rich in phytochemicals and contained similar structural types. Secondly, 20 and 7 chemical markers were identified from SCL and SFL, respectively. Five oleanane-type triterpene saponins (ciwujianoside C1, C3, D2, E and saniculoside N) and two lupine-type triterpene saponins (1-deoxychiisanoside and 24-hydroxychiisanoside) may be used for rapid identification of SCL and SFL. Thirdly, the contents of rutin, hederacoside D, ciwujianoside B, -C3, -E and ursolic acid in SCL (0.308%, 0.024%, 0.042%, 0.131%, 0.038%, and 0.255%, respectively) were higher than in SFL (0.067%, 0.005%, 0.012%, 0.015%, 0.002%, and 0.087%, respectively). Fourthly, an in vivo bioassay verified that both SCL and SFL could inhibit autonomous activity, shorten sleep latency and prolong sleep duration in a dose-dependent manner. To a certain degree, SCL showed a higher and more stable effect. The hypnotic effect could be inhibited by flumazenil (FLU). The two leaves not only had an obvious antagonism action of p-chlorophenoxyacetic acid (pCPA) but also showed a synergistic hypnotic effect with 5-hydroxytryptophan (5-HTP). The beneficial bioactivity may be mediated by 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA). Finally, network pharmacology analysis showed that the undifferentiated and differentiated compounds were the material basis for the similar and the different activities of two leaves. Some typical chemical markers (such as saniculoside N, hederacoside D, ciwujianoside C3, -E and ursolic acid, 24-hydroxychiisanoside and 1-deoxyisochiisanoside) were the potential active compounds and could be used as quality markers in the future. The present study furnished a basis for the further development and utilization of the leaves of these two Acanthopanax species.

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.

Facile preparation of magnetic covalent organic framework-metal organic framework composite materials as effective adsorbents for the extraction and determination of sedatives by high-performance liquid chromatography/tandem mass spectrometry in meat samples.

RATIONALE: Sedatives, which are prone to cause residues in animals, have been abused in modern animal husbandry. Long-term consumption of contaminated meat products would be unfavorable to the human nervous system. Taking into account public health and food safety, it was essential to develop an effective method for the enrichment and detection of sedatives in meat. METHODS: Fe3 O4 @TbBd@ZIF-8 composites were synthesized by using Fe3 O4 nanoparticles as a magnetic core and 1,3,5-triformylbenzene (Tb) and benzidine (Bd) as two building blocks to form Fe3 O4 @TbBd. Furthermore, the zeolitic imidazolate framework-8 (ZIF-8) was modified on the surface of the Fe3 O4 @TbBd. In addition, Fe3 O4 @TbBd@ZIF-8 was used as a magnetic solid-phase extraction (MSPE) adsorbent of typical animal sedatives in pork samples. Mass spectrometry analysis was conducted by electrospray ionization triple-quadrupole mass spectrometry in positive-ion multiple reaction monitoring mode. RESULTS: By combining the optimized MSPE approach with high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS), an accurate and sensitive method for the determination of sedatives was developed. The method exhibited good linearity in the range of 0.03-70 µg/kg with the correlation coefficient (R2 ) ranging from 0.9982 to 0.9999, high sensitivity with limits of detection (LODs) ranging from 0.04 to 0.2 µg/kg, and high precision with relative standard deviation (RSD) less than 5.5%. The adsorption behaviors of Fe3 O4 @TbBd@ZIF-8 towards sedatives were more suitably described by a pseudo-second-order kinetic and Freundlich isotherm model. CONCLUSIONS: The proposed MSPE-HPLC/MS/MS method was successfully applied to the determination of sedatives in real samples and showed excellent applicability. Several sedatives were detected in the selected meat samples. The developed method was shown to be facile, sensitive and accurate for sedative detection and also showed great prospects for determination of sedatives from other complex samples.

Distribution of zopiclone and main metabolites in hair following a single dose.

