Simultaneous trace-level monitoring of seven opioid analgesic drugs in biological samples by pipette-tip micro solid phase extraction based on PVA-PAA/CNT-CNC composite nanofibers followed by HPLC-UV analysis.

Electrospun poly(vinyl alcohol)-(PVA)-poly(acrylic acid) (PAA)/carbon nanotubes(CNTs)-cellulose nanocrystal (CNC) (PVA-PAA/CNT-CNC) composite nanofibers were prepared and characterized using Fourier transform-infrared spectroscopy and field emission scanning electron microscopy. The resultant composite was used as an effective and novel sorbent for pipette-tip micro-solid phase extraction (PT-µSPE) of seven opioid analgesics (OAs) in biological samples followed by HPLC-UV analysis. Addition of CNT-CNC with the high specific surface area and plenty of OH-functional groups endows the nanofibers with considerable extraction efficiency. Under the optimum conditions, the linearity was obtained in the range 1.5 to 700.0 ng mL-1 for morphine, codeine, oxycodone, and tramadol, and 0.5 to 1000.0 ng mL-1 for nalbuphine, thebaine, and noscapine with coefficient of determination (r2) ≥ 0.9990. Detection limits (LODs) based on S/N = 3 were in the range of 0.15-0.50 ng mL-1. The relative standard deviations (RSDs) of 4.1-5.4% (intra-day, n = 5) and 5.2-6.4% (inter-day, n = 3) for three consecutive days were achieved. Finally, the efficiency of the PT-µSPE-HPLC-UV method was evaluated for the determination of OAs in human plasma and urine samples with good recoveries (87.3 to 97.8%). A: Schematic illustration for the preparation of PVA-PAA/CNT-CNC composite nanofibers. B: Schematic presentation of applying PVA-PAA/CNT-CNC composite nanofibers as the sorbent in pipette-tip micro solid-phase extraction (PT-µSPE) for the preconcentration of seven opioid analgesic drugs in biological samples before HPLC-UV analysis.

Antinociceptive effect of the hydroethanolic leaf extract of Calotropis procera (Ait) R. Br. (Apocynaceae): Possible involvement of glutamatergic, cytokines, opioidergic and adenosinergic pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Pain remains real and still a major problem in clinical medicine which requires new agents with improved efficacy for more therapeutic benefits. Plant sources can serve as a basis for the search for some novel drugs hence the analgesic effects of the hydroethanolic extract of Calotropis procera (CPE) which is widespread in Ghana and other tropical areas and used in folkloric medicine for painful and inflammatory conditions was evaluated. MATERIALS AND METHODS: The analgesic properties of orally administered CPE at doses of 30, 100, and 300 mg/kg were evaluated in thermal (tail immersion), chemical (acetic acid-writhing, formalin-induced paw licking, glutamate-induced nociception) and mechanical (Randall-Selitto) tests for analgesia. The involvement of tumour necrosis factor-alpha (TNF-α), interleukin 1ß (IL 1ß), bradykinin, and prostaglandin E2 (PGE2) on the analgesic effects of CPE were also evaluated in hypernociception assays measuring mechanical pain thresholds. RESULTS: The latency of tail withdrawal in the tail immersion test was significantly increased (p = 0.0001) while writhing induced by acetic acid was significantly reduced (p < 0.0001) on treatment with CPE (30-300 mg/kg). The extract also significantly inhibited both phase 1 and phase 2 nociceptive states induced by formalin comparable to morphine (p < 0.0001). Furthermore, the extract significantly attenuated hyper-nociception induced by TNF-α (p < 0.0001), interleukin 1ß (p = 0.0102), bradykinin (p < 0.0001), and prostaglandin E2 (p < 0.0001). Additionally, glutamate-induced paw licking was reduced significantly (p < 0.05). The antinociceptive effects exhibited by CPE (100 mg/kg) in the formalin test was reversed by systemic administration of naloxone (2 mg/kg) and theophylline (5 mg/kg) but not glibenclamide (8 mg/kg), granisetron (2 mg/kg), atropine (3 mg/kg), yohimbine (3 mg/kg, p.o.) nor nifedipine (10 mg/kg). CONCLUSION: Overall, the hydroethanolic leaf extract of Calotropis procera possesses analgesic properties that is mediated possibly through the glutaminergic, opioidergic, and adenosinergic pathways.

