LC-MS/MS screening method for designer amphetamines, tryptamines, and piperazines in serum

Since the late 1990s and early 2000s, derivatives of well-known designer drugs as well as new psychoactive compounds have been sold on the illicit drug market and have led to intoxications and fatalities. The LC-MS/MS screening method presented covers 31 new designer drugs as well as cathinone, methcathinone, phencyclidine, and ketamine which were included to complete the screening spectrum. All but the last two are modified molecular structures of amphetamine, tryptamine, or piperazine. Among the amphetamine derivatives are cathinone, methcathinone, 3,4-DMA, 2,5-DMA, DOB, DOET, DOM, ethylamphetamine, MDDMA, 4-MTA, PMA, PMMA, 3,4,5-TMA, TMA-6 and members of the 2C group: 2C-B, 2C-D, 2C-H, 2C-I, 2C-P, 2C-T-2, 2C-T-4, and 2C-T-7. AMT, DPT, DiPT, MiPT, DMT, and 5MeO-DMT are contained in the tryptamine group, BZP, MDBP, TFMPP, mCPP, and MeOPP in the piperazine group. Using an Applied Biosystems LC-MS/MS API 365 TurboIonSpray it is possible to identify all 35 substances. After addition of internal standards and mixed-mode solid-phase extraction the analytes are separated using a Synergi Polar RP column and gradient elution with 1 mM ammonium formate and methanol/0.1% formic acid as mobile phases A and B. Data acquisition is performed in MRM mode with positive electro spray ionization. The assay is selective for all tested substances. Limits of detection were determined by analyzing S/N-ratios and are between 1.0 and 5.0 ng/mL. Matrix effects lie between 65% and 118%, extraction efficiencies range from 72% to 90%.

Castration of lambs: a welfare comparison of different castration techniques in lambs over 10 weeks of age

Seventy male lambs over 10 weeks of age were castrated using Burdizzo, rubber rings, or surgery to assess the acute and long-term effects of castration. All castrations were performed under local anaesthesia. The surgically castrated lambs were additionally sedated with xylazine and the sedation reversed with tolazoline. The frequency of abnormal postures and immediate behavioural responses indicated that surgically castrated lambs were most distressed; the lambs castrated using Burdizzo and rubber rings were not dissimilar to those of the control group. Between 1.5 and 9h after castration, signs of pain and distress were at a lower level in lambs anaesthetised with bupivacaine compared with those treated with lidocaine. Due to the markedly faster wound healing, Burdizzo castration seemed to be preferable (fewer signs of long-term pain) when compared to the rubber ring technique. It was concluded that local anaesthesia with bupivacaine, followed by the Burdizzo method is the preferable technique for the castration of lambs older than 10 weeks of age.

Continuous extradural analgesia in a cow with complex regional pain syndrome

A chronic pain syndrome, similar to the complex regional pain syndrome (CRPS) described in human beings, was diagnosed in a cow with persisting severe pelvic limb lameness. Diagnosis was based on the disproportionate relationship between the severity and duration of pain and the lesion, the failure of conventional analgesic and surgical therapy and the presence of characteristic clinical features. Multimodal therapy, i.e. a mixture of methadone, ketamine and bupivacaine was administered continuously for 17 days via an extradural catheter to counteract nociceptive hypersensitization. Doses were adjusted daily after assessing the effect, using a composite pain score. Physiotherapy was also performed. The diagnosis of CRPS in cattle is unusual. In this case, treatment was successful and the cow was discharged mildly lame and in improving physical condition. Long-term extradural analgesia proved to be safe and effective in the treatment of this syndrome, which was nonresponsive to conventional therapy.

Effects of an alveolar recruitment maneuver on cardiovascular and respiratory parameters during total intravenous anesthesia in ponies