In forensic investigations, such as drug-facilitated crimes, reference values are useful for interpretation of hair results. The aim of this study was to establish levels of zopiclone and two main metabolites, N-desmethylzopiclone and zopiclone N-oxide, in hair after the administration of a single dose of zopiclone, as very limited data are published. A controlled study was performed, where 16 volunteers consumed either 5 or 10mg zopiclone. Hair was sampled prior to consumption and 14, 30, 60, and 120 days after intake. The deposition of drug in hair segments of all sampling time points was followed in small hair segments of 5-mm, using a validated ultra-high performance liquid chromatography-tandem mass spectrometry method. In all participants, hair segments corresponding to the time of intake were positive for zopiclone, but also with lower concentrations in the neighbouring segments. The highest zopiclone concentrations were detected in samples collected 30 or 60 days after intake. For all sampling time points maximum values for the 5-mg dose ranged from 5.0-370pg/mg for zopiclone and 5.4 to 300pg/mg for N-desmethylzopiclone, where the maximum values for the 10-mg dose ranged from 17 to 590pg/mg for zopiclone and 25-410pg/mg for N-desmethylzopiclone for all sampling time points. No significant difference in concentrations was found between the two dosing groups for either zopiclone or N-desmethylzopiclone. Almost half of the participants showed lower levels 14 days after intake than in the later sampling time points. The metabolite to parent drug ratio of N-desmethylzopiclone to zopiclone varied from 0.6 to 3.4 (median=1.2) for the maximum levels of all sampling time points. N-desmethylzopiclone are suggested to serve as an additional marker to confirm the intake of zopiclone. Traces of zopiclone N-oxide were detected in hair from only eight participants. This study showed, that it was possible to follow zopiclone and N-desmethylzopiclone in hair for 4 months even though the drugs was divided into several segments in the latest collected hair samples, and no obvious wash-out effect between the sampling time points by e.g. personal hygiene could be discerned because the cumulated amount at each sampling time point was similar. We conclude that the analysis of short segments e.g. segments of 5-mm can help determine the time of a single intake of zopiclone and that obtaining a sample 1-2 months after a drug exposure provide the best conditions to detect and interpret the results.

Development of a LC-MS/MS method for determination of propofol-glucuronide in hair and preliminary study on relationships between dose and hair concentration.

Propofol abuse has been reported worldwide, suggesting the need to establish analytical methods for human biological samples to investigate the abuse of propofol. This study aimed to investigate the relationship between dose and hair concentration using a simple and rapid analytical method developed and validated in this study. In the sample preparation, hair samples were washed with distilled water and methanol and extracted in methanol during 16h at room temperature. After centrifugation and evaporation, the residue was reconstituted and filtered through a 0.22µm membrane filter before LC-MS/MS analysis. The precursor-to-product ion transitions were 353 → 175, 113 for propofol glucuronide and m/z 370 → 175, 113 for internal standard(propofol glucuronide-d17). The calibration curves were satisfactory (R2=0.9997) and the limits of detection and quantification were 2 and 5pg/mg, respectively. In addition, this study collected the history of propofol use from subjects using a questionnaire and analyzed subjects' hair samples using a validated analytical method. As a result, the concentrations of propofol glucuronide ranged from 7 to 122pg/mg (mean : 51pg/mg). There were cases of positive relationships, but generally there was no correlation between dose and hair concentration.

A Rapid Detection Method for On-site Screening of Estazolam in Beverages with Au@Ag Core-shell Nanoparticles Paper-based SERS Substrate.