GC-MS analysis of underivatised new psychoactive substances in whole blood and urine.

Herein a method was develop and validated for the detection and quantification of five new psychoactive substances (NPS) belonging to three categories: synthetic cathinones (mephedrone, 3,4-MDPV), opioids (AH-7921) and cannabinoids (JWH-018, AM-2201) by EI GC-MS. Target analytes were quantified in whole blood; in urine the same compounds plus methylone were detected. Liquid-liquid extraction by MTBE - butyl acetate (1:1, v/v) in blood and butyl acetate in urine was applied for the recovery of analytes, while no derivatization was necessary for their sensitive detection and quantification. The method showed good linearity for all analytes within a concentration range from 0.25 to 2 µg/mL for mephedrone, from 0.02 to 0.16 µg/mL for 3,4-MDPV and AH-7921 and from 0.005 to 0.04 µg/mL for JWH-018 and AM-2201. LOD ranged from 0.002 µg/mL (JWH-018 and AM-2201 in blood and urine), to 0.08 µg/mL (mephedrone in urine). LOQ in blood ranged from 0.005 µg/mL for JWH-018 and AM-2201 to 0.25 µg/mL for mephedrone. Accuracy was within acceptable limits with % bias ranging from +20% to -17.98% for intra-assay study and from +18.87% to -11.16% for inter-assay study. Precision was found to be between 2.60% and 17.17% (CV%) for intra-assay study and from 6.03% to 13.72% (CV%) for inter-assay study. An intra laboratory comparison provided proof of the method robustness. The developed method can be used for the reliable and fast quantification of five NPS in blood and the detection of six NPS in urine within the practice of a clinical or forensic toxicology laboratory.

Determination of 37 fentanyl analogues and novel synthetic opioids in hair by UHPLC-MS/MS and its application to authentic cases.

The recent emergence of new fentanyl analogues and synthetic opioids on the drug market poses a global public health threat. However, these compounds cannot typically be identified using existing analytical methods. In this study, we aimed to develop and validate a rapid and sensitive method based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of 37 fentanyl analogues and novel synthetic opioids in hair samples. Hair samples (20 mg) were extracted by cryogenic grinding in an extraction medium of methanol, acetonitrile, and 2 mmol/L ammonium acetate (pH 5.3). Following centrifugation of the samples, the analytes were separated using a WATERS Acquity UPLC HSS T3 column. The limits of detection (LODs) and limits of quantification (LOQs) ranged from 0.5 to 2.5 pg/mg and from 2 to 5 pg/mg, respectively. The intraday and interday precisions were within 13.32% at LOQ, low, medium, and high levels. The accuracies were within the range of 85.63-116.1%. The extraction recoveries were in the range of 89.42-119.68%, and the matrix effects were within the range of 44.81-119.77%. Furthermore, the method was successfully applied to the detection and quantification of fentanyl and sufentanil in hair samples from two authentic cases. Thus, this method has great potential for detecting fentanyl analogues and novel synthetic opioids in forensic work.

Rates of Fentanyl Positivity in Neonatal Urine Following Maternal Analgesia During Labor and Delivery.

BACKGROUND: Fentanyl is commonly given as an analgesic during labor and delivery. The extent of transplacental drug transfer and fetal exposure is not well studied. We analyzed the relationship between neonatal urine fentanyl results and various peripartum factors. METHODS: A total of 96 neonates with urine toxicology screening between January 2017 and September 2018 were included in the study. Medical record review was used to obtain maternal, neonatal, and anesthesia parameters. A subset of 9 specimens were further tested for levels of fentanyl and norfentanyl by liquid chromatography-tandem mass spectrometry. RESULTS: In 29% (n = 24) of cases associated with fentanyl-containing labor analgesia, neonatal toxicology screens were positive for the presence of fentanyl. Positive test results strongly correlated with the cumulative dose and duration of labor analgesia (P < 0.001). The odds of positive neonatal fentanyl screen results increased 4-fold for every 5 hours of maternal exposure to labor analgesia. Importantly, however, neonatal outcomes for infants with positive and negative urine fentanyl screens were the same. CONCLUSIONS: Our study establishes that maternal fentanyl analgesia is strongly associated with positive neonatal urine fentanyl screens and suggests that more judicious use of these laboratory tests may be warranted.