OBJECTIVE: To evaluate pulmonary and cardiovascular effects of a recruitment maneuver (RM) combined with positive end-expiratory pressure (PEEP) during total intravenous anesthesia in ponies. ANIMALS: 6 healthy adult Shetland ponies. PROCEDURE: After premedication with detomidine (10 microg/kg, IV), anesthesia was induced with climazolam (0.06 mg/kg, IV) and ketamine (2.2 mg/kg, IV) and maintained with a constant rate infusion of detomidine (0.024 mg/kg/h), climazolam (0.036 mg/kg/h), and ketamine (2.4 mg/kg/h). The RM was preceded by an incremental PEEP titration and followed by a decremental PEEP titration, both at a constant airway pressure difference (deltaP) of 20 cm H2O. The RM consisted of a stepwise increase in deltaP by 25, 30, and 35 cm H2O obtained by increasing peak inspiratory pressure (PIP) to 45, 50, and 55 cm H2O, while maintaining PEEP at 20 cm H2O. Hemodynamic and pulmonary variables were analyzed at every step of the PEEP titration-RM. RESULTS: During the PEEP titration-RM, there was a significant increase in PaO 2 (+12%), dynamic compliance (+ 62%), and heart rate (+17%) and a decrease in shunt (-19%) and mean arterial blood pressure (-21%) was recorded. Cardiac output remained stable. CONCLUSIONS AND CLINICAL RELEVANCE: Although baseline oxygenation was high, Pa(O2) and dynamic compliance further increased during the RM. Despite the use of high PIP and PEEP and a high tidal volume, limited cardiovascular compromise was detected. A PEEP titration-RM may be used to improve oxygenation in anesthetized ponies. During stable hemodynamic conditions, PEEP titration-RM can be performed with acceptable adverse cardiovascular effects.

Insulin-like growth factor type-1 receptor down-regulation associated with dwarfism in Holstein calves

Perturbations in endocrine functions can impact normal growth. Endocrine traits were studied in three dwarf calves exhibiting retarded but proportionate growth and four phenotypically normal half-siblings, sired by the same bull, and four unrelated control calves. Plasma 3,5,3'-triiodothyronine and thyroxine concentrations in dwarfs and half-siblings were in the physiological range and responded normally to injected thyroid-releasing hormone. Plasma glucagon concentrations were different (dwarfs, controls>half-siblings; P<0.05). Plasma growth hormone (GH), insulin-like growth factor-1 (IGF-1) and insulin concentrations in the three groups during an 8-h period were similar, but integrated GH concentrations (areas under concentration curves) were different (dwarfs>controls, P<0.02; half-siblings>controls, P=0.08). Responses of GH to xylazine and to a GH-releasing-factor analogue were similar in dwarfs and half-siblings. Relative gene expression of IGF-1, IGF-2, GH receptor (GHR), insulin receptor, IGF-1 type-1 and -2 receptors (IGF-1R, IGF-2R), and IGF binding proteins were measured in liver and anconeus muscle. GHR mRNA levels were different in liver (dwarfs

CE provides evidence of the stereoselective hydroxylation of norketamine in equines

CE with multiple isomer sulfated-CD as selector was used for the simultaneous analysis of the stereoisomers of ketamine, norketamine, 5,6-dehydronorketamine and hydroxylated metabolites of norketamine in liquid/liquid extracts of (i) in vitro incubations with ketamine or norketamine and equine liver microsomes and (ii) plasma and urine of ponies receiving a target-controlled infusion of ketamine under isoflurane anesthesia. Hydroxynorketamine metabolites with the hydroxy group at the cyclohexanone ring could be shown to be formed stereoselectively both in vitro and in vivo. Due to the lack of standard compounds, urinary extracts were fractionated by HPLC followed by characterization of the collected fractions with CE and LC-MS(n) with 0.7 mmu mass discrimination. Comparison of LC-MS(n) data obtained with the fractions, an in vitro microsomal sample, and both pony urine and hydrolyzed pony urine led to the identification of four hydroxylated norketamine metabolites with hydroxylation at the cyclohexanone ring, two with hydroxylation at the aromatic ring and four hydroxylated metabolites of ketamine. Due to the lower detection sensitivity, only the four hydroxynorketamine metabolites with hydroxylation at the cyclohexanone ring were observed by CE. The data suggest that demethylation of ketamine followed by hydroxylation of norketamine at the cyclohexanone ring is the major metabolic pathway in equine species and that the ketamine metabolism is highly stereoselective.