Estazolam (EST) is a common sedative-hypnotic drug with a risk of abuse. Therefore, rapid on-site detection of EST is necessary to control the abuse of EST. In this paper, a fast, simple, and sensitive method is demonstrated for the detection of EST in both water and beverages, using surface-enhanced Raman spectroscopy (SERS) techniques. Au@Ag core-shell nanoparticles (NPs) assembled on the filter paper as a SERS substrate exhibit good applicability and practicality. At the same time, density functional theory (DFT) is used to assign the vibration mode of the EST molecules, which can be used as a guide for subsequent experiments. The lowest detectable concentration of EST in aqueous solution can be as low as 5 mg/L, and signal uniformity is excellent (RSD687 = 5.56%, RSD1000 = 4.35%). In addition, EST components artificially added to orange juice and pomegranate juice can be effectively detected by simple pretreatment with a minimum detection concentration as low as 10 mg/L. Therefore, this study found that the use of Au@Ag core-shell nanoparticles paper-based SERS substrate provides a quick and easy method for the detection of illegally added drugs in beverages.

A review of drug abuse in recently reported cases of driving under the influence of drugs (DUID) in Asia, USA, and Europe.

Driving Under the Influence of Drugs (DUID) is considered a serious issue related to the abuse of illegal drugs. DUID cases, including deaths, are being continuously reported in Asia, USA, and Europe. This literature review focuses on illegal drug abuse in recent DUID cases reported in Asia, USA, and Europe. To determine illegal drug abuse in DUID suspects, previous studies collected and analyzed biological samples, such as blood, urine, oral fluids, and hair. In addition, there were forensic autopsies and surveys for investigation of illegal drugs in DUID cases and drivers. In previous studies, ketamine, morphine, methamphetamine (MA), and khat were mainly reported in Asia, whereas amphetamine, benzodiazepines (BZDs), and cannabinoids were mainly reported in USA, and synthetic cannabinoids (SCs), opiates, and cocaine were mainly reported in Europe. Since DUID suspects related to illegal drugs have been frequently reported in Asia, USA, and Europe, there is a need to plan for national monitoring for drivers or motor vehicles to regulate and prevent drug abuse and relevant DUID cases.

Incorporation of zolpidem and methoxyphenamine into white hair strands after single administrations: Influence of hair pigmentation on drug incorporation.

In order to investigate the influence of pigmentation on the incorporation of drugs into hair, time-course changes in drug distribution along non-pigmented (white) hairs as well as pigmented (black) hairs plucked from the same subject was observed following single administrations of two basic drugs with different properties, zolpidem and methoxyphenamine. These drugs in 1-mm sections of single hair specimens were each determined by a liquid chromatography-tandem mass spectrometric procedure. During the early stage (12-36 h) after intake, for black hairs, both drugs were detected over the entire area of hair root (4-5 mm in length), in which notable concentration of these drugs in the hair bulb (0-1-mm segment from the bottom of hair root, Region 1) and lower concentrations in the upper dermis zone (1-2-mm to 3-4-mm or to 4-5-mm segments, Region 2) were commonly observed. Meanwhile, for white hairs, high drug concentrations in Region 1 as detected in black hairs were not observed although only small amounts of these drugs were detected over Region 2. Subsequent time-course changes in the concentration of drugs in hair demonstrated that the drugs once incorporated into white hair via Region 2 decreased gradually over the period from 24 h to 35 days after intake, but those of black hairs remained almost unchanged. These findings revealed here suggest that hair pigments have two important roles in the distribution of drugs: (1) incorporation of drugs into hair via Region 1, and (2) retention of already incorporated drugs in the hair tissue. These findings would be useful for discussing individual drug-use history based on hair analysis in the forensic fields.

[Hair Testing for Drugs in the Field of Forensics].