Kratom (Mitragyna Speciosa) Liver Injury: A Comprehensive Review.

Kratom (Mitragyna speciosa) leaves contain the mu opioid partial agonists mitragynine and 7-hydroxymitragynine. The US Drug Enforcement Agency considers it a 'drug of concern', and the US FDA is reviewing kratom, but there is a paucity of information regarding health effects. Liver injury is often cited as a potential health consequence, however the same few case reports are repeatedly referenced, without a broader context. Furthermore, reports have largely lacked standardized causality assessment methods. The objective is to evaluate causality in kratom liver injury, through a comprehensive scoping review of human cases, and by reviewing epidemiologic, animal, and mechanistic reports that relate to kratom liver injury. Hepatotoxicity causality was systematically examined using the Roussel Uclaf Causality Assessment Method (RUCAM) for case reports. Biopsy findings, potential pathophysiologic mechanisms, and management options are discussed. This review identified 26 case reports and abstracts, in addition to 7 cases reported from the Drug-Induced Liver Injury Network, 25 in FDA databases, and 27 in internet user forums. Latency periods to symptom onset had a median of 20.6 days and mean of 21 days (range 2-49). Common presenting signs and symptoms were abdominal discomfort, jaundice, pruritis, and dark urine. Histologic findings were predominantly cholestatic, although, biochemically, the condition was heterogenous or mixed; the median R ratio was 3.4 and the mean was 4.6 (range 0.24-10.4). Kratom likely causes liver injury based on the totality of low-quality human evidence, and, in the context of epidemiologic, animal, and mechanistic studies. It remains unclear which subgroups of users are at heightened risk.

Highly selective extraction and voltammetric determination of the opioid drug buprenorphine via a carbon paste electrode impregnated with nano-sized molecularly imprinted polymer.

An electrochemical sensor for the opioid drug buprenorphine (BUP) is described. Molecularly imprinted polymer nanoparticles (nanoMIP) were prepared and used to modify a carbon paste electrode (CPE). The BUP-imprinted polymer was synthesized using precipitation polymerization. The resulting polymer along with multiwalled carbon nanotubes (MWCNT) was used to fabricate the modified CPE which exhibited an anodic peak at about +0.73 V (vs. Ag/AgCl) for BUP. The MIP on the CPE functions as selective recognition element with an imprinting factor of 5.6. The assay consists of two-steps, viz. analyte extraction at the electrode surface and differential pulse voltammetric determination of BUP. The effects of various parameters on the electrochemical signal were optimized, and the selectivity of the modified CPE over cross reactants was studied. At optimum experimental conditions, the response is linear in the 1 nM to 50 µM BUP concentration range, and the detection limit is 0.6 nM (at S/N = 3). This method was applied to the determination of BUP in spiked urine with acceptable relative standard deviations (3.2-4.4%). Graphical abstract Schematic representation of buprenorphine (BUP) recognition and voltammetric determination at the surface of carbon paste electrode modified with imprinted polymer and carbon nanotubes.

Kratom Use and Toxicities in the United States.

BACKGROUND: Kratom is an herbal supplement containing alkaloids with opioid properties. This review was conducted to determine toxicities associated with kratom use in the United States in order to provide insight into its safety as a dietary supplement. METHODS: We conducted a retrospective review of kratom exposures reported to the National Poison Data System to determine the toxicities associated with kratom use. We also reviewed records from a county medical examiner's office in New York State to identify kratom-associated fatalities. RESULTS: A total of 2312 kratom exposures were reported, with 935 cases involving kratom as the only substance. Kratom most commonly caused agitation (18.6%), tachycardia (16.9%), drowsiness (13.6%), vomiting (11.2%), and confusion (8.1%). Serious effects of seizure (6.1%), withdrawal (6.1%), hallucinations (4.8%), respiratory depression (2.8%), coma (2.3%), and cardiac or respiratory arrest (0.6%) were also reported. Kratom was listed as a cause or contributing factor in the death of four decedents identified by the county medical examiner's office. CONCLUSIONS: Kratom use is increasing and is associated with significant toxicities. Our findings suggest kratom is not reasonably expected to be safe and poses a public health threat due to its availability as an herbal supplement.

Quantification of Designer Opioids by Liquid Chromatography-Tandem Mass Spectrometry.