Experiences with piglet castration using injectable anaesthetic in Switzerland

The aim of this study was to describe the practical implementation of the painless castration under injection anaesthesia in Switzerland. 30 swine farms were visited and 60 farmers answered a questionnaire. 34 % of the piglets showed movements during castration under injection anaesthesia and 17 % had excitations during recovery from anaesthesia. After 48 minutes half of the piglets were in sternal position and after 112 minutes half of them showed coordinated movements. The body temperature decreased by 3.1 °C until 60 minutes after castration, especially small piglets reached critical temperature levels. 38 % of the piglets showed strong bleeding after castration. The healing of the wound was good according to 82 % of the farmers. 83 % of the farmers reported piglet losses, especially at the beginning of the anaesthesia period. The anaesthesia may be improved by using butorphanol in addition to the combination of ketamine and azaperone. The recovery of the piglets should be in a warm place without any risk of injury by obstacles or the sow. Increased bleeding can be controlled by using an emasculator.

Etomidate for critically ill patients. Con: do you really want to weaken the frail?

Etomidate is an imidazole-derived hypnotic agent preferentially used for rapid sequence induction of anaesthesia because of its favourable haemodynamic profile. However, 11β-hydroxylase inhibition causes adrenal insufficiency with potentially fatal consequences in specific populations. We review the arguments against the liberal administration of etomidate in critically ill, and especially septic, patients. This review considered only high-quality and prospective studies with a low risk of bias. Three major effects have been observed with the clinical use of a single dose of etomidate. First, independent of the clinical setting, etomidate causes adrenal dysfunction via 11β-hydroxylase inhibition ranging from 12 to 48 h, making the drug unsuitable for use in elective interventions. Second, in a systematic review with meta-analyses, including 3715 septic patients, the relative risk of death with etomidate was 1.22 (95% confidence interval 1.11 to 1.35). Based on this statistically significant and clinically relevant increase in mortality, a single dose of etomidate has to be avoided in patients with septic shock. Third, in small randomised controlled trials, a single dose of etomidate in trauma patients was associated with an increased incidence of pneumonia (56.7 vs. 25.9% in controls), prolonged intensive care stay (6.3 vs. 1.5 days) and prolonged hospital stay (11.6 vs. 6.4 days). Based on these randomised controlled trials, the use of etomidate should be avoided in unstable trauma patients. Midazolam and ketamine are two valid alternatives with similar intubation and haemodynamic conditions as etomidate but without its adverse effects. Therefore, for safety reasons, etomidate should be avoided in the critical conditions of sepsis and trauma

Cardiovascular changes after administration of aerosolized salbutamol in horses: five cases.

Prevention and treatment of intraoperative hypoxemia in horses is difficult and both efficacy and safety of therapeutic maneuvers have to be taken into account. Inhaled salbutamol has been suggested as treatment of hypoxia in horses during general anesthesia, due to safety and ease of the technique. The present report describes the occurrence of clinically relevant unwanted cardiovascular effects (i.e. tachycardia and blood pressure modifications) in 5 horses undergoing general anesthesia in dorsal recumbency after salbutamol inhalation. Balanced anesthesia based on inhalation of isoflurane in oxygen or oxygen and air and continuous rate infusion (CRI) of lidocaine, romifidine, or combination of lidocaine and guaifenesine and ketamine was provided. Supportive measures were necessary to restore normal cardiovascular function in all horses but no long-term adverse effects were noticed in any of the cases.

Nerve Stimulator–Guided Sciatic-Femoral Block in Pet Rabbits (Oryctolagus cuniculus) Undergoing Hind Limb Surgery: A Case Series

This article describes the clinical applicability of a nerve stimulator–guided technique, previously described in dogs, to block the sciatic and the femoral nerves in 4 pet rabbits (Oryctolagus cuniculus) undergoing hind limb surgeries. Preanesthetic intramuscular doses of medetomidine (0.08 mg/kg), ketamine (15 mg/kg), and buprenorphine (0.03 mg/kg) were administered to the rabbit patients. The rabbits were intubated and general anesthesia was maintained using isoflurane in oxygen. The sciatic-femoral nerve block was performed with 2% lidocaine at a volume of 0.05 mL/kg/nerve. Sciatic-femoral block was feasible in rabbits, and the motoric responses following electrical stimulation of both nerves were consistent with those reported in dogs after successful nerve location. Iatrogenic complications, namely nerve damage and local anesthetic toxicity, did not occur. Based on these results, the authors conclude that the sciatic-femoral nerve block described in dogs can be safely performed in rabbits. Clinical trials are required to assess the analgesic efficacy of the combined sciatic-femoral nerve block in rabbits as a part of multimodal pain management.

