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A high-performance liquid chromatography method for the determination of benzodiazepines and their metabolites in whole blood and serum using mass spectrometry (MS) and photodiode array (PDA) detection is presented. The combination of both detection types can complement each other and provides extensive case relevant data. The limits of quantification (LOQ) with the MS detection lie between 2 and 3 microg/l for the following benzodiazepines or metabolites: 7-amino-flunitrazepam, alprazolam, desalkyl-flurazepam, desmethyl-flunitrazepam, diazepam, flunitrazepam, flurazepam, alpha-hydroxy-midazolam, lorazepam, midazolam, nitrazepam, nordazepam and oxazepam, respectively 5 microg/l for lormetazepam and 6 microg/l for bromazepam. The LOQ of clobazam determined with the PDA detector is 10 microg/l. A convenient approach for determining the measurement uncertainty of the presented method--applicable also for other methods in an accreditation process--is presented. At low concentrations (<10 microg/l), measurement uncertainty was estimated to be about 50%, and at concentrations >180 microg/l, it was estimated to be about 15%. One hundred and twenty-eight case data acquired over 1 year are summarised.

Objective: To examine the physiologic and pharmacokinetic effects of a technique of total intravenous anesthesia in ponies., Animals: 6 healthy ponies., Procedure: Ponies were premedicated with acepromazine (0.03 mg/kg of body weight, IV) and xylazine (1.0 mg/kg, IV). Two minutes later, anesthesia was induced with ketamine (2.0 mg/kg, IV) followed by climazolam (0.2 mg/kg, IV). Anesthesia was maintained for 120 minutes by an infusion of climazolam (0.4 mg/kg/h) and ketamine (6.0 mg/kg/h). Oxygen (5 L/min) was supplemented. 20 minutes after the infusion was stopped sarmazenil (0.04 mg/kg, IV) was administered. Cardiovascular and respiratory function measurements were taken before and after premedication, and during anesthesia. Plasma cortsol, ACTH, and catecholamine concentrations were used to assess adrenal and pituitary gland function Ketamine and climazolam kinetics were calculated, on the basis of plasma drug concentrations., Results: There were no significant changes from pre-xylazine values in heart rate, respiratory rate, arterial blood pressure, cardiac index, systemic vascular resistance, or arterial PO2, PCO2, and pH. Plasma cortisol concentration decreased during anesthesia, but plasma ACTH and catecholamine concentrations did not change. Recovery was fairly smooth, but some excitement and ataxia were noted in 2 ponies., Conclusion: Ketamine-climazolan infusion appeared suitable for maintenance of anesthesia in ponies, although recovery was not ideal in 2 of 6 ponies.

A simple, selective high-performance liquid chromatographic method with ultraviolet detection at 220 nm is described for quantitation of midazolam and its primary metabolite 1-hydroxymidazolam in 100 microL plasma samples from premature infants. A mobile phase of acetonitrile:tetrahydrofuran:phosphate buffer (0.01 M, pH 6.7) (35:5:60 v/v) was pumped at 1 mL/min through a C8 Symmetry (150 x 3.9 mm) column. Plasma (100 microL) was extracted with 10% v/v isopropyl alcohol in dichloromethane containing 25 ng/mL climazolam (internal standard, IS) followed by back extraction into phosphoric acid (0.02 M). 1-Hydroxymidazolam, midazolam, and climazolam (IS) were eluted at 4.9, 7.4, 8.4 min, respectively. Recoveries were > 70%. Calibration curves in blank plasma were linear (r > 0.999) from 12.5 to 800 ng/mL. Within-day and between-day imprecision (CV%) was 1.8-6.5%, and 4.1-8.8%, respectively. Inaccuracy was 12.3%. Application of the method was demonstrated by a pharmacokinetic analysis of midazolam and 1-hydroxymidazolam in plasma drawn from 15 premature neonates after a single intravenous dose of midazolam (0.1 mg/kg).

Aim of the study was the clinical use of a completely antagonisable anaesthesia in guinea pigs. The dosage based on experimental studies. Fentanyl, climazolam and xylazine (0.05/2.0/2.0 mg/kg) were injected i.m. for combination anaesthesia and naloxone, sarmazenil and yohimbine (0.03/0.3/2.0 mg/kg) i.v. were used for antagonisation. 18 guinea pigs (17 female, one male) with an average body weight of 1177 g aged between eight months and five years were anaesthetised for various surgical procedures. The following clinical parameters were measured: body temperature, muscle relaxation, analgesia, hyperacusia, righting-, interdigital- and palpebral reflex. After antagonisation the return of righting reflex, the appearance of muscle trembling, the first walking without ataxia and the first food uptake were observed. The investigations showed that this kind of anaesthesia is suitable for surgical procedures in the guinea pig and is very safe because of the complete antagonisation of the depressing respiratory and circulatory effects of anaesthesia.

The cardiopulmonary effects of 15 mg ketamine/kg b. wt. and 15 mg ketamine/kg in combination with 1 mg climazolam/kg b. wt. or in combination with 18.5 mg Xylazine/kg b. wt. were tested after intramuscular injection in 17 pigs. Blood pressure, heart rate, respiration rate, arterial pH, pO2, pCO2, lactate in venous blood and body temperature were evaluated over a period of 2 hours. The activities of CK and ASAT in blood plasma were measured until 24 hours after the injections. There was little influence on heart rate and blood pressure after ketamine alone and also after one of the two combinations. Ketamine produced a tachypnea which was seen to a smaller extent also following injection of combinations of the drugs. This tachypnea produced a slight respiratory alkalosis. The metabolic acidosis, indicated by increasing lactate levels, occurring initially and during the recovery period was largely compensated by the respiratory alkalosis. The increased levels of CK and ASAT are explained by the destruction of muscle tissue at the locus of the injection.