Opioids including heroin and commonly prescribed drugs such as oxycodone and fentanyl are among the most commonly abused drugs. In recent years, the abuse of opioids has spread beyond these commonly encountered analytes and now includes novel psychoactive drugs such as AH-7921 and U47700 and a variety of fentanyl-related compounds such as acetyl fentanyl and furanyl fentanyl. The assay described is for the quantitative determination of 19 designer opioids in serum, plasma, and whole blood. Also included is a discussion on the challenges of keeping an analytical method current as new analytes appear on the illicit drug market.

Nature's first "atypical opioids": Kratom and mitragynines.

WHAT IS KNOWN AND OBJECTIVE: Advances in pain research have led to an understanding that many pains are driven by more than one underlying (patho)physiologic cause (ie, they are "multimechanistic") and that better pain relief is obtained with fewer adverse effects when an analgesic is correspondingly multimechanistic. At least two of the more-modern analgesics combine opioid and non-opioid mechanisms, and have become known as "atypical opioids." Less well known is that just as Nature evolved opioids, it also evolved atypical opioids, presaging modern drug discovery efforts. COMMENT: Traditional (typical) opioids are extracts or analogs of substances derived from the poppy plant. They produce their analgesic and adverse effects primarily through a single, opioid mechanism (albeit with individual differences). Two most recent analgesics were developed to have both an opioid mechanism and, a second, non-opioid mechanism of action (inhibition of monoamine neurotransmitter reuptake). Little known is that Nature had already evolved a plant source of compounds with the same properties. WHAT IS NEW AND CONCLUSION: As debate about the use and abuse potential of kratom swirls, conflicting, often contradicting, opinions are expressed. A review of the basic pharmacology of kratom reveals the explanation for the bifurcation in viewpoints: kratom has both opioid and non-opioid properties. Fascinatingly, just as the poppy plant (Papaver) evolved the typical opioids, Mitragyna evolved the mitragynines-Nature's "atypical opioids."

Recent trends and analytical challenges in plant bioactive peptide separation, identification and validation.

Interest in research into bioactive peptides (BPs) is growing because of their health-promoting ability. Several bioactivities have been ascribed to peptides, including antioxidant, antihypertensive and antimicrobial properties. As they can be produced from precursor proteins, the investigation of BPs in foods is becoming increasingly popular. For the same reason, production of BPs from by-products has also emerged as a possible means of reducing waste and recovering value-added compounds suitable for functional food production and supplements. Milk, meat and fish are the most investigated sources of BPs, but vegetable-derived peptides are also of interest. Vegetables are commonly consumed, and agro-industrial wastes constitute a cheap, large and lower environmental impact source of proteins. The use of advanced analytical techniques for separation and identification of peptides would greatly benefit the discovery of new BPs. In this context, this review provides an overview of the most recent applications in BP investigations for vegetable food and by-products. The most important issues regarding peptide isolation and separation, by single or multiple chromatographic techniques, are discussed. Additionally, problems connected with peptide identification in plants and non-model plants are discussed regarding the particular case of BP identification. Finally, the issue of peptide validation to confirm sequence and bioactivity is presented. Graphical representation of the analytical workflow needed for investigation of bioactive peptides and applied to vegetables and vegetable wastes Graphical Abstract.

Dispersion of magnetic graphene oxide nanoparticles coated with a deep eutectic solvent using ultrasound assistance for preconcentration of methadone in biological and water samples followed by GC-FID and GC-MS.

Magnetic graphene nanoparticles coated with a new deep eutectic solvent (Fe3O4@GO-DES) were developed for efficient preconcentration of methadone. The extracted methadone was then analyzed by gas chromatography-flame ionization detection (GC-FID) or gas chromatography-mass spectrometry (GC-MS). Fe3O4@GO-DES were characterized by Fourier transform IR and X-ray diffraction techniques. Ultrasound was used to enhance the dispersion of the sorbent, with a high extraction recovery. Some parameters affecting the extraction recovery, such as pH, type of deep eutectic solvent, sample volume, amount of sorbent, extraction time, and type of eluent, were investigated. Under optimum conditions, the method developed was linear in the concentration range from 3 to 45,000 µg L-1 for GC-FID and from 0.1 to 500 µg L-1 for GC-MS, with a detection limit of 0.8 µg L-1 for GC-FID and 0.03 µg L-1 for GC-MS. The relative standard deviations (n = 6) as the intraday and interday precisions of the methadone spike at a concentration of 100 µg L-1 were 5.8% and 8.4% respectively for GC-FID. The preconcentration factor was 250. Relative recoveries from spiked plasma, urine, and water samples ranged from 95.1% to 101.5%.