Do different anesthesia regimes affect hippocampal apoptosis and neurologic deficits in a rodent cardiac arrest model?

Background Different anesthesia regimes are commonly used in experimental models of cardiac arrest, but the effects of various anesthetics on clinical outcome parameters are unknown. We conducted a study in which we subjected rats to cardiac arrest under medetomidine/ketamine or sevoflurane/fentanyl anesthesia. Methods Asystolic cardiac arrest for 8 minutes was induced in 73 rats with a mixture of potassium chloride and esmolol. Daily behavioral and neurological examination included the open field test (OFT), the tape removal test (TRT) and a neurodeficit score (NDS). Animals were randomized for sacrifice on day 2 or day 5 and brains were harvested for histology in the hippocampus cornus ammonis segment CA1. The inflammatory markers IL-6, TNF-α, MCP-1 and MIP-1α were assessed in cerebrospinal fluid (CSF). Proportions of survival were tested with the Fisher’s exact test, repeated measurements were assessed with the Friedman’s test; the baseline values were tested using Mann–Whitney U test and the difference of results of repeated measures were compared. Results In 31 animals that survived beyond 24 hours neither OFT, TRT nor NDS differed between the groups; histology was similar on day 2. On day 5, significantly more apoptosis in the CA1 segment of the hippocampus was found in the sevoflurane/fentanyl group. MCP-1 was higher on day 5 in the sevoflurane/fentanyl group (p = 0.04). All other cyto- and chemokines were below detection threshold. Conclusion In our cardiac arrest model neurological function was not influenced by different anesthetic regimes; in contrast, anesthesia with sevoflurane/fentanyl results in increased CSF inflammation and histologic damage at day 5 post cardiac arrest.

The Prevention of Delirium and Complications Associated with Surgical Treatments (PODCAST) study: protocol for an international multicentre randomised controlled trial

INTRODUCTION Postoperative delirium is one of the most common complications of major surgery, affecting 10-70% of surgical patients 60 years and older. Delirium is an acute change in cognition that manifests as poor attention and illogical thinking and is associated with longer intensive care unit (ICU) and hospital stay, long-lasting cognitive deterioration and increased mortality. Ketamine has been used as an anaesthetic drug for over 50 years and has an established safety record. Recent research suggests that, in addition to preventing acute postoperative pain, a subanaesthetic dose of intraoperative ketamine could decrease the incidence of postoperative delirium as well as other neurological and psychiatric outcomes. However, these proposed benefits of ketamine have not been tested in a large clinical trial. METHODS The Prevention of Delirium and Complications Associated with Surgical Treatments (PODCAST) study is an international, multicentre, randomised controlled trial. 600 cardiac and major non-cardiac surgery patients will be randomised to receive ketamine (0.5 or 1 mg/kg) or placebo following anaesthetic induction and prior to surgical incision. For the primary outcome, blinded observers will assess delirium on the day of surgery (postoperative day 0) and twice daily from postoperative days 1-3 using the Confusion Assessment Method or the Confusion Assessment Method for the ICU. For the secondary outcomes, blinded observers will estimate pain using the Behavioral Pain Scale or the Behavioral Pain Scale for Non-Intubated Patients and patient self-report. ETHICS AND DISSEMINATION The PODCAST trial has been approved by the ethics boards of five participating institutions; approval is ongoing at other sites. Recruitment began in February 2014 and will continue until the end of 2016. Dissemination plans include presentations at scientific conferences, scientific publications, stakeholder engagement and popular media. REGISTRATION DETAILS The study is registered at clinicaltrials.gov, NCT01690988 (last updated March 2014). The PODCAST trial is being conducted under the auspices of the Neurological Outcomes Network for Surgery (NEURONS). TRIAL REGISTRATION NUMBER NCT01690988 (last updated December 2013).

Effects of opioids on human serotonin transporters.