Hair testing for drugs has been used extensively in the field of forensics since the 1990s as a means of obtaining firm evidence of drug ingestion. In addition to its longer detection windows, hair is the only specimen that can provide chronological information on individual drug use. Illicit drugs and hypnotics account for the majority of substances involved in crimes; they are usually analyzed to prove an addictive use or an exposure to drugs in drug-facilitated crimes. The mechanism of drug incorporation into hair has been intensively investigated to properly interpret the results of hair analysis. However, the exact mechanism remains under much discussion, despite the growing application of hair tests. Recently, the authors have applied matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging and sectional hair analysis of 1-mm segments using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for single-strand hair, to investigate the incorporation pathways of drugs into hair. Time-course changes in drug distribution along single-strand hair suggest that the incorporation of drugs occurs in two regions of the hair root, the hair bulb and the upper part of hair root, and suggest that incorporation from the hair bulb continues for about 2 weeks. Distribution profiles of different drugs in hair additionally revealed that the main incorporation pathway varies (i.e., via the hair bulb or the upper part of hair root) depending on the properties of the drug/metabolite. These findings should be taken into account upon discussing individual drug-use history based on the results of hair analysis.

Establishment of pressurized liquid extraction followed by HPLC-MS/MS method for the screening of adrenergic drugs, steroids, sedatives, colorants and antioxidants in swine feed.

A facile and sensitive multi-residue detection approach of pressurized liquid extraction following high-performance liquid chromatography tandem mass spectrometry was established to detect the residues of adrenergic drugs, steroids, sedative, colorant and antioxidant in feed. The conditions employed for pressurized liquid extraction involved acetonitrile/ethyl acetate (1:1, v/v) as the extracting solvent, the temperature 80°C, two cycles and a static time of 10 min. The extraction was followed by a solid-phase extraction clean-up step. The separation of samples was done by C18 column with the mobile phase of 5 mM ammonium acetate solution and acetonitrile with 0.1% formic acid. The limits of quantification ranged from 0.03 to 1 µg/kg, limits of detection were in a range of 0.01-0.5 µg/kg, and average recoveries were 70.4-98.6%. The pressurized liquid extraction procedure was optimized and overall method was validated in terms of sensitivity, linearity, selectivity, matrix effect, accuracy, recovery and stability of the target drugs in the pressurized liquid extraction extracts solution. The screening method was proved to be fast, selective, accurate and sensitive for screening drugs.

Alprazolam and Zolpidem in Skeletal Tissue of Decomposed Body Confirms Exposure.

In several medico-legal cases, bone samples analysis may provide the only source of toxicological information. This case study reports the analysis of a human bone specimen, belonging to a 46-year-old man, found 3 months after his death due to cervical-thoracic injuries in a motorcycle accident. Bone specimen was the only available material for toxicological analysis, among few skull hair and rotten skin. Analysis was performed by a newly developed and validated ultra-high-pressure liquid chromatography-mass spectrometry/mass spectrometry (UHPLC-MS/MS) method, following simple and efficient sample pretreatment. The results were in accordance with the man's medical record: Alprazolam and zolpidem were found at 2.2 and 5.4 ng/g of bone, respectively. Both these drugs were prescribed to the deceased.

Segmental Hair Analysis-Interpretation of the Time of Drug Intake in Two Patients Undergoing Drug Treatment.

The present study involved segmental testing of hair in two clinical cases with known dosage histories. Hair analysis confirmed the first patient's exposure to the prescribed sertraline and citalopram for several months. Citalopram was generally distributed along the hair shaft in accordance with the drug ingestion period. By contrast, "false" positive results were observed for sertraline in distal hair segments, corresponding to a period of no sertraline exposure, which may indicate incorporation from sweat or sebum, which transport the drugs along the hair surface. The second patient received various drugs during her treatment for brain cancer. Metoclopramide, morphine, oxazepam, paracetamol, sumatriptan, tramadol, and zopiclone, which had been part of the therapy, were all detected in the proximal hair segment. The results of these two cases indicated that results-especially concerning the time of drug intake-must be interpreted with caution and allow for the possibility of incorporation from sweat or sebum.

Toxicological findings in 1000 cases of suspected drug facilitated sexual assault in the United States.