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether-methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 +/- 5.4% (S.D.) and 90.3 +/- 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration-time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].

14C-labeled 8-chloro-6-(2-chlorophenyl)-4H-imidazo [1,5a][1,4]-benzodiazepine-3-carboxamide hydrochloride, 1 X HCl, was administered iv to two dogs. 14C-1 X HCl was extensively metabolized; in one dog, only 2% of the administered radioactivity was excreted as unchanged 1. The 4-hydroxy derivative of 1, compound 2, accounted for an additional 5% of the dose. Three urinary metabolites were identified by enzyme hydrolysis and NMR spectroscopy as conjugates of the phenolic 3'-, 4'-, and 5'-hydroxy derivatives of 1. In both dogs, the 4'-hydroxy derivative was the major identified metabolite (21 and 15% of the dose). Oral administration of 100 mg/kg/day of unlabeled 1 to dogs for 11 weeks resulted in the excretion of four urinary metabolites, the conjugated 3'-hydroxy and 4'-hydroxy derivatives of both 1 and 2. This prominent excretion of conjugated phenolic metabolites does not fit the usual pattern of 1,4-benzodiazepine metabolism in the dog.

A method is described for measuring imidazobenzodiazepine-3-carboxamide, a new anxiolytic agent, in human plasma. A tetradeuterated analogue of the analyte is used as the internal standard. The drug and its internal standard are (1) extracted from plasma at pH 9 with benzene containing 20% 1,2-dichloroethane, (2) derivatized with pentafluoropropionic anhydride in the presence of triethylamine and (3) the nitrile derivative of the analyte and internal standard are analyzed by gas chromatography (GC)-negative chemical-ionization mass spectrometry (CIMS) using methane as both GC carrier gas and CI reagent gas. The mass spectrometer is set to monitor the intense (M-HCl)- ions of imidazobenzodiazepine-3-nitrile and its tetradeuterated analogue at m/z 316 and m/z 320, respectively. Quantitation of an experimental plasma sample is based on the comparison of the m/z 316 to m/z 320 ion ratio in each sample to that obtained from the analyses of control plasma spiked with various amounts of the drug and a fixed amount of internal standard. The limit of quantitation of the method is approximately 100 pg ml-1 of plasma and the precision (relative standard deviation) at a plasma concentration of 1 ng ml-1 is 4%.

In dwarf goats fasted for 2 h, i.v. administration of the benzodiazepine (BZ) agonists diazepam (60 micrograms/kg), brotizolam (2 and 4 micrograms/kg) and climazolam (100 micrograms/kg) induced hyperphagic effects, whereas i.v. injections of the BZ-antagonist flumazenil (R degrees 15-1788; 0.5 mg/kg), the anthelmintic ivermectin (0.1 mg/kg), the 5-HT2 antagonist ritanserine (0.1 mg/kg), ACTH (10 micrograms/kg) and prednisolone (1 mg/kg) were inactive in a 30-min feeding test. Both the BZ-antagonist R degrees 15-3505 (greater than or equal to 0.1 mg/kg) and the opiate receptor antagonist naloxone (0.1 mg/kg) had anorectic effects in dwarf goats given 30 min access to a palatable pelleted concentrate. The hyperphagic effects of climazolam and brotizolam were not antagonized by flumazenil, whereas similar doses of this drug completely reversed muscle incoordination and ataxia induced by much higher doses of these BZ-agonists. In the combination experiments with naloxone and BZ-agonists, naloxone antagonized the hyperphagic effects of both diazepam and brotizolam. Similarly, in the diazepam-R degrees 15-3505 study, there was a significant effect of diazepam and a significant inhibition of this effect by R degrees 15-3505 (50 micrograms/kg). In the diazepam-ivermectin combination experiment no evidence for drug potentiation was found. These results and the mode of action of the above mentioned drugs are discussed in relation to feeding behaviour.

This contribution describes a modern technique of producing general anesthesia in the Göttingen minipig. The method of anesthesia selected is dependent on the duration and type of surgery. The drugs used to produce anesthesia should not interfere with the experimental aims, therefore only drugs with well known actions and a standardized anesthesiological procedure should be used. Climazolam, Ketamine and atropine are preferred for induction of anesthesia. After endotracheal intubation, inhalation anesthetic mixed with oxygen and nitrous oxide is used to maintain general anesthesia. Controlled ventilation is necessary to prevent the animals from developing respiratory acidosis. Postoperative analgesia is provided by orally administering acetylsalicylate or morphine-sulfate pentahydrate at 12 hour intervals.

A trial of the sedative effects of a novel benzodiazepine, climazolam, and of a mixture of climazolam and fentanyl-fluanisone was undertaken in male and female Sprague Dawley and Lewis strain rats (Rattus norvegicus) of different ages. At the recommended intravenous therapeutic dosage of 0.25 mg/kg, climazolam sedated the rats but failed to inhibit the righting reflex or responses to painful stimuli. Even at 25 times the recommended dosage, there was no anaesthetic effect. Predictable and satisfactory surgical anaesthesia was not obtained with the mixture of climazolam and fentanyl-fluanisone administered intraperitoneally. In contrast with diazepam and midazolam, climazolam failed to potentiate the sedative analgesic properties of fentanyl-fluanisone: indeed many animals retained their tail and pedal pinch reflexes. The results also revealed significant differences in effect related to strain and age but not to sex.