The serotonin (5-hydroxtryptamine, 5-HT) system plays a role in analgesia and emesis. The aim of this study was to test whether opioids or ketamine inhibit the human 5-HT transporter and whether this increases free plasma 5-HT concentrations. HEK293 cells, stably transfected with the human 5-HT transporter cDNA, were incubated with morphine, hydromorphone, fentanyl, alfentanil, pethidine (meperidine), tramadol, ketamine, and the reference substance citalopram (specific 5-HT transporter inhibitor). The uptake of [(3)H]5-HT was measured by liquid scintillation counting. In a second series of experiments, study drugs were incubated with plasma of ten healthy blood donors and change of 5-HT plasma-concentrations were measured (ELISA). The end point was the inhibition of the 5-HT transporter by different analgesics either in HEK293 cells or in human platelets ex vivo. Tramadol, pethidine, and ketamine suppressed [(3)H]5-HT uptake dose-dependently with an IC50 of 1, 20.9, and 230 μM, respectively. These drugs also prevented 5-HT uptake in platelets with an increase in free plasma 5-HT. Free 5-HT concentrations in human plasma were increased by citalopram 1 μM, tramadol 20 μM, pethidine 30 μM, and ketamine 100 μM to 280 [248/312]%, 269 [188/349]%, and 149 [122/174]%, respectively, compared to controls without any co-incubation (means [95 % CI]; all p < 0.005). No change in both experimental settings was observed for the other opioids. Tramadol and pethidine inhibited the 5-HT transporter in HEK293 cells and platelets. This inhibition may contribute to serotonergic effects when these opioids are given in combination, e.g., with monoamine oxidase inhibitors or selective serotonin reuptake inhibitors.

Role of calcium, glutamate and NMDA in major depression and therapeutic application

Major depression is a common, recurrent mental illness that affects millions of people worldwide. Recently, a unique fast neuroprotective and antidepressant treatment effect has been observed by ketamine, which acts via the glutamatergic system. Hence, a steady accumulation of evidence supporting a role for the excitatory amino acid neurotransmitter (EAA) glutamate in the treatment of depression has been observed in the last years. Emerging evidence indicates that N-methyl-D-aspartate (NMDA), group 1 metabotropic glutamate receptor antagonists and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) agonists have antidepressant properties. Indeed, treatment with NMDA receptor antagonists has shown the ability to sprout new synaptic connections and reverse stress-induced neuronal changes. Based on glutamatergic signaling, a number of therapeutic drugs might gain interest in the future. Several compounds such as ketamine, memantine, amantadine, tianeptine, pioglitazone, riluzole, lamotrigine, AZD6765, magnesium, zinc, guanosine, adenosine aniracetam, traxoprodil (CP-101,606), MK-0657, GLYX-13, NRX-1047, Ro25-6981, LY392098, LY341495, D-cycloserine, D-serine, dextromethorphan, sarcosine, scopolamine, pomaglumetad methionil, LY2140023, LY404039, MGS0039, MPEP, 1-aminocyclopropanecarboxylic acid, all of which target this system, have already been brought up, some of them recently. Drugs targeting the glutamatergic system might open up a promising new territory for the development of drugs to meet the needs of patients with major depression.

Optimized on-line enantioselective capillary electrophoretic method for kinetic and inhibition studies of drug metabolism mediated by cytochrome P450 enzymes.

Pharmacokinetic and pharmacodynamic properties of a chiral drug can significantly differ between application of the racemate and single enantiomers. During drug development, the characteristics of candidate compounds have to be assessed prior to clinical testing. Since biotransformation significantly influences drug actions in an organism, metabolism studies represent a crucial part of such tests. Hence, an optimized and economical capillary electrophoretic method for on-line studies of the enantioselective drug metabolism mediated by cytochrome P450 enzymes was developed. It comprises a diffusion-based procedure, which enables mixing of the enzyme with virtually any compound inside the nanoliter-scale capillary reactor and without the need of additional optimization of mixing conditions. For CYP3A4, ketamine as probe substrate and highly sulfated γ-cyclodextrin as chiral selector, improved separation conditions for ketamine and norketamine enantiomers compared to a previously published electrophoretically mediated microanalysis method were elucidated. The new approach was thoroughly validated for the CYP3A4-mediated N-demethylation pathway of ketamine and applied to the determination of its kinetic parameters and the inhibition characteristics in presence of ketoconazole and dexmedetomidine. The determined parameters were found to be comparable to literature data obtained with different techniques. The presented method constitutes a miniaturized and cost-effective tool, which should be suitable for the assessment of the stereoselective aspects of kinetic and inhibition studies of cytochrome P450-mediated metabolic steps within early stages of the development of a new drug.