The purpose of this study was to identify the extent and types of drugs found in alleged drug facilitated sexual assaults (DFSA) in 37 states and 1 territory of the United States. In total, 1000 cases were reviewed. Between the cases that gender was provided (613), most of the victims (91.68%) were woman, mean age of 26.8 years old. Blood and/or urine samples were tested. Twenty-one point six percent of the cases were negative for intoxicating substances. A hundred and one different substances were detected. Overall, ethanol was the most prevalent substance, detected in 30.9% of the cases (309 cases), followed by cannabinoids (THC/THCCOOH/11-OH-THC) (28.8% of cases), amphetamine/methamphetamine (16.5% of cases), cocaine/metabolites (10.4% of cases), and clonazepam/metabolite (7.6% of cases). The mean, median and range concentrations of ethanol in blood (n = 309) were 98.6 mg/dL, 82.0 mg/dL and 9.2-366 mg/dL, respectively. Ethanol and cannabinoids were the most frequent combination found. The absence of alcohol and drugs in some cases may represent delay in collecting samples.

Multiclass screening method to detect more than fifty banned substances in bovine bile and urine.

A multiclass screening method to detect fifty-three forbidden substances by liquid-chromatography coupled to hybrid high-resolution mass spectrometry (LC-Q-Orbitrap) was developed and validated in bovine bile and urine. Eight classes of compounds were included in the method's scope (ß-agonists, corticosteroids, nitroimidazoles, progestins, resorcylic acid lactones (RALs), sedatives, steroids and stilbenes) plus chloramphenicol and dapsone. After hydrolysis, the sample was divided in two aliquots, which followed two parallel purification steps. The reunified extracts were injected and two chromatographic runs performed in positive and negative ionization mode, respectively. The validation data (60 different samples per matrix) proved that the method was fit for purpose with detection capabilities lower than 1 µg L-1 in both matrices. The combined application of accurate mass acquisition and two-stage mass spectrometry (parallel reaction monitoring) was crucial to achieve suitable selectivity, which is the most critical parameter mainly for urines. Finally, the long-standing problem of the high rate of false positive results for RALs, due to the natural ingestion of mycotoxin, zearalenone, was taken on including all their labelled standards. That allowed a very satisfactory management of this screening test.

Romaine Lettuce/Skullcap Mixture Improves Sleep Behavior in Vertebrate Models.

The aim of this study is to investigate the effects of romaine lettuce leaves extract (RE), skullcap root extract (SE) and their mixture on sleep behaviors in vertebrate models. HPLC analysis showed that RE contains lactucopicrin (0.02±0.01 mg/g extract), chlorogenic acid (4.05±0.03 mg/g extract), caffeic acid (2.38±0.03 mg/g extract), and chicoric acid (7.02±0.32 mg/g extract) as main phenolic compounds, while SE includes baicalin (99.4±0.5 mg/g extract), baicalein (8.28±0.21 mg/g extract), and wogonin (3.09±0.32 mg/g extract). The mixture of RE (100 mg/g extract) and SE (40 mg/g extract) increased total sleep time by 50.9% compared with the control in pentobarbital-induced sleep model. In electroencephalography (EEG) analysis, RE/SE mixture significantly increased Non-Rapid Eye Movement (NREM), in which delta wave was enhanced by around 40% compared with normal control, leading to the increase of sleep time. In caffeine-induced wake model, RE/SE mixture greatly decreased (53%) caffeine-induced wake time, showing a similar level to normal control. In addition, caffeine-induced decreased of NREM and delta wave effectively increased with RE/SE mixture; NREM and delta wave increased by 85% and 108%, respectively. Furthermore, RE/SE mixture was shown to bind to a gamma-aminobutyric acid type A (GABAA)-benzodiazepine (BZD) receptor stronger than RE or SE single extract. Taken together, RE/SE mixture effectively improved sleep behavior with the increase of NREM via GABAA-BZD receptor binding. RE/SE mixture can be used as an herbal agent for sleep disorders.