Simultaneous quantitation of meperidine, normeperidine, tramadol, propoxyphene and norpropoxyphene in human plasma using solid-phase extraction and gas chromatography/mass spectrometry: Method validation and application to cardiovascular safety of therapeutic doses.

RATIONALE: Several opioid analgesics have been related to the prolongation of cardiac repolarization, a condition which can be fatal. In order to establish a correct estimation of the risk/benefit balance of therapeutic doses of meperidine, normeperidine, tramadol, propoxyphene and norpropoxyphene, it was necessary to develop an analytical method to determinate plasma concentrations of these opioids. METHODS: Here we describe a method which incorporates strong alkaline treatment to obtain norpropoxyphene amide followed by a one-elution step solid-phase extraction, and without further derivatization. Separation and quantification were achieved by gas chromatography/electron ionization mass spectrometry (GC/EI-MS) in selected-ion monitoring mode. Quantification was performed with 500 µL of plasma by the addition of deuterated analogues as internal standards. RESULTS: The proposed method has been validated in the linearity range of 25-1000 ng/mL for all the analytes, with correlation coefficients higher than 0.990. The lower limit of quantification was 25 ng/mL. The intra- and inter-day precision, calculated in terms of relative standard deviation, were 2.0-12.0% and 6.0-15.0%, respectively. The accuracy, in terms of relative error, was within a ± 10% interval. The absolute recovery and extraction efficiency ranged from 81.0 to 111.0% and 81.0 to 105.0%, respectively. CONCLUSIONS: A GC/MS method for the rapid and simultaneous determination of meperidine, normeperidine, tramadol, propoxyphene and norpropoxyphene in human plasma was developed, optimized and validated. This procedure was shown to be sensitive and specific using small specimen amounts, suitable for application in routine analysis for forensic purposes and therapeutic monitoring. To our knowledge, this is the first full validation of the simultaneous determination of these opioids and their metabolites in plasma samples.

Air assisted emulsification liquid-liquid microextraction based on deep eutectic solvent for preconcentration of methadone in water and biological samples.

Nowadays, a deep eutectic solvent (DES) has recently been considered as a green ion liquid analogue. In this study, a new compound of DES has been synthesized as an extraction solvent in air assistedemulsification liquid-liquid microextraction method (DES-AAELLME) for preconcentration and extraction of methadone followed by gas chromatographyflame ionization detector (GC-FID). To obtain an efficient water-miscible deep eutectic solvent, choline chloride (ch-cl) and 5,6,7,8-Tetrahydro-5,5,8,8-tetramethylnaphthalen-2-ol (TNO) were mixed at a molar ratio of 1:2 and tetrahydrofuran (THF) was used as a demulsifier solvent into homogeneous solution for providing a turbid state. The solution was rapidly sucked up and injected 10 times using a 10-mL glass syringe to enhance the turbidity of solution and disperse the aggregated DES droplets into aqueous phase. Some important parameters affecting extraction recovery were investigated. Under optimum conditions, the calibration curve was linear in the concentration range from 2 to 8000µgL-1. The limit of detection and the limit of quantification were found 0.7µgL-1 and 2.3µgL-1 respectively with preconcentration factor of 270. The precision, as the relative standard deviation (RSD) (n=6), was <6% respectively. This method was successfully applied to determine methadone in water and biological samples with an appropriate recovery about 98.4-101.2%.

Development of solid-phase microextraction coupled with liquid chromatography for analysis of tramadol in brain tissue using its molecularly imprinted polymer.

In this work, performance of a molecularly imprinted polymer (MIP) as a selective solid-phase microextraction sorbent for the extraction and enrichment of tramadol in aqueous solution and rabbit brain tissue, is described. Binding properties of MIPs were studied in comparison with their nonimprinted polymer (NIP). Ten milligrams of the optimized MIP was then evaluated as a sorbent, for preconcentration, in molecularly imprinted solid-phase microextraction (MISPME) of tramadol from aqueous solution and rabbit brain tissue. The analytical method was calibrated in the range of 0.004 ppm (4 ng mL-1 ) and 10 ppm (10 µg mL-1 ) in aqueous media and in the ranges of 0.01 and 10 ppm in rabbit brain tissue, respectively. The results indicated significantly higher binding affinity of MIPs to tramadol, in comparison with NIP. The MISPME procedure was developed and optimized with a recovery of 81.12-107.54% in aqueous solution and 76.16-91.20% in rabbit brain tissue. The inter- and intra-day variation values were <8.24 and 5.06%, respectively. Finally the calibrated method was applied for determination of tramadol in real rabbit brain tissue samples after administration of a lethal dose. Our data demonstrated the potential of MISPME for rapid, sensitive and cost-effective sample analysis.

Identification of the First Marine-Derived Opioid Receptor "Balanced" Agonist with a Signaling Profile That Resembles the Endorphins.

Opioid therapeutics are excellent analgesics, whose utility is compromised by dependence. Morphine (1) and its clinically relevant derivatives such as OxyContin (2), Vicodin (3), and Dilaudid (4) are "biased" agonists at the µ opioid receptor (OR), wherein they engage G protein signaling but poorly engage ß-arrestin and the endocytic machinery. In contrast, endorphins, the endogenous peptide agonists for ORs, are potent analgesics, show reduced liability for tolerance and dependence, and engage both G protein and ß-arrestin pathways as "balanced" agonists. We set out to determine if marine-derived alkaloids could serve as novel OR agonist chemotypes with a signaling profile distinct from morphine and more similar to the endorphins. Screening of 96 sponge-derived extracts followed by LC-MS-based purification to pinpoint the active compounds and subsequent evaluation of a mini library of related alkaloids identified two structural classes that modulate the ORs. These included the following: aaptamine (10), 9-demethyl aaptamine (11), demethyl (oxy)-aaptamine (12) with activity at the δ-OR (EC50: 5.1, 4.1, 2.3 µM, respectively) and fascaplysin (17), and 10-bromo fascaplysin (18) with activity at the µ-OR (EC50: 6.3, 4.2 µM respectively). An in vivo evaluation of 10 using δ-KO mice indicated its previously reported antidepressant-like effects are dependent on the δ-OR. Importantly, 17 functioned as a balanced agonist promoting both G protein signaling and ß-arrestin recruitment along with receptor endocytosis similar to the endorphins. Collectively these results demonstrate the burgeoning potential for marine natural products to serve as novel lead compounds for therapeutic targets in neuroscience research.

QuEChERS sample preparation prior to LC-MS/MS determination of opiates, amphetamines, and cocaine metabolites in whole blood.

Modern LC-MS/MS instruments have sensitivity and scanning velocity high enough to analyze many different compounds in single runs. Consequently, the sample preparation procedure has become the bottleneck for developing efficient, rapid, and cheap multi-compound methods. Here, we examined one-step sample preparation based on quick, easy, cheap, effective, rugged, and safe (QuEChERS) salts to set up and validate a LC-MS/MS method for the simultaneous determination of 35 drugs of abuse and their metabolites in whole blood. Despite large differences in physicochemical properties, this simplified QuEChERS extraction method yielded satisfactory recoveries (until 96%) for the 35 molecules. The amounts of QuEChERS salts had no influence on extraction yield. Chromatographic separation was obtained in less than 6 min. LLOD and LLOQ were 3 and 5 ng/mL, respectively. The procedure was successfully validated and then applied to 253 cases of driving under the influence of drugs (DUID), collected over a 6-month period.

Validation of a fast UPLC-MS/MS method for quantitative analysis of opioids, cocaine, amphetamines (and their derivatives) in human whole blood.

BACKGROUND: Conventional methods for analysis of drugs of abuse require multiple assays which can be both expensive and time-consuming. This work describes a novel, rapid, simple and sensitive method for the quantification of 14 illicit drugs and their metabolites in whole blood. Results/methodology: This method employed a rapid liquid-liquid sample extraction of whole blood followed by UPLC-MS/MS analysis. Calibration curves were validated for analysis of appropriate concentrations. Inter- and intra-assay variations were <14.8%. Deviation of accuracy was <14.9% from target concentration for each quality control level. CONCLUSION: This work described the development and the full validation of a precise, sensitive and accurate assay. After validation, this new assay was successfully applied to routine toxicological analysis.

In vitro opioid receptor affinity and in vivo behavioral studies of Nelumbo nucifera flower.

ETHNOPHARMACOLOGICAL RELEVANCE: Nelumbo nucifera Geartn., known as sacred lotus, has been used traditionally in South East Asia as a traditional medicine for various CNS disorders including stress, fever, depression, insomnia, and cognitive conditions. AIM OF THE STUDY: To investigate the in vitro cannabinoid and opioid receptor binding affinities, and in vivo behavioral actions of Nelumbo flower extracts and to isolate the potential compounds to treat CNS associated disorders. MATERIALS AND METHODS: The white and pink flowers of N. nucifera were extracted with 95% EtOH, followed by acid-base partitioning using CHCl3 to give acidic and basic partitions. These partitions were subjected to Centrifugal Preparative TLC (CPTLC) to yield benzyltetrahydroisoquinoline (BTIQ) alkaloids and long chain fatty acids, identified by physical and spectroscopic methods. In addition, EtOH extracts and partitions were analyzed for chemical markers by UHPLC/MS and GC/MS. In vitro neuropharmacological effects were evaluated by cannabinoid (CB1 and CB2) and opioid [delta (δ), kappa (ĸ), and mu (µ)] competitive radioligand binding and GTPγS functional assays. The in vivo behavioral effect was studied through the use of the mouse tetrad assay at 10, 30, 75 and 100mg/kg/ip doses that revealed the effect on locomotion, catalepsy, body temperature, and nociception of acidic and basic CHCl3 partitions, fractions, and compounds. RESULTS: Three aporphines, nuciferine (1), N-nor-nuciferine (2), asimilobine (3), and five BTIQs, armepavine (4), O-methylcoclaurine (5), N-methylcoclaurine (6), coclaurine (7), neferine (10), and a mixture of linoleic and palmitic acids (LA and PA), were identified and evaluated for cannabinoid and opioid receptor displacement activities. Compounds 5-7 showed binding affinities for the ĸ opioid receptor with equilibrium dissociation constant (Ki) values of 3.5 ± 0.3, 0.9 ± 0.1, 2.2 ± 0.2 µM, respectively. Compound 10 displayed affinities for δ-and µ- opioid receptors with Ki values of 0.7 ± 0.1 and 1.8 ± 0.2 µM, respectively, and was determined to be a weak δ agonist by GTPγS functional assay. The mixture of LA and PA (1:1) showed an affinity for δ opioid receptor with a Ki value of 9.2 ± 1.1 µM. The acidic and basic CHCl3 partitions, compounds 1 and 7, and 5-7 mixture were subjected to the tetrad assay, of which the acidic partition displayed decreased locomotion and increased catalepsy, antinociception, and hypothermia in animal at doses of 75-100 mg/kg/ip, and also showed clonic-tonic seizures upon touch at 100mg/kg. CONCLUSION: Bioassay-guided isolation revealed compounds 5-7, 10, and the mixture of LA and PA displayed various degrees of opioid receptor radioligand displacement affinities. The in vivo tetrad assay of acidic CHCl3 partition, enriched with aporphines 1 and 2, displayed actions on all four points of behavioral parameters. It can be concluded that the in vivo mild canabimimetic-type effect observed for the CHCl3 partition is likely mediated through other CNS mechanisms since the extracts, partitions, and isolated compounds had no affinity for the in vitro CB1 and CB2 receptors. This work, along with traditional use and the reported bioactivities of the BTIQ alkaloids, suggested further studies on N. nucifera are needed to understand the roles that the extracts and/or individual compounds might contribute to the behavioral effects.

Biopeptides in Milk: Opiate and Antithrombotic Effects.

Recently, the concept of functional food can be seen worldwide, and there are several local wisdoms on health-conscious. In this context, the protein fractionation of\milk has attracted interests. Casein-derived bioactives have been identified as showing several health beneficial bioactivities including opiate, antithrombotic, antioxidative, antimicrobial, osteoprotective, anticariogenic and growth-promoting. Peptides have to be absorbed from the intestine and reach the target cells in sufficient concentrations or act via receptors; then they can show health effects. In this review paper, the milk-derived peptides and their therapeutic effects are introduced. Also the opiate and antithrombotic effects of these peptides are described.